Enzyme-based compositions and methods for removing dental calculi

Abstract

Compositions and formulations for removing dental calculi are disclosed comprising an enzyme or other biocatalyst that cleaves (i) a surface accessible nucleic acid polymer and (ii) a glycoprotein carbohydrate chain at galactose residues. Optionally, one or more proteolytic enzymes are included. The composition disrupts the structural integrity of the stone so that it is easy to remove without the need for special treatment by a training primordial dental professional. Also disclosed are methods for removing dental calculi using the disclosed compositions and formulations.

Description

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 478,641, filed January 5, 2023, entitled “Enzyme-based Compositions and Methods for Removing Dental Calculi,” the contents of which are hereby incorporated by reference in their entirety. Technical Field

[0002] This disclosure relates to compositions and methods for reducing and removing dental plaque and tartar in humans and non-human animals.

[0003] Merging of sequence lists This application contains a sequence list, which has been filed with the Patent Center in .XML format and is hereby incorporated herein by reference in its entirety. The WIPO sequence list was created on January 4, 2024, named “119879-783212 Sequence Listing.xml”, and is 8 kilobytes in size. Background Technology

[0004] Dental plaque, also known as tartar, is a petrified / mineralized substance that, if left unremoved, gradually accumulates and eventually leads to periodontal diseases such as gingivitis and periodontitis. This results in a chronic inflammatory state that can induce diabetes or heart disease in humans and non-human animals (including but not limited to dogs and cats). Currently, tartar removal is performed by trained dental professionals, ideally once or twice a year, and is generally limited to scraping the tooth surface with metal alloy dental probes or ultrasonic devices to break down and remove the tartar from the enamel. Dental cleaning agents used in routine daily oral hygiene practices, such as toothpaste and mouthwash, are not effective at removing tartar.

[0005] The World Health Organization estimates that oral diseases affect nearly 3.5 billion people globally, with untreated dental caries being the most common health condition and 1 billion people suffering from severe periodontal disease. The estimated number of cases of oral disease worldwide exceeds the combined total of mental disorders, cardiovascular diseases, diabetes, chronic respiratory diseases, and cancer by approximately 1 billion. Furthermore, it is the most prevalent health problem in high-income countries. In the United States, data from the 2018 NHANES estimates that 42% of American adults with teeth have periodontitis and over 25% have untreated cavities. However, oral health problems can extend beyond the oral cavity to systemic manifestations such as diabetes, cardiovascular disease, and premature infants with low birth weight. This link is not precisely defined but is generally associated with chronic inflammatory states stemming from periodontal inflammation. Oral biofilms are a major cause of various oral diseases, such as dental caries and periodontal disease. A key characteristic of biofilms, including dental plaque, is extracellular DNA (eDNA), which contributes to the gel-like properties of plaque formation. Bacteria living in dental biofilms are a major cause of periodontal disease. Furthermore, bacterial species living in tooth biofilms also metabolize sugars and produce acids, which lowers the pH of the biofilm fluid. This leads to a mineral difference between the acidic biofilm fluid and the teeth, resulting in a loss of tooth mineral composition, clinically manifested as tooth structure loss and cavitation. Controlling and preventing the accumulation of tooth biofilm, plaque, and tartar are goals for a healthy oral environment.

[0006] Oral hygiene, especially brushing teeth, is a basic and daily habit for almost everyone, even in developing countries, and its purpose is to remove plaque. However, more than 40% of plaque cannot be removed, even by trained individuals, and may mineralize to become tartar. Current toothpastes are not very effective at preventing tartar formation but rather at slowing its rate.

[0007] Therefore, there is a critical unmet need for products intended to reduce, remove, and / or prevent tartar buildup for routine household use. Summary of the Invention

[0008] In various aspects of this disclosure, a first dental composition is provided which comprises or is substantially composed of at least two enzymes that target calculi. Other aspects and features of this disclosure are detailed below.

[0009] In one aspect, this disclosure includes a dental composition comprising a DNase, a chymotrypsin or a functional variant or derivative thereof, and an orally acceptable additive, carrier or excipient, or any combination thereof. In some aspects, the dental composition is a dental cleaning agent.

[0010] In some respects, dental compositions may not include β-galactosidase.

[0011] In some aspects, the dental composition may include an amount of DNase of about 50,000 to about 250,000, or about 100,000 to about 200,000, or about 150,000 to about 200,000, or 150,000, or 160,000, or 170,000, or 180,000, or 185,000, or 190,000, or 200,000 Kunitz units / mL.

[0012] In some aspects, dental compositions may also include DNase, wherein the DNase is DNase I or a functional variant or derivative thereof.

[0013] In some aspects, the dental composition may also include chymotrypsin or a functional variant or derivative thereof, wherein the chymotrypsin or functional variant or derivative is present in an amount of about 100 to about 5,000, or about 500 to about 4,000, or about 1,000 to about 3,000, or 1,500, or 1,600, or 1,700, or 1,800, or 1,900, or 2,000, or 2,100, or 2,200, or 2,300, or 2,400, or 2,500, or 2,600, or 2,700, or 2,800, or 2,900 or 3,000 units / mL.

[0014] In some aspects, the dental composition may also include orally acceptable additives, wherein the orally acceptable additives are thickeners and / or gelling agents selected from gellan gum (low-acyl or high-acyl), glycerin, silica, guar gum, xanthan gum, polyethylene glycol, polyvinylpyrrolidone and copolymers thereof, polylactic acid, polyglycolic acid, long-chain fatty acid alcohols, cellulose-based polymers, acrylate polymers, and any combination thereof. In some aspects, the dental composition is a dental cleaning agent.

[0015] In some respects, dental compositions may also include oral-acceptable carriers, such as ethanol, isopropanol, glycerin, sorbitol, or any combination thereof.

[0016] In some respects, dental compositions may further include surfactants, such as cetearyl alcohol polyether or stearyl alcohol polyether.

[0017] In some aspects, the dental composition comprises DNase 1 or a functional variant or derivative thereof, chymotrypsin or a functional variant or derivative thereof, and one or more of 10-20% glycerol, 0.00001-0.000001% cetearyl alcohol polyether-25, 0.1%-0.5% xanthan gum, 1-10% sorbitol, and 5-25% silica. The composition may further comprise water.

[0018] In some respects, the dental composition is in the form of a paste or gel, such as toothpaste or dental floss, or tooth powder, or mouthwash. In some respects, the dental composition is a dental floss. In some respects, the dental composition is combined with a dental tray, capsule applicator, oral pick, or dental floss to provide an oral hygiene device.

[0019] In some aspects, this disclosure also includes the use of a dental composition or an apparatus comprising a dental composition for the removal of tartar in a subject in need of it. In some aspects, the dental composition is used as a dental cleaning agent. In some aspects, the subject is a mammal. In some aspects, the subject is a human.

[0020] In some aspects, this disclosure provides a method for reducing or removing dental calculus in a subject in need of it. The method includes contacting a tooth surface with an effective amount of a dental composition comprising DNase I or a functional variant or derivative thereof, and chymotrypsin or a functional variant or derivative thereof. In some aspects, the dental composition is formulated as a dental cleaning agent.

[0021] In some respects, the dental composition does not contain β-galactosidase.

[0022] In some respects, the composition further comprises an orally acceptable additive, carrier or excipient, or a combination thereof.

[0023] In some aspects, the composition comprises a paste or liquid and contact with the tooth surface includes application of the composition using a brush, a tray, a mouthwash, or any combination thereof. In some aspects, the dental composition is formulated as a dental cleaning agent.

[0024] The method may also include the application of the method at least once a day, at least twice a day, or at least three times a day for a period of time of at least one day or longer, one week or longer, one month or longer, or one year or longer.

[0025] In some aspects, this disclosure provides a method for reducing or removing dental calculus in subjects who require it, wherein the method comprises contacting a tooth surface with an effective amount of a dental composition (e.g., a dental cleaning agent).

[0026] In some respects, the subjects were mammals. In some respects, the subjects were dogs, horses, cats, or primates. In some respects, the subjects were humans.

[0027] In some aspects, this disclosure also includes a kit comprising a dental composition and instructions for use. In some aspects, the kit contains the dental composition as a dental cleaning agent. The kit may further include at least one applicator selected from toothbrushes, oral cleaning sticks, dental floss, and dental trays.

[0028] Other technical features are readily apparent to those skilled in the art from the following figures, description and claims. Attached Figure Description

[0029] The aspects of this disclosure are illustrated in the following diagram: Figure 1 A schematic diagram of the design for a proof-of-concept in vivo study. A randomized trial was conducted to evaluate the safety and efficacy of an exemplary formulation for removing existing stone deposits over a 4-week trial period.

[0030] Figure 2 Provides an overview of the scoring associated with the Volpe-Manhold Index. The score is based on the cumulative calculus score, which is the sum of the lingual (tongue-facing) surfaces of the six lower anterior (incisor) teeth.

[0031] Figure 3 As estimated by the Volpe-Manhold score, this formulation significantly reduced total tartar.

[0032] Figure 4 The percentage change in tartar was shown across all subjects in the trial. The change in tartar was consistent across all subjects; tartar increased in all but two control patients and decreased in all but one subject.

[0033] Figure 5 This indicates a positive response from the subjects to the disclosed formulation.

[0034] Figure 6 An overview of the research group's daily brushing schedule.

[0035] Figure 7 This shows the individual V-MI values ​​at baseline and 4 weeks in different treatment groups.

[0036] Figure 8 This displays the percentage of patients who gave a favorable score (4 or 5 on a 5-point scale) to how clean their teeth felt. The score is based on a 5-point scale, where "1" is the least clean you can imagine and "5" is the cleanest you can imagine. Detailed Implementation

[0037] This disclosure provides novel dental compositions and formulations for reducing or removing tartar in humans and non-human animals (including, but not limited to, dogs and cats), methods for reducing or removing tartar, and kits comprising the compositions and formulations.

[0038] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. The following references provide general definitions for many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise stated, the following terms have the meanings assigned to them.

[0039] When elements of this disclosure or their preferred embodiments are introduced, the articles “a,” “an,” “the,” and “said” are intended to mean the presence of one or more elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that additional elements besides those listed may be present.

[0040] A range may be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another embodiment includes from one particular value and / or to another particular value. Similarly, when a value is expressed as an approximation using the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each range are significant both relative to and independent of the other endpoint. It is also understood that many values ​​are disclosed herein, and each value, in addition to itself, is also disclosed herein as “about” that particular value. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed, “less than or equal to” that value, “greater than or equal to” that value, and possible ranges between the values ​​are also disclosed, as properly understood by a person skilled in the art. For example, if the value “10” is disclosed, then “less than or equal to 10” and “greater than or equal to 10” are also disclosed. It is also understood that throughout the application, data is provided in many different formats, and that this data represents a range of endpoints and starting points, as well as any combination of data points. For example, if specific data point "10" and specific data point 15 are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as those between 10 and 15, are disclosed. It is also understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0041] As used herein, the terms “optional” or “optionally” mean that the event or situation described below may or may not occur, and the description includes both the scenario in which the event or situation occurs and the scenario in which it does not occur.

[0042] As used herein, the terms “calculus” or “tartar” are used interchangeably and refer to a form of hardened dental plaque. It is caused by the deposition of minerals from saliva and gingival crevicular fluid in plaque on the teeth. This deposition process kills bacterial cells within the plaque, but the resulting rough and hardened surface provides an ideal surface for further plaque formation. This leads to calculus buildup, which can damage the health of the gums (dental gingiva). Calculus can form along the gingival line (where it is called supragingival) and within the narrow gingival sulcus that exists between the teeth and gums (where it is called subgingival).

[0043] As used herein, the term "stone-targeting enzyme" refers to any of the following: (i) an enzyme that hydrolyzes the sugar-phosphate bonds of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); (ii) a proteolytic enzyme that hydrolyzes amino acid linkages in a protein backbone; and optionally (iii) an enzyme that hydrolyzes glycoprotein carbohydrates at linkages containing galactose, N-acetyl-glucosamine, fucose, N-acetyl-galactosamine, or sialic acid. Stone-targeting enzymes include, but are not limited to, DNases, chymotrypsin, and other proteases, including, but not limited to, the specific enzymes disclosed elsewhere herein. In all respects, stone-targeting enzymes are not β-galactosidases, i.e., compositions as disclosed herein specifically exclude β-galactosidases.

[0044] As used interchangeably herein, the terms “polypeptide” and “protein” refer to a polymer of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be discontinuously segmented by non-amino acid components. These terms also include polymers of amino acids that are native or modified by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-native amino acids), and other modifications known in the art. For example, as used herein, the protein or polypeptide sequence of an enzyme (as a non-limiting example, DNase I (also known as DNase I)) or chymotrypsin also includes derivatives and variants of the polypeptide, and modifications as provided herein.

[0045] The terms “derivative” or “variant,” when used herein to refer to a polypeptide, mean a polypeptide that is related to the wild-type polypeptide, for example, by its amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzyme activity), and / or function. A derivative or variant of a polypeptide may contain one or more amino acid changes (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof, compared to the wild-type polypeptide. As used herein, the term “functional variant or derivative” includes proteins that have the same or enhanced / reduced functional level compared to their wild-type counterpart.

[0046] Throughout this specification, unless the context otherwise requires, the words “comprise” and “include” and their variations (e.g., “comprises”, “comprising”, “includes”, “including”) shall be understood to mean including the said component, feature, element or step, or a group of components, features, elements or steps, but not excluding any other integer or step, or a group of integers or steps.

[0047] As used herein, “and / or” should be understood to specifically disclose each of two specified features or components, and or not together with the other. For example, “A and / or B” should be considered as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein.

[0048] Furthermore, this disclosure also contemplates the exclusion or omission of any feature or combination of features set forth herein in some embodiments. For illustration, if the specification describes a complex as comprising components A, B, and C, it is specifically intended that any one or any combination of A, B, or C may be omitted or denied, individually or in any combination.

[0049] Unless otherwise stated herein, the listing of value ranges herein is intended only as a shorthand for individually referring to each individual value falling within that range, and each individual value is incorporated into the specification as if it were listed separately herein. For example, if the concentration range is stated as 1%–50%, it is expected that values ​​such as 2%–40%, 10%–30%, or 1%–3% are explicitly listed herein. These are merely examples of what is specifically expected, and it is believed that all possible combinations of values ​​between (including) the listed minimum and maximum values ​​are explicitly stated in this disclosure.

[0050] As used herein, the terms “individual,” “subject,” “host,” and “patient” are used interchangeably and refer to any mammalian subject for diagnosis, treatment, prevention, or therapy, such as a human, pet, livestock, horse, or other animal. As used herein, the terms “subject” and “patient” are used interchangeably and refer to both human and non-human animals. The term “non-human animal” in this disclosure includes all vertebrates, such as mammals and non-mammals, such as non-human primates, dogs, cats, etc. In some respects, the subject may be a human.

[0051] As used herein, the terms “administer,” “administering,” or “administration” refer to the oral administration of a compound or composition, regardless of its form. For example, the methods disclosed herein include administering an effective amount of a compound or composition to achieve a desired or stated effect. The compositions disclosed herein may optionally be administered to the oral cavity or a specific portion of the oral cavity (e.g., a brush, toothbrush), floss or dental flosser (e.g., dental floss), irrigator (e.g., a water flosser), cleaning stick (e.g., a dental probe or toothpick), scraper (e.g., a tongue cleaner or tongue scraper), spatula, rod, retainer, mouthguard, or mouth protector, etc., using one or more devices known in the art.

[0052] As used herein, the terms “treat,” “treating,” or “treatment” mean the partial or complete relief, suppression, improvement, or reduction of a disease or condition suffered by a subject. This means any manner in which one or more symptoms of a disease or disorder (e.g., calculus) may be improved or otherwise beneficially altered. As used herein, improvement of symptoms of a particular disorder (e.g., calculus) means any reduction, whether permanent or temporary, persistent or transient, attributable to or related to treatment by the compositions and methods of this disclosure. In some aspects, treatment may promote or result in, for example, a reduction in the amount of calculus (e.g., in the subject) relative to the amount of calculus before treatment; or a reduction in one or more symptoms in the subject associated with one or more periodontal diseases (e.g., gingivitis) relative to the subject's symptoms before treatment.

[0053] As used herein, the terms “prevent,” “preventing,” and “prevention” refer to a reduction in the occurrence of disease or a reduction in the risk of disease or its associated symptoms in a subject. Prevention can be complete, such as the complete absence of disease in a subject. Prevention can also be partial, resulting in fewer, later, or slower-developing diseases in a subject than would occur without this disclosure.

[0054] Since various changes can be made to the above-described units and methods without departing from the scope of this disclosure, it is intended that everything contained in the above description and the examples given below be interpreted in an illustrative rather than restrictive sense.

[0055] (I) Calculus Stones have a complex composition that varies from individual to individual, depending on diet, genetics (subtle variations in saliva composition), and the composition of the oral microbiome, but the mechanisms of their deposition remain to be determined. A recent report (Aghanashini, S. et al., J. of Health Sciences and Res. (2016) 7:42-50) reviewed six theories of stone formation: the Booster mechanism; the Epitactic concept; the inhibition theory; the conversion theory; the bacterial theory; and the enzymatic theory. All theories except the bacterial theory focus on inorganic components. The bacterial theory posits that bacteria adhering to the tooth surface are responsible for stone formation. Research from the United States in the 1960s and 1970s focused on the composition of stone hydrolysis products and did not venture to identify large organic molecules (if any). Since then, no further progress has been made in the United States. Therefore, the field has not consolidated around a single perspective on stone development.

[0056] Recent studies of dental calculus collected from ancient teeth have evaluated the presence of DNA and shown it to be an extremely robust molecule. DNA is negatively charged, and in addition to DNA, calculus contains salivary glycoproteins, among which mucin is the most abundant negatively charged member. Calcium is positively charged and abundant in saliva, mediating salt bridges between the negative charges in DNA and mucin. Another component of calculus formation is the protein network structure generated by transglutaminase activity, which crosslinks glutamine residues on a protein with lysine residues on adjacent protein molecules. This process is thought to originate at the tooth / gingival interface, where surface proteins of the epithelial surface become crosslinked with soluble salivary proteins, creating a network structure extending from the epithelium to the enamel surface. Thus, at the tooth / gingival interface, epithelial surface proteins are crosslinked with other salivary and food proteins by transglutaminase, providing additional building blocks for mineralization.

[0057] The model disclosed herein begins with chemically cleaned tooth enamel in a normal oral environment. It is not intended to be bound by theory, as it is believed that DNA and mucins will interact with enamel hydroxyapatite (Ca). 10Positively charged calcium ions bind to the outer surface of (PO4)6(OH)2, and salivary calcium further associates with the outer surface of enamel-associated DNA and mucin, initiating lamination by attracting more calcium, DNA, and mucin. It is further thought that the less abundant but much larger DNA from the oral environment will associate with available hydroxyapatite calcium and with calcium bound to the outer surface of immobilized mucin. DNA and mucin can be approximated as cylindrical structures with charged groups arranged radially. Therefore, only a small fraction of the charged groups will occupy the molecular surface interacting with the underlying matrix (1 / 4-5 in the case of DNA). Thus, the effectiveness of the co-interaction depends on molecular length to provide the strength required for the interaction to have sufficient duration in a highly hydrated environment. The strength of the co-interaction is reduced by decreasing the length of the DNA polymer or by cleaving negatively charged protein and / or carbohydrate chains terminated on the glycoprotein peptide backbone. With the gradual reduction in the length of the negatively charged polymer due to DNA cleavage and the increased cleavage of glycoprotein chains from the glycoprotein peptide backbone, the co-interaction essential to the calculus and plaque structure is lost, allowing components to be flushed away.

[0058] DNA is generally considered highly resistant to cleavage. Chemical hydrolysis of DNA requires boiling in acid. While the length of DNA relative to a bacterial cell is on a macroscopic scale, the roles of DNA and glycoproteins, such as mucins, in plaque and stone formation are on a molecular scale. The number of association events achievable between mucins and DNA is enormous, contributing to the strength of stone aggregates. Therefore, the extensive length and chemical impermeability of DNA make it a strategic component of stones. Although glycoproteins are not as robust as DNA, they are strong enough to play important structural roles. The association of both glycoproteins and DNA with immobilized calcium is enhanced by synergy. In this case, synergy is analogous to base pairing in double-stranded DNA. Although DNA base pair interactions are established through hydrogen bonds (weak, compared to the dominant ionic salt bridges in stones), the precise geometry of the double helix and the large number of base pairs make separating the two strands difficult enough to require near-boiling temperatures. In the case of stone formation, when a salt bridge loosens through competition with water, the remaining upstream and downstream salt bridges will keep the two loosened bridge partners close enough to facilitate their rapid reassociation.

[0059] Purely chemical methods for tartar removal require a chemically extreme environment that causes oral damage. However, biological / enzymatic methods offer a non-toxic, effective, and efficient way to disintegrate tartar. DNA can be cleaved at accessible sugar-phosphate bonds anywhere along its length by deoxyribonucleases (DNases), reducing the length of the DNA polymer to short DNA fragments (oligonucleotides) with a proportional loss of cohesion. The elements of calcium-associated mucin glycoproteins are sulfate and sialic acid groups (both negatively charged) at the ends of the carbohydrate chains. Salivary mucins have diverse and large carbohydrate chains. They terminate with neutral sugars (56%), sialic acid (26%), and sulfates (19%), with average chain lengths of 13, 17, and 41 units, respectively (Thomsson, KA, Glycobiology (2002) 12: 1-14). The carbohydrate chains are primarily composed of galactose, fucose, and N-acetylglucosamine, with a smaller amount of N-acetylglucosamine. Mucin glycoproteins dimerize end-to-end and then continue to form higher-order structures. The microbes of the oral and gut microbiome have evolved to utilize mucin carbohydrates as a nutrient source. They secrete N-acetyl-glucosaminease, β-galactosidase, N-acetyl-galactosidase, fucoidosidase, and neuraminidase (sialic acidase) to cut carbohydrate chains into smaller sizes for nutrient absorption (Derrien, M., Gut Microbes (2010) 1:254-268). Although the most accessible regions for cleaving carbohydrate chains with β-D-galactosidase and β-N-acetyl-D-glucosaminease are at the galactose and N-acetyl-glucosamine residues in the middle of the chain, any cleavage that removes acidic sulfate and sialic acid units from the protein chain will eliminate the synergy, which is important in stone and plaque structures.

[0060] During the formation of DNA / mucin / calcium complexes, they mix with other salivary proteins and dietary proteins, which are continuously cross-linked by transglutaminase. Over time, more calcium diffuses into the aggregates, further compacting the stones during their mineralization. This underscores the role of additional enzymes (e.g., chymotrypsin) targeting the polypeptide components of these glycoproteins, as also found in this paper, in effectively enhancing the breakdown of stone and plaque structures.

[0061] Since gallstones are solid, they only expose a two-dimensional surface to the environment. Therefore, the activity of a single enzyme, such as DNase, is limited to digesting the DNA accessible to the surface. It cannot reach the DNA buried beneath the exposed layer of cross-linked proteins. The same is true if only proteases, such as chymotrypsin, are used. They digest the exposed cross-linked protein network until what remains on the surface is a completely impermeable salt-bridged DNA / mucin layer. This necessitates the use of a combination of enzymes to gradually attack the surface of the gallstone.

[0062] (II) Dental compositions and formulations The compositions and formulations according to this disclosure comprise at least two calculus-targeting enzymes, and may include combinations of two or more calculus-targeting enzymes, each enzyme having different catalytic activities capable of hydrolyzing different chemical components in the calculus structure. The calculus-targeting enzymes include enzymes that hydrolyze any of the following: (i) sugar-phosphate bonds of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA); (ii) proteins, including glycoproteins; and (iii) glycoprotein carbohydrates. The calculus-targeting enzymes, including DNases, RNases, and proteases, act on readily accessible nucleic acid and glycoprotein elements on the surface of dental plaque and calculus. For example, when surface-accessible DNA is cleaved into shorter fragments by a DNase, the shorter fragments lose their ability to co-associate with the underlying matrix, thereby allowing water to displace the cleaved DNA and proteins. Similarly, when surface-accessible glycoprotein carbohydrate chains are cleaved by β-galactosidases, for example, the underlying sialic acid / sulfate-calcium associations become singular, no longer attached to the protein backbone or part of the cohesive structure, and are effectively replaced by large amounts of water. However, unexpectedly, this function can also be provided by the presence of proteases that perform proteolysis and promote glycoprotein degradation, thus eliminating the need for β-galactosidases. As surface-accessible nucleic acids and glycoproteins are cleaved and replaced, the nucleic acids and glycoproteins below the cleaved nucleic acids and glycoproteins are now accessible by DNases or RNases, as well as proteolytic enzymes. These enzymes repeat a progressive cycle of cleavage and replacement, thereby disintegrating the three-dimensional plaque and stone structure.

[0063] Therefore, in one aspect, the dental composition according to this disclosure comprises at least two enzymes targeting calculi: a first enzyme or other biocatalyst selected from those having catalytic activity of hydrolyzing phosphate ester bonds in nucleic acids, including DNA and RNA; and a second enzyme or other biocatalyst having catalytic activity of hydrolyzing proteins, and any combination thereof. In each aspect, the protein-hydrolyzing enzyme is chymotrypsin or a functional variant or derivative thereof.

[0064] In the disclosed compositions, at least one stone-targeting enzyme or other biocatalyst may be a DNase and / or RNase having catalytic activity of hydrolyzing phosphate ester bonds in DNA and RNA. In one aspect, at least one stone-targeting enzyme is a DNase, such as DNase I. In another aspect, at least one stone-targeting enzyme is an RNase. In yet another aspect, at least one stone-targeting enzyme is a nuclease that is both a DNase and an RNase, such as NuCLEANase food grade (commercially available from c-LEcta GmbH, Leipzig, Germany).

[0065] The amount of nuclease (DNase and / or RNase) can vary from about 50,000 Kunitz units / mL to about 800,000 Kunitz units / mL. For example, in various aspects, the nuclease is present in the composition in amounts of about 100,000 Kunitz units / mL, about 200,000 Kunitz units / mL, about 300,000 Kunitz units / mL, about 400,000 Kunitz units / mL, about 500,000 Kunitz units / mL, about 600,000 Kunitz units / mL, about 700,000 Kunitz units / mL, or about 750,000 Kunitz units / mL. In some aspects, the amount of nuclease can be from about 200 Kunitz units / mL to about 100,000 Kunitz units / mL. For example, in various aspects, the nuclease is present in the composition in amounts of about 200 Kunitz units / mL, about 300 Kunitz units / mL, about 400 Kunitz units / mL, about 500 Kunitz units / mL, about 600 Kunitz units / mL, about 700 Kunitz units / mL, about 800 Kunitz units / mL, about 900 Kunitz units / mL, about 1000 Kunitz units / mL, about 1500 Kunitz units / mL, about 2000 Kunitz units / mL, about 2500 Kunitz units / mL, about 3000 Kunitz units / mL, about 4000 Kunitz units / mL, about 5000 Kunitz units / mL, about 6000 Kunitz units / mL, about 7000 Kunitz units / mL, about 8000 Kunitz units / mL, about 9000 Kunitz units / mL, or about 100,000 Kunitz units / mL.

[0066] In some respects, nucleases from any suitable source organism may be used. In some respects, the nuclease may be from a recombinant source. In some respects, the nuclease may be synthetic. A non-limiting example of a DNase is bovine pancreatic deoxyribonuclease (i.e., "deoxyribonuclease I"; SEQ. ID. NO.: 1) or a functional variant or derivative thereof. In some respects, the DNase comprises a polypeptide sequence having at least about 60% to about 75%, or about 75% to about 80%, or about 80% to about 85%, or about 85% to about 90%, or about 90% to about 95%, or about 95% to about 100% identity with SEQ ID NO: 1. In some respects, the polypeptide has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ IDNO: 1.

[0067] In another aspect, the composition further comprises a protease, such as chymotrypsin. Chymotrypsin is also known as EC 3.4.21.1, chymotrypsin A and B, α-chymotrypsin, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, α-chymar, and α-chymotrypsin A. α-chymotrypsin is a serine protease produced by the pancreas that hydrolyzes peptide bonds of tryptophan, leucine, tyrosine, and phenylalanine. Chymotrypsin preferably cleaves peptide amide bonds, wherein the N-terminal amino acid side chain (P1 position) of the readily cleaved amide bond is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). These amino acids contain an aromatic ring in their side chain, which is inserted into the hydrophobic pocket of the enzyme (S1 position). Chymotrypsin also hydrolyzes other amide bonds in the peptide at a slower rate, particularly those containing leucine and methionine at the P1 position.

[0068] Chymotrypsin is produced in vivo as an inactive precursor, chymotrypsinogen (e.g., bovine chymotrypsinogen, SEQ. ID. NO: 2). It is activated in its active form by another enzyme called trypsin. This active form, known as π-chymotrypsin, is used to produce α-chymotrypsin. Trypsin cleaves the peptide bond between arginine-15 and isoleucine-16 in chymotrypsinogen. This produces two peptides within the π-chymotrypsin molecule, linked together by disulfide bonds. One of the π-chymotrypsins acts on the other by breaking the leucine and serine peptide bonds. The activated π-chymotrypsin reacts with other π-chymotrypsin molecules, cleaving two dipeptides: serine-14-arginine-15 and threonine-147-asparagine-148. The reaction produces α-chymotrypsin, which contains chymotrypsin A, chymotrypsin B and chymotrypsin C (SEQ ID NO: 3, 4 and 5, respectively), which are linked by disulfide bonds to form active α-chymotrypsin.

[0069] In some aspects, chymotrypsin from any suitable source organism may be used. In some aspects, the chymotrypsin may be from a recombinant source. In some aspects, the chymotrypsin may be synthetic. In some aspects, the composition comprises a polypeptide containing one or more of SEQ ID NO: 3, 4, or 5, or a variant or derivative thereof. In some aspects, the chymotrypsin is a recombinant chymotrypsin optimized for commercial production or commercial use. In some aspects, the recombinant chymotrypsin is optimized for use in dental products. In some aspects, the composition comprises a polypeptide sequence having at least about 60% to about 75%, or about 75% to about 80%, or about 80% to about 85%, or about 85% to about 90%, or about 90% to about 95%, or about 95% to about 100% identity with one or more of SEQ ID NO: 3-5. In some aspects, the composition comprises one or more polypeptides having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 3-5.

[0070] In some respects, the amount of chymotrypsin is present in functional units based on the hydrolysis rate of the chromogenic substrate, in amounts of about 100 to about 5,000, or about 500 to about 4,000, or about 1,000 to about 3,000, or 1,500, or 1,600, or 1,700, or 1,800, or 1,900, or 2,000, or 2,100, or 2,200, or 2,300, or 2,400, or 2,500, or 2,600, or 2,700, or 2,800, or 2,900 or 3,000 units / mL.

[0071] In some respects, dental compositions may contain additional proteolytic enzymes. Non-limiting examples of proteolytic enzymes include aminopeptidase M, bromelain, carboxypeptidase A, carboxypeptidase B, carboxypeptidase Y, cathepsin C, chymotrypsin, collagenase, dispersin, endopeptidase Arg-C, endopeptidase Asp-N, endopeptidase Glu-C, endopeptidase Lys-C, enterokinase, factor Xa, figokinase, kallikrein, papain, pepsin, plasmin, streptomycin, proteinase K, subtilisin, thermophilic protease, thrombin, or trypsin.

[0072] In some aspects, the dental composition may further comprise a calculus-targeting enzyme that hydrolyzes carbohydrate polymers of glycoproteins. In all aspects, the calculus-targeting enzyme that hydrolyzes carbohydrate polymers of glycoproteins is not a β-galactosidase.

[0073] In some aspects, the dental compositions disclosed herein comprise an effective amount of a nuclease (DNase and / or RNase) and an effective amount of chymotrypsin or a functional variant or derivative thereof. In some aspects, the dental compositions disclosed herein comprise an effective amount of a DNase and an effective amount of chymotrypsin or a functional variant or derivative thereof. In some aspects, the dental compositions disclosed herein comprise an effective amount of DNase I and an effective amount of chymotrypsin or a functional variant or derivative thereof. In some aspects, the dental compositions disclosed herein comprise an effective amount of a nuclease (DNase and / or RNase), an effective amount of chymotrypsin or a functional variant or derivative thereof, an orally acceptable additive, carrier, or excipient, or any combination thereof. In some aspects, the dental compositions disclosed herein comprise an effective amount of a DNase, an effective amount of chymotrypsin or a functional variant or derivative thereof, an orally acceptable additive, carrier, or excipient, or any combination thereof. In some aspects, the dental compositions disclosed herein comprise an effective amount of DNase I, an effective amount of chymotrypsin or a functional variant or derivative thereof, an orally acceptable additive, carrier, or excipient, or any combination thereof.

[0074] In some aspects, the dental compositions disclosed herein do not contain β-galactosidase. In some aspects, the dental compositions consist essentially of an effective amount of a nuclease (DNase and / or RNase) and an effective amount of chymotrypsin or a functional variant or derivative thereof. In some aspects, the dental compositions consist essentially of an effective amount of a DNase and an effective amount of chymotrypsin or a functional variant or derivative thereof. In some aspects, the dental compositions consist essentially of an effective amount of DNase I and an effective amount of chymotrypsin or a functional variant or derivative thereof.

[0075] The units of enzymes disclosed herein should be understood according to the customary usage of each enzyme in the art. For example, for DNase, one unit is defined as the amount of enzyme required to produce an absorbance increase of 0.001 / min / mL at 260 nm by highly polymerized DNA at 25 °C under conditions of HCl (pH 7.5), 50 mM MgCl2, and 13 mM CaCl2. For chymotrypsin, one unit is the amount of enzyme required to hydrolyze 1 μmol of N-benzoyl-L-tyrosine ethyl ester per minute at pH 7.8 and 25 °C. For proteinase K, one unit is the amount of enzyme required to digest urea-denatured hemoglobin per minute at pH 7.5 and 37 °C using Folin & Ciocalteu phenol reagent to produce an absorbance equivalent to 1.0 µmol of L-tyrosine (6). (See, for example, www.worthington-biochem.com / PROK / cat.html). Those skilled in the art will appreciate how units of other proteolytic enzymes are defined. In the following examples, amylase and RNase without DNA enzyme were used as controls.

[0076] Stable enzymes used in the preparation of dental compositions and formulations are readily available from commercial enzyme suppliers such as Worthington Biochemical Corp. (Lakewood, NJ) as purified lyophilized powders. Enzymes may be derived from animal tissues, such as the pancreas of an animal (e.g., a cow), or produced using recombinant methods. The lyophilized powder is dissolved in an aqueous solvent, which may be water. Sufficient solvent is added to a commercially supplied vial of lyophilized powder to fill it approximately halfway, and the remainder is filled with glycerol to produce a moderately stable 50 / 50 water / glycerol solution. This solution can be kept in a refrigerator for at least several weeks. Approximately 50 μl to approximately 200 μl of each enzyme solution is extracted using a plastic or glass pipette or other instrument. The enzyme or other biocatalyst solution, or a combination of enzyme or other biocatalyst solutions, or a composition or formulation containing the enzyme, is then applied to the teeth, paying particular attention to the gingival margin, using any of the various known oral application methods or tools. For example, the enzyme solution or a composition or formulation containing one or more enzymes may be applied to a toothbrush, or preferably a small interdental cleaning stick with a brush. Alternatively, multiple enzymes can be prepared as described, then combined in a single solution and applied to an application brush, or each enzyme solution can be applied to the brush individually. Alternatively, a composition or formulation containing one or more enzymes can be prepared as described and then applied to an application brush. The brush is used to apply the enzyme solution to the tooth surface, paying particular attention to the gingival margin, where tartar tends to form.

[0077] As described above, any one or more calculus-targeting enzymes can be prepared as simple solutions or combined with orally acceptable additives, carriers, or excipients to prepare liquid, paste, or gel forms, which help maintain contact between the enzyme and the tooth surface for a longer period of time than is permitted by liquids. As disclosed herein, enzyme solutions can be combined with or added to mouthwash or toothpaste compositions as known in the art. Non-limiting examples of orally acceptable additives, carriers, or excipients are generally as known in the art and include thickeners or gelling agents, binders, stabilizers, preservatives, flavoring agents, fluoride salts, surfactants, abrasives, tartar control agents, calcium chelating agents, and colorants. Additives such as flavoring agents and colorants can be generally as known in the art and are readily commercially available.

[0078] Non-limiting examples of adhesives include carboxyvinyl polymers (such as polyacrylic acid crosslinked with polyallyl sucrose or polyallyl pentaerythritol), hydroxyethyl cellulose, hydroxypropyl cellulose, water-soluble salts of cellulose ethers (such as sodium carboxymethyl cellulose and sodium carboxymethyl hydroxyethyl cellulose), and natural gums (such as carrageenan, guilarin, guar gum, xanthan gum, gum arabic, and tragacanth gum).

[0079] Non-limiting examples of thickeners and gelling agents include gellan gum (low-acyl or high-acyl), glycerin, silica, guar gum, xanthan gum, polyethylene glycol, polyvinylpyrrolidone and its copolymers, polylactic acid, polyglycolic acid, long-chain fatty acid alcohols, cellulose-based polymers and acrylate polymers.

[0080] Non-limiting examples of carriers include orally acceptable alcohols such as ethanol, isopropanol, and glycerin. Non-limiting examples of tartar control agents are generally those known for use in readily available commercial dental cleaning products, such as pyrophosphates and their salts, polyphosphates, polyphosphonates, and mixtures thereof. Pyrophosphates include dialkali metal and tetraalkali metal pyrophosphates and mixtures thereof. Non-limiting examples of antibacterial agents and preservatives include quaternary ammonium salts, their polymers, chlorhexidine and its salts, polyhexamethylene biguanide, oxytetracycline, organic acids, chelating agents such as calcium chelators (e.g., ethylenediaminetetraacetic acid (EDTA)), essential oils, and parabens. Non-limiting examples of antibiotics include penicillin and tetracycline. Non-limiting examples of orally acceptable abrasives include silica, including fumed silica, or other inorganic particles, synthetic polymer particles, or organic particles, such as plant-derived particles.

[0081] Any orally acceptable surfactant can be used, most of which are anionic, nonionic, or amphoteric. In some aspects, the choice and amount of surfactant ensure that it does not interfere with enzyme activity. Non-limiting examples of nonionic surfactants include cetearyl alcohol polyether, stearyl alcohol polyether, polyethoxylated fatty acid sorbitan esters, ethoxylated fatty acids, esters of polyethylene glycol, ethoxylated monoglycerides and diglycerides of fatty acids, and ethylene oxide / propylene oxide block polymers. In some aspects, the surfactant is selected from polyethylene glycol ethers of fatty alcohols, particularly polyethylene glycol ethers having 20-40 ethylene oxide groups per unit. In some exemplary aspects, the surfactant is cetearyl alcohol polyether-25 or stearyl alcohol polyether-30. One or more surfactants are optionally present in a total amount of about 0.000001% to about 5%, depending on the formulation used and the type of surfactant. In some respects, the amount of surfactant is about 0.000001% to about 0.00001% by weight of the composition, or about 0.00001% to about 0.0001%, or about 0.0001% to about 0.001%, or about 0.001% to about 0.01%, or about 0.01% to about 1%, or about 1% to about 5% by weight of the composition.

[0082] In some aspects, dental compositions or formulations further comprise water and / or polyols. Typical polyols used in oral care compositions, particularly dental floss or toothpaste compositions, include humectants such as glycerin, sorbitol, polyethylene glycol, polypropylene glycol, propylene glycol, xylitol (and other edible polyols), hydrogenated partially hydrolyzed polysaccharides, and mixtures thereof. In some aspects, the polyol is glycerin and / or sorbitol. In some aspects, the amount of water and / or polyol depends on the nature of the formulation. In some exemplary aspects, the amount of water and / or polyol is at least 10 wt%, or at least 20 wt%, or at least 30 wt%, or at least 40 wt%, or at least 50 wt%, or at least 60 wt%, or at least 70 wt%, or at least 80 wt% based on the total weight of the composition. In some aspects, the level of water and / or polyol should be less than 95 wt%, or less than 90 wt%, or less than 85 wt%, or less than 80 wt% of the total composition.

[0083] In some aspects, the formulations or compositions disclosed herein contain about 5% to about 90% glycerol. In some aspects, glycerol may be present in the composition or formulation at about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%. In some aspects, glycerol may be present in the composition or formulation at about 10% to about 20%.

[0084] In some aspects, the formulations or compositions disclosed herein contain about 0.0000001% to about 0.01% of cetearyl alcohol polyether-25. In some aspects, cetearyl alcohol polyether-25 may be present in the composition or formulation at about 0.0000001%, 0.0000005%, about 0.000001%, about 0.000005%, about 0.00001%, about 0.00005%, about 0.0001%, about 0.0005%, about 0.001%, or about 0.01%. In some aspects, about 0.000001% to about 0.00001% of cetearyl alcohol polyether-25 may be present in the composition or formulation.

[0085] In some aspects, the formulations or compositions disclosed herein contain about 0.01% to about 10% xanthan gum. In some aspects, xanthan gum may be present in the formulation or composition at about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some aspects, about 0.1% to about 0.5% xanthan gum may be present in the composition or formulation.

[0086] In some aspects, the formulations or compositions disclosed herein contain about 0.1% to about 30% sorbitol. In some aspects, sorbitol may be present in the formulation or composition at about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%. In some aspects, about 1% to about 10% sorbitol may be present in the composition or formulation.

[0087] In some aspects, the formulations or compositions disclosed herein contain about 0.1% to about 50% silica. In some aspects, the silica is present in amounts of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, or about 2%. It is present in the composition or formulation at 1%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%.

[0088] In some aspects, the formulations or compositions disclosed herein may further comprise a solvent, such as water. In some aspects, water may be present in the composition or formulation at a concentration of about 0.01% to about 99%. In some aspects, water may be present in the concentrations of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 39%. 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% exist.

[0089] In some respects, the pH of the compositions disclosed herein can be adjusted with pharmaceutically acceptable acids, bases, or buffer solutions. The pH of the compositions disclosed herein is preferably in the range of about 6.0 to about 9.5, and more preferably in the range of about 6.1 to about 7.0 (e.g., about 6.1 to about 6.3 or about 6.7 to about 7.0).

[0090] In some respects, the compositions or formulations provided herein can be prepared and used in various forms suitable for oral cavity preparation, such as in the form of dental fumigants, including toothpaste, tooth powder, teething gel, mouthwash, oral spray, lozenges, chewing gum, dental paste, gingival massage cream, mouthwash tablets, or liquid formulations. In some respects, the formulation is a dental fumigant. The term "dental fumigant" generally refers to a formulation used to clean the surfaces of the oral cavity. A dental fumigant is an oral composition that is not intentionally swallowed for the purpose of systemic administration of a therapeutic agent, but rather applied to the oral cavity for the treatment of the oral cavity and then spat out. In some respects, dental fumigants are used with cleaning tools such as toothbrushes, typically by applying them to the bristles of the toothbrush and then brushing the accessible surfaces of the oral cavity. In some respects, dental fumigants are in the form of pastes or gels (or combinations thereof).

[0091] The dental cleaning composition according to this disclosure may contain a mixture of water and polyol as an aqueous continuous phase in various relative amounts, wherein the amount of water is generally in the range of 10% to 50% by weight (based on the total weight of the dental cleaning agent), and the amount of polyol is generally in the range of about 20% to about 60% by weight (based on the total weight of the dental cleaning agent).

[0092] Oral care compositions, particularly toothpaste or dental cleaning compositions, may further contain additional abrasive materials. In some aspects, the abrasive material may be present in an amount of about 0.5% to about 75%, or about 3% to about 60% by weight of the total paste. Suitable abrasive cleaners include silica dry gels, hydrogels, and aerogels, as well as precipitated particulate silica; calcium carbonate, dicalcium phosphate, tricalcium phosphate, calcined alumina, sodium metaphosphate and potassium metaphosphate, sodium pyrophosphate and potassium pyrophosphate, sodium trimetaphosphate, sodium hexametaphosphate, particulate hydroxyapatite, and mixtures thereof. In some respects, the abrasive material is silicon dioxide, which is present in an amount of at least about 10%-15%, or 15%-20% or 20%-25%, or 25%-30% or 30%-35%, or 35%-40%, or 40%-45%, or 45%-50%, or 50%-55%, or 55%-60%, or 60%-70% or 70%-80% by weight.

[0093] In some aspects, the compositions disclosed herein may contain zinc ions. Preferably, the sources of zinc ions are zinc chloride, zinc acetate, zinc gluconate, zinc sulfate, zinc fluoride, zinc citrate, zinc lactate, zinc oxide, zinc monoglyceride, zinc tartrate, zinc pyrophosphate, zinc maleate, and mixtures thereof.

[0094] In some respects, the compositions disclosed herein may further contain optional ingredients conventional in the art, such as fluoride ion sources, buffers, flavoring agents, sweeteners, coloring agents, emulsifiers, preservatives, ant sensitivity agents, bleaching agents, and antimicrobial agents.

[0095] In some aspects, the compositions disclosed herein may be substantially liquid in nature, such as mouthwash or cleansing agents. In such formulations, the medium is generally a water-alcohol mixture, and desiccants are desired to include the wetting agents described below. Typically, the weight ratio of water to alcohol is in the range of about 1:1 to about 20:1. In this type of formulation, the total amount of the water-alcohol mixture is generally in the range of about 70% to about 99.9% by weight of the formulation. The alcohol is generally ethanol or isopropanol.

[0096] In some aspects, the composition may be a solid, semi-solid, gel, or paste, such as tooth powder, dental tablets, or toothpaste (dental cream) or gel dental cleaning agents. The medium of such solid or paste-like oral preparations typically contains a dentally acceptable polishing material. Non-limiting examples of polishing materials may include water-insoluble sodium metaphosphate, potassium metaphosphate, tricalcium phosphate, calcium phosphate dihydrate, anhydrous dicalcium phosphate, calcium pyrophosphate, magnesium orthophosphate, trimagnesium phosphate, calcium carbonate, hydrated alumina, calcined alumina, aluminum silicate, zirconium silicate, silica, bentonite, and mixtures thereof. Other suitable polishing materials may include particulate thermosetting resins, such as melamine-, phenolic, and urea-formaldehyde, as well as cross-linked polyepoxides and polyesters. In some aspects, the polishing material may include a particle size of up to about 5 micrometers, an average particle size of up to about 1.1 micrometers, and a surface area of ​​up to about 50,000 cm². 2 / g of crystalline silica, silica gel or colloidal silica, and composite amorphous alkali metal aluminosilicates.

[0097] Polishing materials are typically present in solid or paste compositions at a concentration of about 10% to about 99% by weight. In some cases, polishing materials may be present in toothpaste at a concentration of about 10% to about 75%, and in tooth powder at a concentration of about 70% to about 99%. In toothpaste, when the polishing material is silica in nature, it is typically present at a concentration of about 10% to 30% by weight. Other polishing materials are generally present at a concentration of about 30% to 75% by weight.

[0098] In toothpaste, the liquid carrier may generally contain water and a wetting agent in an amount ranging from about 10% to about 80% by weight of the formulation. Glycerin, propylene glycol, sorbitol, and polypropylene glycol are examples of suitable wetting agents / carriers. In some applications, a liquid mixture of water, glycerin, and sorbitol may be used. In transparent gels where refractive index is an important consideration, about 2.5% to 30% w / w water, 0% to about 70% w / w glycerin, and about 20% to about 80% w / w sorbitol may be used.

[0099] Toothpaste, cream, and gel generally contain natural or synthetic thickeners or gelling agents in a proportion of about 0.1 to about 10, preferably about 0.5 to about 5% w / w. Suitable thickeners may be synthetic lithium montmorillonite, a synthetic colloidal magnesium alkali metal silicate composite clay, for example, that can be obtained as Laponite (e.g., CP, SP 2002, D) sold by Laporte Industries Limited. Laponite D contains, by weight, about 58.00% SiO2, 25.40% MgO, 3.05% Na2O, 0.98% Li2O, and some water and trace metals. Other suitable thickeners, non-limiting examples, may include Irish moss, iota carrageenan, tragacanth gum, starch, polyvinylpyrrolidone, hydroxyethylpropyl cellulose, hydroxybutyl methylcellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose (e.g., available as Natrosol), sodium carboxymethyl cellulose, and colloidal silica, such as finely ground Syloid (e.g., 244). Also included may be solubilizers, such as wetting polyols like propylene glycol, dipropylene glycol, and hexanediol; cellosols like methyl cellosol and ethyl cellosol; vegetable oils and waxes containing at least about 12 carbons in their linear chains, such as olive oil, castor oil, and petrolatum; and esters like amyl acetate, ethyl acetate, and benzyl benzoate.

[0100] Furthermore, the toothpaste compositions disclosed herein may further contain pharmaceutical ingredients. Common toothpaste compositions contain appropriate pharmaceutical ingredients depending on their intended use, and fluoride, which forms a fluoride film on teeth to make them resistant to acids such as lactic acid, is recently recognized as indispensable for toothpaste compositions. Lactic acid is a metabolic product of bacteria that cause tooth decay. In some aspects, the composition may contain sodium fluoride or sodium monofluorophosphate, alone or in combination. Additionally, for the prevention of periodontitis, water-soluble salts such as sodium chloride, sodium bicarbonate with high osmotic pressure, aminocaproic acid, allantoin and allantoin derivatives, as well as various vitamins, may be used concurrently.

[0101] Furthermore, the toothpaste composition disclosed herein may further include a binder. A binder that prevents the separation of the solid powder components and the liquid components is indispensable for ointment-type toothpastes, and any water-soluble macromolecule can be used. Commonly used binders include sodium carboxymethyl cellulose synthesized from tree cellulose, carrageenan extracted from seaweed, and xanthan gum obtained from microbial metabolism.

[0102] Furthermore, the toothpaste composition disclosed herein may further include a foaming agent. The foaming agent enhances the usability of the product, aids in cleaning and promotes the dispersion and penetration of pharmaceutical ingredients, and reduces surface tension, thereby facilitating the separation of foreign substances. The primary foaming agent used is the anionic surfactant sodium lauryl sulfate, and depending on the nature of the formulation, nonionic surfactants such as polyoxyethylene-polyoxypropylene copolymer (poloxam), polyoxyethylene-cured castor oil, and polyoxyethylene-sorbitan fatty acid esters may be used as supplementary agents.

[0103] Various other materials, such as whitening agents, preservatives, silicones, chlorophyll compounds, and / or ammoniating materials such as urea, diammonium phosphate, and mixtures thereof, may be incorporated into the oral formulation in the presence of these adjuvants in amounts that substantially do not adversely affect the desired properties and characteristics.

[0104] Any suitable flavoring or sweetening material may also be used. Non-limiting examples of suitable flavoring ingredients include flavoring oils such as spearmint, peppermint, wintergreen, sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon, and orange oils, as well as methyl salicylate. Suitable sweeteners may include sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclohexanesulfonate, perilla, AMP (methyl aspartate), saccharin, etc. Appropriately, flavorings and sweeteners may each or together constitute approximately 0.1% to 5% or more of the formulation.

[0105] The compositions disclosed herein may also be incorporated into tablets, or into chewing gum or other products, for example by stirring into a warm gum base or coating the outer surface of a gum base. Illustrative examples include jelutong, rubber latex, vinyl resin, etc., desirablely together with conventional plasticizers or softeners, sugars or other sweeteners, or such as glucose, sorbitol, etc.

[0106] In some respects, oral preparations may be appropriately labeled for sale or otherwise distributed. For example, a bottle of mouthwash will have a label that essentially describes it as a mouthwash or rinsing solution, and will include its instructions for use; and toothpaste, cream, or gel will typically be in a collapsible tube, usually made of aluminum, lead-lined, or plastic, or other squeeze, pump, or pressurized dispenser for metering the contents, with a label that essentially describes it as toothpaste, gel, or dental cream.

[0107] In another aspect, this disclosure provides a composition comprising the above-described composition, a fluoride ion source, and a carrier. Such a composition may be a dental composition in the form of a gel, liquid, solid, powder, cream, or lozenge.

[0108] In some aspects, this disclosure includes an oral hygiene device comprising the disclosed composition or formulation. In some aspects, the dental composition or formulation may be used with a dental tray, capsule applicator, oral cleaning stick, or dental floss.

[0109] In some aspects, a dental tray may include an article shaped to at least partially cover one or more teeth, gums, or dental implants. In some aspects, a dental tray may be arched, round, or semi-circular. In some aspects, a dental tray may be a dental appliance (e.g., an orthodontic appliance or retainer), a night guard, a mouthguard, a treatment tray, full or partial dentures, braces, etc. In some aspects, a dental tray may include a flexible polymer material that generally conforms to the patient's teeth and may be transparent, translucent, or opaque.

[0110] In some aspects, the dental trays of this disclosure can be prepared using casting and curing methods. In some aspects, the dental trays can be prepared by a method comprising the steps of: (a) preparing a polymerizable composition; (b) depositing the polymerizable composition in an amount sufficient to fill a master mold cavity onto a master mold surface; (c) filling the cavity by moving beads of the polymerizable composition between a pre-formed substrate (e.g., a PET film) and the master mold, wherein at least one of the substrate and the master mold is flexible; and (d) curing the composition.

[0111] In some aspects, dental trays may be made of polymeric materials, which may include one or more of the following: amorphous thermoplastic polymers, semi-crystalline thermoplastic polymers and transparent thermoplastic polymers such as polycarbonate, thermoplastic polyurethane, acrylic, polysulfone, polypropylene, polypropylene / ethylene copolymer, cyclic olefin polymers / copolymers, poly-4-methyl-1-pentene or polyester / polycarbonate copolymers, styrene-based polymers, polyamides, polymethylpentene, polyetheretherketone, polyurethane, polyolefins including metallocene polyolefins, polyesters such as elastomeric polyesters (e.g., Hytrel), biodegradable polyesters such as polylactic acid, polylactic acid / glycolic acid, succinic acid and glycol copolymers, fluoropolymers including fluoroelastomers, polyacrylates, polymethacrylates or any combination thereof. In another aspect, the material may be selected from transparent or substantially transparent semi-crystalline thermoplastics, crystalline thermoplastics, and composite materials, such as polyamides, polyethylene terephthalate, polybutylene terephthalate, polyester / polycarbonate copolymers, polyolefins, cyclic olefin polymers, styrene copolymers, polyetherimides, polyetheretherketones, polyethersulfones, polypropylene terephthalate, and mixtures and combinations thereof. In some aspects, the material may be a polymeric material selected from polyethylene terephthalate, polyethylene terephthalate glycol, polycyclohexylenedimethyleneterephthalate glycol, and mixtures and combinations thereof. In some respects, dental trays may comprise materials including styrene-acrylonitrile, cellulose acetate butyrate, cellulose acetate propionate, cellulose triacetate, polyethersulfone, polymethyl methacrylate, polyurethane, polyester, polycarbonate, polyvinyl chloride, polystyrene, polyethylene naphthalate, copolymers or blends based on naphthalic acid, polycyclic olefins, polyimide, and glass. In some respects, dental trays may be made of biodegradable materials.Non-limiting examples of suitable biodegradable polymers include polycarboxylic acids; polyanhydrides such as maleic anhydride polymers; polyorthoesters; poly-amino acids; polyethylene oxide; polyphosphazene; polylactic acid, polyglycolic acid, and their copolymers and mixtures such as poly(L-lactic acid). (PLLA), poly(D,L,-lactide), poly(lactic-co-glycolic acid) and 50 / 50 weight ratio (D,L-lactide-co-glycolic acid); polydioxanone; polypropylene fumarate; polyphenolic peptides; polycaprolactone and its copolymers and mixtures such as poly(D,L-lactide-co-caprolactone) and polycaprolactone co-butyl acrylate; polyhydroxybutyrate valerate and its mixtures; polycarbonates such as tyrosine-derived polycarbonates and acrylates, polyiminocarbonates and polydimethyltrimethylcarbonate; cyanoacrylates; calcium phosphate; polyglycosaminoglycans; macromolecules such as polysaccharides (including hyaluronic acid, cellulose and hydroxypropyl methylcellulose); gelatin; starch; dextran; and alginates and their derivatives, proteins and peptides; and mixtures and copolymers of any of the above. Biodegradable polymers can also be surface-erodible polymers, such as polyhydroxybutyrate and its copolymers, polycaprolactone, polyanhydrides (both crystalline and amorphous), and maleic anhydride. In some respects, dental trays can be made from a single polymer material or can comprise multiple layers of different polymer materials.

[0112] In some respects, the compositions or formulations provided herein can be prepared and used in various forms suitable for animals. In this respect, the compositions or formulations provided herein can be prepared and used in various forms, non-limiting examples of which may include pet toothbrushes and toothpaste, chew sticks, rubber toys, chew toys, treats, bite or nibble toys, pet food, pet snacks, pet teething gels, nuggets, powders, or dental appliances (e.g., dentures). The compositions or formulations provided herein can be incorporated into any disclosed form suitable for animals using methods known in the art.

[0113] (II) Methods of removing calculus This disclosure includes methods for reducing or removing tartar from the tooth surface in subjects in whom it is needed. In some aspects, the subjects are human or non-human animals, including but not limited to cats, dogs, cattle, horses, or primates.

[0114] In some respects, methods for reducing or removing tartar include, for example, contacting the surface of a subject's teeth with an effective amount of at least two tartar-targeting enzymes as disclosed herein. Contact can be, for example, by applying a solution (e.g., an aqueous solution), a dental cleaning agent, or a dental composition or formulation as disclosed herein to the tooth surface, waiting for a period of time, and then rinsing the mouth (usually with water). It will be appreciated that the amount of time can vary depending on a range of factors, including the extent and severity of tartar buildup, the individual being treated, whether the treatment is performed in a professional office by a dental professional or at home, and other factors. For example, the solution may be applied to the tooth surface for periods of less than about 60 minutes, less than about 30 minutes, less than about 15 minutes, less than about 15 minutes, less than about 10 minutes, less than about 5 minutes, less than about 2 minutes, less than about 1 minute, or about 30 seconds.

[0115] Contact can be repeated and / or performed periodically, such as once or twice daily or more frequently, once every two days, once every three days, once every four days, once every five days, once every six days, once weekly, once every two weeks, once every three weeks, or once monthly, or once or twice a year, at approximately regular intervals. Further, contact can be made before, during, or after contact with oral care agents such as toothpaste or mouthwash, or with their treatment. Following contact with enzymes or other biocatalyst solutions or compositions, disintegrated or loosened tartar can be further removed from the tooth surface and oral cavity by rinsing with water or another reagent, manual scraping or rubbing, brushing, and / or wiping. In some aspects, the method may include the use of the dental compositions provided herein, as well as additional dental treatments, routines, and / or procedures.

[0116] In some aspects, this disclosure includes a method for reducing or removing dental calculus in subjects who require it, the method comprising contacting a tooth surface with an effective amount of the composition. In this aspect, the method for reducing or removing dental calculus in subjects who require it comprises contacting a tooth surface with an effective amount of the composition comprising DNase I or a functional variant or derivative thereof and chymotrypsin or a functional variant or derivative thereof. In some aspects, the method reduces or removes dental calculus by about 1% or about 2% in subjects compared to the average of subjects or a group of subjects who do not use the disclosed composition or formulation. Approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47% Approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, approximately 100% or more. In some aspects, the disclosed formulations or compositions reduce or remove stones by at least 40%.

[0117] In some aspects, the formulations or compositions disclosed herein can be used in methods for preventing calculus in subjects who require them. In such aspects, preventing dental calculus in subjects who require it comprises contacting the tooth surface with an effective amount of a composition comprising DNase I or a functional variant or derivative thereof and chymotrypsin or a functional variant or derivative thereof. In some respects, compared with subjects or the mean of a subject population that did not use the disclosed compositions or formulations, this method prevented tartar buildup in subjects by approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately... 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, approximately 100% or more.

[0118] In some aspects, the formulations or compositions disclosed herein can be used in methods for improving palatability in subjects who require them. In this aspect, methods for improving palatability in subjects who require them include contacting a tooth surface with an effective amount of a composition comprising DNase I or a functional variant or derivative thereof and chymotrypsin or a functional variant or derivative thereof. In some respects, compared with subjects or the mean of a subject population that did not use the disclosed compositions or formulations, the method improved palatability in subjects by approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, approximately 100% or more.

[0119] In some aspects, the compositions or formulations disclosed herein may be administered to a subject after professional dental cleaning. In some aspects, the compositions or formulations disclosed herein may be administered to a subject prior to a scheduled professional dental cleaning. In some aspects, the compositions or formulations disclosed herein may be administered to a subject during professional dental cleaning.

[0120] In some aspects, the method of application includes brushing teeth with the formulation or composition of this disclosure for about 10 seconds to about 120 seconds. In some aspects, brushing teeth may include brushing teeth with the formulation or composition for about 10 seconds, about 11 seconds, about 12 seconds, about 13 seconds, about 14 seconds, about 15 seconds, about 16 seconds, about 17 seconds, about 18 seconds, about 19 seconds, about 20 seconds, about 21 seconds, about 22 seconds, about 23 seconds, about 24 seconds, about 25 seconds, about 26 seconds, about 27 seconds, about 28 seconds, about 29 seconds, about 30 seconds, about 31 seconds, about 32 seconds, about 33 seconds, about 34 seconds, or about 35 seconds. Approximately 36 seconds, approximately 37 seconds, approximately 38 seconds, approximately 39 seconds, approximately 40 seconds, approximately 41 seconds, approximately 42 seconds, approximately 43 seconds, approximately 44 seconds, approximately 45 seconds, approximately 46 seconds, approximately 47 seconds, approximately 48 seconds, approximately 49 seconds, approximately 50 seconds, approximately 51 seconds, approximately 52 seconds, approximately 53 seconds, approximately 54 seconds, approximately 55 seconds, approximately 56 seconds, approximately 57 seconds, approximately 58 seconds, approximately 59 seconds, approximately 60 seconds, approximately 61 seconds, approximately 62 seconds, approximately 63 seconds, approximately 64 seconds, approximately 65 seconds Approximately 66 seconds, approximately 67 seconds, approximately 68 seconds, approximately 69 seconds, approximately 70 seconds, approximately 71 seconds, approximately 72 seconds, approximately 73 seconds, approximately 74 seconds, approximately 75 seconds, approximately 76 seconds, approximately 77 seconds, approximately 78 seconds, approximately 79 seconds, approximately 80 seconds, approximately 81 seconds, approximately 82 seconds, approximately 83 seconds, approximately 84 seconds, approximately 85 seconds, approximately 86 seconds, approximately 87 seconds, approximately 88 seconds, approximately 89 seconds, approximately 90 seconds, approximately 91 seconds, approximately 92 seconds, approximately 93 seconds, approximately 94 seconds, approximately 9 5 seconds, approximately 96 seconds, approximately 97 seconds, approximately 98 seconds, approximately 99 seconds, approximately 100 seconds, approximately 101 seconds, approximately 102 seconds, approximately 103 seconds, approximately 104 seconds, approximately 105 seconds, approximately 106 seconds, approximately 107 seconds, approximately 108 seconds, approximately 109 seconds, approximately 110 seconds, approximately 111 seconds, approximately 112 seconds, approximately 113 seconds, approximately 114 seconds, approximately 115 seconds, approximately 116 seconds, approximately 117 seconds, approximately 118 seconds, approximately 119 seconds, or approximately 120 seconds. In some cases, brushing teeth includes brushing with a preparation or composition for approximately 30 seconds.

[0121] In some aspects, the administration method includes using a dental tray containing a formulation or composition of the present disclosure for about 1 minute to about 60 minutes. In some aspects, the dental tray containing the formulation or composition may administer for about 1 minute, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, or about 33 minutes. Approximately 34 minutes, 35 minutes, 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 minutes, 55 minutes, 56 minutes, 57 minutes, 58 minutes, 59 minutes, or 60 minutes. In some cases, a dental tray containing a preparation or composition may be given for approximately 30 minutes.

[0122] In some aspects, the compositions or formulations of this disclosure may be administered at least once daily. In some aspects, the compositions or formulations may be administered at least twice daily. In some aspects, the compositions or formulations may be administered at least three times daily. In some aspects, the compositions or formulations may be administered more than three times daily.

[0123] In some aspects, the method includes brushing teeth with a formulation or composition of the present disclosure, followed by brushing with commercially available toothpaste.

[0124] In some aspects, the method includes sequentially brushing teeth with the compositions or formulations of the present disclosure, and using a dental tray containing the formulations or compositions of the present disclosure. Brushing teeth with the formulations or compositions of the present disclosure, and then using a dental tray containing the formulation. In some aspects, the method includes using a dental tray containing the formulations or compositions of the present disclosure, and then brushing teeth with the formulations or compositions of the present disclosure. In some aspects, the method includes brushing teeth with the formulations or compositions of the present disclosure, and then using a dental tray containing the formulations or compositions of the present disclosure, and then brushing teeth with the formulations or compositions of the present disclosure.

[0125] In some aspects, the treatment methods may include single, multiple, and repeated administration of the compositions or formulations disclosed herein as needed for the prevention or treatment of a disease or condition (e.g., stones) suffered by the subject. In some aspects, the treatment methods may include evaluating stone levels in the subject before, during, or after treatment. In some aspects, treatment may continue until a reduction in stone levels can be detected in the subject.

[0126] In some aspects, the effective dose of the compositions or formulations disclosed herein may include, but is not limited to, for example, about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5, 1.75, 2, 2.5 or 3-10,000 mg / kg / day, or as required by a particular composition or formulation.

[0127] For the disclosed compositions or formulations, the dosage may also be in International Units (IU) per day (lU / day) and is approximately 100 lU / day, 200 lU / day, 300 lU / day, 400 lU / day, 500 lU / day, 600 lU / day, 700 lU / day, 800 lU / day, 900 lU / day, 1000 lU / day, 1100 lU / day, 1200 lU / day, 1300 lU / day, 1400 lU / day, 1500 lU / day, 1600 lU / day, 1700 lU / day, 1800 lU / day, 1900 lU / day, 2000 lU / day, 2100 lU / day, 2200 lU / day, 2300 lU / day, 2400 lU / day, 2500 lU / day. lU / day, 2600 lU / day, 2700 lU / day, 2800 lU / day, 2900 lU / day, 3000 lU / day, 3100 lU / day, 3200 lU / day, 3300 lU / day, 3400 lU / day, 3500 lU / day, 3600 lU / day, 3700 lU / day, 3800 lU / day, 3900 lU / day, 4000 lU / day, 4500 lU / day, 5000 lU / day, 5500 lU / day, 6000 lU / day, 6500 lU / day, 7000 lU / day, 7500 lU / day, 8000 lU / day, 9000 lU / day, 10,000 lU / day, 20,000 lU / day, 30,000 lU / day, 40,000 lU / day, 50,000 lU / day, 60,000 lU / day, 70,000 lU / day, 90,000 lU / day, 100,000 lU / day, 200,000 lU / day, 300,000 lU / day, 400,000 lU / day, 500,000 lU / day, 600,000 lU / day, 700,000 lU / day, 800,000 lU / day, 900,000 lU / day, 1,000,000 lU / day, 1,100,000 lU / day, 1,200,000 lU / day, 1,300,000 lU / day, 1,400,000 lU / day lU / day or 1,500,000 lU / day.

[0128] The dosage and interval can be adjusted individually to provide the desired effect, or to be sufficient to maintain the therapeutic or preventative effect.

[0129] The specific dosage and treatment regimen for any particular subject will depend on a variety of factors, including the activity of the specific enzyme used, the severity of the stones in the subject and the process of stone accumulation, the condition or symptoms, the patient's management of the condition or symptoms, and the judgment of the treating physician.

[0130] The effective amount may be administered, applied, or dosed once or multiple times. The therapeutically effective amount (i.e., the effective dose) of the formulation depends on the type of composition or formulation chosen. Furthermore, treatment of a subject with the effective amount of the formulation or composition described herein may include a single treatment or a series of treatments. For example, the effective amount may be administered at least once. The composition may be administered once or multiple times daily to once or multiple times weekly; including every other day. Those skilled in the art will recognize that certain factors can affect the dose and duration required to effectively treat a subject, including but not limited to the severity of the stones, prior treatment, the subject's overall health or age, and any other pre-existing conditions.

[0131] After administration, the subject may be assessed to detect, evaluate, or determine their stone level. In some cases, treatment may continue until a change in the disease level (e.g., a decrease) can be detected in the subject. After improvement in the patient's condition (e.g., a change in the stone level in the subject (e.g., a decrease)), a maintenance dose of the formulation or composition disclosed herein may be given, if necessary. Subsequently, the dose or frequency, or both, may be reduced, for example as a function of stone accumulation, to maintain the improved condition level. However, after any recurrence of stone accumulation, the patient may require long-term intermittent treatment.

[0132] In some aspects, the compositions disclosed herein may include other compounds, drugs, or agents for treating calculus, plaque, and / or periodontal disease. For example, in some cases, the compositions disclosed herein may be combined with one or more (e.g., one, two, three, four, five, or fewer than ten) compounds. When administered co-administered, the formulations or compositions disclosed herein may act together with other agents for treating calculus, plaque, and / or periodontal disease to produce a mechanically additive or synergistic therapeutic effect.

[0133] (III) Kits A further aspect of this disclosure provides kits comprising an amount of any one or any combination of the dental compositions or formulations detailed above. The kit optionally includes one or more brushes, toothpicks, dental trays, and / or applicators for applying any one of the enzyme solution, composition, or formulation to the teeth and then removing disintegrating tartar from the tooth surface. Kit components may be provided together with other kit components in a suitable container, such as any commercially available container or packaging. Kits provided herein typically include instructions for carrying out the methods detailed herein. Instructions included in the kit may be affixed to packaging material or included as a packaging insert. Although instructions are generally written or printed material, they are not limited thereto. This disclosure contemplates any medium capable of storing and transmitting such instructions to an end user. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, tape cases, chips), optical media (e.g., CD-ROMs), etc. As used herein, the term "instructions" may include the address of the website providing the instructions.

[0134] Not expected to be bound by theory, it is believed that the current compositions and methods are so effective in breaking down and removing tartar, partly due to the way tartar accumulates and is structured. The inventors' work demonstrates that dental plaque has a layered structure, including an internal gel or gel-like structure layered with one or more layers of calcium-crosslinked extracellular DNA (eDNA) and proteins in a cross-linked network. The internal gel or gel-like portion contains aqueous contents similar to those of salivary glands. These aqueous contents may include inorganic substances, calcium, phosphates, hydroxyapatite, proteins and glycoproteins, amylases, polyproline proteins, and mucins. Additionally, dental plaque may contain contents from non-salivary gland sources, including transglutaminase from the oral epithelial surface and chymotrypsin from the soft palate. Dental plaque may further include contents from non-host sources, such as bacterial cells from the oral microbiome, eDNA secreted from the microbiome, and proteins and polysaccharides from food. The outermost layer of plaque contains calcium-cross-bridged eDNA (approximately 0.01%) and proteins (approximately 0.04%–0.05%), including negatively charged mucin glycoproteins and cross-linked network structures of transglutaminase.

[0135] Furthermore, the inventors determined that the plaque calcification process can begin immediately, with plaque becoming approximately 50% calcified within 2 days and 60%-90% calcified within approximately 12 days. This calcification process is also gradual, thereby adding continuous layers and thus transforming the stone structure from a more two-dimensional to a more three-dimensional structure. While the network structure of calcium-crosslinked eDNA and mucin glycoproteins is reversible, the network structure formed by transglutaminase crosslinking proteins is irreversible. The compositions and methods of this disclosure have been developed to effectively target the contents and formation process of stone-forming structures, aiding in the treatment or prevention of stones, as described in the following examples. Example

[0136] Example 1: Anti-stone enzyme formulation containing DNase 1 and chymotrypsin As provided, an anti-stone enzyme composition containing DNase 1 and chymotrypsin was formulated. Two batches were prepared, namely Formulation I and Formulation II, containing different levels of the nonionic surfactant cetearyl alcohol polyether and water.

[0137] For formulation I, thoroughly mix the chemical components disclosed in Table 1.

[0138] Table 1: Chemical composition of formulation I Stock solution Volume / amount Glycerin 90 ml 10% ceteth-25 36 ml 2% xanthan gum 90 ml 80% sorbitol 36 ml Water 30 ml Silica 72 g Discard approximately 110 ml of the combined volume. Then, batch-mix the enzymes into the solution. Add 5 g of DNase 1 (4270 Kunitz units / mg) to the combined solution and mix until homogeneous. Add 5 g of chymotrypsin (66.6 units / mg) to the solution and mix until homogeneous. Repeat this step 6 times to obtain an approximate DNase activity of 185,652.17 units / ml and a chymotrypsin activity of 2,895.65 units / ml. Fill capped syringes (1 ml / syringe) with the preparation and refrigerate until administration.

[0139] Similarly, formulation II was prepared by initial dissolution of the chemical components provided in Table 2.

[0140] Table 2: Chemical composition of formulation II Stock solution Volume / amount Glycerin 90 ml 10 ceteth-25 0.36 ml 2% xanthan gum 90 ml 80% sorbitol 36 ml Silica 72 g To control volume, the volume of this formulation was compensated to 200 ml with water. The enzyme was then mixed in batches into the solution. 5 g of DNase 1 (3900 Kunitz units / mg) was added to the solution and mixed until homogeneous. Next, 5 g of chymotrypsin (66.6 units / mg) was added to the solution and mixed until homogeneous. This step was repeated 6 times, with an additional 30 ml of water added each time. To obtain sufficient volume for the experiment, 50 ml of formulation I was added to this formulation. The approximate DNase 1 activity was determined to be 183,300 Kunitz / ml and the chymotrypsin activity to be 114 units / ml. The formulation was filled into capped syringes (1 ml / syringe) and refrigerated until administration.

[0141] In clinical studies, syringes filled with formulation II were used to evaluate the efficacy and safety of stone treatment in human subjects.

[0142] Example 2: In vivo study evaluating the anticalculus activity and safety characteristics of dental formulations The safety and efficacy of formulations containing DNase 1 and chymotrypsin in removing existing stone deposits were investigated in subjects. Intergroup and intragroup comparisons were performed over a 4-week trial period. Figure 1 The study design is illustrated in the diagram.

[0143] Recruitment These trials recruited 40 participants. Eligibility for the trials was based on the following eligibility criteria.

[0144] Inclusion criteria To be included in the study, each participant must: 1. Provide written informed consent to participate in the research; 2. Must be 18 years of age or older; 3. You agree not to participate in any other oral / dental product studies during this study and to use only the designated oral hygiene products (including toothbrushes, toothpaste, home remedies, thread, or other products such as chewing gum, mouthwash, tongue cleaners, etc.) throughout the study. 4. Agree to avoid the use of any selective dental procedures (including non-study dental prevention) until the study is completed; 5. Agree to avoid using any non-research-based oral hygiene and whitening products; 6. Good overall health, as determined by the investigator / designated personnel based on a review of the participant's health history / current condition; 7. Has 6 mandibular anterior teeth, without crowns or veneers; 8. Agree to comply with the research procedures and schedule, including follow-up; 9. There is at least 9 mm of tartar on the first 6 teeth of the lower jaw and they reported having dental cleanings in the previous 2-6 months.

[0145] Exclusion criteria Participants will be excluded from the study if they meet any of the following criteria: 1. Having a medical condition that requires preoperative antibiotic treatment before dental procedures; 2. Use chlorhexidine mouthwash regularly; 3. Any oral condition or pathology that may interfere with study compliance and / or examination procedures (e.g., extensive caries, long-term neglect, advanced periodontitis); 4. Having a current history of oral or oropharyngeal cancer; 5. Subjects reported pregnancy or breastfeeding; 6. Not brushing teeth regularly; 7. Any circumstances that may make participation in the study unsafe shall be determined at the discretion of the researcher.

[0146] Delay criteria Participants will withdraw from the study and be excluded from the study analysis if they meet any of the following criteria: 1. Participated in any other oral / dental product research since the last visit; 2. Has received any elective dental treatment since the last visit; 3. Use of any oral hygiene products other than the designated research products; 4. Unable or unwilling to comply with the product instructions for use for any reason.

[0147] After participants met the inclusion / exclusion criteria, their calculus status was examined. Only participants with at least 9 mm of calculus accumulated on the lingual surfaces of the six mandibular anterior teeth were eligible. Protocol details were discussed with each potential participant, and written informed consent was obtained from all participants before any activities related to the clinical study. Participants may withdraw their consent at any time without bias. A Participant Responsibility Form (CRF) was completed for each participant. If a participant did not complete the study for any reason, an explanation was entered on the CRF.

[0148] Study visits The following visits were conducted during the study period: Visit 1 (informed consent, baseline, and impressions): During Visit 1, participants received an explanation of the nature and procedure of the study, signed an informed consent form, and received a copy of the signed consent form. Following the signing of informed consent, researchers conducted oral examinations and reviewed inclusion / exclusion criteria to ensure eligibility. Demographic details (including age, sex, and race), medical history, and concomitant medications were assessed in eligible participants. Demographic information and inclusion / exclusion criteria were also obtained and captured from appropriate Case Report Forms (CRFs). Participants were assessed to ensure at least 9 mm of calculus was present on the lingual surface of all six mandibular anterior teeth. Eligible participants were randomized to the treatment group.

[0149] Participants were randomly assigned to one of three study groups using a randomization protocol developed and maintained by an independent statistician. Participants were stratified according to their baseline lingual V-MI calculus score and sex (see [link to study group]). Figure 2 ).

[0150] During Visit 1, all randomized subjects (except the custom tray test group who returned the next day) underwent their first use under supervision at the study site.

[0151] Participants used the product at home twice daily for four weeks. Each use was performed according to the group's instructions. Participants received a diary card to record their home therapy experiences.

[0152] Subjects randomized to the enzyme composition gel group with a custom-made mouthguard received an impression at this visit. Impressions of the lower jaw teeth were used to construct individual mouthguards for use during clinical trials. The mouthguards were constructed from vacuum-formed mouthguard plastic. Each mouthguard was trimmed to include only the tooth and gingival margins and eliminated from contact with the cervical margin of each tooth to insulate the enzyme composition gel from contact with the robust tooth surface.

[0153] Visits 2 and 3 (2-week and 4-week check-ups): During visits 2 and 3 (after treatment 2 and 4 weeks, respectively), continuity criteria were evaluated and recorded on the appropriate CRF. After verification of continuity eligibility, safety was assessed via dental examination, and adherence to the study protocol was evaluated. Stone deposits were evaluated to replicate the assessment made during visit 1.

[0154] Table 3 provides an overview of the visits: Table 3: Research Procedures and Timeline Identification of study product The products used during this study are listed below: 1. ADA Reference Soft Manual Toothbrush: (Colgate® Classic [Pokey], Colgate-Palmolive Co., New York, NY) Use with ADA toothpaste.

[0155] 2. ADA Reference Soft Manual Toothbrush: Use only with formulations.

[0156] 3. Enzyme formulation: Formulation II, as provided in this article.

[0157] 4. ADA-approved standard fluoride toothpaste: Crest® Cavity Protection Cool Mint Gel, 0.243% sodium fluoride, Procter & Gamble, Cincinnati, OH 45202.

[0158] Calculus measurement 1. Calculate the Volpe-Manhold index (V-MI) on the lingual surfaces of the six lower anterior teeth (see...). Figure 2 Volpe-Manhold Index (V-MI) 2 Calculus present on the lingual surfaces of six lower anterior teeth was measured. A standard periodontal probe, graduated in millimeters, was used. After the teeth were dried with airflow, the probe was placed at the lowest edge of the visible calculus, and measurements were taken on the following three planes: (a) Divide the center of the side of the tongue in two; (b) Diagonally across the mesial incisal angle of the tooth, through the area of ​​maximum calculus height; and (c) Diagonally across the distal corner of the tooth, through the area of ​​maximum calculus height.

[0159] The examiner then assigned a score to each measurement plane. Measurements were made in increments of 0.5 mm, starting at 0.5. A score of (0) indicated the absence of stones at the measurable site. The V-MI for each subject was calculated by summing the millimeter scores for all rated sites.

[0160] Take digital photos of the lingual sides of the six lower anterior teeth.

[0161] Methodology and study design This study was conducted as a randomized, single-blind, single-center, parallel-group clinical trial with three treatments to evaluate the safety and efficacy of the enzyme formulation in removing stone accumulation. Participants underwent a Volpe-Manhold Index (V-MI) stone examination as described below at each visit.

[0162] According to the eligibility criteria, forty (40) subjects with at least 9 mm of calculus on the lingual surface of their mandibular anterior teeth were eligible to continue participating in the study. Subjects were randomly assigned to one of three groups (a control group of 20 subjects and each test group of 10 subjects).

[0163] All treatment groups brushed their teeth twice daily for 2 minutes with an ADA-approved toothbrush and dental floss. All three groups followed the following protocol: Test Group A: Brush teeth for two minutes with a control toothbrush and Crest toothpaste, twice a day.

[0164] Test Group B: Using a toothbrush, brush teeth for 1 minute with half the amount of enzyme formulation in the syringe (1 ml / syringe). Subjects were instructed to start brushing on the lingual (tongue) side of their lower incisors with a separate toothbrush and finish on the same teeth. Then, subjects brushed with control toothpaste, spat it out, reapplied the remaining amount in the syringe, emptied the syringe containing the enzyme formulation, and brushed again for another minute, starting on the lingual side of their lower incisors and ending there after 1 minute of brushing, then spat it out without rinsing. Brushing with the enzyme formulation was completed at least 30 minutes before the control brushing. Subjects were instructed to spit out the enzyme formulation without rinsing after use for at least 30 minutes. Subjects then brushed their teeth twice daily for two minutes with both the control toothbrush and Crest toothpaste. In total, subjects brushed their teeth four times daily, twice with the enzyme formulation and twice with Crest toothpaste. The control toothbrush was rinsed daily with hot water until clean.

[0165] Test Group C: Before using the custom-made tray, subjects brushed their teeth for 1 minute using a separate toothbrush designated only for the enzyme formulation, applying half a syringe (1 ml / syringe), then spitting it out without rinsing. A custom-made tray was filled with the enzyme formulation from a second syringe and worn once daily for 30 minutes. The tray was removed, and subjects then brushed their teeth for 1 minute with the remaining enzyme formulation from the first syringe, spitting it out, without rinsing for at least 30 minutes. Subjects completed their oral care routine by brushing their teeth twice daily for two minutes with a control toothbrush and toothpaste (Crest). The control toothbrush was rinsed daily with hot water until clean.

[0166] Participants were stratified based on calculus levels and sex obtained during screening / baseline examination. During the 4-week testing period, participants brushed their teeth twice daily under unsupervised supervision and returned at weeks 2 and 4 for safety and V-MI checks.

[0167] Evaluation and data collection The study testing phase comprised a total of 56 treatment sessions and 3 clinical visits over a 4-week period. Evaluation data were collected for each participant at baseline, 2 weeks, and 4 weeks. The mean dataset for each group was calculated.

[0168] Study endpoints and efficacy evaluation: The following endpoints were set for this study: Primary endpoint: 1. After 4 weeks of treatment, the number of stones in the test group was significantly reduced compared with the control group.

[0169] 2. Compared with the longitudinal group values, the number of stones in the test group was significantly reduced.

[0170] 3. Safety assessment based on adverse events in either test group after 4 weeks of treatment.

[0171] 4. Stones are reduced by removing existing deposits and / or preventing new deposits.

[0172] Secondary endpoints: 1. Prevention of stone accumulation in the treatment group, as evidenced by no significant increase in stone score compared to baseline.

[0173] 2. After 2 weeks of treatment, the number of stones in the test group was significantly reduced compared with the control group.

[0174] Exploratory endpoints 1. Any difference in taste when a patient rubs their tongue against the back of their incisors? 2. Any improvement in the patient's breath? 3. Any changes in the appearance or color of the teeth. Analysis An analysis set was performed, including all subjects who used the experimental gel at least once. The performance analysis set consisted of all subjects who provided at least one post-treatment performance measure. Only observed data were used; missing data were not entered.

[0175] Subject disposition The safety analysis set was used to describe the treatment of subjects.

[0176] Subject management was tabulated, outlining the number of subjects enrolled, exposed, prematurely terminated, and completed. A withdrawal / termination list was prepared, including the reason for withdrawal and the time of withdrawal.

[0177] Safety analysis Safety analysis was descriptive in nature, with SAEs and AEs tabulated by body system, preference, severity, and relationship to the study gel. In relevant cases, events were correlated with remote log file data. Additionally, safety complaints reported by participants were described and investigated.

[0178] Oral examination was performed according to the OSHT (Oral Soft & Hard Tissue Assessment). The evaluation of oral soft and hard tissues involved a visual examination of the oral cavity and perioral region using a standard dental lamp, dental mirror, and gauze. Structures examined included the gingiva (free and attached), hard and soft palate, oropharynx / uvula, buccal mucosa, tongue, floor of mouth, labial mucosa, buccal / labial mucosal folds, lips, teeth, and perioral region. All abnormalities that might be product-related and were noticed after product dispensing, whether not documented at baseline or present at baseline but worsened during product use, were recorded on the AECRF.

[0179] Performance analysis Efficacy was determined by the reduction of calculus within the treatment group (longitudinal analysis) and between the treatment and control groups after 4 weeks of treatment. Furthermore, efficacy was determined by the prevention of calculus accumulation in the treatment group, as evidenced by the absence of a significant increase in calculus score compared to baseline.

[0180] The primary outcome was calculated using the Volpe-Manhold index, and all other variables were considered secondary. Stone scores from this index were summed to provide a total score for each oral cavity at each clinical examination. Although analyses were performed at other measurement points during the study period, the primary time point was at the end of the trial, four weeks after product use.

[0181] Analysis of variance was used to examine differences between product groups, and the statistical significance of the mean data for age and rating index was determined parametrically. If necessary, inter-group comparisons for each parameter were performed using multiple range tests, with gender compensation applied. Data from all subjects who completed the final evaluation were used in the analysis.

[0182] The data for each scoring index were also analyzed using analysis of covariance with baseline data as covariates. Including covariates (baseline data) in the statistical model improves the accuracy of determining the impact of the test product on the scores. The adjusted mean generated by this procedure compensates for any differences present in the baseline data between treatment groups. This reduces variability and increases efficacy, and also adjusts for baseline imbalances caused by participant attrition.

[0183] A one-sample t-test was used to compare the mean VMI longitudinally (i.e. within treatment) based on the change from baseline to the final examination.

[0184] All comparisons were performed using two-tailed tests at an overall significance level of 0.05. Summary statistics were provided for all time points. Safety data were summarized. Adverse events experienced by all subjects who received the tested product were included in the safety analysis. Adherence to the study protocol, as well as accuracy and completeness, were examined for all cases. Cases that did not meet these requirements were excluded from the initial analysis.

[0185] Post-power analyses were performed on endpoints that were determined to be non-statistically significant. This determined the appropriate sample size required for the driven study.

[0186] All reported adverse events were listed, with details of duration, outcome, severity, and possible relationship to the investigational product. The frequency of subjects experiencing one or more adverse events during the trial was tabulated and compared across treatment groups. If necessary, the likelihood ratio chi-square test was used to compare the frequency of adverse events experienced by subjects in each treatment group.

[0187] Results Study population / enrollment: The final list of enrolled subjects is provided in Table 4: Table 4: Enrollment and Exit Data Subjects # Recruitment / scheduled 44 Withdrawals / no shows 0 Subject consent 44 Screening / baseline failures 4 Randomized to product 40 Received product 40 Disqualified / withdrawn 2 Completed 38 Subject withdrawals / disqualifications 2 All participants who completed the study demonstrated satisfactory adherence. Forty participants were randomized, and 38 completed the study. Of the randomized participants, 22 were female and 18 were male. The mean age was 50.4 years, with a minimum age of 23 years and a maximum age of 74 years. Baseline stone scores were similar across all treatments.

[0188] Oral tissue examination An oral examination was performed, including the gingiva (free and attached), hard and soft palate, oropharynx, buccal mucosa, tongue, floor of mouth, labial mucosa, buccal / labial mucosal folds, lips, and perioral region. No evidence of hard or soft tissue injury related to the use of the test product was observed and / or reported. An overview of adverse events observed during the examination or self-reported by the subjects is provided in Table 5.

[0189] Table 5: Overview of Adverse Events # Group Examiner or self-reported AE AE rating Product-related Resolved within 5 days Treatment-based resolution 1019 A Examiner Gingival margin abrasion (toothbrush) #26 Mild Likely caused by brushing Yes Yes 1024 B Examiner Gingival margin erythema #27 Mild Likely caused by brushing Yes Yes 1032 B Examiner Gingival margin erythema #24 & 28 Mild Likely caused by brushing Yes Yes 1044 B Examiner Inflammation of the papillae on the anterior side of the tongue Mild Possible Yes No 1013 C Examiner Gingival margin erythema #23-26 Moderate Likely caused by brushing Yes Yes 1044 Self-reported Burning sensation of the tongue NA Possible Yes No 1012 C Self-reported Gingival sensitivity NA Possible Yes Yes 1012 C Self-reported Lip sensitivity NA Possible Yes Yes All adverse events were resolved within a 5-day reassessment period. All adverse events observed in test groups B and C (inspector and self-reported) were attributed to the test product. Erythema was most likely caused by a brushing procedure of 4 times per day compared to the control group who brushed twice daily. Adverse events in subject 1019 were toothbrush abrasion.

[0190] Clinical study results A summary of the Volpe-Manhold index scores and changes from baseline to week 4 for each of the three treatments is provided in Tables 6 and 7. Figure 3 In the 4-week study, both enzyme formulations reduced stone size. Enzyme formulation alone reduced stone size by 40.0%, while enzyme formulation plus a custom tray containing the enzyme formulation reduced stone size by 38.1%. Longitudinal within-group analysis showed statistically significant stone reduction in both enzyme formulation groups over the 4-week study (p<0.001). The reductions were similar between the two groups. In contrast, the control group showed a statistically significant increase in stone size of 12.2% over the 4-week study (p<0.001). The reduction at 2 weeks was 0.8% in the enzyme formulation group and 1.6% in the enzyme formulation plus custom tray group.

[0191] Table 6: Volpe-Manhold Index Scores Table 7: Changes in Volpe-Manhold Index Score over 4 Weeks Table 8 shows that, compared with the control group, the two enzyme-based treatments showed statistically superior reduction in stones (p<0.001).

[0192] Table 8: ANCOVA scores of VM-I stone scores at week 4 Table 9 provides the week 4 analysis of the covariance results. As shown in Table 7, neither group treated with the active enzyme formulation showed a clinically or statistically significant reduction at week 2. The confidence intervals provided in Table 8 indicate that the group receiving dental treatment was statistically superior to the control group in terms of calculus reduction.

[0193] Table 9: Week 4 Analysis of Covariance Results for the Volpe-Manhold Index Effect Estimate Standard error T-value Pr>|t| α Lower limit Upper limit A minus C 8.7642 0.8490 10.32 <.0001 0.05 7.0370 10.4914 A minus B 8.7942 0.8854 9.93 <.0001 0.05 6.9929 10.5955 B minus C 0.0300 1.0287 0.03 0.9769 0.05 -2.0629 2.1229 Safety (Week 2 and Week 4) Oral soft and hard tissue examinations (OSHT) were performed at baseline, week 2, and week 4. As shown in Table 5, except for four participants (#1019, #1024, #1032, #1013), all findings at week 2 and week 4 were continuations of the prior symptoms noted at baseline.

[0194] Example 7: Results Overview The results from the study indicate that the study participants tolerated the use of the disclosed enzyme formulation well.

[0195] In the 4-week study, participants showed a clinically significant and statistically significant reduction in stones. Figure 4 Furthermore, participants in the active treatment group gave more favorable responses to how clean their teeth felt. Compared to the control group, 75% of group B (enzyme formulation) and 67% of group C (enzyme formulation with tray) gave positive feedback, while only 50% of participants in the control group gave positive feedback (data not shown). Additionally, groups B and C reported that their teeth felt cleaner, smoother, and less gritty. Figure 5 ).

[0196] Example 8: Analysis of the composition and structure of dental plaque / tartar Calculus collected from the tooth using a metal alloy dental probe was placed in 0.5 mL of water in a 1.5 mL microcentrifuge tube. The calculus was then crushed using a Teflon pestle attached to the microcentrifuge tube. The contents were centrifuged, the supernatant was aspirated and resuspended in 1 mL of water, and 100 μL aliquots were collected and placed in one or more microcentrifuge tubes. The contents of each tube were centrifuged, the supernatant was aspirated and then resuspended in 300 μL of water in a tube containing one of the following: No substances were added (water control). trypsin chymotrypsin DNA enzyme 1 nuclease "Genius nuclease" The contents of each tube were mixed and incubated at approximately 37°C for 30 minutes. The contents were centrifuged, and the supernatant was removed. The presence and amount of protein, DNA, and calcium were analyzed. The original quantifications were multiplied by a dilution factor (e.g., 1:3 for calcium, 1:5 for DNA, or 1:5 for protein) to obtain the total amount of protein, DNA, or calcium isolated from the sample. Table 11 shows the results obtained. The results shown in Table 11 indicate that chymotrypsin and DNase 1 are more efficient than trypsin or other nucleases in extracting protein, DNA, or calcium from gallstones.

[0197] Table 11: Percentage of Observed Maximum Product Output Protein (% of control) DNA (% of control) Calcium (% of control) Control 100 100 100 Trypsin 397 691.7 241.6 Chymotrypsin 7225.2 1125 550 DNase 1 3137.8 900 258.3 Nuclease 557.4 300 50 "Genius Nuclease" 173.8 0 25 Example 9: Human Clinical Study Method As a parallel-group, examiner-blinded, randomized, three-treatment clinical trial, a proof-of-principle human clinical study was conducted to evaluate the safety and efficacy of a novel enzyme formulation (i.e., Tartarase) in removing existing calculus deposits within one month, measured using the Volpe-Manhold Index (V-MI) on the lingual surfaces of the six lower anterior teeth. Secondary objectives included a safety assessment evaluated by examination of gingival tissue (free and attached), hard and soft palate, oropharynx, buccal mucosa, tongue, floor of mouth, labial mucosa, buccal / labial mucosal folds, lips, and perioral region. Additionally, a questionnaire assessing oral cleanliness using a five-point subjective scale (1 = least clean and 5 = cleanest) was selected and included as an exploratory endpoint. Safety assessments and oral examinations were performed at all visits. Forty participants consented to enrollment, with 20 randomized to the control group (Crest Cavity Protection) and 20 assigned to the Tartarase treatment group.

[0198] This study was approved by the USIRB (USIRB2022SRI / 10) in accordance with ICH Guideline E6 and the US Food and Drug Administration, and conducted by Salus Research, Inc. (an American Dental Association (ADA) Qualified Independent Research Site). The study product was fluoride-free, and the IRB required all participants to brush their teeth twice daily with fluoride toothpaste.

[0199] Adult participants with six (6) mandibular anterior teeth, known as rapid tartar builders, were recruited from the Salus Research database. These participants had at least 9 mm of tartar measured on their six mandibular anterior teeth, as evaluated using V-MI (dental cleaning completed between 2 and 6 months prior to study start). Participants who did not brush regularly, used chlorhexidine mouthwash, or required antibiotic prophylaxis prior to dental procedures were excluded. Participants withdrew from the study if they did not follow product instructions or used any oral hygiene product other than the designated study product. Flossing, electric toothbrushes, and mouthwash were prohibited.

[0200] During the initial visit, participants were evaluated to ensure at least 9 mm of calculus on the lingual surfaces of the six mandibular anterior teeth, and demographics, medical history, and concomitant medications were assessed for eligible participants. Forty eligible participants were randomly assigned to one of three study groups using a randomization protocol developed and maintained by an independent statistician. Participants were stratified by baseline lingual V-MI calculus score and sex. Half of the test participants (n=20) were randomized to the control group, and 10 were assigned to each Tartarase group (Tartarase Brush only and Tartarase Brush + tray). All participants underwent their first use under supervision at the study site, followed by unsupervised use during the 4-week test period. For each participant, evaluation data were collected at baseline, 2, and 4 weeks (Table 12). All test participants used the same ADA reference soft manual toothbrush, brushing twice daily with control toothpaste Crest® Cavity Protection Cool Mint Gel (0.243% sodium fluoride). Test group assignments are as follows: 1. Control group (20 participants): Brushed their teeth twice a day with an ADA toothbrush and Crest toothpaste for two minutes each time.

[0201] 2. Tartarase Brush (10 participants): Brush teeth twice daily with Tartarase and twice daily with Crest.

[0202] 3. Tartarase Tray (10 participants): Custom-made trays were created for all participants using the Tartarase tray. Participants brushed their teeth with Tartarase for 1 minute, then applied Tartarase using the custom tray (worn once daily for 30 minutes). The procedure was then repeated with another 1 minute of Tartarase brushing. The Tartarase brushing / tray / brushing was performed once daily. Participants also brushed their teeth with Crest twice daily.

[0203] Table 12: Baseline characteristics, including the mean Volpe-Manhold index (V-MI) for different treatment groups, demographic data for different treatment groups, and normal brushing habits for different treatment groups.

[0204] Tartarase formulation The formulation was prepared in two batches, each sufficient for 20 participants to receive 2.0 ml / day for 14 consecutive days. Contains: cetearyl alcohol polyether-25 (0.23%), glycerol (13.6%), xanthan gum (0.28%), sorbitol (4.4%), and hydrated silica (11%). Also contains: DNase 1 (197,326 Kunitz units / ml) and chymotrypsin (3,333 units / ml). These enzymes were obtained from Worthington Biochemicals.

[0205] For this one-month study, the test product was formulated using twice the level per unit volume that had previously shown effectiveness in preclinical studies using canine stones.

[0206] Efficacy evaluation and statistical analysis Efficacy was determined by changes in calculus within the treatment group (longitudinal analysis) and between groups compared to the control group after 4 weeks of treatment. Additionally, the efficacy of the Tartarase treatment group was determined by prevention of calculus accumulation, assessed by no significant increase in calculus score compared to baseline. The primary outcome was calculus abundance using the Volpe-Manhold index, with all other variables considered secondary. Measurements were taken on the lingual surfaces of the six mandibular anterior teeth. Calculus scores from this index were summed to provide a total score for each oral cavity at each clinical examination. The primary time point was at the end of the trial after 4 weeks of product use.

[0207] Data from all subjects who completed the final assessment were used in the analysis. Data for each scoring index were also analyzed using analysis of covariance with baseline data as covariates. Covariates (baseline data) were included in the statistical model to improve the accuracy of determining the impact of the test product on the scores. Adjusted means generated by this procedure compensated for any differences between treatment groups present in the baseline data. This reduced variability and increased power, and also adjusted for baseline imbalances due to subject attrition. Longitudinal (i.e., within-treatment) comparisons of the V-MI means were performed using one-sample t-tests based on changes from baseline to the final assessment. All comparisons were performed using two-tailed tests at an overall significance level of 0.05.

[0208] Safety assessment Oral examinations were performed at all visits, including the gingiva (free and attached), hard and soft palate, oropharynx, buccal mucosa, tongue, floor of mouth, labial mucosa, buccal / labial mucosal folds, lips, and perioral region. Additionally, subjects were asked about any adverse events (AEs) since the previous visit. AEs were categorized as serious or non-serious based on severity, whether expected or unexpected, and their likely relationship to the test product. All AEs (examiner and self-reported) were assessed at subsequent visits to determine if they had been resolved.

[0209] result An overview of the research group's daily brushing schedule is shown in Figure 6 Forty (40) participants were randomized and thirty-seven (37) completed the study. One patient refused to comply with the study procedures and was removed from the study; two patients withdrew their consent forms. Of the randomized participants, there were 22 females and 18 males. The mean age was 50.4 years, with a minimum age of 23 years and a maximum age of 74 years. Caucasians comprised 33 (82.5%) of the 40 participants, and none of the patients identified as Hispanic or Latino. Baseline calculus scores were similar across all treatments. Participants reported similar daily oral hygiene routines (twice daily).

[0210] Efficacy: Dental calculus In the 4-week study, both treatments with Tartarase reduced tartar. Specifically, the Tartarase brushing group alone removed 40.0% of tartar, and the Tartarase brushing + tray group removed 38.1%. Longitudinal within-group analysis showed statistically significant tartar reduction in both Tartarase groups during the 4-week study (p<0.001), and statistically different from the Crest control group (Table 13). Results were similar in both Tartarase treatment groups, and all 17 samples assigned to the experimental product showed tartar reduction. Figure 7 In contrast, during the 4-week study, the Crest control group resulted in a statistically significant increase in kidney stones of 12.2% (p<0.001). Furthermore, the Crest control group showed increased stone deposition in the tested subjects. Figure 7 During the 2-week evaluation period, no test group showed significant changes.

[0211] Table 13: Mean baseline and 4-week V-MI measurements in different treatment groups, as well as percentage changes and directions of V-MI. Oral hygiene questionnaire Compared to the control group, participants in the Tartarase treatment group reported more favorable responses regarding how clean their teeth felt. 75% of those brushing with Tartarase alone and 67% of those brushing with Tararase plus the tray self-reported a positive rating of "4" or "5" (5 = cleanest). Figure 8 The control group reported 50% positive approval for either category "4" or "5". Test subjects were given the opportunity to provide their overall impression of the assigned Tartarase product regarding oral cleanliness, including: 1. "I can feel every single tooth in my mouth, and they all feel smooth." 2. "I can feel that my individual teeth are better, and my teeth—especially my lower inner teeth—feel cleaner." 3. "I feel like my mouth stays clean and fresh all day. My breath smells better." Security issues No safety issues were observed with Tartarase, and no serious adverse events (AEs) or severe acute exacerbations (SAEs) were reported. Tartarase was well tolerated. Based on oral examination, there were 5 AEs (4 AEs were attributed to toothbrush abrasion and the other to mild inflammation of the anterior papillae of the tongue). In total, there was 1 AE in the control group, 3 AEs in the Tartarase brushing group, and 1 AE in the Tartarase tray group (Table 14). No changes in oral hard tissues were observed during the 4-week trial period. Three test subjects self-reported AEs, with 2 reporting gingival or lip sensitivity (Tartarase tray group) and 1 experiencing tongue burning (Tartarase brushing group). All AEs (examiner observation and self-report) resolved within a 5-day reassessment period (Table 14).

[0212] Table 14: Overview of Adverse Events Overview of Example 9 After 4 weeks, Tartarase significantly reduced tartar buildup, with both Tartarase test groups showing a reduction of approximately 40%, while the Crest control group showed a 12% increase in tartar deposits. Furthermore, after 4 weeks of unsupervised daily use, every Tartarase test subject experienced a reduction in tartar. Not wanting to be bound by theory, the results of this study support the theory that DNA and proteins together provide structural support for tartar, and that enzymes targeting DNA and denatured proteins are particularly effective in disrupting the structure of tartar, thereby reversing tartar buildup. Equally important, the results showed no evidence of further tartar buildup, indicating that the Tartarase formulation effectively prevents tartar formation after teeth cleaning. Tartarase test subjects showed approximately a 40% reduction in tartar over 4 weeks, while the control group experienced a 12% increase in tartar. Consideration should be given to creating custom trays for application.

[0213] Since the active ingredient in Tartarase is an enzyme, it is important to consider the overall characteristics of the enzyme. Enzymes are protein catalysts. They promote normally occurring chemical reactions to occur at an enhanced rate. Additionally, they allow reactions to occur in both forward and reverse directions. Therefore, for these types of enzymes, accumulated products inhibit the enzyme's catalysis of its forward reaction. This may explain why the Tartarase tray group did not outperform the Tartarase brushing group. Furthermore, in vitro studies have found that enzymes act extremely rapidly, with reactions completed in <5 minutes, indicating that the 30-minute incubation time in the tray group did not provide an advantage.

[0214] Preclinical studies have supported the importance of dual-enzyme formulations. It is hypothesized that the only way to attack calculus is on its outer two-dimensional surface. The model of calculus structure consists of a calcified complex of a basic biopolymer. If an enzyme is specific to only one complex biopolymer, it cannot proceed further once the target biopolymer has been consumed, as the remaining target is masked by biopolymers that are not the target of that single enzyme. Therefore, the combined use of enzymes allows for a progressive mechanism that systematically peels calculus from its outermost surface, eventually reaching the enamel.

[0215] Clinical studies have demonstrated its ability to reduce tartar. During a 4-week trial period, the tested Tartarase toothpaste showed significant tartar removal in test subjects who were known tartar builders and had cleaned their teeth between 2 and 6 months prior to entering the study. This trial suggests that the Tartarase formulation has the potential to reduce tartar when used with routine daily oral hygiene.

[0216] This study was designed to evaluate the safety and efficacy of a novel enzyme formulation (Tartarase) in removing existing tartar deposits, measured using the Volpe-Manhold Index (V-MI) on the lingual surfaces of six lower anterior teeth, over a one-month period. The test formulation, containing a high excess of the active enzyme in a common mixture of inactive ingredients, was compared to Crest Cavity Protection as a control. A total of 40 randomized participants began the study, with 20 assigned to the control group and 20 to the Tartarase group (one group brushed with Tartarase twice daily, and the other group brushed with Tartarase and wore a Tartarase-filled denture for 30 minutes, followed by brushing with Tartarase again). Results showed a 12% increase in tartar in the Crest control group compared to a 40% reduction in tartar in the Tartarase group.

[0217] Table 15: Sequences

Claims

1. A dental composition comprising: a. DNA enzymes and / or RNA enzymes; b. Chymotrypsin or its functional variants or derivatives; and c. Orally acceptable additives, carriers or excipients, or any combination thereof.

2. The dental composition according to claim 1, wherein the composition does not contain β-galactosidase.

3. The dental composition according to any one of claims 1 or 2, wherein the DNase is present in an amount of about 50,000 to about 250,000, or about 100,000 to about 200,000, or about 150,000 to about 200,000, or 150,000, or 160,000, or 170,000, or 180,000, or 185,000, or 190,000, or 200,000 Kunitz units / mL.

4. The dental composition according to any one of claims 1-3, wherein the DNase is DNase I or a functional variant or derivative thereof.

5. The dental composition according to any one of claims 1-4, wherein chymotrypsin or a functional variant or derivative thereof is present in an amount of about 100 to about 5,000, or about 500 to about 4,000, or about 1,000 to about 3,000, or 1,500, or 1,600, or 1,700, or 1,800, or 1,900, or 2,000, or 2,100, or 2,200, or 2,300, or 2,400, or 2,500, or 2,600, or 2,700, or 2,800, or 2,900 or 3,000 units / mL based on the hydrolysis rate of the chromogenic substrate.

6. The dental composition of claim 1, wherein the orally acceptable additive is a thickener and / or gelling agent selected from gellan gum (low-acyl or high-acyl), glycerin, silica, guar gum, xanthan gum, polyethylene glycol, polyvinylpyrrolidone and its copolymers, polylactic acid, polyglycolic acid, long-chain fatty acid alcohols, cellulose-based polymers, acrylate polymers and any combination thereof.

7. The dental composition of claim 1, wherein the orally acceptable carrier is selected from ethanol, isopropanol, glycerol, or sorbitol.

8. The dental composition of claim 1, further comprising a surfactant selected from cetearyl alcohol polyether or stearyl alcohol polyether.

9. The dental composition of claim 1, wherein the composition comprises: a. DNA enzyme I or its functional variants or derivatives a. Chymotrypsin or its functional variants or derivatives; and one or more of the following, c. 10%-20% glycerin, d. 0.000001%-0.00001% cetearyl alcohol polyether-25, e. 0.1%-0.5% xanthan gum, f. 1%-10% sorbitol, and g.5-25% silicon dioxide.

10. The dental composition according to claim 9, further comprising water.

11. An oral hygiene device comprising the composition according to any one of claims 1-10, wherein the composition is combined with a dental tray, capsule applicator, oral cleaning stick, or dental floss.

12. Use of the composition or device according to any one of claims 1-11 for the removal of dental calculus in a subject in need of it.

13. Use of the composition according to claim 12, wherein the subject is a mammal.

14. Use of the composition according to claim 12, wherein the subject is a human.

15. A method for reducing or removing dental calculus in a subject in need of it, said method comprising contacting a tooth surface with an effective amount of a composition comprising: a. DNA enzyme I or its functional variants or derivatives, and b. Chymotrypsin or its functional variants or derivatives.

16. The method of claim 15, wherein the composition does not contain β-galactosidase.

17. The method of claim 15, wherein the composition further comprises an orally acceptable additive, carrier, or excipient, or a combination thereof.

18. The method according to any one of claims 15-17, wherein the composition comprises a paste or liquid, and contact with the tooth surface comprises applying the composition using a brush, a tray, a mouthwash, or any combination thereof.

19. The method of claim 18, wherein the method comprises applying the medication at least once a day, at least twice a day, or at least three times a day for a period of at least one week.

20. A method for reducing or removing dental calculus in a subject in need of it, the method comprising contacting a tooth surface with an effective amount of the composition of any one of claims 1-10.

21. The method according to any one of claims 15-20, wherein the subject is a mammal.

22. The method of claim 21, wherein the subject is a dog, horse, cat, or primate.

23. The method of claim 21, wherein the subject is a human.

24. A kit comprising: a. The composition according to any one of claims 1-10; b. Instructions for use.

25. The kit of claim 24, further comprising at least one applicator selected from toothbrushes, oral cleaning sticks, dental floss, and dental trays.

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