AU2019100471A4 - Methods and compositions for reducing tooth, root and dentin sensitivity - Google Patents

Abstract

Abstract Methods of reducing tooth, root and dentin sensitivity by administering an oral care product (a dentifrice or a mouthwash) that consists essentially of a sea salt, xylitol and aloe vera leaf juice.

Description

Methods and Compositions for Reducing Tooth, Root and Dentin Sensitivity

2019100471 02 May 2019

Field

Oral care products (mouthwashes, toothpastes) that reduce tooth, root and dentin sensitivity as well as reduce plaque and tartar, reduce inflammation/gingivitis, and remineralize tooth enamel. Methods of protecting the oral microbiome by neutralizing toxins secreted by bacteria in the human oral cavity - Gingipains, Leukotoxin A, and Lipopolysaccharides - with clinicallyeffective amounts of natural and naturally-derived, non-cytotoxic and non-bactericidal ingredients.

Background .0 Essential oils have been used for the treatment of a variety of ailments since ancient times. The safety and efficacy of essential oils in dentistry have been reported in numerous clinical studies. See, e.g., Namrata Dagli et al., Essential oils, their therapeutic properties, and implication in dentistry: A Review J. Int. Soc. Prev. Community Dent. Vol. 5, No. 5 (2015), pp. 335-340.

Enamel is a quasi-translucent structure that serves protects the teeth from decay. Improper .5 brushing habits, consuming acidic foods and beverages, using at-home peroxide-containing whitening products, and clenching or grinding teeth all cause teeth sensitivity. These activities wear away and break down the enamel. There has been and remains a need for oral care products that reduce teeth sensitivity, improve plaque control, reduce gingivitis, but that do not cause dysbiosis in the oral microbiome. These needs are met by the methods and compositions 20 of the present invention.

There also exists a need to maintain a healthy oral microbiome by administering a noncytotoxic oral care product, preferably in the form of a solution (also known in the dental arts as a mouthwash), and thereby reducing levels of glucansucrases, Leukotoxin A, Gingipains, and lipopolysaccharides from P. gingivalis without causing a decrease in the levels of common 25 bacteria in the oral cavity - Streptococcus mutans, Porphyromonas gingivalis, Staphylococcus aureus, Fusobacterium nucleatum, subsp. nucleatum Knorr and Fusobacterium nucleatum

Strain W1481. These needs are also met by the methods and compositions of the present invention.

2019100471 02 May 2019

Summary

The present invention is directed to methods for reducing dentin sensitivity by administering oral care products consisting essentially of (i) a Dead Sea salt, at a concentration of from 0.5% to 3.0%; (ii) at least one essential oil selected from the group consisting of peppermint oil, Wintergreen oil, or spearmint oil; (iii) xylitol; and (iv) preferably, aloe vera leaf juice.

A key attribute of certain preferred oral care products used in practicing methods of the present invention is the absence of fluoride.

.0 Additional key attributes of oral care products used in practicing methods of the present invention:

(i) non-cytotoxicity within the framework of ISO 10993-5 Biological Evaluation of Medical Devices - Tests For In Vitro Cytotoxicity, as described below;

(ii) not being bactericidal, as described detail below;

.5 (iii) not containing: artificial colors, dyes or flavors; paraben or formaldehyde preservatives; sodium lauryl sulfate or sodium laureth sulfate; petroleum-derived glycerin; or genetically modified organisms (GMOs).

A further key aspect of mouthwash embodiments used in practicing the methods of the present invention is the absence of ethyl alcohol. Such mouthwashes are sometimes described as alcohol free - a term that is to be understood by the person having ordinary skill in the art as referring to ethyl alcohol. (Alcohol free mouthwashes may contain other alcohols, including menthol or glycerol.)

Detailed Description

2019100471 02 May 2019

Dentinal hypersensitivity (also referred to in the dental arts as dentin sensitivity and in the present application as tooth sensitivity or tooth, root or dentin sensitivity) is defined as a condition characterized by short, sharp pain arising from exposed dentine in response to stimuli, typically thermal, evaporative, tactile, osmotic or chemical and which cannot be ascribed to any other dental defect or pathology. See GR Holland et al., Guidelines for the design and conduct of clinical trials on dentine hypersensitivity. J Clin. Periodontol. Vol, 24, pp. 808-13 (1997). Dentin sensitivity can be assessed clinically by a jet of air or using an exploratory probe on the exposed dentin, in a mesio-distal direction, examining all the teeth in .0 the area in which the patient complains of pain. The extent of pain can be quantified according to categorical scale (i.e., slight, moderate or severe pain) or using a visual analogue scale.

By the term dentifrice is meant a preparation for cleansing and polishing the teeth, that may, and preferably does provide one or more therapeutic benefits (as described below). As will be understood by the skilled artisan, a dentifrice (also referred to in the art and in this application .5 as a toothpaste) may be formulated as a paste, gel or powder and is preferably applied with a toothbrush.

Dentifrice embodiments of the present invention are administered by dispensing at least about 0.25 grams of a dentifrice of the present invention (commonly described in the scientific literature and in recommendations from dental professionals and toothpaste manufacturers as 20 a pea sized amount), and more preferably at least 0.50 grams of dentifrice, and brushing the teeth for at least 30 seconds, preferably for at least 60 seconds, and still more preferably for at least 120 seconds, at least once per day, in the evening prior to going to sleep. In certain preferred embodiments, the dose of toothpaste per administration ranges from about 0.4 to about 0.6 grams.

By brushing is meant placing the bristles of a toothbrush in contact with the teeth, preferably at an angle of about 45 degrees to the gum line (where the gums and teeth meet), and moving the bristles in gentle, short strokes along the outer surfaces (cheek side), the inside surfaces (tongue side) and the chewing surfaces of all teeth. The strokes may be in a back-and-forth

2019100471 02 May 2019 motion (side-to-side, or up-and-down) or a circular motion. After brushing, the dentifrice is expectorated.

Still more preferably, dentifrice embodiments of the present invention are administered by brushing the teeth for at least 30 seconds, preferably for at least 60 seconds, and still most preferably for at least 120 seconds, twice daily - once in the evening prior to going to sleep; and once in the morning after breakfast.

By the term mouthwash is meant a solution that is swished, preferably vigorously, around the mouth, and then expectorated, thereby cleaning the mouth and making the breath smell pleasant.

.0 Mouthwash embodiments of the present invention are administered by swishing about 15-20 ml (about 0.5-0.75 fluid ounces) in the mouth, for a period of time sufficient to contact the teeth, the gums, the roof of the mouth and the tongue. Preferably, mouthwash is swished for at least 30 seconds, preferably for at least 60 seconds, at least once per day, in the evening prior to going to sleep.

.5 Still more preferably, mouthwash embodiments of the present invention are administered for at least 30 seconds, preferably for at least 60 seconds, most preferably for at least 120 seconds, at least once per day twice daily -once in the evening, after brushing prior to going to sleep; and once in the morning after brushing after breakfast.

In especially preferred embodiments, a person practicing a method of the present invention (e.g., a consumer or a patient) brushes his/her teeth with a dentifrice of the invention for at least 30 seconds, more preferably for at least 60 seconds, and then swishes a mouthwash of the invention in his/her mouth for a period of at least 30 seconds, preferably at least 60 seconds - a period of time sufficient for the mouthwash to contact the teeth, the gums, the roof of the mouth and the tongue.

Persons practicing a method of the present invention are instructed to abstain from drinking or eating for at least 30 minutes after administering the oral care compositions of the invention; and, since oral care compositions of the invention are not intended for ingestion, to

2019100471 02 May 2019 expectorate the oral care composition after use (i.e., brushing in the case of a dentifrice, or swishing in the case of a mouthwash).

Non-cytotoxicity of oral care products of the present invention is confirmed within the framework of ISO 10993-5:2009 based on the widely-used Trypan blue exclusion test. (Trypan blue is a colorant which stains dead cells, i.e., cells with loss of membrane integrity.) More particularly, Balb/c 3T3 clone A31 cells (ATCC CCL 163; 86th passage) are seeded in multi-well plates (24 wells, each 15.5 mm in diameter) at the starting density of 30,000 cells/cm2 in culture medium - Dulbecco's Modified Eagle Medium (DMEM) - supplemented with 10 % (v/v) fetal calf serum (FCS). No antibiotics are used. Cultures are incubated at 37°C in a humidified .0 atmosphere containing 5% (v/v) CO2, for 24 hours, and are examined with a microscope to verify a sub-confluent monolayer with less than de minimus alteration in morphology. Culture medium is withdrawn and replaced with a solution of one of the following: oral care products of the present invention at 5,000 qg/mL, as well as dilutions 1,500 qg/mL, 500 qg/mL and 150 qg/mL; a negative control (phenol, Chemical Abstract Service No. 108-95-2, 0.64mg/mL); and .5 a positive control (DMEM supplemented with 10 % (v/v) FCS and 1% antibiotics (v/v). A positive control is defined as statistically significant (30% or greater) inhibition of cell growth as compared to the negative control.

Wells are incubated at 37°C in a humidified atmosphere containing 5 % (v/v) CO2, for a 24-hour period. Photomicrographs are taken (320x magnification) showing the cell layer in contact with :0 the negative control, the positive control and the oral care product of the present invention.

Morphology and cell density of the cultures are observed. At the end of the incubation period, culture medium is removed. Cells are detached for two minutes using 250 μΙ_ trypsin (0.05 % (w/v) in Hank's balanced solution Ca++ and Mg++ free supplemented with 1 mM EDTA. 250 μΙ_ of a Trypan blue solution at 0.2 % (w/v) in 0.15 M NaCI and 10 % (v/v) FCS are added. Cells are 25 incubated for two minutes. Living cells (uncolored) are counted using a hemocytometer. Cell morphology and cell density of medium treated with 5,000 μg/mL of the oral care product of the present invention are observed to be comparable to those of negative control; neither

2019100471 02 May 2019 shows statistically significant (30% or greater) inhibition of cell growth. In contrast, cells treated with the positive control show greater than 50% inhibition on cell growth.

Oral care products of the present invention are “non-bactericidal” by which is meant that when administered at a concentration of 5 mg/ml in a 10 ml solution also containing 0.1 ml of artificial saliva (AFNOR NF S91-141, Pickering Laboratories, Inc. Mountain View, California) with 1 x 10⁵ - 1 x 10⁶ colony forming units (“CFUs”) of a bacteria selected from the group of S. mutans, P. gingivalis, S. aureus, Fusobacterium nucleatum subsp. nucleatum Knorr (ATCC® 25586D-5™), and Fusobacterium nucleatum Strain W1481, the bacterial count (i.e., number of CFUs) does not decrease.

.0 Numbers used in describing quantities of ingredients and/or reaction conditions are to be understood as being modified in all instances by the term about.

Unless otherwise indicated, percentages, parts and ratios are to be understood as based upon the total weight of the composition.

Numerical ranges are meant to include numbers within the recited range, and combinations of .5 subranges between, the given ranges. For example, a range from 1-5, includes 1, 2, 3, 4 and 5, as well as subranges such as 2-5, 3-5, 2-3, 2-4, 1-4, etc.

At least one means one or more, and also includes individual components as well as mixtures/combinations.

Dead Sea salt is a mixture of natural hygroscopic minerals and micronutrients found in the Dead

Sea and is comprised of sodium chloride, potassium chloride, calcium chloride and magenesium bromide. S. Halevy et al., J. Eur. Acad. Dermatol. Venereol., Vol. 9, pp. 237-242 (1997). Dead Sea salt also contains separate ions: Br-, Rb+, Li+, Fe3+, AI3+, SO42-, Sr2+ and Mn2+. See, e.g.,

I.L. Schamberg, Treatment of psoriasis at the Dead Sea, Int J Dermatol, Vol. 17, No. 6 (1978), pp. 524-525; Paz and Shani, supra.

Dead Sea salt is present in oral care products of the invention at a concentration of less than about 3%, preferably a concentration of from about 0.1% to 2%, still more preferably a

2019100471 02 May 2019 concentration of from about 0.5% to about 1.5%.

Xylitol is the pentahydric alcohol that conforms to the formula:

CHoOH

I

HCOH

HOCH

HCOH

CH₂OH

Xylitol is present in oral care products of the invention at a concentration of at least about 7.5%, more preferably at least about 10%, or at a dose of about 0.1 g/brushing or rinsing.

Aloe vera leaf juice useful in the present invention preferably contains (i) glycosides at a concentration of at least about 1%, preferably at least about 2%, and still more preferably at about 3%, as well as (ii) at least two, preferably three, anti-inflammatory agents selected from .0 the group of anthraquinones, sterols, auxins and gibberellins and (iii) and immunomodulatory muccopolysachharides, preferably Acemannan.

An essential oil is a mixture of terpenic hydrocarbons, especially monoterpenes and sesquiterpenes, and oxygenated derivatives such as aldehydes, ketones, epoxides, alcohols, and esters.

^5 Oral care products ofthe present invention contain one or more essential oils selected from the group consisting of spearmint oil, Wintergreen oil, and peppermint oil.

Spearmint oil is the volatile oil obtained from the leaves of Mentha viridis (also known as Mentha spicata).

Wintergreen oil is the volatile oil obtained from the leaves of Gaultheria procumbens.

Peppermint oil is a volatile oil obtained from the whole plant Mentha piperita.

In one set of preferred embodiments, oral care products of the present invention include at

2019100471 02 May 2019 least one essential oil in the genus Mentha, selected from Mentha piperita (peppermint) oil and

Mentha viridis (spearmint) leaf oil.

In yet another preferred embodiment, oral care products of the present invention include Gaultheria procumbens (wintergreen) leaf oil.

In one even more preferred embodiment, oral care products of the present invention include peppermint oil and one of wintergreen oil or spearmint oil.

In another even more preferred embodiment, oral care products of the present invention include wintergreen oil and one of peppermint oil or spearmint oil.

In a still further even more preferred embodiment, oral care products of the present invention .0 include spearmint oil and one of peppermint oil or wintergreen oil.

In especially preferred embodiments, the oral care products of the present invention contain spearmint oil, peppermint oil, and wintergreen oil.

Menthol, an alcohol that can be isolated from peppermint or other mint oils, can also be used in oral care products of the present invention.

.5 Oral care products of the present invention also preferably include one or both of of Ocimum basilicum (basil) oil and/or Eugenia caryophyllus (clove flower) oil.

In certain preferred embodiments of the invention, basil oil is present at a concentration of up to about 0.5%.

In certain preferred embodiments of the invention, clove oil is present at a concentration of at least about .005%. In other preferred embodiments of the invention, clove oil is present at a concentration of at least about 0.01%. In certain of these preferred embodiments, clove oil is preferably at a concentration of up to about 0.02%.

Other essential oils that may be included in oral care products of the present invention include Melaleuca alternifolia (tea tree) leaf oil, the oil distilled from the leaves of the Melaleuca alternifolia, and Zingiber officinale (ginger) root oil, which is obtained from the dried rhizomes

2019100471 02 May 2019 of Zingiber officinale.

Dentifrice embodiments of the present invention may include mild abrasives (to remove debris and residual surface stains), humectants (to prevent water loss in the toothpaste), thickening products, also known in the art as binders (to stabilize the toothpaste formula), flavoring products (for taste) and detergents (to create foaming action).

Mild abrasives suitable for use in the toothpaste embodiments of the present invention include calcium carbonate, dehydrated silica gels, hydrated aluminum oxides, magnesium carbonate, phosphate salts and silicates. Silica, also called silicone dioxide, bentonite clay and hydrated silica are minerals. Some toothpastes of the present invention preferably contain hydrated .0 silica.

Humectants that may be, and preferably are, ingredients in toothpastes of the present invention include glycerin, preferably vegetable glycerine, propylene glycol, and sorbitol.

Glycerin, a sugar alcohol that can be synthesized or obtained from natural sources, is an especially preferred humectant used in toothpastes of the invention.

.5 Non-limiting examples of thickening products that may be, and preferably are included in toothpaste embodiments of the present invention include gums and colloids. Preferred colloids are of marine origin, even more preferably seaweeds.

Two preferred gums are xanthan gum and biosaccharide gum-1; both are polysaccharides derived from the fermentation of carbohydrates. Xanthan gum is derived from glucose or corn 20 syrup. Biosaccharide gum-1 is derived from sorbitol.

Carrageenan, a polysaccharide hydrocolloid obtained from edible red seaweeds in the Gigartinaceae or Solieriaceae families, may be, and preferably is, present in toothpastes of the invention.

Examples

2019100471 02 May 2019

The following examples illustrate compositions and methods of practicing of the present invention in some of its embodiments; the examples should not be construed as limiting the scope of the invention. Other embodiments will be apparent to one skilled in the art from consideration of the specification and examples. It is intended that the specification, including 5 the examples, is considered exemplary only without limiting the scope and spirit of the invention.

Some of the examples illustrate preferred embodiments of the invention. Variations of these preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as .0 appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, unless otherwise indicated herein or otherwise clearly contradicted by context, the inventions include all modifications and equivalents of the subject matter disclosed and recited in the claims appended hereto as permitted by applicable law.

.5 Dentifrice Examples

Dentifrices of the present invention are formulated for use by persons having sensitive teeth

- and contain the following ingredients at the following concentrations:

Dead Sea salt is present in toothpastes of the invention at a concentration of less than about 3%, preferably from about 0.5% to 2%, most preferably from about 0.75% to about 1.5%.

Xylitol is present in toothpastes of the present invention at a concentration of at least 10% or at a dose of 0.1 g/brushing.

Aloe vera leaf juice is present in toothpastes of the present invention at a concentration of at least about 10%, preferably at least about 20%, and most preferably at least about 40%.

Mentha piperita (peppermint) oil is present in toothpastes of the invention at a concentration 25 of less than 1%, preferably less than about .075%, more preferably at a concentration of less than about 0.05%, and even more preferably at a concentration of about 0.03%.

2019100471 02 May 2019

Mentha viridis (spearmint) oil is present in toothpastes of the invention, preferably at a concentration of up to about 4%, preferably up to about 2%, more preferably up to about 1%, and still more preferably less than about 0.5%.

Gaultheria procumbens (wintergreen) leaf oil is present in toothpastes of the invention, preferably at a concentration of up to about 1%, preferably from about 0.25% to about 0.5%.

In some preferred toothpaste embodiments, the weight ratio of wintergreen oil to peppermint oil is about 10:1.

In other preferred toothpaste embodiments, the weight ratio of spearmint oil to peppermint oil is about 3:1.

.0 In still other preferred toothpaste embodiments, the weight ratio of wintergreen oil to spearmint oil is about 3:1.

In one preferred mouthwash embodiment, the weight ratio of wintergreen to peppermint is from about 6:1 to about 5:1.

In another preferred mouthwash embodiment, the weight ratio of spearmint to peppermint is .5 about 5:3.

In still another preferred mouthwash embodiment, the weight ratio of wintergreen to spearmint is about 4:1.

Basil oil (also known as Ocimum tenuiflorum oil) is present in toothpastes of the invention, preferably at a concentration of up to about .05%.

Clove oil is present in toothpastes of the invention, preferably at a concentration of up to about 0.05%.

Glycerin is present in toothpastes of the present invention at a concentration of from about 2.5% to about 20%, preferably from about 5.0% to about 15%.

Carrageenan may be, and preferably is, present in toothpastes of the invention, preferably at a

2019100471 02 May 2019 concentration of at least about 0.05%. preferably about 0.1%. Even more preferably, carrageenan is food-grade.

Xanthan gum may be, and preferably is present in toothpastes of the invention, preferably at a concentration of at least about 0.10.

Titanium dioxide may be present in certain toothpastes of the invention; when present, titanium dioxide is present at a concentration of up to about 0.6%.

Hydrated silica may be, and preferably is, present in toothpastes of the invention, at a concentration of from about 10% to about 25%.

Toothpastes of the present invention contain a foaming anionic other than sodium lauryl .0 sulfate, preferably sodium methyl cocoyl taurate or sodium lauroyl sarcosinate. In certain preferred embodiments, sodium methyl cocoyl taurate is present in toothpastes of the invention at a concentration of up to about 2%.

Mouthwash Examples

Dead Sea salt is present in mouthwashes of the present invention at a concentration of from .5 about .5% to about 5%, preferably from about .75% to about 3%, and most preferably from about 1% to about 2%.

Xylitol is present in mouthwashes formulated for use in the present invention at a concentration of from about 5% to about 30%, preferably from about 7% to about 15%, and most preferably from about 8% to about 12%.

Aloe vera leaf juice is present in mouthwashes of the present invention at a concentration of from about 10% to about 90%, preferably from about 20% to about 85%, and most preferably from about 50% to about 70%.

Clove flower oil is present in mouthwashes of present invention at a concentration of from about .005% to about .075%, preferably from about .01% to about .04%, and most preferably 25 from about .01% to about .03%.

2019100471 02 May 2019

Basil oil is preferably present in mouthwashes of the present invention at a concentration of from about .005% to about .5%, preferably from about .01% to about .2%, and most preferably from about .02% to about .1%.

Peppermint oil is present in mouthwashes of the present invention at a concentration of from about .005% to about .12%, preferably from about .01% to about .1%, and most preferably from about .02% to about .09%.

Spearmint oil is present in mouthwashes of the present invention at a concentration of from about .01% to about 1%, preferably from about .02% to about .17%, and most preferably from about .05% to about .15%.

.0 Wintergreen oil is present in mouthwashes of the present invention at a concentration of from about .03% to about 1%, preferably from about .05% to about .5%, and most preferably from about .1% to about .45%.

Clinical Study

Effect of Mouthwash of Invention on Remineralization .5 10 healthy volunteers (7 females and 3 males), ages 18-45, each with a minimum of 16 clinically and radiographically healthy teeth as defined by clinical examination, and with an absence of any apparent pathology, were recruited to participate in a randomized, double-blind clinical study with three study arms (i.e., segments), each lasting five days. The study objective was to compare remineralization of eroded (i.e., demineralized) enamel by use of two mouthwashes: (i) the mouthwash of the present invention (Mouthwash of Invention); and (ii) a prior art mouthwash, Sensodyne® Pronamel® Fluoride Rinse (Prior Art Mouthwash) containing, as an active ingredient, sodium fluoride at a concentration of 0.05% (0.02% w/v fluoride ion).

Three hundred (300) enamel chips were created from extracted teeth classified as healthy, which were extracted by an experienced dentist. More particularly, 150 pairs of chips were cut from the same area of each extracted tooth. A first group of 150 chips (one from each pair)

2019100471 02 May 2019 were eroded by 6-hour immersion in 40 ml of an acidic solution (buffered to pH of approximately 4.4) as described by Stookey GK, et al. The Featherstone laboratory pH cycling model: a prospective, multi-site validation exercise Am. J. Dent. Vol. 24, No. 5, pp. 322-328 (2011) (2.0mmol/l calcium, 2.0mmol/l phosphate, and 75mmol/l acetate). A second group of 5 150 chips (the corresponding/remaining tooth from each pair) was designated as controls, and stored in sealed double-walled containers, impervious to ambient light, in de-mineralized water at a temperature of 4°C and 100% humidity.

Thirty (30) intra-oral retainers were fabricated from standard alginate impressions of the upper teeth) - three for each study participant. Five enamel chips from the first group, eroded as .0 described above, were attached to the palatal area of each retainer; the chips were spaced approximately 0.75 to 1.5 cm apart. A new retainer was made prior to each of the three study segments.

Total study duration was 36 days:

• Days 0-7: one-week washout period .5 · Days 8 through 12: Study Segment 1 (intra-oral retainer worn; no mouthwash) • Days 13 - 19: one-week washout period • Days 20 - 24: Study Segment 2 (intra-oral retainer worn; Mouthwash of Invention used) • Days 25 - 31: one-week washout period • Days 32 - 36: Study Segment 3 (intra-oral retainer worn; Prior Art Mouthwash used)

Subjects were supplied with toothpaste (CREST®Total Care, Procter & Gamble, Cincinnati, OH) in a quantity sufficient to brush their teeth twice daily for the duration of the study (36 days) as well as with a new soft bristled toothbrush (ORAL B®, GSK, Warren, NJ) at the outset of the study (Day 0) and at beginning of the second and third study segments (Study Days 20 and 32). Subjects were instructed not to use any oral care products (e.g., floss or baking soda) other than those provided by the study staff.

Throughout the study - from the initial washout period through completion of Study Segment 3

- participants (i) brushed their teeth twice daily, each time for two minutes, and (ii) wore the

2019100471 02 May 2019 retainers for a minimum of 22 hours per day. Neither the retainer nor the enamel specimens were brushed. Retainers were removed when eating solid foods; at which time the retainers were placed in a sealed container. Subject compliance (wearing of retainers for 22 hours/day) was confirmed by diary entries completed on a daily basis.

For Segment 2, subjects were randomly assigned mouthwash either the Mouthwash of the Invention or Prior Art Mouthwash. For Segment 3, subjects were assigned the other mouthwash. Subjects that used Mouthwash of the Invention in Segment 2, used the Prior Art Mouthwash in Segment 3. Subjects that used the Prior Art Mouthwash in Segment 2, used the Mouthwash of the Invention in Segment 3.

.0 In both Segments 2 and 3, after each brushing, participants rinsed with the assigned mouthwash for sixty seconds; thereafter, the mouthwash was expectorated. For thirty minutes following expectoration, subjects did not rinse, drink or eat.

At the end of each segment, a total of fifty enamel chips were collected (five chips from the retainers worn by each of the ten study participants) and were tested for microindentation .5 hardness (also known in the art, and referred to in this application, as microhardness); fifty controls were tested at the same time. More particularly, three microhardness measurements were taken on each chip using the Knoop test, an established and standard technique for measuring enamel mineralization, by applying a fixed force (load) using a pyramid-shaped diamond indenter for a specified dwell time period: one in an area of typical appearance; one 20 in an area with the healthiest (best) appearance; and one in an area with the most damaged (worst) appearance. See ASTM Standard E384-16 (ASTM International, West Conshohocken, PA). See also Craig RG and Peyton FA, The Microhardness of Enamel and Dentin J Dent Res, Vol. 37, No. 4, pp. 661-668 (1958). Microhardness measurements were compared using the Kruskal-Wallis one-way analysis of variance with post-hoc Tukey's test.

The microhardness of the eroded (i.e., demineralized) enamel chips in Segments 2 and 3 was substantially the same. There were no statistically significant differences (p>0.05) between microhardness of enamel chips treated (i.e., rinsed) with the Mouthwash of the Invention and

2019100471 02 May 2019 the Prior Art Mouthwash. Put differently, demineralized enamel chips were observed to have substantially regained their original microhardness after rinsing with both mouthwashes.

In vivo Effects of Whitening Strip of Invention on Tooth, Root and Dentin Sensitivity and Gingival Irritation

Ten participants ages 18-45 (mean 29.4 years) - each with (i) a minimum of 16 clinically and radiographically healthy teeth, as determined by clinical examination and (ii) an absence of any pathological symptoms, gingival recession or tooth, root or dentin sensitivity prior to enrollment - were recruited to participate in a 10-day, randomized, double-blinded, clinical study.

.0 At the commencement of the study, each subject received forty (40) identically packaged, coded whitening strips - 20 Test Strips (as defined above) and 20 Prior Art Strips (as defined above) - as well as a new soft-bristled ORAL B® toothbrush and CREST® Total Care toothpaste. Subjects brushed their teeth twice daily and used only the oral hygiene products as described above. For example, no mouthwash or floss was used. Subjects applied four whitening strips .5 - one on each designated quadrant of the mouth (i.e., upper left, lower left, upper right, lower right) - to the outer surface of the teeth for thirty (30) minutes each day. After treatment, subject rinsed their mouths with water. Subjects were instructed to wait at least 22 hours before each subsequent application. After each application, on a daily basis, subjects completed a questionnaire to report tooth, root or dentin sensitivity using the 5-point Likert z0 Scale, and gingival irritation (yes or no). At the conclusion of the study, cumulative selfreported scores were calculated for both endpoints - tooth, root or dentin sensitivity and gingival irritation. Additionally, the two endpoints were evaluated at baseline, and on Study Days 5 and 10 by a blinded clinician. Sensitivity was measured (yes/no) after a 3 second burst of pressurized air. Gingival irritation was determined by visual inspection on a scale ranging 25 from 0 to 3.

Quadrants of the oral cavity to which the Test Strips were applied were reported to have lower levels of gingival irritation. Sensitivity was reported to be comparable in the two treatment groups, both as self-evaluated by the subject and by clinical evaluation by a trained observer

2019100471 02 May 2019 (e.g., using a jet of air or an exploratory probe on the exposed dentin) at baseline and on Days 5 and 10.

Adherent Deposition Without Structural Change

Without wishing to be bound by a particular theory, applicant believes that the inventive method of the present invention can be explained in terms of temporary occlusion of the dentinal tubules, where by temporary occlusion is meant deposition of salt, preferably Dead Sea salt, on the surface of the teeth in a manner that deposits salt crystals on the surface of the teeth, blocking openings to the dentinal tubules, but not changing the structure or function of the teeth.

.0 Ten extracted teeth that appeared healthy to the naked eye of a trained observer were stored in artificial saliva at body temperature 36.5-37.5 °C (97.7-99.5 °F). Artificial saliva was prepared using conventional equipment and laboratory protocols routinely used by the person having ordinary skill in the dental research. One non-limiting example of artificial saliva has a pH of about 7.0 and contains the following ingredients at the concentrations indicated in .5 parentheses: sodium chloride (0.381 g/L); calcium chloride di-hydrate (0.213 g/L); potassium dihydrogen phosphate (0.738 g/L); potassium chloride (1.114 g/L); and gastric mucin (2.20 g/L). Another non-limiting example of artificial saliva is a ready-to-use solution having a pH of 6.75 ±0.05(5)25±0.4°C available from Pickering Laboratories, Inc. (Mountain View, California).

Twice daily, samples were removed from the saliva solution, and treated (i.e. swished) for 60 seconds in 20 mL of mouthwash of the invention to simulate twice-daily intra-oral use. Then samples were imaged under the light microscope at a 13x magnification. Next samples were washed with air/water spray for 30 seconds, before being returned to artificial saliva solution, which was replaced daily. This was performed daily for a period of 2 weeks.

Photographs and microscope images (13x magnification) were taken at baseline, after the first treatment, and after one and two weeks of treatment. Baseline tooth images show that the tooth surface is healthy and clean. Immediately after swishing in mouthwash of the invention, a small clear granular deposit was visible on the tooth surface. No residual deposits or tooth surface changes were visible during inspection under the light microscope after one and two weeks of treatment (Figures 3 and 4).

2019100471 02 May 2019

Claims (4)

1. Claims

   1. A method of reducing tooth, root, and dentin sensitivity comprising administering to a subject an effective amount of an oral care product comprised of (a) Dead Sea salt, at a concentration of from about 0.5% to about 3.0%; and (b) xylitol; and (c) aloe vera leaf juice.
2. 2. The method of claim 2 wherein the oral care product contains at least one essential oil selected from the group of peppermint oil, spearmint oil and Wintergreen oil.
3. 3. The method of claim 3 wherein the oral care product contains at least one essential oil selected from the group of peppermint oil, spearmint oil and Wintergreen oil.
4. 4. The method of claim 1 wherein the oral care product is a dentifrice or a mouthwash that is administered for at least 30 seconds at least one time daily