Assay for Oral Inflammation

Abstract

Methods and kits for determining the presence or severity of oral inflammation, such as gingivitis or periodontitis wherein the determination method comprises: (a) obtaining a test sample of oral material from said subject; (b) determining the amount of polymorphonuclear neutrophil indicator substance in said test sample; and (c) comparing the amount of polymorphonuclear neutrophil indicator substance in the test sample to a control sample.

Claims

1. A method of detecting the presence, absence or severity of oral inflammation, or monitoring the oral inflammation disease state, in a subject comprising (a) obtaining a test sample of oral material from said subject, (b) determining the amount of polymorphonuclear neutrophil indicator substance in said test sample, (c) comparing the amount of polymorphonuclear neutrophil indicator substance in the test sample to a control sample.

2. The method of claim 1 wherein the control sample is a sample from a healthy subject or from a plurality of healthy subjects.

3. The method of claim 1 wherein the oral material is saliva.

4. The method of claim 1 wherein an increased amount of polymorphonuclear neutrophil indicator substance relative to the control sample indicates the presence of oral inflammation.

5. The method of claim 1 wherein an increased level of polymorphonuclear neutrophil indicator substance in the test sample indicates ineffective oral inflammation treatment and a decreased level of polymorphonuclear neutrophil indicator substance in the test sample indicates effective oral inflammation treatment.

6. The method of claim 1 wherein the oral inflammation is gingivitis.

7. The method of claim 1 wherein the oral inflammation is periodontitis.

8. The method of claim 1 wherein an associated condition of the oral inflammation is halitosis.

9. The method of claim 1 wherein the test sample is obtained with a swab.

10. The method of claim 1 wherein the test sample is obtained by expectoration.

11. The method of claim 1 wherein the polymorphonuclear neutneutrophil indicator substance is lactoferrin.

12. The method of claim 1 wherein the polymorphonuclear neutrophil indicator substance is leukocyte esterase.

13. The method of claim 1 wherein the polymorphonuclear neutrophil indicator substance is calprotectin.

14. The method of claim 1 wherein the oral material is contacted with an absorbent material containing reagents to determine the presence or amount of POLYMORPHONUCLEAR NEUTROPHIL indicator substance.

15. The method of claim 14 wherein the absorbent material is a strip or in a dipstick.

16. A method for selecting a treatment regimen in a subject determined to have oral inflammation from claim 1 comprising (a) obtaining a test sample of an oral material such as saliva from said subject, (b) determining the amount of POLYMORPHONUCLEAR NEUTROPHIL, or POLYMORPHONUCLEAR NEUTROPHIL indicator substance, in said test sample, (c) comparing the amount of POLYMORPHONUCLEAR NEUTROPHIL, or POLYMORPHONUCLEAR NEUTROPHIL indicator substance, in the test sample to a control, (d) selecting a treatment regimen based on the comparison made in step (c).

17. The method of claim 16 including the additional steps of the subject administering a therapeutically effective amount of a dentifrice composition.

18. The method of claim 16, wherein the dentifrice composition comprises of an active agent selected from the group consisting of antimicrobial agent, antiinflammatory agent, fluoride ion source, a basic amino acid and mixtures thereof.

19. The method of claim 16 wherein the treatment regimen is selected from toothbrushing twice daily, toothbrushing more than twice daily, treating the subject with antiflammaory agents, treating the subject with antimicrobial agents, or a combination thereof.

20. The method of claim 16 wherein the antimicrobial agent is selected from the group consisting of halogenated diphenyl ether, triclosan, herbal extracts, essential oils, rosemary extract, thymol, menthol, eucalyptol, methyl salicylate, bisguanide antiseptics, chlorhexidine, alexidine, octenidine, phenolic antiseptics, hexetidine, povidone iodine, delmopinol, salifluor, metal ions and their salts, zinc chloride, zinc lactate, zinc citrate, stannous fluoride, and stannous chloride, sanguinarine, propolis, oxygenating agents, hydrogen peroxide, buffered sodium peroxyborate, peroxycarbonate, cetyl pyridinium chloride, magnolia extract, magnolol, honokiol, butyl magnolol, propyl honokiol, and mixtures thereof; and the anti-inflammatory agent is selected from the group consisting of steroidal agents, flucinolone, hydrocortisone, nonsteroidal agents (NSAIDs), ketorolac, flurbiprofen, ibuprofen, naproxen, indomethacin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, ketoprofen, fenoprofen, piroxicam, nabumetone, aspirin, diflunisal, meclofenamate, mefenamic acid, oxyphenbutazone, phenylbutazone and combinations thereof.

21. The method of claim 16 wherein the active agent is selected from the group consisting of triclosan, chlorhexidine, fluoride ion source, arginine and mixtures thereof.

22. The method of claim 16 which is an at home method.

23. A diagnostic kit for detecting the presence, absence or severity of oral inflammation, or monitoring the oral inflammation disease state, comprising a saliva absorbent material which contains reagents to determine the presence and/or amount of POLYMORPHONUCLEAR NEUTROPHIL indicator substance together with instructions for application of the saliva absorbent material to the oral cavity and instructions for a treatment method based on the amount of POLYMORPHONUCLEAR NEUTROPHIL indicator substance detected.

24. The diagnostic kit of claim 23, wherein the instructions for application of the saliva absorbent material to the oral cavity comprise: (a) obtaining a test sample of oral material from said subject, (b) determining the amount of polymorphonuclear neutrophil indicator substance in said test sample, (c) comparing the amount of polymorphonuclear neutrophil indicator substance in the test sample to a control sample.

25. The diagnostic kit of claim 23 wherein the treatment method is the administration of a dentifrice composition which comprises an active agent selected from the group consisting of antimicrobial agent, antiinflammatory agent, fluoride ion source, a basic amino acid and mixtures thereof.

26. The diagnostic kit of claim 23 wherein the treatment regimen is selected from toothbrushing twice daily, toothbrushing more than twice daily, treating the subject with antiflammaory agents, treating the subject with antimicrobial agents, or a combination thereof.

27. The diagnostic kit of claim 23 wherein the antimicrobial agent is selected from the group consisting of halogenated diphenyl ether, triclosan, herbal extracts or essential oils, rosemary extract, thymol, menthol, eucalyptol, methyl salicylate), bisguanide antiseptics, chlorhexidine, alexidine, or octenidine), phenolic antiseptics, hexetidine, povidone iodine, delmopinol, salifluor, metal ions and their salts, zinc chloride, zinc lactate, zinc citrate, stannous fluoride, and stannous chloride, sanguinarine, propolis, oxygenating agents, hydrogen peroxide, buffered sodium peroxyborate, or peroxycarbonate, cetyl pyridinium chloride, magnolia extract, magnolol, honokiol, butyl magnolol, propyl honokiol, and mixtures thereof; and the anti-inflammatory agent is selected from the group consisting of steroidal agents, flucinolone, hydrocortisone, nonsteroidal agents (NSAIDs), ketorolac, flurbiprofen, ibuprofen, naproxen, indomethacin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, ketoprofen, fenoprofen, piroxicam, nabumetone, aspirin, diflunisal, meclofenamate, mefenamic acid, oxyphenbutazone, phenylbutazone and combinations thereof.

28. The diagnostic kit of claim 23 wherein the active agent is selected from the group consisting of triclosan, chlorhexidine, fluoride ion source, arginine and mixtures thereof.

Description

DETAILED DESCRIPTION

[0011] The following description of the preferred embodiment(s) is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.

[0012] As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any value within the range can be selected as the terminus of the range. In addition, all references cited herein are hereby incorporated by referenced in their entireties. In the event of a conflict in a definition in the present disclosure and that of a cited reference, the present disclosure controls. Unless otherwise specified, all percentages and amounts expressed herein and elsewhere in the specification should be understood to refer to percentages by weight. All percentages expressed herein are on a weight by dry matter basis unless specifically stated otherwise.

[0013] In the context of the invention, the term treating or treatment, as used herein, means reversing, alleviating, mitigating or inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. As used herein and in the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictates otherwise.

[0014] The term normal control refers to a subject or pool of subjects that does not have oral inflammation.

[0015] The term swab refers to any device suitable for collecting oral fluid formed from an absorbent material. Preferably, the absorbent material is attached to a holder, but the term swab, as used herein, includes free standing devices made from absorbent material that may be placed in the mouth or mouth surface to absorb the saliva located therein.

[0016] The term oral material refers to any substance in the oral cavity that can be sampled, e.g., saliva, gingival crevicular fluid, oral scrapings from the tongue or cheek, gingival plaque, and the like. A preferred oral material for the methods of the invention is saliva.

[0017] As used herein, measure or determine refers to any qualitative or quantitative determinations.

[0018] In the methods of the invention, the subject can be any animal, and is preferably a mammal such as a human, mouse, rat, cat, dog, horse, cow, donkey, sheep, or pig. Humans are most preferred.

[0019] In some aspects, the methods comprise obtaining a test sample of oral material such as saliva from the subject. The saliva can be obtained by any means suitable in the art. For example, various oral fluid collection devices and kits are known in the art and are commercially available. The oral fluid collection device can be comprised of or otherwise shaped or formed as particles, fibers, plates, and the like. Saliva can be collected by aspiration or absorption. Suitable oral fluid collection devices are commercially available from Oasis Diagnostics Corp., Vancouver, Wash., such as Super-SAL, VerOFy, Versi-SAL, and UltraSal-2.

[0020] In some aspects, the oral material, e.g., saliva, is obtained by means of a device such as a swab. The swab can be comprised of any material suitable in the art such as alginate, sponge, capillary matrix, filter paper, calcium fibers, cellulose-based materials, or synthetic polymers such as a polyurethane, polyester, rayon, or a polymer of ethylene glycol and terephthalic acid. Polymers of ethylene glycol and terephthalic acid (i.e., polyethylene terephthalate) sold under the trade name DACRON (DuPont) are particularly suited to the present invention. It is preferred that the swab not contain cotton or other natural cellulose-based materials, because cotton may be contaminated with metals from the soil in which the cotton plant was grown, thereby potentially contaminating the assay and producing inaccurate or false positive results. The absorbent material of the swab can be impregnated with salts or a hypertonic solution to facilitate absorption. The absorbent material can also be impregnated with a flavorant to make the sampling more pleasant to the subject. The swab can be on the end of a holder or an applicator made from plastic, wood, aluminum, and the like, with synthetic materials such as plastic being preferred. The swab can then placed in an aqueous solution, preferable sterile and buffered. The swab can be swirled, vortexed, etc. to dislodge the PMN and/or their derivatives or components from the swab material into the aqueous solution.

[0021] In another embodiment the saliva is obtained by expectoration. For example, a subject rinses the oral cavity with an aqueous solution, preferably sterile and/or buffered. Examples of buffered solutions include Hanks Balanced Salt solution, phosphate buffered saline, TRIS buffer, and the like. The contents of the oral cavity, i.e, salivary samples, are then expectorated into containers, preferable sterile.

[0022] The number of PMN can be determined in the samples via conventional techniques known in the art, e.g., via cell counters, microscopy, and the like. If microscopy is used, the salivary samples can then be concentrated, e.g., by centrifugation, filtration, etc. and then resuspended. Cell stains, conventional or fluorescent, such as acridine orange, and the like can be used to stain the PMN to make them more visible. In one embodiment the number of PMN can be determined by the methodology disclosed in the paper: Bender J S, Thang H, Glogauer M, (2006) Novel rinse assay for the quantification of oral neutrophils and the monitoring of chronic periodontal disease, J. Periodontal Res. 2006, 41:214-220, incorporated herein by reference.

[0023] The time required to collect saliva from the subject can vary. In some aspects, the oral fluid collection device or buffered solution is to remain in the subject's mouth for at least about 5 seconds, or at least about 10 seconds, or at least about 15 seconds, or at least about 20 seconds, or at least about 30 seconds. In some aspects, the oral fluid collection device or buffer solution can remain in the subject's mouth for at least about 1 minute, or more.

[0024] The method of the invention is for aiding in the diagnosis, prognosis and disease monitoring of oral inflammation, such as gingivitis or periodontitis, and associated conditions, e.g., bad breadth or halitosis. The methods of the invention can also be used for evaluating potential therapeutic agents to treat and/or ameliorate the symptoms associated with oral inflammation. The methods of the invention can be used to determine the presence, absence or severity of oral inflammation.

[0025] In one embodiment of the invention the amount of PMN or PMN indicator substance in a test sample is compared to a control. If the method of the invention is to detect the presence, absence or severity of oral inflammation, the control is from normal healthy subjects, i.e., subjects that are known to not have oral inflammation.

[0026] In a particular aspect, the invention provides a method for diagnosing oral inflammation, gingivitis or peridontitis, and/or determining the severity thereof, in a test subject, by providing an oral material sample such as a saliva sample from a normal healthy subject or oral materials samples such as saliva samples from a plurality of normal healthy subjects, determining the amount of PMN or PMN indicator substance in the healthy subject(s) samples, and obtaining one or more samples from the test subject and determining the amount of PMN is the test subject oral material, and comparing the amount of PMN or PMN indicator substance in the normal oral material sample(s) to the amount of PMN or PMN indicator substance in the test oral material sample(s),

[0027] If the method of the invention is to monitor the effectiveness of oral inflammation treatment or monitoring the oral inflammation disease state, then the control is a test sample taken from the same subject at a prior time, which can be before or after treatment is initiated. The results obtained from practicing the monitoring method of the invention can be used to adjust or modify the oral inflammation treatment, that is, change the practices, behavior, regimen, dose and/or substance, e.g., dentifrice or medicament, used to treat the inflammation.

[0028] In a further aspect, the invention provides a method for monitoring the disease state in a subject having oral inflammation, by comparing the level of PMN or PMN indicator substance in one or more oral material samples at a first time point to the level of PMN or PMN indicator substance at a second time point; and correlating a decrease in the level of PMN or PMN indicator substance at the second time point as compared to the first time point with an improvement in the subject's disease state, and/or correlating an increase in the level of PMN or PMN indicator substance at the second time point as compared to the first time point with an increase in the severity of the subject's disease state.

[0029] The present invention encompasses measuring PMN indicator substances instead of measuring PMN directly. The amounts of PMN indicator substances are any substances that can be measured in oral materials such as saliva wherein the amount of substance is correlated with the amounts of PMN. Such PMN indicator substances include, for example, metabolites, components, enzymes, cytokines, PMN excretions, PMN breakdown products, and the like. Specific examples of PMN indicator substances include salivary lactoferrin, calprotectin, and salivary leukocyte esterase. In most embodiments, the correlation with PMN is positive, e.g., the greater the amount of lactoferrin and/or leukocyte esterase, the greater the amount of PMN. However, it is also contemplated that a negative correlation may exist, i.e., the greater the amount of a particular measured substance, the lesser amount of PMN. In either case, standards can be developed by techniques known in the art, e.g., development of data tables or standard curves, to compare the amount of a particular substance in the oral cavity to the presence, absence or severity of oral inflammation.

[0030] Assays are known in the art for determining the presence and/or amount of leukocyte esterase, calprotectin, and/or lactoferrin in biological samples. Such assays can be based on detecting reactions of the substance to be measured, or complexes formed therewith, by color changes, fluorescence, luminescence, radioisotopic detection, ELSIA, and the like.

[0031] U.S. Pat. No. 5,776,780, which is incorporated herein by reference, discloses a reagent system for detecting and measuring leukocyte esterase. The color generating mechanism or indicator of the reagent system is the result of leukocyte esterase acting upon compatible esters. This ester/esterase reaction produces a relatively unstable indoxyl moiety that is oxidized to form an indigo color that is monitored by monochromatic spectrophotometry. The addition of dehydrogenase to the reagent will enhance the speed of reaction and completeness of the reaction of the indoxyl moiety. The dehydrogenase oxidizes the alcohol group on the indoxyl group and promotes formation of a ketone. This transitional indoxyl ketone radical enhances color development, specificity, and accuracy and sensitivity of the reaction. The reagent system may contain one or more of the following compounds, 2,4-dinitrophenylhydrazine, hydroxylamine, or semicarbizide, which in the presence of indoxide ketones will give color development that can be monitored at the same wavelength as the indigo. A further enhancement of the method concerning the indoxyl intermediate, is the addition of p-dimethylaminobenzaldehyde or p-nitro-benzenediazonium tetrafluroborate or other azo indicators.

[0032] In one embodiment the invention provides a diagnostic kit for determining the amount of PMN in saliva sample by determining the amount of leukocyte esterase in said sample comprising:

(a) an ester compatible with leukocyte esterase;
(b) means for contacting the ester with the sample to produce indoxyl moieties that from from the reaction of the ester with any leukocyte esterase present in the sample; and
(c) means for measuring production of indoxyl moieties.

[0033] In one embodiment the means for determining production of the indoxyl moieties is by detecting a color change.

[0034] Assays for lactoferrin can be based on detecting antibody/antigen complexes. A further aspect of the invention provides a diagnostic kit for determining the amount of PMN in saliva sample by determining the amount of lactoferrin in said sample specifically reactive with an antibody specific for lactoferrin, comprising:

(a) a lactoferrin specific antibody;
(b) means for contacting the lactoferrin specific antibody with the sample to produce complexes comprising the lactoferrin specific antibody and any lactoferrin present in the sample; and
(c) means for measuring production of the complexes.

[0035] The lactoferring specific antibodies are commercially available or can be obtained though techniques known in the art, for example:

(a) administering lactoferrin to at least one mouse to produce at least one immunized mouse;
(b) removing B-lymphocytes from the at least one immunized mouse;
(c) fusing the B-lymphocytes from the at least one immunized mouse with myeloma cells, thereby producing hybridomas;
(d) cloning the hybridomas;
(e) selecting clones which produce anti-lactoferrin antibody;
(f) culturing the anti-lactoferrin antibody-producing clones; and then
(g) isolating anti-lactoferrin antibodies from the cultures.

[0036] In one embodiment, the lactoferrin is an antigen in immunoassays including enzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzyme linked antibody binding assays or procedures known in the art for the detection of antibodies.

[0037] In ELISA assays, the lactoferrin specific antibody is immobilized onto a selected surface, for example, a surface capable of binding proteins such as filter paper or the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed lactoferrin specific antibody, a nonspecific protein, such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample, may be bound to the selected surface. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific bindings onto the surface. The immobilizing surface is then contacted with a saliva sample to be tested in a manner conducive to immune complex (antigen/antibody) formation. This may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to incubate for from about 2 to 4 hours, at temperatures such as of the order of about 25 to 37 C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution, such as PBS/Tween or a borate buffer. Following formation of specific immunocomplexes between the test sample and the bound lactoferrin specific antibody, and subsequent washing, the occurrence, and even amount, of immunocomplex formation may be determined by subjecting the immunocomplex to a second antibody having specificity for the lactoferrin specific antibody.

[0038] If the lactoferrin specific antibody is of human origin, the second antibody can an antibody having specificity for human immunoglobulins and in general IgG. To provide detecting means, the second antibody may have an associated activity such as an enzymatic activity that will generate, for example, a colour development upon incubating with an appropriate chromogenic substrate. Quantification may then be achieved by measuring the degree of colour generation using, for example, a visible spectra spectrophotometer. Modifications to this procedure can be made via techniques known in the art. For example, sandwich lateral flow chromatographic immunoassays can be employed.

[0039] In other embodiments, the assays for PMN indicator substances, such as lactoferrin, calprotectin and leukocyte esterase, can be miniaturized and/or streamlined. For example, the necessary reagents can be incorporated into a saliva absorbent material, such as a water absorbent material such as cellulose. The saliva absorbent material can be in the form of sheets or strips or be incorporated into devices such as a dipstick that can be directly contacted with an oral material such as saliva. In such embodiments the strip or dipstick can be directly contacted with saliva in the oral cavity, thus obviating the need to collect a separate sample. Is such assays the presence and/or amount of PMN indicator substance can be determined by a color change and comparing the test color to a standard color chart.

[0040] Such assays are commercially available for detecting substances in urine or feces, e.g., OnSite FOB Hi Rapid Test-Cassette, available from CTK Biotech, Inc; Urinalysis Reagent Test Strips, available from Tigermedical; CLIA-URS-10 Urine Reagent Strips, available from CLIAwaived, Inc., calprotectin ELISA kits available from Alpha Laboratories such as CALcheck Blue and Quantum Blue; and the like. Some commercially available assays have the capability of testing for PMN indicator substances such as leukocyte esterase, typically concomitantly for other substances as well, and can be used in the methods of the invention without modification. However, in some embodiments the assays are tailored to test for saliva and only for PMN indicator substances.

[0041] The level of lactoferrin in an oral material such as saliva is correlated with the amount of PMN in the oral material such as saliva. Thus the present invention in another embodiment concerns a method for diagnosing oral inflammation, gingivitis or peridontitis, and/or determining the severity thereof, in a test subject, by providing an oral material sample such as a saliva sample from a normal healthy subject or oral material samples such as saliva samples from a plurality of normal healthy subjects, determining the amount of lactoferrin in the healthy subject(s) sample(s), and obtaining one or more samples from the test subject and determining the amount of lactoferrin in the test subject sample(s), and comparing the amount of lactoferrin in the normal sample(s) to the amount of lactoferrin in the test sample(s).

[0042] In another embodiment the invention concerns a method for monitoring the disease state in a subject having oral inflammation, by comparing the level of lactoferrin in one or more oral material samples at a first time point to the level of lactoferrin at a second time point; and correlating a decrease in the level of lactoferrin at the second time point as compared to the first time point with an improvement in the subject's disease state, and/or correlating an increase in the level of lactoferrin at the second time point as compared to the first time point with an increase in the severity of the subject's disease state.

[0043] The level of leukocyte esterase in an oral material such as saliva is correlated with the amount of PMN in the oral material such as saliva. Thus, the present invention concerns a method for diagnosing oral inflammation, gingivitis or peridontitis, and/or determining the severity thereof, in a test subject, by providing an oral material sample such as a saliva sample from a normal healthy subject or samples from a plurality of normal healthy subjects, determining the amount of leukocyte esterase in the healthy subject(s) sample(s), and obtaining one or more samples from the test subject and determining the amount of leukocyte esterase in the test subject sample(s), and comparing the amount of leukocyte esterase in the normal sample(s) to the amount of leukocyte esterase in the test sample(s).

[0044] In another embodiment the invention concerns a method for monitoring the disease state in a subject having oral inflammation, by comparing the level of leukocyte esterase in one or more oral material samples at a first time point to the level of leukocyte esterase at a second time point; and correlating a decrease in the level of leukocyte esterase at the second time point as compared to the first time point with an improvement in the subject's disease state, and/or correlating an increase in the level of leukocyte esterase at the second time point as compared to the first time point with an increase in the severity of the subject's disease state.

[0045] The level of calprotectin in an oral material such as saliva is correlated with the amount of PMN in the oral material such as saliva. Thus, the present invention in another embodiment concerns a method for diagnosing oral inflammation, gingivitis or peridontitis, and/or determining the severity thereof, in a test subject, by providing an oral material sample such as a saliva sample from a normal healthy subject or oral material samples such as saliva samples from a plurality of normal healthy subjects, determining the amount of calprotectin in the healthy subject(s) sample(s), and obtaining one or more samples from the test subject and determining the amount of calprotectin in the test subject sample(s), and comparing the amount of calprotectin in the normal sample(s) to the amount of calprotectin in the test sample(s).

[0046] In another embodiment the invention concerns a method for monitoring the disease state in a subject having oral inflammation, by comparing the level of calprotectin in one or more oral material samples at a first time point to the level of calprotectin at a second time point; and correlating a decrease in the level of calprotectin at the second time point as compared to the first time point with an improvement in the subject's disease state, and/or correlating an increase in the level of calprotectin at the second time point as compared to the first time point with an increase in the severity of the subject's disease state.

[0047] The methods on the invention for monitoring the disease state of a subject can be used to initiate, change or modify oral inflammation treatments.

[0048] In one embodiment the invention concerns a method for selecting a treatment regimen in a subject suspected of having oral inflammation comprising

(a) obtaining a test sample of an oral material such as saliva from said subject,
(b) determining the amount of PMN, or PMN indicator substance, in said test sample,
(c) comparing the amount of PMN, or PMN indicator substance, in the test sample to a control,
(d) selecting a treatment regimen based on the comparison made in step (c).

[0049] In the selecting method, the additional step of the subject carrying out the selected treatment regimen is preferred, in one embodiment, in consultation with a dental professional.

[0050] The treatment regimen will vary depending on the comparasion of the test sample with the control (step(c)). In some instances the comparison will show little or no inflammation, in which case the treatment regimen may comprise toothbrushing with a suitable dentrifrice once or twice per day. If the comparison indicates some degree of oral inflammation, then one or more of several types of treatment regimens may be selected, for example;

1. Toothbrushing more than twice per day, e.g., 3, 4, 5, or 6 times,
2. The use of antimicrobial agents. The antimicrobial agent can in incorporated into a dentifrice, e.g., toothpaste or mouthwash, or can be administered via other means, e.g., in tablet, suppository, transdermal patch, or injectable form. The amount administered will be an effective anti-microbial amount, i.e., an amount sufficient to eliminate or lessen the severity of the oral inflammation.
3. The use of antiflammatory agents. The antiinflammatory agent can in incorporated into a dentifrice, e.g., toothpaste or mouthwash, or can be administered via other means, e.g., in tablet, suppository, transdermal patch, or injectable form. The amount administered will be an effective anti-inflammatory amount, i.e., an amount sufficient to eliminate or lessen the severity of the oral inflammation.

[0051] The antimicrobial agent useful in the present invention is not particularly limited, and may be selected from halogenated diphenyl ether (e.g. triclosan), herbal extracts or essential oils (e.g., rosemary extract, thymol, menthol, eucalyptol, methyl salicylate), bisguanide antiseptics (e.g., chlorhexidine (CHX), alexidine, or octenidine), phenolic antiseptics, hexetidine, povidone iodine, delmopinol, salifluor, metal ions and their salts (e.g., zinc chloride, zinc lactate, zinc citrate, stannous fluoride, and stannous chloride), sanguinarine, propolis, oxygenating agents (e.g., hydrogen peroxide, buffered sodium peroxyborate, or peroxycarbonate), cetyl pyridinium chloride, magnolia extract, magnolol, honokiol, butyl magnolol, propyl honokiol, and mixtures thereof. Anti-attachment agents such as Solrol also can be included in dentifrices, as well as plaque dispersing agents such as enzymes (papain, glucoamylase, etc.).

[0052] Suitable anti-inflammatory agents include without limitation steroidal agents such as flucinolone and hydrocortisone, and nonsteroidal agents (NSAIDs) such as ketorolac, flurbiprofen, ibuprofen, naproxen, indomethacin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, ketoprofen, fenoprofen, piroxicam, nabumetone, aspirin, diflunisal, meclofenamate, mefenamic acid, oxyphenbutazone and phenylbutazone.

[0053] Other known actives for oral care compositions can also be used in the treatment regimen such as a fluoride ion source (e.g. an amine fluoride, sodium fluoride, sodium monofluorophosphate, stannous fluoride, etc.) or a basic amino acid (e.g. arginine, an active ingredient in Colgate's Pro-Argin technology.

[0054] It is an advantage of the present invention that the facile, safe, and non-invasive nature of the methods of the invention allows for, in some embodiments, the methods to be carried out by the subject or patient who can record/report the results. On a routine basis patients need several motivated and compliance based approaches to help maintain and monitor their oral health status. The methods herein off a simple, cost-effective, rapid and easy means to perform routine evaluations of the oral health status in the privacy of their own homes. Thus, the methods herein provide suitable approaches that can be implemented in the home setting, i.e., are at home methods and kits. The methods allow subjects to monitor their oral health routinely and maintain a periodic record of their findings that can be discussed and used in consultation with their professional dental provider. The methods can be used subsequent to a dental visit to monitor and evaluate their oral health status and seek or modulate dental care based on the results of the methods. In some embodiments the results can be computerized, electronically stored, and/or automatically submitted to a dental professional.

[0055] In the methods of the invention, the oral material samples such as saliva samples are compared on the same unit basis, e.g., 1, 0.1, 0.001 or 0.0001 ml. or other volumes of oral material as long as the volumes are the same.

[0056] The invention thus provides in one embodiment, a method (Method 1). For determining the presence, absence, or severity of oral inflammation, or monitoring the oral inflammation disease state, in a subject comprising

(a) obtaining a test sample of an oral material such as saliva from said subject,
(b) determining the amount of PMN, or PMN indicator substance, in said test sample,
(c) comparing the amount of PMN, or PMN indicator substance, in the test sample to a control.

[0057] For example, the invention provides

1.1. The method of method 1 wherein the control is a sample from a normal healthy subject or a plurality of healthy subjects;
1.2. The method of method 1 wherein the control is a different test sample from the same subject taken at a prior time;
1.3. The method of any foregoing method wherein an increased amount of PMN or PMN indicator substance relative to the control indicates the presence of oral inflammation;
1.4. The method of any foregoing method wherein an increased level of PMN or PMN indicator substance in the test sample indicates ineffective oral inflammation treatment and a decreased level of PMN or PMN indicator substance in the test sample indicates effective oral inflammation treatment;
1.5. The method of any foregoing method wherein the oral inflammation is gingivitis;
1.6. The method of any foregoing claim wherein the oral inflammation is periodontitis;
1.7. The method of any foregoing method wherein an associated condition of the oral inflammation is halitosis;
1.8. The method of any foregoing method wherein the test sample is obtained with a swab;
1.9. The method of any foregoing method wherein the test sample is obtained by expectoration;
1.10. The method of any of the foregoing method wherein the amount of polymorphonuclear neutrophils is determined by microscopy;
1.11. The method of any of the foregoing method wherein the polymorphonuclear neutrophil indicator substance is lactoferrin;
1.12. The method of any foregoing method wherein the polymorphonuclear neutrophil indicator substance is leukocyte esterase;
1.13. The method of any foregoing method wherein the polymorphonuclear neutrophil indicator substance is calprotectin;
1.14. The method of any of the foregoing method wherein the amount of PMN indicator substance is determined by contacting saliva with a saliva absorbent material which contains the necessary reagents to determine the presence and/or amount of PMN indicator substance;
1.15. The method of the immediately preceding method wherein the presence and/or amount of PMN indicator substance is identified by a color change.
1.16. A method for selecting a treatment regimen in a subject suspected of having oral inflammation comprising
(a) obtaining a test sample of an oral material such as saliva from said subject,
(b) determining the amount of PMN, or PMN indicator substance, in said test sample,
(c) comparing the amount of PMN, or PMN indicator substance, in the test sample to a control,
(d) selecting a treatment regimen based on the comparison made in step (c);
1.17. The method of 1.16 including one or more of the embodiments of 1.1-1.16;
1.18. The method of 1.16 or 1.17 including the additional step of the subject carrying out the selected treatment regimen;
1.19. The method of 1.16 or 1.17 wherein the treatment regimen is selected from toothbrushing twice daily, toothbrushing more than twice daily, treating the subject with antiflammaory agents, treating the subject with antimicrobial agents, or a combination thereof;
1.20. The method of 1.19 wherein the antimicrobial agent is selected from halogenated diphenyl ether (triclosan), herbal extracts or essential oils (e.g., rosemary extract, thymol, menthol, eucalyptol, methyl salicylate), bisguanide antiseptics (e.g., chlorhexidine, alexidine, or octenidine), phenolic antiseptics, hexetidine, povidone iodine, delmopinol, salifluor, metal ions and their salts (e.g., zinc chloride, zinc lactate, zinc citrate, stannous fluoride, and stannous chloride), sanguinarine, propolis, oxygenating agents (e.g., hydrogen peroxide, buffered sodium peroxyborate, or peroxycarbonate), cetyl pyridinium chloride, magnolia extract, magnolol, honokiol, butyl magnolol, propyl honokiol, and combinations thereof. Anti-attachment agents such as Solrol also can be included, as well as plaque dispersing agents such as enzymes (papain, glucoamylase, etc.).
1.21. The method of 1.19 or 1.20 wherein the anti-inflammatory agent is selected from steroidal agents such as flucinolone and hydrocortisone, and nonsteroidal agents (NTHEs) such as ketorolac, flurbiprofen, ibuprofen, naproxen, indomethacin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, ketoprofen, fenoprofen, piroxicam, nabumetone, aspirin, diflunisal, meclofenamate, mefenamic acid, oxyphenbutazone, phenylbutazone, and combinations thereof
1.22. The method of any foregoing method which is an at home method.

[0058] The invention also features kits for practicing the inventive methods, for example, kits for assaying a subject. In one embodiment a diagnostic kit for identifying a subject having oral inflammation or as being at elevated risk of developing a disease or condition resulting from oral inflammation, comprising a saliva absorbent material which contains the necessary reagents to determine the presence and/or amount of PMN indicator substance together with instructions for use.

[0059] The invention is illustrated by the following non-limiting examples.

Examples

Example 1

[0060] Objective:

[0061] This clinical study evaluated the levels of polymorphonuclear leukocytes (PMN), a type of immune cell in comparison to the clinical parameters of dental plaque (plaque index) and gingivitis (gingival Index) respectively.

[0062] Procedure:

[0063] For this study, subjects were evaluated by these methods at Baseline, Visit 1 (7 days), and Visit 2 (14 days). For the study period, all subjects were provided a commercially available fluoride toothpaste for oral hygiene.

[0064] Dental Plaque Assessment:

[0065] The dentition is disclosed with disclosing solution and plaque is scored at the disto-, mid-, mesio-buccal, and disto-, mid-, mesio-lingual surfaces of each tooth according to the criteria of the modified Quigley and Hein Index (Turesky et al., 1970 and Quigley & Hein, 1962), which is scored on a 0 to 5 scale. Subjectwise scores are calculated by summing all scores for all sites and dividing by the total number of sits scored.

[0066] Gingivitis Assessment

[0067] The degree of gingivitis is scored at 6 sites (same sites listed above) of each tooth according to the criteria of the Gingival Index System (Le and Silness, 1963), which is scored on a 0 to 3 scale. Subjectwise scores are calculated by summing all scores for all sites and dividing by the total number of sites scored.

[0068] PMN Measurement

[0069] Prior to clinical assessments, subjects are instructed to rinse vigorously with Hank's Balanced Salt solution (Gibco HBSS, Life Technologies) for 30 seconds and then expectorated into sterile tubes. Formaldehyde is added to the samples which are then centrifuged. The resulting pellets are resuspended and stained with acridine orange in a dark room at room temperature for 20 minutes prior to analysis by fluorescence microscopy.

[0070] Results:

[0071] One hundred fifty-nine (159) adults (61 men and 98 women; age range 19-61 years) were enrolled in study.

[0072] Subject demographics are shown in Table 1. Summaries from clinical evaluations are shown in Tables 2 through 4 below.

TABLE-US-00001 TABLE 1 Summary of Age and Sex Characteristics for enrolled subjects. Number (Percent) of subjects Age of subjects Male Female Total Mean Range 61 (38.4) 98 (61.6) 159 (100) 30.8 19-61

[0073] In order to compare PMN with Plaque Index and Gingival Index a graphical display and a corresponding correlation analysis was performed. Prior to analysis, due to the skewed distribution of the PMN scores, a log 10 data transformation was employed. The analysis shows that the correlation coefficient of Log(PMN) with Plaque Index is 0.57 and the correlation coefficient of Log(PMN) with Gingival Index is 0.66. Both correlation coefficients are significantly (P<0.0001) different from zero.

[0074] The analysis above demonstrates that Log(PMN) is giving results that are strongly correlated with the Plaque Index and the Gingival Index. The final phase of the analysis is designed to assess the statistical sensitivity of Log(PMN) compared to the Plaque Index and the Gingival Index, with respect to the ability of the measure to detect changes from Baseline. Tables 2, 3, and 4 summarize the data that are analyzed and the results of the analysis.

[0075] In Table 2 the baseline data are summarized for each of the three indices.

TABLE-US-00002 TABLE 2 Baseline Gingival Plaque Index Index Log(PMN) 2.88 0.94 1.37 0.82 5.28 0.56

[0076] In Tables 3 and 4 the change from baseline data to visits 1 and 2, respectively, are summarized and the analysis is summarized through the t-Statistic and P-Value for the significance of the mean change from Baseline. The t-Statistic is an outcome measure of this analysis and is used to determine the P-value used to assess the statistical significance of the change from Baseline.

TABLE-US-00003 TABLE 3 Change from Baseline to Visit 1 (Visit 1 - Baseline) Gingival Plaque Index Index Log(PMN) Mean Change Std. 0.11 0.40 0.25 0.29 0.22 0.45 P-Value 0.0007 <0.0001 <0.0001 Absolute Value of t-Statistic 3.47 10.66 6.19

TABLE-US-00004 TABLE 4 Change from Baseline to Visit 2 (Visit 2 - Baseline) Gingival Plaque Index Index Log(PMN) Mean Change Std. 0.02 0.36 0.41 0.39 0.27 0.46 P-Value 0.5127 <0.0001 <0.0001 Absolute Value of t-Statistic 0.66 13.44 7.34

[0077] The summaries shown in Table 2 give the average and standard deviation of the Baseline value for each measure. In Tables 3 and 4 all analyses show statistically significant (p<0.0001) mean changes from Baseline except for the change between Visit 2 and Baseline for the Plaque Index. However, in both Tables 3 and 4 the largest t-statistic is associated with the Gingival Index. Since the larger the t-Statistic the greater the statistical sensitivity of the measure it can be concluded that in this study the Gingival Index has more statistical sensitivity than the other two measures in terms of detecting changes from baseline. The Log(PMN), while showing more statistical sensitivity than the Plaque Index, shows less sensitivity than the Gingival Index.

[0078] The population of the 159 adults evaluated could be categorized into discrete groups based on plaque index, gingival index and PMN counts with lower number indices being indicative of a healthier oral environment.

TABLE-US-00005 Variable Group Count Mean Plaque index A 45 1.79 B 11 1.99 C 19 3.16 D 38 3.35 E 46 3.64 Gingival index A 45 0.29 B 11 0.65 C 19 1.30 D 38 1.79 E 46 2.29 PMN (Counts per ml) A 45 65556 B 11 139545 C 19 313487 D 38 535230 E 46 690435

Example 2

[0079] Objective:

[0080] This clinical study evaluated the levels of polymorphonuclear leukocytes (PMN) in conjunction with clinical parameters i.e. dental plaque (PI), gingival inflammation (GI) and periodontal pocket probing depth (PD) amongst the following groups of subjects stratified on the basis of their initial clinical status: [0081] healthy: subjects with no gingivitis and in good oral health [0082] gingivitis: subjects with gingivitis based on clinical evaluation [0083] periodontal disease: subjects with periodontal disease based on clinical evaluation

[0084] Shown in table 5 are demographic characteristics of enrolled subjects and results from clinical evaluations (table 6) and levels of polymorphonuclear leukocytes (table 7) prior to and after 28 day use of the triclosan/copolymer toothpaste (Colgate Totaltriclosan and polymethylvinylether/maleic anhydride copolymer) for oral hygiene.

Procedures are described herein or are similar to those described in Example 1.

TABLE-US-00006 TABLE 5 Demographics of enrolled subjects. Parameter Healthy Gingivitis Periodontal disease Number of subjects 17 17 13 Mean Age 29.87 30 47.08 Age SD 8.52 6.54 11.70 Minimum Age 21 22 26 Maximum Age 52 45 64 Number of males 6 12 7 Number of females 11 5 6

TABLE-US-00007 TABLE 6 Results from clinical evaluations of subjects prior to and after 28 day use of the triclosan/copolymer toothpas Before After treatment treatment Average Average Population Clinical assessment scores scores p value Healthy Dental plaque (PI) 0.77 0.45 0.000 Healthy Gingival score (GI) 0.47 0.38 0.046 Healthy Pocket Depth (PD) 2.36 2.10 0.000 Gingivitis Dental plaque (PI) 1.35 0.68 0.000 Gingivitis Gingival score (GI) 1.43 0.59 0.000 Gingivitis Pocket Depth (PD) 2.67 2.25 0.000 Periodontal Dental plaque (PI) 1.75 0.94 0.000 Disease Periodontal Gingival score (GI) 1.78 0.88 0.000 Disease Periodontal Pocket Depth (PD) 3.58 2.65 0.000 Disease
Results indicate significant reductions in each evaluated clinical parameter for all groups of evaluated subjects.

TABLE-US-00008 TABLE 7 Counts for polymorphonuclear leukocytes prior to and after 28 day use of the triclosan/copolymer dentifrice. After treatment Before Average treatment numbers Average of PMN Comparing before numbers of (10.sup.4) treatment to after PMN (10.sup.4) Cells % treatment scores (p Population Cells per ml per ml reduction value) Healthy 1.84 1.313 28.6 <0.05 (Significant) Gingivitis 6.56 2.813 57.1 <0.05 (Significant) Periodontitis 28.37 16.5 41.8 <0.05 (Significant)
Results indicate significant reductions in PMN scores in each group of evaluated subjects.

Example 3

[0085] Objective:

[0086] This clinical study evaluated the levels of polymorphonuclear leukocytes (PMN), amongst subjects assigned either a commercially available fluoride toothpaste or the triclosan/copolymer toothpaste for oral hygiene. Subjects were evaluated prior to and after 30 day use of each toothpaste.

[0087] Shown in table 8 are results from this study.

[0088] Procedures are as described herein or are similar to the prior examples.

TABLE-US-00009 TABLE 8 Counts for polymorphonuclear leukocytes amongst subjects prior to and after 30 days use of a fluoride toothpaste or the triclosan/copolymer dentifrice. Treatment group # of subjects Baseline score SEM Day 30 score SEM % reduction Fluoride toothpaste 37 14.05 2.06 11.65 1.6 17.08 (Crest) Triclosan/copolymer 37 20.03 4 13.08 2.85 34.69 t-Test No significant differences Significant differences Results between treatments between treatments at Day at baseline. P value = 30. 0.100 P value 0.00014

[0089] Results indicate significant reductions in PMN scores amongst those provided the triclosan/copolymer toothpaste for oral hygiene. A similar effect was not observed amongst those using the fluoride toothpaste.

Example 4

[0090] This clinical study evaluates the levels of poylmorphonuclear leukocytes (PMN), in conjunction with the following well accepted clinical and microbiological parameters prior to and after 2 week use of a 0.12% chlorhexidine (CHX) mouthrinse for oral hygiene:

[0091] Clinical Parameters: [0092] Malodor based on organoleptic and halimeter evaluations. [0093] An evaluation of tongue coating based on a tongue coat clinical index.

[0094] Microbiological Parameters: [0095] Total viable organisms of the dental plaque, saliva and tongue surface. [0096] Malodor organisms in the dental plaque, saliva and tongue surface.
Shown in table 9 are demographics of enrolled subjects.

TABLE-US-00010 TABLE 9 Patient Demographics Status N Mean Age Control 15 39.6 Gingivitis 21 44.76 Halitosis 18 54.72

[0097] The numbers of polymorphonuclear leukocytes (PMN) from all subjects prior to and after use of the CHX mouthinse are determined. Results indicate reductions in PMN scores for all groups of subjects after two week use of the CHX mouthrinse.

[0098] Summarized in tables 10-12 are results from clinical evaluations of subjects by a halimeter, organoleptic scores and by the tongue coat index.

TABLE-US-00011 TABLE 10 Halitosis evaluation (using a Halimeter) Mean Mean halitosis baseline scores after 2 week scores for use of 0.12% Status halitosis chlorhexidine Control 79.53 52.87 Gingivitis 111.62 51 Halitosis 253.5 107.7

TABLE-US-00012 TABLE 11 Halitosis evaluation (Organoleptic Scores) Mean halitosis scores after 2 Mean baseline week use of scores for 0.12% Status halitosis chlorhexidine Control 1.467 0.6 Gingivitis 2.476 1.19 Halitosis 3.684 1.632

TABLE-US-00013 TABLE 12 Tongue Coat Index Mean Mean scores after 2 baseline week use of 0.12% Status scores chlorhexidine Control 1.667 0.667 Gingivitis 2.619 1.619 Halitosis 3.632 2.053

[0099] Results indicate significant reductions in each clinical score after subjects were assigned the CHX rinse.

[0100] Summarized in tables 13-18 are results from microbiological evaluations of subejcts for viable organisms in dental plaque, saliva and tongue surface in additiona o maloor organisms in each of these samples.

TABLE-US-00014 TABLE 13 Total Viable Bacteria in Dental Plaque (Log CFU/ml) Mean score Mean after 2 week baseline use of 0.12% Status scores chlorhexidine Control 7.18 5.86 Gingivitis 7.49 6.25 Halitosis 7.58 6.15

TABLE-US-00015 TABLE 14 Viable Dental Plaque Malodor Bacteria (Log CFU/ml). Mean scores Mean after 2 week baseline use of 0.12% Status scores chlorhexidine Control 6.7 5.71 Gingivitis 6.86 5.86 Halitosis 6.83 5.4

TABLE-US-00016 TABLE 15 Total Viable Bacteria on Tongue Surface (Log CFU/ml). Mean scores Mean after 2 week baseline use of 0.12% Status scores chlorhexidine Control 7.26 6.5 Gingivitis 7.63 6.67 Halitosis 7.72 6.83

TABLE-US-00017 TABLE 16 Viable Tongue Surface Malodor Bacteria (Log CFU/ml) Mean scores Mean after 2 week baseline use of 0.12% Status scores chlorhexidine Control 6.75 6.13 Gingivitis 6.95 6.25 Halitosis 7.18 6.09

TABLE-US-00018 TABLE 17 Total Viable Salivary Bacteria (Log CFU/ml) Mean scores Mean after 2 week baseline use of 0.12% Status scores chlorhexidine Control 7.81 6.97 Gingivitis 7.86 7.01 Halitosis 7.78 7.16

TABLE-US-00019 TABLE 18 Viable Salivary Malodor Bacteria (Log CFU/ml) Mean scores Mean after 2 week baseline use of 0.12% Status scores chlorhexidine Control 7.03 6.2 Gingivitis 7.09 6.09 Halitosis 7.1 6.13

[0101] Subjects demonstrated significant reductions in all types of organisms in each oral sample evaluated following the use of the CHX rinse.

Example 5

[0102] This study determines how quickly subjects brushing with Total or fluoride toothpaste begin to show changes in oral PMN and clinical outcomes.

Study Design:

[0103] 1. Subjects brushed with either a fluoride toothpaste or with the Total toothpaste.
2. Subjects were evaluated at study entry (baseline or prior to issuing any toothpaste) and were also evaluated after brushing with provided paste for 1 week and 2 weeks.
The following evaluations were conducted for all subjects at baseline and after 1 week and 2 week use of assigned toothpaste:

1. PMN

[0104] 2. Clinical parameters (dental plaque index, gingivitis index, bleeding index).

TABLE-US-00020 TABLE 19 Summary of Age &Gender For Subjects Who Completed the Clinical Study Number of Subjects Age.sup.3 Treatment Male Female Total.sup.3 Mean Range Test Toothpaste Group.sup.1 6 19 25 44.8 19-70 Control Toothpaste Group.sup.2 7 18 25 42.7 19-65 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY) .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY) .sup.3No statistically significant difference was indicated between the treatment groups with respect to either gender or age.

TABLE-US-00021 TABLE 20 Subject Mean (SD) Polymorphonuclear Leukocytes (Log Cells/ml) Samples at Baseline, 1- Week and 2-Weeks For Subjects Who Completed the Clinical Study Baseline 1-Week 2-Week Summary Summary Summary Parameter Treatment n (Mean S.D.) (Mean S.D.) (Mean S.D.) Polymorphonuclear Test Toothpaste Group.sup.1 25 5.17 0.32 4.93 0.23 4.77 0.28 Leukocytes (Log Control Toothpaste 25 5.08 0.40 5.08 0.36 5.11 0.38 Cells/ml) Group.sup.2 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3No statistically significant difference was indicated between the two treatment groups at baseline with respect to polymorphonuclear leukocytes (Log Cell/ml) samples.

TABLE-US-00022 TABLE 21 Subject Mean (SD) Gingival Index, Bleeding Index, Plaque Index Scores and Pocket Depth at Baseline and 2-Weeks For Subjects Who Completed the Clinical Study Baseline 2-Week Summary Summary Parameter Treatment n (Mean S.D.) (Mean S.D.) Gingival Index Test Toothpaste 25 1.42 0.32 1.01 0.35 Group.sup.1 Control 25 1.38 0.26 1.32 0.25 Toothpaste Group.sup.2 Bleeding Index Test Toothpaste 25 0.55 0.22 0.35 0.25 Group.sup.1 Control 25 0.65 0.33 0.59 0.30 Toothpaste Group.sup.2 Plaque Index Test Toothpaste 25 2.17 0.57 1.81 0.49 Group.sup.1 Control 25 2.16 0.51 2.02 0.49 Toothpaste Group.sup.2 Pocket Depth Test Toothpaste 25 2.18 0.46 1.92 0.43 Group.sup.1 Control 25 2.06 0.41 2.02 0.38 Toothpaste Group.sup.2 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3No statistically significant difference was indicated among the two treatment groups at baseline with respect to gingival index, bleeding index, plaque index scores and pocket depth (mm).

TABLE-US-00023 TABLE 22 Baseline-Adjusted Subject Mean (SE) Polymorphonuclear Leukocytes (Log Cells/ml) Samples at 1-Week For Subjects Who Completed the Clinical Study 1-Week Within Treatment Between-Treatment Summary Analysis Comparisons Index Treatment n (Mean S.E.) Reduction.sup.3 Sig..sup.4 Difference.sup.5 Sig..sup.6 Polymorphonuclear Test 25 4.91 0.05 38.3% P = 0.001 35.4% P = 0.009 Leukocytes (Log Toothpaste Cells/ml) Group.sup.1 Control 25 5.10 0.05 4.5% P = 0.752 Toothpaste Group.sup.2 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3Reduction exhibited by the 1-week mean relative to the baseline mean. A positive value indicates a reduction in polymorphonuclear leukocytes (Log Cells/ml) at the 1-week examination. .sup.4Significance of paired t-test comparing the baseline and 1-week examinations. .sup.5Difference between the 1-week means expressed as a reduction of the 1-week mean for the Control Toothpaste Group. A positive value indicates a reduction in polymorphonuclear leukocytes (Log Cells/ml) for the Test Toothpaste Group relative to Control Toothpaste Group. .sup.6Significance of ANCOVA comparison of baseline-adjusted means.

TABLE-US-00024 TABLE 23 Baseline-Adjusted Subject Mean (SE) Polymorphonuclear Leukocytes (Log Cells/ml) Samples at 2-Weeks For Subjects Who Completed the Clinical Study 2-Week Within Treatment Between-Treatment Summary Analysis Comparisons Index Treatment n (Mean S.E.) Reduction.sup.3 Sig..sup.4 Difference.sup.5 Sig..sup.6 Polymorphonuclear Test 25 4.75 0.05 57.3% P < 0.001 59.3% P < 0.001 Leukocytes Toothpaste (Log Cells/ml) Group.sup.1 Control 25 5.14 0.05 4.7% P = 815 Toothpaste Group.sup.2 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3Reduction exhibited by the 2-week mean relative to the baseline mean. A positive value indicates a reduction in polymorphonuclear leukocytes (Log Cells/ml) at the 2-week examination. .sup.4Significance of paired t-test comparing the baseline and 2-week examinations. .sup.5Difference between the 2-week means expressed as a reduction of the 2-week mean for the Control Toothpaste Group. A positive value indicates a reduction in polymorphonuclear leukocytes (Log Cells/ml) for the Test Toothpaste Group relative to Control Toothpaste Group. .sup.6Significance of ANCOVA comparison of baseline-adjusted means.

TABLE-US-00025 TABLE 24 Baseline-Adjusted Subject Mean (SE) Gingival Index, Bleeding Index, Plaque Index Scores and Pocket Depth For Subjects Who Completed the Clinical Study 2-Week Within Treatment Between-Treatment Summary Analysis Comparisons Treatment N (Mean S.E.) Reduction.sup.3 Sig..sup.4 Difference.sup.5 Sig..sup.6 Gingival Index Test 25 0.99 0.05 29.3% P < 0.001 25.6% P < 0.001 Toothpaste Group.sup.1 Con 25 1.33 0.05 5.0% P = 0.008 Index trol Toothpaste Group.sup.2 Bleeding Index Test 25 0.39 0.03 35.0% P < 0.001 29.1% P < 0.001 Toothpaste Group.sup.1 Control 25 0.55 0.03 8.3% P = 0.045 Toothpaste Group.sup.2 Plaque Index Test 25 1.80 0.05 16.7% P < 0.001 11.3% P = 0.004 Toothpaste Group Control 25 2.03 0.05 6.0% P < 0.001 Toothpaste Group.sup.2 Pocket Depth Test 25 1.87 0.04 0.25 mm P < 0.001 0.2 mm P = 0.001 (mm) Toothpaste Group.sup.1 Control 25 2.07 0.04 0.05 mm P = 0.218 Toothpaste Group.sup.2 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3Reduction exhibited by the 2-week mean relative to the baseline mean. A positive value indicates a reduction in index scores at the 2-week examination. .sup.4Significance of paired t-test comparing the baseline and 2-week examinations. .sup.5Difference between the 2-week means expressed as a reduction of the 2-week mean for the Control Toothpaste Group. A positive value indicates a reduction in index scores for the Test Toothpaste Group relative to Control Toothpaste Group. .sup.6Significance of ANCOVA comparison of baseline-adjusted means.

Results Summary:

[0105] subjects brushing with Total begin to show reductions in PMN after only one week use of this toothpaste. [0106] additional reductions in PMN amongst subjects brushing with Total in the second week of use. In other words, the effects of Total get better from the week 1 to week 2.

Significance of Results:

[0107] 1. PMN (or neutrophil) changes commence in the initial days following use of an effective toothpaste (Total).
2. Furthermore, the % changes for PMN are higher for Total than a fluoride toothpaste.
3. The % differences for PMN in the Total group are far higher than those for conventional clinical indices i.e. gingivitis index, bleeding index and plaque index.

Example 6

[0108] Study Objective:

[0109] Determine how quickly subjects brushing with Total or fluoride toothpaste begin to show changes in oral PMN and clinical outcomes.

Study Design:

[0110] 1. Subjects brushed with either a fluoride toothpaste or with the Total toothpaste.
2. Subjects were evaluated at study entry (baseline or prior to issuing any toothpaste) and were also evaluated after brushing with provided paste for 1 week and 2 weeks.
The following evaluations were conducted for all subjects at baseline and after 1 week and 2 week use of assigned toothpaste:

1. PMN

[0111] 2. Clinical parameters (dental plaque index, gingivitis index, bleeding index).

[0112] The results are in the tables below.

CONCLUSIONS

[0113] subjects brushing with Total begin to show reductions in PMN after only one week use of this toothpaste.

[0114] additional reductions in PMN amongst subjects brushing with Total in the second week of use, i.e. the effects of Total get better from

the week 1 to week 2.

TABLE-US-00026 TABLE 25 Summary of Age &Gender For Subjects Who Completed the Clinical Study Number of Subjects Age.sup.3 Treatment Male Female Total.sup.3 Mean Range Test Toothpaste Group.sup.1 6 19 25 44.8 19-70 Control Toothpaste Group.sup.2 7 18 25 42.7 19-65 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY) .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY) .sup.3No statistically significant difference was indicated between the treatment groups with respect to either gender or age.

TABLE-US-00027 TABLE 26 Subject Mean (SD) Polymorphonuclear Leukocytes (Log Cells/ml) Samples at Baseline, 1-Week and 2-Weeks For Subjects Who Completed the Clinical Study Baseline 1-Week 2-Week Summary Summary Summary (Mean (Mean (Mean Parameter Treatment n S.D.) S.D.) S.D.) Polymorphonuclear Test Toothpaste 25 5.17 0.32 4.93 0.23 4.77 0.28 Leukocytes (Log Group.sup.1 Cells/ml) Control Toothpaste 25 5.08 0.40 5.08 0.36 5.11 0.38 Group.sup.2 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3No statistically significant difference was indicated between the two treatment groups at baseline with respect to polymorphonuclear leukocytes (Log Cell/ml) samples.

TABLE-US-00028 TABLE 27 Subject Mean (SD) Gingival Index, Bleeding Index, Plaque Index Scores and Pocket Depth at Baseline and 2-Weeks For Subjects Who Completed the Clinical Study Baseline 2-Week Summary Summary Pa- (Mean (Mean rameter Treatment n S.D.) S.D.) Gingival Test Toothpaste Group.sup.1 25 1.42 0.32 1.01 0.35 Index Control Toothpaste Group.sup.2 25 1.38 0.26 1.32 0.25 Bleeding Test Toothpaste Group.sup.1 25 0.55 0.22 0.35 0.25 Index Control Toothpaste Group.sup.2 25 0.65 0.33 0.59 0.30 Plaque Test Toothpaste Group.sup.1 25 2.17 0.57 1.81 0.49 Index Control Toothpaste Group.sup.2 25 2.16 0.51 2.02 0.49 Pocket Test Toothpaste Group.sup.1 25 2.18 0.46 1.92 0.43 Depth Control Toothpaste Group.sup.2 25 2.06 0.41 2.02 0.38 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3No statistically significant difference was indicated among the two treatment groups at baseline with respect to gingival index, bleeding index, plaque index scores and pocket depth (mm).

TABLE-US-00029 TABLE 28 Baseline-Adjusted Subject Mean (SE) Polymorphonuclear Leukocytes (Log Cells/ml) Samples at 1-Week For Subjects Who Completed the Clinical Study 1-Week Within Treatment Between-Treatment Summary Analysis Comparisons Index Treatment n (Mean S.E.) Reduction.sup.3 Sig..sup.4 Difference.sup.5 Sig..sup.6 Polymorphonuclear Test 25 4.91 0.05 38.3% P = 0.001 35.4% P = 0.009 Leukocytes (Log Toothpaste Cells/ml) Group.sup.1 Control 25 5.10 0.05 4.5% P = 0.752 Toothpaste Group.sup.2 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3Reduction exhibited by the 1-week mean relative to the baseline mean. A positive value indicates a reduction in polymorphonuclear leukocytes (Log Cells/ml) at the 1-week examination. .sup.4Significance of paired t-test comparing the baseline and 1-week examinations. .sup.5Difference between the 1-week means expressed as a reduction of the 1-week mean for the Control Toothpaste Group. A positive value indicates a reduction in polymorphonuclear leukocytes (Log Cells/ml) for the Test Toothpaste Group relative to Control Toothpaste Group. .sup.6Significance of ANCOVA comparison of baseline-adjusted means.

TABLE-US-00030 TABLE 29 Baseline-Adjusted Subject Mean (SE) Polymorphonuclear Leukocytes (Log Cells/ml) Samples at 2-Weeks For Subjects Who Completed the Clinical Study 2-Week Within Treatment Between-Treatment Summary Analysis Comparisons Index Treatment n (Mean S.E.) Reduction.sup.3 Sig..sup.4 Diff..sup.5 Sig..sup.6 Polymorphonuclear Test 25 4.75 0.05 57.3% P < 0.001 59.3% P < 0.001 Leukocytes (Log Toothpaste Cells/ml) Group.sup.1 Control 25 5.14 0.05 4.7% P = 815 Toothpaste Group.sup.2 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer, 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3Reduction exhibited by the 2-week mean relative to the baseline mean. A positive value indicates a reduction in polymorphonuclear leukocytes (Log Cells/ml) at the 2-week examination. .sup.4Significance of paired t-test comparing the baseline and 2-week examinations. .sup.5Difference between the 2-week means expressed as a reduction of the 2-week mean for the Control Toothpaste Group. A positive value indicates a reduction in polymorphonuclear leukocytes (Log Cells/ml) for the Test Toothpaste Group relative to Control Toothpaste Group. .sup.6Significance of ANCOVA comparison of baseline-adjusted means.

TABLE-US-00031 TABLE 30 Baseline-Adjusted Subject Mean (SE) Gingival Index, Bleeding Index, Plaque Index Scores and Pocket Depth For Subjects Who Completed the Clinical Study 2-Week Within Treatment Between-Treatment Summary Analysis Comparisons Index Treatment n (Mean S.E.) Reduction.sup.3 Sig..sup.4 Difference.sup.5 Sig..sup.6 Gingival Test 25 0.99 0.05 29.3% P < 0.001 25.6% P < 0.001 Index Toothpaste Group.sup.1 Control 25 1.33 0.05 5.0% P = 0.008 Toothpaste Group.sup.2 Bleeding Test 25 0.39 0.03 35.0% P < 0.001 29.1% P < 0.001 Index Toothpaste Group.sup.1 Control 25 0.55 0.03 8.3% P = 0.045 Toothpaste Group.sup.2 Plaque Test 25 1.80 0.05 16.7% P < 0.001 11.3% P = 0.004 Index Toothpaste Group.sup.1 Control 25 2.03 0.05 6.0% P < 0.001 Toothpaste Group.sup.2 Pocket Test 25 1.87 0.04 0.25 mm P < 0.001 0.2 mm P = 0.001 Depth Toothpaste (mm) Group.sup.1 Control 25 2.07 0.04 0.05 mm P = 0.218 Toothpaste Group.sup.2 .sup.1Dentifrice formulation containing 0.3% triclosan, 2.0% copolymer (polymethylvinylether/maleic anhydride copolymer), 0.243% sodium fluoride in a silica base (Colgate-Palmolive Co., New York, NY). .sup.2Colgate Cavity Protection Great Regular Flavor Toothpaste (Colgate-Palmolive Co., New York, NY). .sup.3Reduction exhibited by the 2-week mean relative to the baseline mean. A positive value indicates a reduction in index scores at the 2-week examination. .sup.4Significance of paired t-test comparing the baseline and 2-week examinations. .sup.5Difference between the 2-week means expressed as a reduction of the 2-week mean for the Control Toothpaste Group. A positive value indicates a reduction in index scores for the Test Toothpaste Group relative to Control Toothpaste Group. .sup.6Significance of ANCOVA comparison of baseline-adjusted means.