AU700252B2 - Composition for dental use comprising krill enzyme - Google Patents

Description

WO 95/33470 PCT/SE94/00549 COMPOSITION FOR DENTAL USE COMPRISING KRILL ENZYME Technical Field Regular oral hygiene such as tooth brushing is normally insufficient to provide adequate control of dental microbial plaque. Consequently, the incorporation of antimicrobial and plaque dissolving agents in dental products such as dentifrices has been proposed by dental researchers for a long time review by P.D. Marsh, J. Clin. Periodont., 18, 462-67 (1991).

Apart from certain fluorides most compounds of potential use as antiplaque agents have turned out to be incompatible with either vehicle substances employed or the oral environment.

Furthermore, recent research demonstrates that antiplaque agents should be designed to control rather than eliminate the dental plaque microflora Marsh, Brit. Dent. J. 9, 174-77 (1991). Like other parts of the digestive tract the oral cavity possesses a natural microflora which is beneficial to the host. Changes in the natural microflora may result in caries and periodontal disease.

A number of antibacterial and surface active substances have been evaluated and tested for plaque control (Table i).

Bisquanides and phenols (Chlorohexidine and Triclosan) have proved to be most effective. However, although showing a strong antibacterial action, they have undesirable short-term effects. Furthermore, possible long-term effects are causing concern.

Recently, surface active agents such as amino alcohols have been introduced with the purpose of affecting the adhesion of bacteria to oral surfaces. Again, little is known about possible adverse long-term effects. Furthermore, bacteria are known to develop resistance to chemotherapeutic agents on prolonged use.

PCT/SE94/00 5 49 WO 95/3347 0 2 Table 1: Classes and examples of antimicrobial antiplaque agents proposed for use in dentifrices.

Class of inhibitor Some examples Antibiotics Tetracycline, penicillin Antibiguanidines Chlorhexidine, alexidine 0 EnBisbiguanidines Mutanase, amyloglucosidase/ Enzyes glucose oxidase Essential oils Thymol, eucalyptol Fluoride Monofluorophosphate M etal ions Zinc, stannous copper Metal l o n s Sanguinarine Plant extracts Sanguinarine Phenols Triclosan Quaternary ammonium Quaternary ammonium Cetylpyridinum chloride compounds Sodium lauryl, sulphate Surfactants o Several enzymes have been employed as antiplaque agents.

However, in most cases it has not been possible to confirm promising laboratory tests in clinical trials. Proteases, 25 mutanases and dextranases have been used primarily to dissolve the plaque matrix which allows bacteria to adhere to tooth surface. An enzyme dentifrice which is presently being marketed (Zendium) contains an aminoglycosidase, the effect of which is based on an indirect antibacterial action. How- 30 ever, the clinical effects seems to be too limited.

The enzyme mixture from the digestive tract of the krill schrimp, Euphuasia superba, has proved to be unique in composition and amazingly potent in action when used on organic substrates as compared to previously known digestive enzymes.

Thus, a method of therapeutic an non-therapeutic cleaning is described in EP-B1-0,107,6 34 Disclosure of Invention In accordance with a first aspect of the present invention, there is provided a method for decreasing the adhesive ability of plaque bacteria in a human subject and preventing dental plaque formation in the human subject comprising administering WO 95/33470 3 PCT/SE94/00549 in the oral cavity of said subject a prophylactically effective amount of enzymes from Antarctic krill (Euphausia superba) in a carrier adapted for prophylactic treatment.

In accordance with a second aspect of the present invention, there is provided the use of enzymes from Antarctic krill (Euphausia superba) in the manufacture of a prophylactic composition for decreasing the adhesive ability of plaque bacteria in a human subject and preventing dental plaque formation in the human subject.

Thus, in general, the invention concerns the use of these krill enzymes (KE) to prevent dental plaque on natural and artificial teeth, dental appliances, implants and soft tissues of the 15 mouth. By applying krill enzymes to these surfaces, plaque formation, caries, yeast-cell infections and soft-tissue inflammation will be prevented.

Compositions of enzymes from Antarctic krill (Euphausia 20 superba) have been studied as potential oral prophylactic antiplaque/agents. A number of in vitro/in vivo studies have demonstrated that krill enzymes: retard plaque accumulation both in vitro and in vivo more effectively than Alcalase do not exhibit an antibacterial effect; and show mild antifungal 25 properties against Candida albicans, common in oral opportunistic infections. Hence, it is concluded that krill enzymes offer highly promising properties as an antiplaque agent due to the high efficiency of krill enzymes in dissolving plaques and apparent biocompatibility.

Oral diseases such as caries, gingivitis/periodontitis and candidiasis are caused when microorganisms of the natural oral microflora accumulate on retentive surfaces such as teeth and artificial restorations, thereby forming dental plaque Poor or inadequate oral hygiene causes this plaque formation.

Mechanical toothbrushing is still the most efficient way to remove dental plaque WO 95/33470 4 PCT/SE94/00549 As dental plaque consist of oral bacteria/microbes which aggregate on dental/oral surfaces by adherence, growth and production of extracellular organic matrix enzymes for debriding this organic debris have for a long time been considered an ideal dental plaque remover Peptidase, pancrease, dextranases and Alcalase® (12) are all different enzymes tried over the years as dental plaque debriders with limited clinical success, the main reason for failure being weakness in catalyst action and in retaining adequate and sufficient enzyme concentrations on substrates in the salivarich oral environment 9 The concept of chemical plaque control has consequently been concentrated on antibacterial substances such as chlor- 15 hexidine 16) and surface active agents These substances, though, have some adverse side effects and the long term effect on the organism is uncertain. (19).

When enzymes are employed to remove plaque or to facilitate mechanical removal by toothbrushing they will merely have a i limited effect on the inflammatory reactions in the gingiva 1 soft tissue 3) since there is strong evidence that food debris has its strongest devastating effect on teeth in the earliest phase after food consumption.

The adherence of yeast-cells (Candida albicans) to dentures and oral soft tissue causes inflammatory reactions which are difficult to cure. The main reason is, again, poor denture hygiene Enzyme-containing denture-cleaners are already on the market Alcalase® being the more prominent (8, 9, 10, 12).

As shown in the examples of this application, krill enzymes Shave proved effective in prevention of dental plaque and yeast-cell adherence affecting adherence and metabolism of oral microorganisms and further improving the reduction of underlying inflammations in soft tissues.

WO 95/33470 PCT/SE94/00549 The proteolytic activity krill enzymes is often quantified in casein units. In a krill enzyme mixture, the proteolytic activity is measured as a part of the total enzymatic activity, which may comprise carbohydrate-splitting and lipolytic activity Example 1 Preparation of the enzyme composition Krill enzymes were extracted from the raw material, krill (Euphausia superba) according to standard procedures described in (EP-A 107,634, EP-A 177,605). In short, krill was frozen immediately after caught and stored at -30 0

C

before preparation. The thawed krill was mixed with water, homogenised and centrifuged in the cold. The supernatant was decanted and saved. Lipids were removed by extraction with organic solvents, the water phase concentrated and freezedried thus resulting in a "crude krill enzyme preparation" suitable for dental use.

Exampel 2 Detachement of yeast cells adhered to acrylic surfaces In accordance with a Japanese study design strains of Candida albicans were incubated in 4 acrylic beakers for 24 h at 37°C. The yeast cell then adhered to the acrylic surfaces which could be seen as a matt coating in the beakers. This model resembles yeast-cell infections on dental protheses.

Solutions of krill enzymes (1 resp were added to two beakers while two other beakers served as controls. 1 h later stereo microscopy showed that >90% of countable yeast cells had detached from acrylic surfaces of krill enzyme treated beakers compared to controls. The results were photodocumented (Table 1) WO 95/33470 PCT/SE94/00549 6 Example 3 Retardation of plaque formation in dogs Two healthy beagle dogs were used in this study. In this split-mouth design, teeth on the control side were treated with a water solution applied 2 times daily for 2 minutes at 6 h intervals. Teeth on the active side were treated in the same way with krill enzymes in a 2% buffer solution. Baseline status of the teeth was gingival health (GI=0) and no plaque (PLI=0) obtained by thorough cleaning twice daily for two weeks prior to test start. The test run lasted for 3 weeks during which time the dogs were fed a carbohydrate-rich diet.

The dogs were briefly sedated at treatment and registration.

Gingival index according to Loe and plaque index according to Silness L6e were used for registration of plaque and gingival inflammation. Plaque on teeth was made visible with eosin colorin (Diaplac). Teeth no P3 P4 and canines were used for registration. In addition to clinical registration all registrations were photodocumented.

From the photos the amount of plaque on teeth buccal and approximal surfaces was assessed. The amount of plaque was less on active side compared to control teeth. Some of these results were statistically significant at p< 0.05 using the sign test.

Example 4 Retardation of plaque formation in humans A test subject started with clinically clean and healthy teeth and gingiva (GI=0 PLI=0) restrained from all oral hygiene for one week. During this time a water and a krill enzyme 2% buffer solution was applied to control and active sides, respectively, in a split mouth design 3 times daily WO 95/33470 PCT/SE94/00549 7 for 2 minutes. At the end of test period the result was photodocumented after that plaque on teeth had been visualized with eosin color (Diaplac). There was a noticable difference in plaque accumulation and associated gingival inflammation between control and test sides.

Example A dental paste or gel is prepared with the following composition: Sodium monofluorophosphate, dicalcium phosphate dihydrate, glycerol, sorbitol, sodium citrate, sodium lauryl sulphate, aromatic flavour, sodium saccharinate, methylhydroxy-4-benzoate, krill enzyme extract, citric acid colouring agents.

Example 6 A chewing gum is prepared with the following composition: Chewing gum base, sodium fluoride, krill enzyme extract.

Example 7 A mouth wash and prosthesis cleaner is prepared with the following composition: Base, carrier, saline water solution, krill enzyme extract.

WO 95/33470 PCT/SE94/00549 8 References 1. L6e, H, Theilade, e Jensen, S.B. (1965) Experimental gingivitis in man. J. of Periodontology 36, 177-1887.

2. Budts-Jbrgensen, E. (1974) The significance of Candida albicans in denture stomatitis. Scand J Dent Res 82 151.

3. Socransky, S.S. (1970) Relationship of bacteria to the aethiology of periodontal disease. J of Dent Res 49, 223-228.

4. Hull, P.S. (1980) Chemical inhibition of plaque. J of Clin Periodontology 7, 431-442.

5. Budtz-Jbrgensen, Theilade, E, Theilade, J. (1983) Quantitative relationship between yeasts and bacteria in denture-induced stomatitis. Scand J Dent Res 91, 134.

6. Arendorf, T, Walker, D.M. (1979) Oral candidal populations in health and disease. Br Dent J 147 267.

7. Connor, Schoenfels, Taylor, R.L. (1977) An evaluation of an enzyme denture cleanser. J Prosthet Dent 37, 147.

8. Budtz-Jbrgensen, E. (1979) Material methods for cleansing dentures. J Prosthet Dent 42, 619.

Budts-J6rgensen, E Kelstrup, J. (1977) Enzymes as denture cleansers. Scan J Dent Res 85, 209.

11. Axelsson, P, Lindhe, J. (1974) The effect a preventive programme on dental plaque, gingivitis and caries in schoolchildren J of Clin Periodontology 1, 126-138.

WO 95/33470 PCT/SE94/00549 9 12. Budtz-J6rgensen, E, Kelstrup, J Poulsen, S. (1983) Reduction of formation of denture plaque by a protease (Alcalase). Acta Odontol Scand 41, 93-98 13. Lang, N.P. Cumming, B.R. Lbe, H. (1973) Toothbrush frequency is it is related to plaque development and gingival health. J of Periodontology 44, 396-405.

14. Kornman, (1986) The role of supragingival plaque in the prevention and treatment of periodontal diseases Reveiw. J of Periodont Res 21, Suppl., 5-22.

Axelsson, P, Lindhe, J Nystr6m, B, (1991) On the prevention of caries and periodontal disease. Results of a year longitudinal study in adults. J of Clin Periodontology 18, 182-189.

16. Marsh, P.D. (1991) Dentifrices containing new agents for the control of plaque. J Clin Periodontaol 18, 462-467.

17. Lbvdal, A et al., Combined effect of subgingival scaling and controlled oral hygiene in the incidence og gingivitis.

Acta Odontol Scand 19, 537-555 (1961).

18. Krembel, J et al, (1969) Fractionation of human dental plaques. Arch oral Bio 14, 563-565.

19. FlUtra, L Gjarmo P et al (1971) Side effects of chlorhexidrive mouthwashes Scand J of Dent Res 79 119-125.

Collaert, Bruno (1992) Chemotherapeutic supragingival plaque control with delmopinol hydrochloride. Thesis.

21. Shogo Minagi et al. (1987) Objective testing of the efficiency of denture-cleansing agents. J of Prosthetic Dentistry 58 595-598.

Claims (5)

1. Use of enzymes from Antarctic krill (Euphausia superba) in the manufacture of a prophylactic composition for decreasing the adhesive ability of plaque bacteria in a human subject and preventing dental plaque formation in the human subject.

2. Use as claimed in claim 1, wherein the composition comprises 0.01 to 100 units of said enzymes per 100 g of composition.

3. A method for decreasing the adhesive ability of plaque bacteria in a human subject and preventing dental plaque formation in the human subject comprising administering in the oral cavity of said subject a prophylactically effective amount of enzymes from Antarctic krill (Euphausia superba) in a carrier adapted for prophylactic treatment.

4. A method as claimed in claim 3, wherein the amount administered is 1 to 3 g, twice daily. *20

5. A method as claimed in claim 3 or 4 wherein a composition comprising 0.01 to 100 units of said enzymes per 100 g of composition is administered. DATED this 21st day of October 1998 KRISTIAN HELLGREN, LARS HELLGREN, VIGGO MOHR and JAN VINCENT, By their Patent Attorneys, E. F. WELLINGTON CO., By:ruce Welli (Bruce Wellington) A/BA/4566