EP0817606A1 - Oral care compositions - Google Patents
Oral care compositionsInfo
- Publication number
- EP0817606A1 EP0817606A1 EP96908026A EP96908026A EP0817606A1 EP 0817606 A1 EP0817606 A1 EP 0817606A1 EP 96908026 A EP96908026 A EP 96908026A EP 96908026 A EP96908026 A EP 96908026A EP 0817606 A1 EP0817606 A1 EP 0817606A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- oral care
- protease
- mouth
- care composition
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/66—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
Definitions
- the present invention relates to oral care compositions and products comprising certain proteases.
- proteases for e . g . oral care purposes, and a method for using oral care products of the invention.
- One of the major events for newly hatched parents is the appearance of the first tooth in the mouth of their baby. From that day on the caring parents include dental care as a natural part of taking care of their baby.
- the teeth must at least be brushed once every day using toothpaste or the like. Further the mouth should regularly be rinsed with a mouth wash.
- the brushing of the teeth primarily helps removing food particles from the teeth, and constitutes an important first step in preventing dental caries, which may cause dental holes, and further in preventing dental diseases.
- a good dental hygiene should also be obtained to prevent formation of plaque and tartar, outbreak of oral diseases, and adherence of stains from tea, coffee and tobacco smoking, and the like, which will remove the basis for obtaining a good appearance when e.g. smiling.
- a number of unpleasant incidences can be avoided by using oral care products for obtaining and/or maintaining e.g. a fresh breath, as most people dislike a in general bad breath, or e.g. an occasional garlic smelling breath.
- dental caries arise when cariogenic microorganisms, such as Streptococcus mutans or Streptococcus sanguis, grow in oral cavities.
- Sucrose derived from foods, can be converted into water-soluble and insoluble polysaccharides by glucosyltransferase (GTF) produced by e.g the above mentioned cariogenic microorganisms.
- GTF glucosyltransferase
- These polysaccharides coat the surface of the cariogenic microorgan ⁇ isms and other bacteria, and finally adhere onto the tooth surface to form a dental plaque.
- Bacteria contained in the dental plaque can degrade the polysaccharides to acids, such as lactic acid, which can lyse the tooth enamel, and may result in dental holes.
- plaque As plaque continues to accumulate rock hard white or yellowish deposits may arise. These deposits are called calcified plaque, calculus or tartar, and are formed in the saliva from plaque and minerals, such as in particular calcium. Accumulation of tartar below the gum line may cause periodontal disease.
- oral care compositions and products comprising enzymes, includ ⁇ ing proteases, glucose oxidase, amylases, lipase, peroxidases, have been suggested.
- proteolytic enzymes are today successfully used in connection with cleaning of dentures, and have also been suggested for oral care products e.g. for removing food remnants containing proteins, and for dissolving the proteinaceous matrix of tartar, which is attached to the dentin and enamel.
- oral care products e.g. for removing food remnants containing proteins, and for dissolving the proteinaceous matrix of tartar, which is attached to the dentin and enamel.
- the use in oral care products has not been successful as said use is connected with serious disadvantages.
- First of all proteases have a damaging impact on the gums (and teeth) in the mouth when being exposed for a period of time.
- proteases are inhibited by compounds, such as H 2 0 2 , which can be used advantageously in oral care products.
- proteolytic enzymes for oral care compositions and products include carboxypeptidase, pepsin, trypsin, pancreatic chymotrypsin, cathepsin, papain, and certain fungal derived proteases (see e.g. DE paten- 734,765 and GB patent 1,033,229)
- GB patent 1,197,164 relates to preparations for dental care and/or mouth care comprising proteolytic enzymes obtained from Bacillus subtllis as therapeutics.
- the use of said proteolytic enzymes improves the storage stability.
- FR patent 1,448,385 concerns oral care products comprising proteolytic enzymes, such as animal derived carboxypeptidase, pepsin, trypsin, pancreatic chymotrypsin, cathepsin, and papain derived of vegetable origin, and other enzymes, such as Upases
- US patent 4,364,926 discloses a novel alkali ⁇ ne protease M 3 useful for preventing dental caries.
- the protease has a strong alkaline pH-optimum between 9 and 12.5, is not inhibited by a number of chemical compounds, and has glucosyl- transferase (GTF) inhibitory activity.
- GTF glucosyl- transferase
- US patent 4,986,981 (Glace et al.) concerns a pH neutral tooth ⁇ paste having low abrasion.
- the toothpaste comprises papain in an amount which is stated to effectively clean away plaque, mucin, and tartar.
- Oral care products comprising proteases may inflict bleedings in the mouth, and may damage or irritate the mouths mucus mem ⁇ brane. Even after rinsing the mouth with water, small amounts of protease constitutes such a risk.
- the object of the present invention is to overcome the above mentioned problem by providing oral care compositions and products, such as dentifrice compositions, comprising a protease as an active oral care component, which does not inflict any damages or irritations in the mouth cavity.
- the invention relates to an oral care composition
- a protease which protease is substan- tially inactive in the natural environment prevailing in the mouth.
- the invention relates to oral care products, such as toothpastes and mouth washes, comprising an oral care composi- tion of the invention, or an essentially purified protease substantially inactive in the natural environment prevailing in the mouth, when the oral care composition or product is absent.
- Another object of the invention is the use of proteases, substantially inactive in the natural environment prevailing in the mouth, in e.g. oral care compositions and products.
- a final object of the invention is to provide a method for using said oral care products.
- Figure 1 shows the pH activity profile for protease I and protease II described in WO 95/02044.
- Figure 2 shows the protein removing ability of protease I and protease II described in WO 95/02044 in comparison to said ability of Subtilisin Carlsberg, in a 3% H 2 0 2 solution, pH 3.5, 4 hours, 25°C.
- the present invention solves the above mention problem related to oral care compositions and products comprising proteases.
- the inventor has perceived that certain proteases advantage ⁇ ously can be used in oral care compositions and products.
- a suitable environment is defined and created in the mouth, to facilitate the oral cleaning and/or treating process.
- proteases As one of the problems connected to prior art use of proteases in oral care compositions and products relate to the action of proteases after use, proteases, which are substantially inac- tive in the natural environment prevailing in the mouth, can be useful.
- parameters responsible for the protease inactivation may be any physical parameter, such as pH, temperature, humidity, ionic strength, etc.
- pH Especially contemplated is however the pH, as it is a relatively easy parameter to control. Having in mind that the pH naturally prevailing in the mouth lies within the range from about 6 to 8, especially about pH 6.4 to 7.0 (pH of saliva), proteases being inactive within this pH-ranges do not inflict any damage or irritation in the mouth.
- the advantageous known oral care action of proteases can be activated by adjusting the pH in the mouth to a suitable pH, where the protease has a suitable activity.
- the pH in the mouth is to be decreased. This can be done by adding suitable pH-decreasing agents to the oral care composition or product. However, if the pH of the oral care composition or product in it self is acidic, the pH adjustment is not necess- ary.
- the pH must be increased (if necessary) , e.g. by adding pH-increasing agents.
- the first aspect of the invention is to provide an oral care composition comprising a protease, wherein the protease is substantially inactive in the natural environment prevailing in the mouth, when the oral care composition is absent.
- the protease is active during use of the oral care composition, e.g. in connection with brushing the teeth or washing the mouth. After use any remains of active protease in the mouth will be inactivated, as the environment in the mouth will return to the natural stage, i.e. an environment primarily created by the saliva. Said return may optionally be speeded up by rinsing the mouth with water.
- said protease is subsrantially inactive in the natural pH-environment prevailing in the mouth, when the oral care composition is absent.
- the pH of saliva ranges from about 6.4 to 7.0.
- the pH in the mouth may be transposed a little up or down e.g. in connection with eating and drinking. Therefore, proteases being substantially inactive in a close range of about pH 7 , better within the pH-range from about 6.4 to 7.0, and even better within the pH-range from about pH 6 to 8, can be used as the protease component in an oral care composition or product of the invention.
- Proteases having a suitable relative activity (more than 70%) outside said range are preferred.
- the temperature in the mouth is usually in the range of 20°C up to at most 37°C. Therefore, proteases having a high relative activity (more that 70%) within this temperature interval are preferred according to the invention. However, all proteases being active in the mouth within this temperature interval are contemplated according to the invention.
- substantially inactive define proteases having an activity so low that the proteases do not inflict any damages or irritations in the mouth, when the pH lies in a close range of 7, preferably within the pH-range of saliva (about pH 6.4 to 7.0), more preferably in the pH-range from about 6 to 8.
- the relative protease activity (determined from the protease activity described in the Methods and Materials section) must not exceed about 70%, and should preferably be below about 50%, more preferably below about 30%, especially below about 10%, even better below about 5%, such as below about 2%, determined on the basis of the proteolytic activity at the pH-optimum.
- the protease(s) used in a composition of the invention is(are) derived from microorgan ⁇ isms, such as bacteria or fungal microorganisms.
- proteases derived from a strain of Bacillus sp. in particular a strain of Bacillus subtilis, or fungal strains of a Rhizopu ⁇ sp. , in particular a strain of R . niveus , or a strain of a Schytalidium sp., or a strain of a Sulpholobus sp. , or a strain of a Ther oplas a sp., or a strain of a Aspergillus sp. , in particu ⁇ lar a strain of A . aculeatu ⁇ , or A . niger, or A . aw amori, or A .
- a strain of a Trichoderma sp. in particular a strain of T . harzianum , or T . reesie , or a strain of a Fusarium sp. , in particular a strain of F . oxysporum , or a strain of a Humicola sp..
- the protease may be either an acidic or alkaline protease, within the group of proteases (i.e. enzymes classified under the Enzyme Classification number E.C. 3.4 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)), which is substantially inactive in the natural environment prevailing in the mouth, when the oral care composition or product is absent.
- proteases i.e. enzymes classified under the Enzyme Classification number E.C. 3.4 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)
- IUBMB International Union of Biochemistry and Molecular Biology
- Said modified proteases can be selected by screening for protease variants having a suitable pH-profile. Said variants may be provided by site directed or cassette mutagenesis as described in e.g. EP 130.756 (Genencor) or EP 479.870 (Novo Nordisk A/S) ; or
- the protease has a protease activity pH-optimum at about pH 5.
- proteases are the acidic proteases, exemplified with protease I and protease II, respectively, described in WO 95/02044 (Novo Nordisk A/S) .
- acidic proteases are substantially inactive in the natural environment prevailing in the mouth, including the natural pH-environment prevailing in the mouth.
- Protease I and protease II are surprisingly better than expected, as the proteases are substantially inactive in the natural environment in the mouth.
- Protease I and protease II are substantially inactive within the pH-range from about pH 6 to 8, and have relative protease activities being less than 50% within the pH-range from about 6.4 to 7.0. Within a close range of about pH 7, the relative protease activities are insignifi ⁇ cant for protease I, and substantially reduced for protease II (see figure 1 and example 1) .
- Protease I and protease II have good protein removing activities in the presence of as high as up to 3% H 2 0 2 (see figure 2 and example 2) . This is advantageous as H 2 0 2 is used in oral care products, such as dentifrices, as whitening/bleaching agent.
- a protease is, in the context of the invention, regarded as having "a good protein removing activity" if it is capable of remove significantly more protein from a surface than Subtilisin Carlsberg (available from Novo Nordisk A/S as Alcalase®) at the same conditions in 3% H 2 0 2 .
- the amount of protease needed in an oral care composition or product of the invention depends on the particular compound employed, but ranges generally from 0.0001% to 20%, preferably from about 0.001% to about 10%, and most preferably from about 0.01% to about 5% by weight of the final product.
- the protease may be added as an essentially purified mono-component enzyme preparation, such as an enzyme preparation containing one of the proteases described in WO 95/02044 (Novo Nordisk A/S) , but may also be added as an enzyme preparation exhibiting at least one other activity.
- Contemplated activities include the activity of a reducing enzyme, such as peroxidase, and further utanase, dextranase, lipase, amylase, glucose oxidase activity, and anti-microbial polypeptides, enriched with a protease which is substantially inactive in the natural environment prevailing in the mouth of humans, and animals, such as dogs and cats.
- the oral care compo ⁇ sition comprises protease, dextranase and/or mutanase activity.
- An oral care composition according to the invention may advan ⁇ tageously be used for producing an oral care product having any suitable physical form (i.e. powder, paste, gel, liquid, oint ⁇ ment, tablet etc.).
- Said "oral care product” is defined as a product which effectively can be used for maintaining and/or improving oral hygiene in the mouth of humans and animals, and/or preventing or treating dental diseases.
- oral care products include toothpaste, dental cream, gel or tooth powder, odontic, mouth washes, pre- or post brushing rinse formulations, chewing gum, lozenges, and candy.
- Toothpastes and tooth gels typically include abrasive polishing materials, foaming agents, flavouring agents, humectants, binders, thickeners, sweetening agents, whitening/bleaching/stain removing agents, and water.
- Mouth washes including plaque removing liquids, typically com ⁇ prise a water/alcohol solution, flavour, humectant, sweetener, foaming agent, and colorant.
- said abrasive polishing material includes alumina and hydrates thereof, such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates, such as calcined aluminum silicate and aluminum silicate, calcium carbonate, zirconium silicate, and also powdered plastics, such as polyvinyl chloride, polyamides, polymethyl methacrylate, polystyrene, phenol-formaldehyde resins, melamine-formaldehyde resins, urea-formaldehyde resins, epoxy resins, powdered polyethylene, silica xerogels, hydrogels and aerogels and the like.
- alumina and hydrates thereof such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates, such as calcined aluminum silicate and aluminum silicate, calcium carbonate, zirconium silicate, and also powdered plastic
- abrasive agents are calcium pyrophosphate, water-insoluble alkali metaphosphates, dicalcium phosphate and/or its dihydrate, dicalcium orthophosphate, tricalciu phosphate, particulate hydroxyapatite and the like. It is also possible to employ mix ⁇ tures of these substances.
- the abrasive product may be present in from 0 to 70% by weight, preferably from 1% to 70%.
- the abrasive material content typically lies in the range of from 10% to 70% by weight of the final toothpaste product.
- Humectants are employed to prevent loss of water from e.g. toothpastes.
- Suitable humectants for use in oral care products according to the invention include the following compounds and mixtures thereof: glycerol, polyol, sorbitol, polyethylene glycols (PEG), propylene glycol, 1,3-propanediol, 1,4- butanediol, hydrogenated partially hydrolysed polysaccharides and the like.
- Humectants are in general present in from 0% to 80%, preferably 5 to 70% by weight in toothpaste.
- Silica, starch, tragacanth gum, xanthan gum, extracts of Irish moss, alginates, pectin, cellulose derivatives, such as hydroxyethyl cellulose, sodium carboxymethyl cellulose and hydroxypropyl cellulose, polyacrylic acid and its salts, polyvinylpyrrolidone, can be mentioned as examples of suitable thickeners and binders, which helps stabilizing the dentifrice product.
- Thickeners may be present in toothpaste creams and gels in an amount of from 0.1 to 20% by weight, and binders to the extent of from 0.01 to 10% by weight of the final product.
- foaming agent soap anionic, cationic, non-ionic, amphoteric and/or zwitterionic surfactants can be used. These may be pres ⁇ ent at levels of from 0% to 15%, preferably from 0.1 to 13%, more preferably from 0.25 to 10% by weight of the final product.
- Surfactants are only suitable to the extent that they do not exert an inactivation effect on the present protease.
- Surfactants include fatty alcohol sulphates, salts of sulphonated mono-glycerides or fatty acids having 10 to 20 car ⁇ bon atoms, fatty acid-albumen condensation products, salts of fatty acids amides and taurines and/or salts of fatty acid esters of isethionic acid.
- Suitable sweeteners include saccharin.
- Flavours such as spearmint
- Whitening/bleaching agents include H 2 0 2 and may be added in amounts less that 5%, preferably from 0.25 to 4%, calculated on the basis of the weight of the final product. Water is usually added in an amount giving e.g. toothpaste a flowable form.
- water-soluble anti-bacterial agents such as chlorhexidine digluconate, hexetidine, alexidine, quaternary ammonium anti-bacterial compounds and water-soluble sources of certain metal ions such as zinc, copper, silver and stannous (e.g. zinc, copper and stannous chloride, and silver nitrate) may also be included.
- certain metal ions such as zinc, copper, silver and stannous (e.g. zinc, copper and stannous chloride, and silver nitrate) may also be included.
- anti-calculus agents include compounds which can be used as fluoride source, dyes/colorants, preservatives, vitamins, pH-adjusting agents, anti-caries agents, desensitiz- ing agents etc.
- a toothpaste produced from an oral care composition of the invention may e.g. comprise the following ingredients:
- Another object of the invention relates to the use of proteases, which are substantially inactive in the natural environment prevailing in the mouth, in e.g. oral care composi- tions and products.
- the proteases are substantially inactive in the natural pH-environment prevailing in the mouth, within a close range of about pH 7, preferably within the pH- range from about 6.4 to 7.0, and even better within the pH- range from about 6 to 8.
- the invention relates to a method for using an oral care product of the invention, wherein a) the oral care product is placed in the mouth, b) contacted with the teeth and/or gums for a period of time, c) removed from the mouth, and d) optionally rinsed with a liquid.
- a tooth brush or the like may advantageously be used for contacting the oral care product with the teeth and/or gums.
- the contact may take place by rinsing the mouth.
- step b) The time period of contact in step b) is optional. However, contacting the oral care product with the teeth and/or gums for between about 1 to 5 minutes will normally be sufficient for obtaining the desired result.
- the oral care product may be removed from the mouth in any suitable way, e.g. by spitting it out.
- the mouth may be rinsed with a liquid, such as tap water. This will secure that the environment, e.g. the pH-environment, in the mouth returns to the natural stage within a short period of time.
- Protease I is described in WO 95/02044
- Protease II is described in WO 95/02044
- Subtilisin Carlsberg is available from Novo Nordisk A/S as Alcalase®.
- 1 haemoglobin protease unit is defined as the amount of enzyme liberating 1 millimole of primary amino groups (deter ⁇ mined by comparison with a serine standard) per minute under standard conditions as described below:
- haemoglobin (bovine, supplied by Sigma) is prepared with the Universal Buffer described by Britton and Robinson, J. Chem. Soc.. 1931, p. 1451), adjusted to a pH of 5.5. 2 ml of the substrate solution are pre-incubated in a water bath for 10 min. at 25°C. 1 ml of an enzyme solution containing b g/ml of the enzyme preparation, corresponding to about 0.2-0.3 hpu/ml of the Universal Buffer (pH 5.5) is added. After 30 min.
- reaction is terminated by the addition of a quenching agent (5 ml of a solution containing 17.9 g of trichloroacetic acid, 29.9 g of sodium acetate and 19.8 g of acetic acid made up to 500 ml with deionized water) .
- a quenching agent (5 ml of a solution containing 17.9 g of trichloroacetic acid, 29.9 g of sodium acetate and 19.8 g of acetic acid made up to 500 ml with deionized water) .
- a blank is prepared in the same way as the test solution with the exception that the quenching agent is added prior to the enzyme solution.
- the reaction mixtures are kept for 20 min. in a water bath after which they are filtered through Whatman 42 paper filters.
- OPA o-phthaldialdehyde
- the OPA test is also performed with a serine standard containing 10 mg of serine in 100 ml of Universal Buffer (pH 5.5) .
- the buffer alone is used as a blank.
- the protease activity is calculated from the OD measurements by means of the following formula:
- hpu/g of enzyme preparation hpu/ml: b
- 0D t , 0D b , OD and 0D B is the optical density of the test solution, blank, serine standard and buffer, respectively
- Cser is the concentration of serine (mg/ml) in the standard ( v in this case 0.1 mg/ml), and M is the molecular weight of serine (105.09).
- Q is the dilution factor for the enzyme solution (in this case 8) and t, is the incubation time in minutes (in this case 30 minutes) .
- proteases I and protease II described in WO 95/02044 were tested.
- the ability of proteases I and protease II described in WO 95/02044 was compared with Subtilisin Carlsberg.
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Abstract
The present invention relates to oral care compositions and products, such as dentifrices. According to the invention oral care compositions and products may advantageously comprise a protease, which is substantially inactive in the natural environment prevailing in the mouth, when the oral care composition or product is absent. Further the invention relates to the use of proteases, substantially inactive in the natural environment prevailing in the mouth, in e.g. oral care compositions or products. A final object of the invention is to provide a method for using said oral care products.
Description
Title: Oral care compositions
FIELD OF THE INVENTION
The present invention relates to oral care compositions and products comprising certain proteases.
Also contemplated is the use of said proteases for e . g . oral care purposes, and a method for using oral care products of the invention.
BACKGROUND OF THE INVENTION
One of the major events for newly hatched parents is the appearance of the first tooth in the mouth of their baby. From that day on the caring parents include dental care as a natural part of taking care of their baby.
To be able to sufficiently guarantee the capability of chewing, e.g. foods, during a whole lifetime it is necessary to keep the teeth in a good condition and to obtain a good oral hygiene.
To obtain and maintain a good dental and oral hygiene the teeth must at least be brushed once every day using toothpaste or the like. Further the mouth should regularly be rinsed with a mouth wash.
The brushing of the teeth primarily helps removing food particles from the teeth, and constitutes an important first step in preventing dental caries, which may cause dental holes, and further in preventing dental diseases.
A good dental hygiene should also be obtained to prevent formation of plaque and tartar, outbreak of oral diseases, and adherence of stains from tea, coffee and tobacco smoking, and
the like, which will remove the basis for obtaining a good appearance when e.g. smiling. Furthermore, a number of unpleasant incidences can be avoided by using oral care products for obtaining and/or maintaining e.g. a fresh breath, as most people dislike a in general bad breath, or e.g. an occasional garlic smelling breath.
Dental caries, plaque and tartar
In general it is believed that dental caries arise when cariogenic microorganisms, such as Streptococcus mutans or Streptococcus sanguis, grow in oral cavities. Sucrose, derived from foods, can be converted into water-soluble and insoluble polysaccharides by glucosyltransferase (GTF) produced by e.g the above mentioned cariogenic microorganisms. These polysaccharides coat the surface of the cariogenic microorgan¬ isms and other bacteria, and finally adhere onto the tooth surface to form a dental plaque. Bacteria contained in the dental plaque can degrade the polysaccharides to acids, such as lactic acid, which can lyse the tooth enamel, and may result in dental holes. As plaque continues to accumulate rock hard white or yellowish deposits may arise. These deposits are called calcified plaque, calculus or tartar, and are formed in the saliva from plaque and minerals, such as in particular calcium. Accumulation of tartar below the gum line may cause periodontal disease.
To prevent the formation of dental caries, plaque, and tartar, oral care compositions and products comprising enzymes, includ¬ ing proteases, glucose oxidase, amylases, lipase, peroxidases, have been suggested.
The use of proteolytic enzymes are today successfully used in connection with cleaning of dentures, and have also been suggested for oral care products e.g. for removing food remnants containing proteins, and for dissolving the proteinaceous matrix of tartar, which is attached to the dentin
and enamel. However, the use in oral care products has not been successful as said use is connected with serious disadvantages. First of all proteases have a damaging impact on the gums (and teeth) in the mouth when being exposed for a period of time.
Further, most proteases are inhibited by compounds, such as H202, which can be used advantageously in oral care products.
Examples of suggested proteolytic enzymes for oral care compositions and products include carboxypeptidase, pepsin, trypsin, pancreatic chymotrypsin, cathepsin, papain, and certain fungal derived proteases (see e.g. DE paten- 734,765 and GB patent 1,033,229)
GB patent 1,197,164 relates to preparations for dental care and/or mouth care comprising proteolytic enzymes obtained from Bacillus subtllis as therapeutics. The use of said proteolytic enzymes improves the storage stability.
US patent 3,194,738 and FR patent 1,377,415 suggest the use of enzymes obtained from Aspergillus oryzae having proteolytic and amylolytic activity for toothpaste, chewing gum and mouth washes.
FR patent 1,448,385 concerns oral care products comprising proteolytic enzymes, such as animal derived carboxypeptidase, pepsin, trypsin, pancreatic chymotrypsin, cathepsin, and papain derived of vegetable origin, and other enzymes, such as Upases
US patent 4,364,926 (Yokogawa et al.) discloses a novel alkali¬ ne protease M3 useful for preventing dental caries. The protease has a strong alkaline pH-optimum between 9 and 12.5, is not inhibited by a number of chemical compounds, and has glucosyl- transferase (GTF) inhibitory activity.
US patent 4,986,981 (Glace et al.) concerns a pH neutral tooth¬ paste having low abrasion. The toothpaste comprises papain in
an amount which is stated to effectively clean away plaque, mucin, and tartar.
Discussion of prior art Oral care products comprising proteases may inflict bleedings in the mouth, and may damage or irritate the mouths mucus mem¬ brane. Even after rinsing the mouth with water, small amounts of protease constitutes such a risk.
Therefore, it is desirable to be capable of providing oral care compositions and products comprising proteases, which do not inflict any risk of causing damages in the mouth.
SUMMARY OF THE INVENTION
The object of the present invention is to overcome the above mentioned problem by providing oral care compositions and products, such as dentifrice compositions, comprising a protease as an active oral care component, which does not inflict any damages or irritations in the mouth cavity.
In the first aspect the invention relates to an oral care composition comprising a protease, which protease is substan- tially inactive in the natural environment prevailing in the mouth.
Further the invention relates to oral care products, such as toothpastes and mouth washes, comprising an oral care composi- tion of the invention, or an essentially purified protease substantially inactive in the natural environment prevailing in the mouth, when the oral care composition or product is absent.
Another object of the invention is the use of proteases, substantially inactive in the natural environment prevailing in the mouth, in e.g. oral care compositions and products.
A final object of the invention is to provide a method for using said oral care products.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows the pH activity profile for protease I and protease II described in WO 95/02044.
Figure 2 shows the protein removing ability of protease I and protease II described in WO 95/02044 in comparison to said ability of Subtilisin Carlsberg, in a 3% H202 solution, pH 3.5, 4 hours, 25°C.
DETAILED DESCRIPTION OF THE INVENTION
The present invention solves the above mention problem related to oral care compositions and products comprising proteases.
The inventor has perceived that certain proteases advantage¬ ously can be used in oral care compositions and products. During use of oral care compositions and products a suitable environment is defined and created in the mouth, to facilitate the oral cleaning and/or treating process.
As one of the problems connected to prior art use of proteases in oral care compositions and products relate to the action of proteases after use, proteases, which are substantially inac- tive in the natural environment prevailing in the mouth, can be useful.
According to the invention parameters responsible for the protease inactivation may be any physical parameter, such as pH, temperature, humidity, ionic strength, etc. Especially contemplated is however the pH, as it is a relatively easy parameter to control.
Having in mind that the pH naturally prevailing in the mouth lies within the range from about 6 to 8, especially about pH 6.4 to 7.0 (pH of saliva), proteases being inactive within this pH-ranges do not inflict any damage or irritation in the mouth.
The advantageous known oral care action of proteases can be activated by adjusting the pH in the mouth to a suitable pH, where the protease has a suitable activity.
Therefore, if the protease is active in acidic environment the pH in the mouth is to be decreased. This can be done by adding suitable pH-decreasing agents to the oral care composition or product. However, if the pH of the oral care composition or product in it self is acidic, the pH adjustment is not necess- ary.
If the protease is active in alkaline environment the pH must be increased (if necessary) , e.g. by adding pH-increasing agents.
When rinsing the mouth with water, after using a protease- containing oral care product, most of the protease will be removed from the mouth. However, even small amounts of remain¬ ing active protease may inflict damages in the mouth. By using an oral care product comprising a composition of the invention such damages can be avoided, as said composition comprises protease(s) which is(are) only active at the pH created by the oral care composition or product, and substantially inactive in the natural environment prevailing in the mouth. After rinsing the mouth with water the pH will, after a short period of time, return to a pH between about 6.4 to 7.0 (pH of salvia) , which will inactivate the protease. Consequently, any amount of remaining protease will do no harm in the mouth.
Consequently the first aspect of the invention is to provide an oral care composition comprising a protease, wherein the protease is substantially inactive in the natural environment
prevailing in the mouth, when the oral care composition is absent.
It is to be understood that according to the invention the protease is active during use of the oral care composition, e.g. in connection with brushing the teeth or washing the mouth. After use any remains of active protease in the mouth will be inactivated, as the environment in the mouth will return to the natural stage, i.e. an environment primarily created by the saliva. Said return may optionally be speeded up by rinsing the mouth with water.
In a preferred embodiment said protease is subsrantially inactive in the natural pH-environment prevailing in the mouth, when the oral care composition is absent.
As mentioned above the pH of saliva ranges from about 6.4 to 7.0. However, the pH in the mouth may be transposed a little up or down e.g. in connection with eating and drinking. Therefore, proteases being substantially inactive in a close range of about pH 7 , better within the pH-range from about 6.4 to 7.0, and even better within the pH-range from about pH 6 to 8, can be used as the protease component in an oral care composition or product of the invention. Proteases having a suitable relative activity (more than 70%) outside said range are preferred.
The temperature in the mouth is usually in the range of 20°C up to at most 37°C. Therefore, proteases having a high relative activity (more that 70%) within this temperature interval are preferred according to the invention. However, all proteases being active in the mouth within this temperature interval are contemplated according to the invention.
The above used terms "substantially inactive" define proteases having an activity so low that the proteases do not inflict any damages or irritations in the mouth, when the pH lies in a
close range of 7, preferably within the pH-range of saliva (about pH 6.4 to 7.0), more preferably in the pH-range from about 6 to 8.
More specifically this means that, within the above mentioned pH-ranges, the relative protease activity (determined from the protease activity described in the Methods and Materials section) must not exceed about 70%, and should preferably be below about 50%, more preferably below about 30%, especially below about 10%, even better below about 5%, such as below about 2%, determined on the basis of the proteolytic activity at the pH-optimum.
In an embodiment of the invention the protease(s) used in a composition of the invention is(are) derived from microorgan¬ isms, such as bacteria or fungal microorganisms.
Especially contemplated according to the invention is proteases derived from a strain of Bacillus sp. , in particular a strain of Bacillus subtilis, or fungal strains of a Rhizopuε sp. , in particular a strain of R . niveus , or a strain of a Schytalidium sp., or a strain of a Sulpholobus sp. , or a strain of a Ther oplas a sp., or a strain of a Aspergillus sp. , in particu¬ lar a strain of A . aculeatuε, or A . niger, or A . aw amori, or A . oryzae, or a strain of a Trichoderma sp. , in particular a strain of T . harzianum , or T . reesie , or a strain of a Fusarium sp. , in particular a strain of F . oxysporum , or a strain of a Humicola sp..
According to the invention the protease may be either an acidic or alkaline protease, within the group of proteases (i.e. enzymes classified under the Enzyme Classification number E.C. 3.4 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)), which is substantially inactive in the natural environment prevailing in the mouth, when the oral care composition or product is absent.
Furthermore, also genetically modified proteases designed for the purpose of the invention are contemplated. Said modified proteases can be selected by screening for protease variants having a suitable pH-profile. Said variants may be provided by site directed or cassette mutagenesis as described in e.g. EP 130.756 (Genencor) or EP 479.870 (Novo Nordisk A/S) ; or by random mutagenesis by using methods well established in the art.
In a specific embodiment of the invention the protease has a protease activity pH-optimum at about pH 5.
Specific examples of such proteases are the acidic proteases, exemplified with protease I and protease II, respectively, described in WO 95/02044 (Novo Nordisk A/S) .
These acidic proteases are substantially inactive in the natural environment prevailing in the mouth, including the natural pH-environment prevailing in the mouth.
Protease I and protease II are surprisingly better than expected, as the proteases are substantially inactive in the natural environment in the mouth. Protease I and protease II are substantially inactive within the pH-range from about pH 6 to 8, and have relative protease activities being less than 50% within the pH-range from about 6.4 to 7.0. Within a close range of about pH 7, the relative protease activities are insignifi¬ cant for protease I, and substantially reduced for protease II (see figure 1 and example 1) . Further, Protease I and protease II have good protein removing activities in the presence of as high as up to 3% H202 (see figure 2 and example 2) . This is advantageous as H202 is used in oral care products, such as dentifrices, as whitening/bleaching agent.
A protease is, in the context of the invention, regarded as having "a good protein removing activity" if it is capable of remove significantly more protein from a surface than
Subtilisin Carlsberg (available from Novo Nordisk A/S as Alcalase®) at the same conditions in 3% H202.
The amount of protease needed in an oral care composition or product of the invention depends on the particular compound employed, but ranges generally from 0.0001% to 20%, preferably from about 0.001% to about 10%, and most preferably from about 0.01% to about 5% by weight of the final product.
In the preparation of an oral care composition or product, the protease may be added as an essentially purified mono-component enzyme preparation, such as an enzyme preparation containing one of the proteases described in WO 95/02044 (Novo Nordisk A/S) , but may also be added as an enzyme preparation exhibiting at least one other activity. Contemplated activities include the activity of a reducing enzyme, such as peroxidase, and further utanase, dextranase, lipase, amylase, glucose oxidase activity, and anti-microbial polypeptides, enriched with a protease which is substantially inactive in the natural environment prevailing in the mouth of humans, and animals, such as dogs and cats.
In a preferred embodiment of the invention the oral care compo¬ sition comprises protease, dextranase and/or mutanase activity.
An oral care composition according to the invention may advan¬ tageously be used for producing an oral care product having any suitable physical form (i.e. powder, paste, gel, liquid, oint¬ ment, tablet etc.). Said "oral care product" is defined as a product which effectively can be used for maintaining and/or improving oral hygiene in the mouth of humans and animals, and/or preventing or treating dental diseases.
Examples of such oral care products include toothpaste, dental cream, gel or tooth powder, odontic, mouth washes, pre- or post brushing rinse formulations, chewing gum, lozenges, and candy.
O 96/29978
11
Toothpastes and tooth gels typically include abrasive polishing materials, foaming agents, flavouring agents, humectants, binders, thickeners, sweetening agents, whitening/bleaching/stain removing agents, and water.
Mouth washes, including plaque removing liquids, typically com¬ prise a water/alcohol solution, flavour, humectant, sweetener, foaming agent, and colorant.
According to the invention said abrasive polishing material includes alumina and hydrates thereof, such as alpha alumina trihydrate, magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates, such as calcined aluminum silicate and aluminum silicate, calcium carbonate, zirconium silicate, and also powdered plastics, such as polyvinyl chloride, polyamides, polymethyl methacrylate, polystyrene, phenol-formaldehyde resins, melamine-formaldehyde resins, urea-formaldehyde resins, epoxy resins, powdered polyethylene, silica xerogels, hydrogels and aerogels and the like. Also suitable as abrasive agents are calcium pyrophosphate, water-insoluble alkali metaphosphates, dicalcium phosphate and/or its dihydrate, dicalcium orthophosphate, tricalciu phosphate, particulate hydroxyapatite and the like. It is also possible to employ mix¬ tures of these substances.
Dependent on the oral care product the abrasive product may be present in from 0 to 70% by weight, preferably from 1% to 70%. For toothpastes the abrasive material content typically lies in the range of from 10% to 70% by weight of the final toothpaste product.
Humectants are employed to prevent loss of water from e.g. toothpastes. Suitable humectants for use in oral care products according to the invention include the following compounds and mixtures thereof: glycerol, polyol, sorbitol, polyethylene glycols (PEG), propylene glycol, 1,3-propanediol, 1,4- butanediol, hydrogenated partially hydrolysed polysaccharides
and the like. Humectants are in general present in from 0% to 80%, preferably 5 to 70% by weight in toothpaste.
Silica, starch, tragacanth gum, xanthan gum, extracts of Irish moss, alginates, pectin, cellulose derivatives, such as hydroxyethyl cellulose, sodium carboxymethyl cellulose and hydroxypropyl cellulose, polyacrylic acid and its salts, polyvinylpyrrolidone, can be mentioned as examples of suitable thickeners and binders, which helps stabilizing the dentifrice product. Thickeners may be present in toothpaste creams and gels in an amount of from 0.1 to 20% by weight, and binders to the extent of from 0.01 to 10% by weight of the final product.
As foaming agent soap, anionic, cationic, non-ionic, amphoteric and/or zwitterionic surfactants can be used. These may be pres¬ ent at levels of from 0% to 15%, preferably from 0.1 to 13%, more preferably from 0.25 to 10% by weight of the final product.
Surfactants are only suitable to the extent that they do not exert an inactivation effect on the present protease. Surfactants include fatty alcohol sulphates, salts of sulphonated mono-glycerides or fatty acids having 10 to 20 car¬ bon atoms, fatty acid-albumen condensation products, salts of fatty acids amides and taurines and/or salts of fatty acid esters of isethionic acid.
Suitable sweeteners include saccharin.
Flavours, such as spearmint, are usually present in low amounts, such as from 0.01% to about 5% by weight, especially from 0.1% to 5%.
Whitening/bleaching agents include H202 and may be added in amounts less that 5%, preferably from 0.25 to 4%, calculated on the basis of the weight of the final product.
Water is usually added in an amount giving e.g. toothpaste a flowable form.
Further water-soluble anti-bacterial agents, such as chlorhexidine digluconate, hexetidine, alexidine, quaternary ammonium anti-bacterial compounds and water-soluble sources of certain metal ions such as zinc, copper, silver and stannous (e.g. zinc, copper and stannous chloride, and silver nitrate) may also be included.
Also contemplated according to the invention is the addition of anti-calculus agents, anti-plaque agents, compounds which can be used as fluoride source, dyes/colorants, preservatives, vitamins, pH-adjusting agents, anti-caries agents, desensitiz- ing agents etc.
A toothpaste produced from an oral care composition of the invention (in weight % of the final toothpaste composition) may e.g. comprise the following ingredients:
Abrasive material 10 to 70%
Humectant 0 to 80%
Thickener 0.1 to 20%
Binder 0.01 to 10% Sweetener 0.1% to 5%
Foaming agent 0 to 15%
Whitener 0 to 5%
Protease 0.0001% to 20%
(according to the invention) Other enzymes 0 to 20%
Another object of the invention relates to the use of proteases, which are substantially inactive in the natural environment prevailing in the mouth, in e.g. oral care composi- tions and products.
In a preferred embodiment the proteases are substantially inactive in the natural pH-environment prevailing in the mouth, within a close range of about pH 7, preferably within the pH- range from about 6.4 to 7.0, and even better within the pH- range from about 6 to 8.
Finally the invention relates to a method for using an oral care product of the invention, wherein a) the oral care product is placed in the mouth, b) contacted with the teeth and/or gums for a period of time, c) removed from the mouth, and d) optionally rinsed with a liquid.
If the oral care product to be used is in solid to flowable form a tooth brush or the like may advantageously be used for contacting the oral care product with the teeth and/or gums. In the case of a liquid oral care product the contact may take place by rinsing the mouth.
The time period of contact in step b) is optional. However, contacting the oral care product with the teeth and/or gums for between about 1 to 5 minutes will normally be sufficient for obtaining the desired result.
After use, the oral care product may be removed from the mouth in any suitable way, e.g. by spitting it out. Optionally the mouth may be rinsed with a liquid, such as tap water. This will secure that the environment, e.g. the pH-environment, in the mouth returns to the natural stage within a short period of time.
MATERIALS AND METHODS
Materials
Protease I is described in WO 95/02044 Protease II is described in WO 95/02044
Subtilisin Carlsberg is available from Novo Nordisk A/S as Alcalase®.
Methods Proteolvtic activity
1 haemoglobin protease unit (hpu) is defined as the amount of enzyme liberating 1 millimole of primary amino groups (deter¬ mined by comparison with a serine standard) per minute under standard conditions as described below:
A 2% (w/v) solution of haemoglobin (bovine, supplied by Sigma) is prepared with the Universal Buffer described by Britton and Robinson, J. Chem. Soc.. 1931, p. 1451), adjusted to a pH of 5.5. 2 ml of the substrate solution are pre-incubated in a water bath for 10 min. at 25°C. 1 ml of an enzyme solution containing b g/ml of the enzyme preparation, corresponding to about 0.2-0.3 hpu/ml of the Universal Buffer (pH 5.5) is added. After 30 min. of incubation at 25°C, the reaction is terminated by the addition of a quenching agent (5 ml of a solution containing 17.9 g of trichloroacetic acid, 29.9 g of sodium acetate and 19.8 g of acetic acid made up to 500 ml with deionized water) . A blank is prepared in the same way as the test solution with the exception that the quenching agent is added prior to the enzyme solution. The reaction mixtures are kept for 20 min. in a water bath after which they are filtered through Whatman 42 paper filters.
Primary amino groups are determined by their colour development with o-phthaldialdehyde (OPA) , as follows: 7.62 g of disodium tetraborate decahydrate and 2.0 g of sodium dodecylsulfate are dissolved in 150 ml of water. 160 mg of OPA dissolved in 4 ml of methanol were then added together with 400 μl of β- mercaptoethanol after which the solution is made up to 200 ml with water. To 3 ml of the OPA reagent are added 400 μl of the filtrates obtained above, with mixing. The optical density (OD) at 340 nm is measured after about 5 min. The OPA test is also performed with a serine standard containing 10 mg of serine in
100 ml of Universal Buffer (pH 5.5) . The buffer alone is used as a blank. The protease activity is calculated from the OD measurements by means of the following formula:
(ODt - ODb) x Cser x Q hpu/ l enzyme solution:
(ODser - 0DB) x Wser x t,
hpu/g of enzyme preparation = hpu/ml: b
wherein 0Dt, 0Db, OD and 0DB is the optical density of the test solution, blank, serine standard and buffer, respectively,
Cser is the concentration of serine (mg/ml) in the standard (vin this case 0.1 mg/ml), and M is the molecular weight of serine (105.09). Q is the dilution factor for the enzyme solution (in this case 8) and t, is the incubation time in minutes (in this case 30 minutes) .
EXAMPLES
Example l
Activity profile To illustrate the existence of suitable proteases for oral care compositions and products of the invention the pH activity profiles in relative percents of protease I and protease II described in WO 95/02044 were determined.
As can be seen from figure 1 the relative protease activity has disappeared at pH 7 for protease I, and has been substantially reduced for protease II.
Example 2
Protease activity in 3% H7Q?
To illustrate the useful effect of suitable proteases in oral care products, the ability to remove protein from a surface in a 3% H202 solution, 25°C, 4 hours, pH 3.5, was tested. The ability of proteases I and protease II described in WO 95/02044 was compared with Subtilisin Carlsberg.
As can be seen from figure 2 both protease I and protease II are far more effective for removing protein than Subtilisin Carlsberg. This makes both proteases highly useful for oral care compositions and products containing H202.
As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without depart¬ ing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.
Claims
1. An oral care composition comprising a protease, character¬ ized in that the protease is substantially inactive in the
5 natural environment prevailing in the mouth, when the oral care composition is absent.
2. The oral care composition according to claim 1, wherein the protease is substantially inactive in the natural pH-
10 environment prevailing in the mouth, when the oral care composition is absent.
3. The oral care composition according to any of claims 1 and 2, wherein the protease is substantially inactive in the
15 close range of about 7, preferably with the range of about pH 6.4 to 7.0, even more preferred between about pH 6 to 8.
4. The oral care composition according to claims 3, wherein the protease is active at temperatures between 20°C and 37°C.
20
5. The oral care composition according to claims 3 and 4, wherein the proteolytic activity is below 70%, preferably below 50%, more preferably below 30%, better below 10%, even better below 5%, such as below 2%, in comparison to the proteolytic
25 activity at the pH-optimum.
6. The oral care composition according to any of claims l to 5, wherein the protease is derived from a microorganism.
307. The oral care composition according to claim 6, wherein the protease is derived from a strain of Bacillus sp. , in particu¬ lar a strain of Bacillus subtilis, or fungal strains of a iRhizopus sp. , in particular a strain of R . niveus, or a strain of a Schytalidium sp. , or a strain of a Sulpholobus sp. , or a
35 strain of a Ther oplasma sp. , or a strain of a Aspergillus sp. , in particular a strain of A . aculeatus, or A . niger, or A. awamori , or A . oryzae , or a strain of a Trichoderma sp. , in particular a strain of T. harzianum , or T. reesie , or a strain of a Fusariu sp. , in particular a strain of F . oxysporum, or a strain of a Humicola sp..
58. The oral care composition according to any of claims 1 to 7, wherein the protease is an acidic protease.
9. The oral care composition according to claim 8, wherein the acidic protease is a genetically modified protease.
10
10. The oral care composition according to any of claims 1 to 9, wherein the protease has a good protein removing activity in the presence of H202.
15 11. The oral care composition according to any of claims l to 8, and 10 wherein the protease is protease I described in WO 95/02044.
12. The oral care composition according to any of claims 1 to 208, and 10, wherein the protease is protease II described in WO
95/02044.
13. The oral care composition according to any of claims 1 to 7, wherein the protease is an alkaline protease.
25
14. The oral care composition according to claim 13, wherein the alkaline protease is a genetically modified protease.
15. The oral care composition according to claims 13 and 14, 30 wherein the protease has a good protein removing activity in the presents of H202.
16. The oral care composition according to any of claims 1 to 15, comprising at least one other activity selected from the
35 group including a reducing enzyme, such as peroxidase, and amylase, dextranase, mutanase, lipase, glucose oxidase, and anti-microbial polypeptides, or a mixture thereof.
17. The oral care composition according to claim 16, comprising dextranase or mutanase, or a mixture thereof.
18. An oral care product, comprising an oral care composition 5 according to any of claims 1 to 17, or a protease being substantially inactive in natural environment prevailing in the mouth, when the oral care product is absent.
19. An oral care product according to claim 18, wherein said 10 protease is substantially inactive the natural pH-environment prevailing in the mouth, when the oral care product is absent.
20. An oral care product according to claims 18 and 19, being a dentifrice.
15
21. The oral care product according to claim 20, wherein the dentifrice is a toothpaste or tooth powder.
22. The oral care product according to claim 20, wherein the 20 dentifrice is a mouth wash.
23. The oral care product according to any of claims 18 to 22, comprising an anti-plaque compound or composition.
2524. The oral care product according to any of claims 18 to 23, comprising an anti-tartar compound or composition.
25. The oral care product according to any of claims 18 to 24, comprising a bleaching or whitening agent.
30
26. The oral care product according to claim 25, comprising H202.
27. The oral care product according to any of claims 18 to 26, 35 comprising at least one other activity selected from the group including a reducing enzyme, such as peroxidase, amylase, dextranase, mutanase, lipase, glucose oxidase or anti-microbial activity, or a mixture thereof.
28. The oral care product according to claim 27, comprising 5 dextranase or mutanase, or a mixture thereof.
29. Use of a protease, which is substantially inactive in the natural environment prevailing in the mouth.
10 30. The use according to claim 29, wherein the protease is substantially inactive in the natural pH-environment prevailing in the mouth.
31. The use according to claim 29 and 30, in oral care composi- 15 tions and/or products.
32. A method for using oral care products according to any of claims 18 to 28, wherein a) the oral care product is placed in the mouth,
20 b) contacted to the teeth and/or gums for a period of time, c) removed from the mouth, and d) optionally rinsed with a liquid.
29. The method according to claim 29, wherein a tooth brush is 5 used for contacting the oral care product with the teeth and/or gums.
30. The method according to claims 29 and 30, wherein the oral care product is contacted with the teeth and/or gums by
30 rinsing.
31. The method according to any of claims 29 to 31, wherein the rinsing liquid in step d) is water.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK32495 | 1995-03-28 | ||
DK32495 | 1995-03-28 | ||
PCT/DK1996/000126 WO1996029978A1 (en) | 1995-03-28 | 1996-03-28 | Oral care compositions |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0817606A1 true EP0817606A1 (en) | 1998-01-14 |
Family
ID=8092267
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP96908026A Withdrawn EP0817606A1 (en) | 1995-03-28 | 1996-03-28 | Oral care compositions |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP0817606A1 (en) |
JP (1) | JPH11502527A (en) |
KR (1) | KR19980703322A (en) |
CN (1) | CN1182359A (en) |
AU (1) | AU5142896A (en) |
BR (1) | BR9607778A (en) |
CA (1) | CA2216928A1 (en) |
WO (1) | WO1996029978A1 (en) |
Families Citing this family (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6376449B2 (en) | 1993-03-27 | 2002-04-23 | Novozymes A/S | Acidic cleaning composition comprising an acidic protease I |
IL109705A (en) * | 1993-06-17 | 1998-07-15 | Allergan Inc | Enzyme compositions and methods for contact lens cleaning |
KR100478211B1 (en) * | 1997-10-01 | 2005-08-11 | 애경산업(주) | Whitening Toothpaste Composition |
US6521215B2 (en) * | 1999-05-28 | 2003-02-18 | Devin Okay | Compositions and methods for tooth treatment |
US8128911B2 (en) | 2002-05-10 | 2012-03-06 | Colgate-Palmolive Company | Antibacterial dentifrice exhibiting enhanced antiplaque and breath freshening properties |
DE10237317B4 (en) * | 2002-08-15 | 2010-04-08 | 3M Espe Ag | Enzyme-containing composition, process for their preparation and their use |
US7931892B2 (en) * | 2003-08-11 | 2011-04-26 | Oragenics, Inc. | Compositions and methods for the maintenance of oral health |
EP1600141B1 (en) | 2004-05-24 | 2013-04-17 | 3M Deutschland GmbH | Collagenolytic active enzyme containing compositions for the treatment of dental caries |
EP1787627A1 (en) | 2005-11-17 | 2007-05-23 | 3M Innovative Properties Company | Anti-microbial dental impression material |
BRPI0906461A2 (en) * | 2008-02-08 | 2015-07-14 | Colgate Palmolive Co | Oral Care Composition and Method for Applying an Oral Care Composition to the Oral Cavity |
TWI455728B (en) * | 2010-01-14 | 2014-10-11 | Colgate Palmolive Co | Dentifrice compositions |
EP2745836B1 (en) * | 2012-12-18 | 2016-08-31 | Sunstar Suisse SA | Topical oral composition for alleviating dry mouth symptoms and for treating mouth ulcers |
CN105330208A (en) * | 2015-10-28 | 2016-02-17 | 九江学院 | Unburned material gradient type curing method |
CN107184414A (en) * | 2017-05-11 | 2017-09-22 | 江苏苏沃尚新材料科技有限公司 | A kind of artificial tooth cleaning agent |
CN109601725B (en) * | 2019-01-31 | 2022-09-06 | 重庆博力生物科技有限公司 | Edible composition |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS56106593A (en) * | 1980-01-24 | 1981-08-24 | Dainippon Pharmaceut Co Ltd | Alkali protease m3 |
ZA875318B (en) * | 1986-08-28 | 1988-01-25 | Warner-Lambert Company | Enzyme containing denture cleansers and method of use |
DK81193D0 (en) * | 1993-07-06 | 1993-07-06 | Novo Nordisk As | ENZYME |
-
1996
- 1996-03-28 CA CA002216928A patent/CA2216928A1/en not_active Abandoned
- 1996-03-28 JP JP8528811A patent/JPH11502527A/en active Pending
- 1996-03-28 WO PCT/DK1996/000126 patent/WO1996029978A1/en not_active Application Discontinuation
- 1996-03-28 BR BR9607778A patent/BR9607778A/en not_active Application Discontinuation
- 1996-03-28 CN CN96193501A patent/CN1182359A/en active Pending
- 1996-03-28 EP EP96908026A patent/EP0817606A1/en not_active Withdrawn
- 1996-03-28 AU AU51428/96A patent/AU5142896A/en not_active Abandoned
- 1996-03-28 KR KR1019970706726A patent/KR19980703322A/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO9629978A1 * |
Also Published As
Publication number | Publication date |
---|---|
MX9707377A (en) | 1997-11-29 |
AU5142896A (en) | 1996-10-16 |
BR9607778A (en) | 1998-07-07 |
CN1182359A (en) | 1998-05-20 |
WO1996029978A1 (en) | 1996-10-03 |
JPH11502527A (en) | 1999-03-02 |
KR19980703322A (en) | 1998-10-15 |
CA2216928A1 (en) | 1996-10-03 |
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