U.S. patent application number 11/000028 was filed with the patent office on 2005-09-22 for use of osteopontin in dental formulations.
Invention is credited to Bertelsen, Hans, Burling, Hans, Graverholt, Gitte, Jorgensen, Anders Steen, Sorensen, Esben Skipper.
Application Number | 20050207996 11/000028 |
Document ID | / |
Family ID | 34655023 |
Filed Date | 2005-09-22 |
United States Patent
Application |
20050207996 |
Kind Code |
A1 |
Burling, Hans ; et
al. |
September 22, 2005 |
Use of osteopontin in dental formulations
Abstract
Use of osteopontin for reducing plaque bacterial growth on tooth
enamel and dental formulations containing osteopontin.
Inventors: |
Burling, Hans; (Lund,
SE) ; Sorensen, Esben Skipper; (Sabro, DK) ;
Bertelsen, Hans; (Videbaek, DK) ; Jorgensen, Anders
Steen; (Arhus C, DK) ; Graverholt, Gitte;
(Arhus N, DK) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
901 NEW YORK AVENUE, NW
WASHINGTON
DC
20001-4413
US
|
Family ID: |
34655023 |
Appl. No.: |
11/000028 |
Filed: |
December 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60525828 |
Dec 1, 2003 |
|
|
|
Current U.S.
Class: |
424/50 |
Current CPC
Class: |
A61K 31/16 20130101;
Y02A 50/30 20180101; A61Q 11/00 20130101; A61P 1/02 20180101; A61P
31/04 20180101; Y02A 50/406 20180101; A61K 8/64 20130101 |
Class at
Publication: |
424/050 |
International
Class: |
A61K 007/16 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 1, 2003 |
DK |
PA 2003 01777 |
Claims
1. Use of osteopontin for reducing or preventing plaque bacterial
growth on tooth enamel.
2. Use of osteopontin according to claim 1 for reducing or
preventing dental caries.
3. Use of osteopontin according to claim 1 for reducing or
preventing calculus dentalis.
4. Use of osteopontin according to claim 1 for reducing or
preventing periodontal disease.
5. Use of osteopontin according to claim 1 for reducing or
preventing demineralization of teeth.
6. A dental formulation containing osteopontin.
7. A dental formulation according to claim 6 in the form of a
toothpaste, toothpowder, tooth gel, dental mouthwash, mouth spray
or chewing gum.
8. A dental formulation according to claim 7 comprising about 5 mg
osteopontin to about 1500 mg osteopontin per kg formulation.
9. A dental formulation according to claim 8 comprising about 100
mg osteopontin to about 1000 mg osteopontin per kg formulation.
10. A dental formulation according to claim 9 comprising about 200
mg osteopontin to about 500 mg osteopontin per kg formulation.
11. A dental formulation according to claim 10 comprising about 350
mg osteopontin per kg formulation.
12. Use of osteopontin for the manufacturing of a pharmaceutical
composition for treating or preventing dental caries, calculus
dentalis or periodontal disease.
13. A method for preventing or inhibiting plaque bacterial growth
on tooth enamel by administration in the mouth of an effective
amount of osteopontin.
14. Method of treatment or preventing of dental caries, calculus
dentalis or periodontal disease by oral administration of an
effective amount of osteopontin to a patient in need of such
treatment.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention concerns dental formulations. In
particular the invention concerns the use of osteopontin (OPN) for
significantly reducing plaque bacterial growth on tooth enamel.
BACKGROUND OF THE INVENTION
[0002] Many problems occur in connection with care of the teeth,
cosmetically as well as therapeutically, such as formation of
dental plaque, bacterial growth in dental plaque, dental caries,
dental calculus (tartar), periodontal disease and demineralization
of teeth.
[0003] Dental plaque is a complex biofilm that accumulates on the
hard tissues (teeth) in the oral cavity. Although over 500
bacterial species comprise plaque, colonization follows a
regimented pattern with adhesion of initial colonizers to the
enamel salivary pellicle followed by secondary colonization through
bacterial adhesion. It is well known that a range of streptococci
species belong to the early colonizers. It is therefore important
to control the adhesion of these bacteria. A variety of adhesins
and molecular interactions underlie the adhesive interactions and
contribute to plaque development and ultimately to diseases such as
caries and periodontal disease.
[0004] The enamel of teeth is comprised of hydroxyapatite. In the
mouth a natural equilibrium exists between, on the one hand,
hydroxyapatite being dissolved from the enamel of the teeth and, on
the other, hydroxyapatite being formed on or in the teeth from
substances occurring naturally in the saliva. When the equilibrium
is such that the hydroxyapatite is dissolved, a cariogenic
condition arises which is referred to as demineralization.
[0005] It has long been known that incorporation of fluoride ions
into enamel protects it from demineralization. Therefore, fluoride
is often incorporated in toothpaste. However, fluoride can give
rise to fluorose, especially if the patient is swallowing the
toothpaste or another dentrifrice or oral hygiene product, which is
often the case, for example for small children. It can also be a
problem in areas with rather high amounts of fluoride in the
drinking water.
[0006] The purpose of this patent application is to propose the use
of an indigenous milk protein, osteopontin, added to a dental
formulation to control or inhibit the growth of bacteria on the
tooth surface and thereby prevent or reduce plaque formation and
caries.
[0007] There are a couple of patents that describe the use of milk
protein fractions, i.e. hydrolysates of casein, Ca
phosphopeptides/CPP, and glucomacropeptide/GMP from renneting of
milk for repair of damage to the enamel with the hypothesis that
they act as a supply of amorphous calcium phosphate to the
enamel.
[0008] WO 03/059304 proposes an oral care composition containing a
fluoride ion source and CPP fraction. This formulation stabilizes
the calcium phosphate added to the oral formulation in an amorphous
form and at the same time stabilizes the fluoride level in the
formulation.
[0009] WO 00/07454 describes GMP which, added to a special food
formulation based on milk, has an anticariogenic effect on
rats.
[0010] In contrast to CPP and GMP peptides, OPN introduces a new
dimension in the oral cavity, through its potential to bioactively
influence the attachment of oral organisms to the enamel and thus
affect the development of caries. Like CPP and GMP, OPN also has an
effect on the levels of amorphous calcium phosphate in the saliva
available for tooth repair.
[0011] OPN thus has several functions in the dental care
context.
[0012] The great advantage of using OPN in e.g. toothpaste and
mouthwash is that it is a natural protein component in bovine milk
and thus there is a limited need for clinical testing.
SUMMARY OF THE INVENTION
[0013] It has now surprisingly been shown that osteopontin used in
oral formulations reduce bacterial adherence and growth on enamel
surfaces.
[0014] The invention is about oral compositions, containing
osteopontin including toothpaste, mouthwash and chewing gum as well
as related compositions.
DETAILED DESCRIPTION OF THE INVENTION
[0015] An in vitro study using a tooth analogue of a finely
polished hydroxyapatite (HA) disc dipped in a dilute solution of
OPN gives a film of OPN on the HA surface, which cannot be washed
away with water. When such treated discs were brought into contact
with human saliva surprisingly we found that the film of OPN
significantly reduced the adherence and growth of bacteria on the
analogue tooth surfaces resulting in a significantly lower plaque
formation. The trials were made using saliva samples from a group
of patients of different ages and oral flora. These results
implicate the potential use of OPN in oral formulations such as
toothpaste and mouthwash but also as an ingredient in chewing
gum.
[0016] Therefore the invention relates to the use of osteopontin
for reducing plaque bacterial growth on tooth enamel and dental
formulations containing osteopontin.
[0017] As used herein the term "osteopontin" or "OPN" means
osteopontin obtained from milk, including naturally occurring
fragments or peptides derived from OPN by proteolytic cleavage in
the milk, or genesplice-, phosphorylation-, or glycosylation
variants as obtainable from the method proposed in WO 01/49741. The
milk can be milk from any milk producing animals, such as cows,
camels, goats, sheep, dromedaries and llamas. However, OPN from
bovine milk is preferred due to the availability. All amounts are
based on native bovine milk OPN, but can easy be corrected to the
corresponding amounts of an active fraction thereof or OPN from
another source. OPN or derivates thereof can also be genetically
prepared.
[0018] The dental formulations can be any dentifrice or related
product of relevance in oral hygiene, such as for example
toothpowder, tooth gel, dental mouthwash, mouth spray or chewing
gum. Osteopontin (OPN) is an acidic, highly phosphorylated, sialic
acid rich, calcium binding protein. OPN binds 28 moles of phosphate
and about 50 moles of Ca per mole. The isoelectric point is about
3.0. The protein exists in many tissues in the body and plays a
role as a signalling and regulating protein. It is an active
protein in biomineralization processes. OPN is expressed by a
number of cell types including bone cells, smooth muscle cells and
epithelial cells.
[0019] OPN is present in bovine milk. A typical concentration is 20
mg per l.
[0020] OPN can relatively simply be isolated by anion
chromatography from e.g. acid whey at pH 4.5 as described by patent
WO 01/497741 A2 or WO 02/28413. Purity of up to 90-95% can easily
be obtained.
[0021] The amount of osteopontin is normally between about 50 mg
OPN and about 1500 mg osteopontin per kg dental formulation.
However, smaller amount will also have an effect. Higher amounts
can be used, but the effect will not be essentially increased. A
useful amount is 100-1000 mg OPN per kg, preferably 200-500 mg, and
most preferred about 350 mg. Larger amounts will presumably not
give better results and is therefore not recommended, because OPN
is a rather expensive ingredient.
[0022] Preferred compositions of the subject invention are as
already mentioned in the form of as tooth-pastes, tooth-gels and
tooth powders. Components of such toothpaste and tooth-gels include
one or more of the following: a dental abrasive (from about 10% to
about 50%), a surfactant (from about 0.5% to about 10%), a
thickening agent (from about 0.04% to about 0.5%), a humectant
(from about 0.1% to about 3%), a flavouring agent (from about 0.04%
to about 2%), a sweetening agent (from 0.1% to about 3%), a
colouring (from about 0.01% to about 0.5%) and water (from 2% to
45%). Anticaries agents contain from 0.001% to about 1% OPN.
Anti-calculus agents contain from about 0.1% to about 13% OPN.
[0023] Tooth powders, of course, are substantially free from all
liquid components.
[0024] Other preferred compositions of the subject invention are
dental mouth washes, including mouth sprays. Components of such
mouth washes and mouth sprays typically include one or more of the
following: water (from about 45% to about 95%), ethanol (from about
0% to about 25%), a humectant (from about 0% to about 50%), a
surfactant (from about 0.01% to about 7%), a flavouring agent (from
about 0.04% to about 2%), a sweetening agent from (from about 0.1%
to about 3%). and a colouring agent (from about 0.001% to about
0.5% anti-caries agent including OPN, from about 0.001% to 1% and
an anti-calculus agent (from about 0.1% to about 13%).
[0025] A third area of application is in chewing gum formulations
of various compositions in general terms.
[0026] The invention is further illustrated by the following
examples and experiments.
EXAMPLES
[0027] Background
[0028] It was the purpose of this study to prove two things:
[0029] OPN binds to hydroxyapatite surfaces in a stable way. The
film cannot be removed by rinsing with water or buffer.
[0030] OPN influences the adherence of bacteria on the tooth
analogue surface.
[0031] The ability of OPN to prevent plaque build-up in-vitro has
been investigated in this study on hydroxyapatite (HA) discs acting
as a model for tooth enamel. The discs were dipped in OPN solution
and subsequent incubated with saliva. Growth on the substrates was
studied with bacterial count of different plaque-forming bacterial
strains at the Faculty of Odontology at Malmo University,
Sweden.
[0032] As a reference for OPN coated HA surfaces, non-coated and
BSA (bovine serum albumin) treated HA discs were used.
[0033] The study was performed on well characterized saliva samples
from 6 donors as described below.
[0034] Materials and Methods
[0035] Chemicals
[0036] Osteopontin (OPN) was prepared by Arla Foods amba with a
chromatographic purity of about 95%. Bovine serum albumin (BSA) was
purchased from Sigma-Aldrich. Sodium dodecylsulphate (SDS) was
obtained from Sigma (St. Louis, Mo., USA, L-6026). All other
chemicals were of analytical grade and the water used was of
Milli-Q quality. Pre-treatments of substrates and adsorption
experiments were performed in phosphate buffer containing 0.01 M
phosphate and 0.05 M sodium chloride at pH 7. All glassware and
pipette tips, etc as well as buffers were sterilised by
autoclavation. Protein solutions used in experiments were
sterilised by filtration (cut-off 0.22 .mu.m).
[0037] Salivary Samples
[0038] Stimulated saliva for the experiments was collected after
chewing a piece of paraffin. A summary of the donors of stimulated
salivary samples is shown in Appendix 1.
[0039] No food or drink was allowed two hours prior to
collection.
[0040] Substrates
[0041] Hydroxyapatite (HA) discs, 10 mm in diameter, were purchased
from Swedish Ceramic Institute, Goteborg. The discs used for SEM
studies were polished on both sides so that they could be cleaned
using ultrasound. Before starting the experiments, substrates
(discs) were treated in mild detergent solution and thoroughly
rinsed in water. Finally, they were rinsed in ethanol and water.
Substrates were stored in 70% ethanol until use when they were
rinsed with water and dried in a flow of nitrogen. After drying,
substrates used in ellipsometry measurements were plasma cleaned in
low pressure residual air (10-30 Pa) using a radio frequency glow
discharge unit (Harrick PDC 3XG, Harrick Scientific Corp.,
Ossining, N.Y.).
[0042] Bacterial Film Formation
[0043] Substrate surfaces were pre-incubated for 1 h in protein
solution at 37.degree. C. Substrates were then rinsed with buffer
and incubated at 37.degree. C. for 40 h. Stirring was applied
during pre-treatment and incubation in the experiments. After
incubation the HA discs were rinsed with buffer.
[0044] Ellipsometry
[0045] The interaction of OPN with salivary proteins on HA
substrates was followed by ellipsometry, which is an optical method
to measure the changes in polarization of light upon reflection at
a surface (3). The instrument used was a Rudolph thin film
ellipsometer, type 436 (Rudolph Research, Fairfield, N.J.),
equipped with a xenon lamp filtered to 4015 .ANG., and operated in
a set-up as described by Landgren and Jonsson (1993). To determine
the ellipsometric angles, .DELTA. and .PSI., for the bare substrate
the position of the intensity minimum was established. From the
changes in .DELTA. and .PSI. upon adsorption the thickness and the
refractive index for the adsorbed protein film were calculated
according to McCrackin et al. (4) assuming a homogeneous film at
the surface. The adsorbed mass, .GAMMA. (mg/m.sup.2), was
calculated according to Cuypers et al. (5). As shown by these
authors, the adsorbed mass can, at low surface coverage, be
determined more accurately than the film thickness and the
refractive index, and has for this reason been presented here. The
values of the ratio between molar weight and molar refractivity and
of the partial specific volume used were 4.1 g/ml and 0.75 ml/g,
respectively. These values are commonly used for proteins and have
previously been applied in a number of studies regarding adsorption
of salivary components (cf. (6, 7)). Surfaces prepared as described
in the substrate section above were placed in the ellipsometer
cuvette containing the buffer. The cuvette was thermostated at
37.degree. C. unless otherwise stated, and the solution was stirred
with a magnetic stirrer. When the ellipsometric angles were stable
saliva was added to a final concentration of 10% (v/v). The
adsorption was measured for 1 h followed by rinsing of the cuvette
with a continuous buffer flow of 12 ml/min for 5 min. The
desorption was then followed for another 25 min. This was the
standard procedure for pellicle formation. In order to obtain a
measure of the cohesive properties of the pellicle the experiments
featured a final addition of SDS. In the standard experiment a
concentration of 17 mM (twice the cmc in water and approximately
nine times the cmc in buffer) was added and after this a final
rinse with buffer was performed 5 min after the SDS addition.
[0046] Methods for Microbiological Analyses
[0047] Culture Medium
[0048] The basic agar media employed for the isolation of micro
organisms were blood agar (8), Mitis Salivarius agar (MSA, Difco
Lab.), Mitis Salivarius-bacitracin (MSB) agar (9), Candida
Selective agar according to Nickerson (Merck), Mac Conkey agar
(Difco Lab) and Staphylococcus medium 110 (Difco Lab).
[0049] Culturing Procedures for Saliva Samples
[0050] Saliva samples were transported in conventional VMG II
(viability preserving) medium, vortex-mixed within 24 h of
collection, diluted and inoculated onto blood, MSA and MSB agars.
Blood agar was incubated in an anaerobic chamber (10% hydrogen, 10%
carbon dioxide in nitrogen) for 4 days and, MSA agar in an
atmosphere of 5% carbon dioxide for 2 days. The total number of
colony-forming units (CFU) on the agar plates was counted with the
aid of a stereomicroscope.
[0051] Removal of Micro Organisms from the Discs
[0052] The micro organisms attached to the discs were removed by
sonication in a Sonics Vibracell with a microtip using 10 pulses of
1 second. Removal of cells by sonication was confirmed to be
efficient by the lack of observed microbial growth.
[0053] Culturing Procedures for Desorbate Samples
[0054] The desorbate samples were vortex-mixed, diluted and
inoculated into the following media: blood agar, MSA, Candida
Selective agar, Mac Conkey agar and Staphylococcus medium 110.
Blood agar was incubated in an anaerobic chamber (10% Hydrogen, 10%
Carbon Dioxide in Nitrogen) for 5 days, the MSA in an atmosphere of
5% Carbon Dioxide for 2 days and, Candida Selective agar, Mac
Conkey agar and Staphylococcus medium 110 aerobically for 2 days.
The total number of CFU on the agars was counted with the aid of a
stereomicroscope. The CFU on MSA was analysed paying special
attention to morphology, size and number of different colony types.
Cells from representative colonies of each morphological type were
Gram-stained and inoculated on blood agar for later identification.
For future characterization, isolates growing on the MSA were
retained at -79C in skim milk (10% skim milk powder in distilled
water, w/v; Oxoid Lab. L31, Hampshire, UK).
[0055] Identification of Streptococcal Isolates
[0056] Gram-positive, catalase negative cocci in chains were
considered to be streptococci and these isolates will be identified
to species and subspecies levels based on characteristics described
previously (10).
[0057] Results and Discussion
[0058] Protein Interaction Study
[0059] In order to investigate the interaction between OPN and HA
and OPN and saliva an ellipsometry study was performed. In FIG. 1
the effect of adsorption temperature is shown. From this figure it
can be seen that the adsorption of OPN on the HA surfaces happens
almost instantaneously. A larger amount of OPN was absorbed at
higher temperature (37.degree. C.), although, rinsing resulted in
some desorption at 37.degree. C. to a stable surface load of 1 mg
per m.sup.2. To simulate the chemical effect of tooth brushing SDS
was added to the cuvette for five minutes, followed by rinsing with
buffer. After this cleaning step there was no significant
difference in adsorbed amount at the two temperatures.
[0060] The results obtained indicate that OPN gives a surface
coverage of the HA discs that is stable although a reduction in
film thickness is obtained after water rinse and SDS treatment.
[0061] Bacterial Film Formation
[0062] The counts of bacteria shown in table 1 and 2 desorbed by
mild sonification show for HA discs treated with OPN solutions with
concentration 0.1 mg/ml and 1 mg/ml respectively a significant
effect on adherence of bacteria both in total counts on blood agar
and MSA agar plates for all salivary donors. HA discs treated with
BSA or saliva exhibited more bacteria on the surfaces. The total
counts of micro organisms were 10- to 1000-fold lower on OPN-coated
discs than on the control discs (saliva or BSA-coated). The
stimulated saliva samples exhibited a highly diverse microbial
composition which is considered to be a common characteristic of
the salivary microflora. Of interest was the finding that
significantly fewer streptococci colonized the OPN-coated than the
control discs.
1TABLE 1 Bacterial analysis of whole discs, polished on both sides
incubated in stimulated saliva after different pre-treatments.
Total count (CFU) on blood agar plates incubated anaerobically.
Pre- Donor treatment BSA Saliva OPN 0.1 OPN 1 ME 33.000 6.800 102 0
K 12.600 5.200 280 48 Br 45.200 71.400 8.800 4.800 ML 19.900 41.000
7.100 13.800 Be 6.700 1.280 3.400
[0063]
2TABLE 2 Bacterial analysis of whole discs, polished on both sides
incubated in stimulated saliva after different pre-treatments.
Total count on Mitis Salivarious Agar (MSA) incubated facultatively
anaerobically. Pre- Donor treatment BSA Saliva OPN 0.1 OPN 1 ME 19
50 15 0 K 9.400 44 11 1 Br 1.220 2.140 480 960 ML 2.190 360 30 90
Be 120 120 30 70
SUMMARY
[0064] The results show a clear effect of OPN pre-treatment of HA
discs on the amount of bacteria after incubation in stimulated
saliva. The anti-adhesive effect seems to be almost concentration
independent in the range investigated 0.1-1 mg/ml of OPN solution
for dipping of the tooth analogues. For a mono-layer coverage a
small amount of OPN is needed.
[0065] It is interesting to note that significantly less
streptococci adherence the OPN-coated discs compared with the
control discs.
* * * * *