Note: Descriptions are shown in the official language in which they were submitted.
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ORAL COMPOSITIONS AND USE THEREOF
This application claims the priority benefit of U.S. Provisional Patent
Application Serial No. 60/255,304 filed December 13, 2001, which is hereby
incorporated by reference in its entirety.
This invention was made, at least'in part, with funding received from
the National Institutes of Health, NIDCR Grant No. R37 DE07907. The U.S.
Government may have certain rights in this invention.
FIELD OF THE INVENTION
The present invention relates to oral compositions and their use for
inhibiting the activity of surface-bound glucosyltransferase; treating or
inhibiting
dental caries, gingivitis, candidiasis, and/or denture stomatitis; inhibiting
the
accumulation of microorganisms on an oral surface; and treating or inhibiting
aphthous ulcerations on an oral surface.
SACI~GROUND OF THE INVENTION
Colonization of tooth surfaces by mutans streptococci is associated
with the etiology and pathogenesis of dental caries in animals and humans
(Fitzgerald
and Keyes, 1960; Loesche, 1986). Glucosyltransferase enzymes ("GTFs") produced
by Streptococcus n2utans have been recognized as virulence factors in the
pathogenesis of dental caries (De Stoppelaar et al., 1971; Tanzer et al.,
1985;
Yamashita et al., 1993). GTFs catalyze the formation of soluble and insoluble
a-
linked glucans from sucrose and contribute significantly to the polysaccharide
composition of dental plaque matrix (Rolls et al., 1983). Dental plaque is
essentially a
biofilm. Glucans promote the adherence and accumulation of cariogenic
streptococci
on the tooth surface, and play an essential role in the development of
pathogenic
dental plaque related to caries activity (Hamada and Slade, 1980; Schilling
and
Bowen, 1992; Yamashita et al., 1993). Streptococcus nautans produces at least
three
GTFs: GTF B, which synthesizes a polymer of mostly insoluble a,1,3-linked
glucan;
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GTF C, which synthesizes a mixture of insoluble a1,3-linked glucan and soluble
a1,6-linked glucan; and GTF D which synthesizes a1,6-linked soluble glucan
(Aoki
et al., 1986; Hanada and Kuramitsu, 1988; Hanada and Kuramitsu, 1989). An
additional enzyme, GTF from S. sahguinis (GTF Ss), may also be involved in the
development of dental plaque (Nyvad and Kilian, 1987; Vacca-Smith et al.,
2000). S.
sanguihis colonizes tooth surface early in plaque formation, and its GTF
catalyzes
predominantly a1,6-linked soluble glucan (Ceska et al., 1972). Enzymatically
active
GTFs are present in the soluble fraction of whole human saliva and are also
incorporated into salivary pellicle that is formed on tooth surfaces (Rolla et
al., 1983;
Scheie et al., 1987). Furthermore, the GTFs incorporated into an experimental
pellicle
demonstrate distinct physical and kinetic properties when compared to the same
enzymes in solution; GTF C and D express enhanced enzymatic activity
(Schilling
and Bowen, 1988; Vacca-Smith et al., 1996; Venkitaraman et al., 1995). A laxge
proportion of the glucans synthesized by these surface-adsorbed GTFs is
retained on
the pellicle and may provide binding sites for mutans streptococci,
contributing to the
i~z situ formation of dental plaque (Schilling and Bowen, 1988; Schilling and
Bowen,
1992; Vacca-Smith and Bowen, 1998). Therefore, inhibition of GTFs both in
solution
and adsorbed to the pellicle of tooth surface is one of the strategies to
prevent dental
. caries and other plaque related diseases.
Propolis, a resinous substance collected by Apis mellifera bees from
various plant sources and mixed with secreted beeswax, is a multifunctional
material
used by bees in the construction, maintenance and protection of their hives
(Burdock,
1998; Ghisalberti, 1979). Propolis is a non-toxic natural product with
multiple
pharmacological effects and a complex chemical composition (Burdock, 1998;
Ghisalberti, 1979). Several compounds have been identified in propolis and
three
distinct chemical groups have been reported to the present: 1) flavonoid
aglycones; 2)
cinnamic acid derivatives; and 3) terpenoids (Bankova et al., 1995; Banskota
et al.,
1998; Park et al., 1998; Tazawa et al., 1998). Among them, flavonoids have
been
considered the main biologically active compounds in propolis (Amoros et al.,
1992;
Bonhevi et al., 1994; Ghisalberti, 1979). Propolis exhibits a wide range of
biological
activities, including antimicrobial, anti-inflammatory, anesthetic, and
cytostatic
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properties (Burdock, 1998; Ghisalberti, 1979). It has been demonstrated
previously
that two chemically distinct types of propolis from Brazil inhibited the
activity of
GTFs and the growth of mutans streptococci in vitro (Koo et al., 2000a; Koo et
al.,
2000b; Koo et al., 2000c). Furthermore, topical application twice daily of
propolis
(Koo et al., 1999) or inclusion in drinking water available ad libitum (Ikeno
et al.,
1991) reduced the incidence of dental caries in rats. Nevertheless,
information on the
biological properties of specific compounds, which could be useful in
prevention of
oral diseases, is sparse. Therefore, it would be desirable to identify
individual
components of propolis as well as other compounds not in propolis, which have
similar activity.
The present invention is directed to overcoming these and other
deficiencies in the art.
SUMMARY OF THE INVENTION
A first aspect of the present invention relates to an oral composition
including: an organoleptically suitable carrier and an amount of a terpenoid
and a
flavonoid, dispersed in the carrier, which is effective to prevent or treat
dental caries,
dental plaque formation, gingivitis, candidiasis, dental stomatitis, aphthous
ulceration,
or fungal infections.
A second aspect of the present invention relates to a method of
inhibiting the activity of surface-bound glucosyltransferase which includes:
contacting
a surface-bound glucosyltransferase with an effective amount of a flavonoid or
a
combination of a flavonoid and a terpenoid, under conditions effective to
inhibit the
glucan-forming activity of the surface-bound glucosyltransferase.
A third aspect of the present invention relates to a method of treating or
inhibiting dental caries, gingivitis, candidiasis, or denture stomatitis,
which method
includes: providing an oral composition of the present invention and
contacting an
oral surface with an effective amount of the oral composition under conditions
effective to treat or inhibit dental taxies, gingivitis, candidiasis, or
denture stomatitis.
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A fourth aspect of the present invention relates to a method of
inhibiting accumulation of microorganisms on an oral surface which includes:
providing an oral composition of the present invention and contacting an oral
surface
with an effective amount of the oral composition under conditions effective to
inhibit
accumulation of a microorganism which promotes dental caries, gingivitis,
candidiasis, denture stomatitis, or formation of dental plaque matrix.
A fifth aspect of the present invention relates to a method of treating or
inhibiting aphthous ulceration which includes: contacting an oral surface with
an
effective amount of a terpenoid, a flavonoid, or a combination thereof, under
conditions effective to treat an existing aphthous ulceration or inhibit
formation of an
aphthous ulceration.
It is believed that the oral compositions of the present invention axe
particularly well suited for use in treating or inhibiting dental caries,
gingivitis,
candidiasis, and denture stomatitis, because oral compositions including
terpenoids
and flavonoids can both disrupt the activity of glucosyltransferases which are
in
solution and/or bound to a solid surface as well as destroy microorganisms
which
produce the glucosyltransferases. They axe similarly well suited to inhibit
accumulation of microorganisms which promote dental caries, gingivitis,
candidiasis,
denture stomatitis, or formation of dental plaque matrix. Additional benefits
include
minimizing the staining of teeth and oral malodors.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph illustrating time-kill curves for mutans streptococci
strains by tt-farnesol at four times the MIC (or MBC). Streptococcus mutaus GS-
5
(~l0), S. mutates UA 159 (~/o), S sobrinus 6715 (~/o).
Figures 2A-B are graphs which illustrate the effects of apigenin on the
activities of streptococcal GTFs in solution (2A) and adsorbed onto saliva-
coated
hydroxyapatite (sHA) surface (2B). GTF B (~), GTF C (~), GTF D (0), GTF Ss
(o).
The data shown are mean values (~SD). The percentage of inhibition was
calculated
considering the control (DMSO:EtOH, final concentration of 7.5% and 1.25%,
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vol./vol.) as maximum GTF activity. At each concentration of apigenin, means
labeled with symbols (* or 8) are not significantly different from each other,
p<0.05.
Figure 3 is a graph illustrating the effects of apigenin (1.33 mM) on the
activities of GTFs in solution and adsorbed onto saliva-coated hydroxyapatite
surface.
Open bars represent solution assays and marked bars represent surface assays.
The
percentage of inhibition was calculated considering the control (solute) as
100% GTF
activity. tt-Farnesol (1.33 mM), chlorhexidine (1.33 mM), and fluoride (250
ppm)
showed either negligible or moderate inhibitory effects.
Figure 4 is a graph illustrating the effects of 1.33 mM tt-Farnesol and
0.12% CHX (1.33mM) on the viability of Streptococcus mutans UA 159 biofilins.
A
similar profile was obtained for S. sobrinus 6715. Apigenin (1.33 mM) and
fluoride
(250 ppm) showed negligible antibacterial activity as measured by the killing
of
bacteria.
Figure 5 is a graph illustrating the effects of treatments on smooth-
surface caries and severity scores. Values capped by symbols are statistically
significantly different from control (p<0.05). ANOVA, comparison for all pairs
using
Tukey-Kramer HSD.
Figure 6 is a graph illustrating the effects of treatments on sulcal caries
and severity scores. Values capped by symbols are statistically significantly
different
from control (p<0.05). ANOVA, comparison for all pairs using Tukey-Kramer HSD.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to oral compositions and their use in
inhibiting the activity of surface-bound glucosyltransferase; treating or
inhibiting
dental caries, gingivitis, candidiasis, and/or denture stomatitis; inhibiting
the
accumulation of microorganisms on an oral surface; and treating or inhibiting
aphthous ulcerations on an oral surface.
Oral compositions which can be used in accordance with the present
invention include an organoleptically suitable carrier and a terpenoid and/or
a
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flavonoid dispersed in the carrier, preferably both a terpenoid and a
flavonoid
dispersed in the carrier.
Suitable terpenoids include, without limitation, terpenes, terpinols,
diterpenic acids, diterpenes, triterpenes, and derivatives thereof. Exemplary
terpenoids include, without limitation, tt-farnesol as well as its
stereoisomers and
derivatives, (3-caryophyllene, terpineol, nerolidol, bisabolol, santatol,
dehydroabietic
acid, abietic acid, (3-amyrine, triterpenic alcohol of amyrine, lanosterol,
cupressic acid
and its derivatives, agathic acid and its derivatives, agathalic acid,
betuletol,
melliferone, moronic acid, anwuweizonic acid, betulonic acid, syringaldehyde,
imbricatoloic acid, communic acid, methyl isocupressate, tremetone, viscidone
and its
derivatives, 8-cadinene, ledol, guajol, a-copaene, (3-selinene, a-elemene,
calamenene,
a-muurolene, y-muurolene, [3-eudesmol, humulene, bulnesol, and combinations
thereof. Many such terpenoids are commercially available or readily
synthesized
according to known procedures.
The terpenoid is present in an amount which is effective to prevent or
to treat dental caries, dental plaque formation, gingivitis, candidiasis,
dental
stomatitis, aphthous ulcerations, and/or fungal infections. Typically, though
not
exclusively, the effective amount of terpenoid present in the oral composition
is less
than about 5 percent by weight/volume. Preferably, the terpenoid is present in
an
amount which is between about 0.01 to about 2 percent by weight/volume, more
preferably about 0.01 to about 1.5 percent by weight/volume.
Suitable flavonoids include flavones, flavonols, dihydroflavonols,
flavonones, and derivatives thereof. Exemplary flavonoids include, without
limitation, apigenin and its derivatives, acacetin, baicalein, chrysin,
luteolin,
tectochrysin, kaempferol, kaempferide, galangin, isorhamnetin, rhamnetin,
myricetin,
fisetin, rutin, pinobanksin, pinobanksin-3-acetate, pinobanksin-7-methyl eter,
pinocembrin, sakuranetin, isosakuranetin, quercetin, hesperitin, naringin,
pinostrobin
and its derivatives, trihydroxymethoxy flavanone, tetraxydroxy flavanone,
tetrahydroxyflavone, ermanin, 3,5,7-trihydroxy-4'-methoxyflavanol, 5,6,7-
trihydroxy-
3,4'-dimethoxyflavone, 3,7-dihydroxy-5-methoxyflavanone, 2,5-dihydroxy-7-
methoxyflavanone, 3-methylquercetin, 8-methylkaempferol, and combinations
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thereof. Many such flavonoids are commercially available or readily
synthesized
according to known procedures.
The flavonoid is present in an amount which is effective to inhibit
soluble or surface-bound glucosyltransferase activity or to prevent or to
treat dental
caries, dental plaque formation, gingivitis, candidiasis, dental stomatitis,
aphthous
ulcerations, and/or fungal infections. Typically, though not exclusively, the
effective
amount of flavonoid present in the oral composition is less than about 5
percent by
weight/volume. Preferably, the flavonoid is present in an amount which is
between
about 0.01 to about 1 percent by weight/volume, more preferably about 0.01 to
about
0.5 percent by weight/volume.
The oral compositions can take the form of a toothpaste or gel, a
powder, a solution (e.g., mouthwash or dental rinse), a suspension, an
emulsion, a
lozenge, a mucoadhesive vehicle, a tablet or a gum. The composition can also
be
presented in a delivery vehicle such as a dental floss impregnated with a
composition
of the present invention.
The particular choice of carrier will depend, at least in part, upon the
desired form which the oral composition will take. The carrier is preferably
both
organoleptically suitable and pharmaceutically acceptable for oral
administration.
Typically, the carrier will include as a major component one or more of the
following:
water, glycerin, alcohols such as ethanol, sorbitol, propylene glycol, etc.,
DMSO,
curable polymers, and powders such as starch.
When water is employed, deionized water is preferred. Typically,
water can comprise from about 10 to about 85 weight percent of the oral
composition,
depending upon the formulation.
Polymeric delivery vehicles can include copolymers of
polyvinylmethylether with malefic anhydride and other similax delivery
enhancing
polymers.
According to a preferred embodiment of the oral composition, the oral
composition includes effective amounts of both a terpenoid and a flavonoid
dispersed
in an organoleptically suitable non-polymeric carrier (i.e., substantially
free from
other propolis components). The terpenoid, flavonoid, and non-polymeric
carrier can
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be of the type described above, present in the amount described above. The
molar
ratio of terpenoid:flavonoid can be between about 0.1 to about 10:1,
preferably about
0.5 to about 5:1.
In addition, the oral composition can include a number of additives,
including without limitation, an abrasive agent, a gelling agent, a humectant,
a
cariostatic agent, a flavoring agent or sweetener, a desensitizing agent, an
anti-
calculus agent, a whitening agent, a surfactant, a binding agent, a
preservative, an
opacifying agent, a coloring agent, a buffering agent, or combinations
thereof.
Abrasive agents are typically employed in dentifrice compositions.
Suitable abrasive agents include, without limitation, aluminum oxide, aluminum
hydroxide, calcium hydrogen phosphate dehydrate or anhydride, silica gel,
zirconosilicate, silicic anhydride, aluminosilicate, calcium carbonate,
calcium
pyrophosphate, aluminum silicate, insoluble sodium metaphosphate, magnesium
tertiary phosphate, magnesium carbonate, calcium sulfate, synthetic resins,
and
combinations thereof. Abrasives can generally be employed in effective amounts
of
between about 20 to about 90 weight percent, more typically about 20 to about
60
weight percent for dentifrices.
Gelling agents (i.e., thickeners) can be used in various compositions to
assist in processing. Suitable gelling agents include, without limitation,
carrageenan,
sodium carboxymethyl cellulose, alkali metal alginates such as sodium
alginate,
gums, polyvinyl alcohol, and vee gum or the like. Typically, the gelling
agents are
employed in amount of about 0.3 to about 5 weight percent.
Humectants can also be employed in the oral compositions,
particularly toothpastes and gels and oral rinses. Suitable humectants include
sorbitol,
glycerin, propylene glycol, 1,3-butylene glycol, polyethylene glycol, xylitol,
maltitol,
lactitol, or the like. The humectant can also be used as the bulk carrier in
many
instances, in which case it can be present in an amount of about 5 to about 90
weight
percent, more typically about 10 to about 60 weight percent.
Cariostatic agents (i.e., non-flavonoid cariostatic agents) can be
provided in each form of the oral composition. Suitable cariostatic agents
include,
without limitation, sodium fluoride, stannous fluoride, aminefluorides, sodium
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monofluorophosphate, sodium trimeta-phosphate, triclosan, casein, or
combinations
thereof. If desired, the cariostatic agent can be present in an amount between
about
0.01 to about 3 weight percent, more typically between about 0.02 to about 1
weight
percent.
Flavoring agents are desired in most oral compositions to enhance the
flavor and palatability of the oral composition and, thus, the likelihood of
their use.
Suitable flavoring agents can be flavoring oils (e.g., oil of spearmint,
peppermint,
wintergreen, sassafras, clove, sage, eucalyptus, cinnamon, lemon, and orange,
methyl
salicylate, etc.) or sweeteners (e.g., sucrose, sucralose, lactose, maltose,
xylitol,
sodium cyclamate, perillartine, aspartyl phenyl alanine methyl ester,
saccharine, etc.).
Flavoring agents can be present, either individually or collectively, in an
amount of
about 0.1 to about 10 weight percent, more typically about 0.1 to about 5
weight
percent.
Desensitizing agents can be introduced in some embodiments of the
oral composition to treat individuals whose teeth are sensitive to thermal
shock,
chemicals, etc. Suitable desensitizing agents include, without limitation,
potassium
citrate, potassium chloride, potassium tartrate, potassium bicarbonate,
potassium
oxalate, potassium nitrate, and strontium salts. Desensitizing agents can be
present,
either individually or collectively, in an amount of about 0.1 to about 5
weight
percent, more typically about 0.1 to about 3 weight percent.
Anti-calculus agents can be introduced in some embodiments of the
oral composition to treat tartar formation. Suitable anti-calculus agents
include,
without limitation, alkali-metal pyrophosphates, hypophosphite-containing
polymers,
organic phosphonates, phosphocitrates, and combinations thereof. Anti-calculus
agents can be present, either individually or collectively, in an amount of
about 0.1 to
about 5 weight percent, more typically about 0.1 to about 3 weight percent. .
Whitening agents can be employed in some forms of the oral
composition. Suitable whitening agent including urea peroxide, calcium
peroxide,
and hydrogen peroxide. Whitening agents can be employed in amounts of about
0.5
to about 5 weight percent.
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Surfactants can also be employed in the various oral compositions.
Any of a variety of types of surfactants can be utilized, including anionic,
nonionic,
cationic and zwitterionic or amphoteric surfactants, or combinations thereof.
Exemplary anionic surfactants include, without limitation, sodium lauryl
sulfate,
sodium lauroyl sarcosinate, oc-olefin sulfonate, taurate, lauryl monoglyceride
sulfate,
lauryl monoglyceride sulfonate, and combinations thereof. Exemplary nonionic
surfactants include, without limitation, TWEEN, lauroyl diethanol amide,
stearyl
monoglyceride, sucrose fatty acid esters, lactose fatty acid esters, lactitol
fatty acid
esters, maltitol fatty acid esters, polyoxyethylene sorbitan monostearate, and
combinations thereof. Exemplary ampholytic surfactants include, without
limitation,
betain and amino acid type surfactants. Surfactants can be present in amount
of about
0.5 to about 15 weight percent, more typically about 0.5 to about 10 weight
percent.
Binding agents can be utilized, typically for tablet or lozenge forms.
Such binding agents include sodium carboxymethyl-cellulose, xanthan gum, gum
arabic, etc. as well as synthetic polymers such as polyacrylates and
caxboxyvinyl
polymers. Binders can be present in amounts of about 0.5 to about 50 weight
percent
depending on the form of the oral composition.
Preservatives can be utilized to enhance the storage properties of the
oral composition. One suitable preservative is benzoate (e.g., sodium
benzoate),
which also possesses a degree of caxiostatic activity.
Opacifying agents can also be added to various oral compositions of
the present invention. Titanium dioxide is a white powder which adds opacity
to the
compositions. Titanium dioxide can be present in an amount of about 0.25 to
about 5
weight percent.
Coloring agents may also be added to the oral compositions of the
present invention. The coloring agent may be in the form of an aqueous
solution, i.e.,
an approximately 1 percent coloring agent in water solution. Color solutions
can be
present in an amount of about 0.01 to about 5 weight percent.
The oral composition may also include buffers and salts to buffer the
pH anionic strength of the oral composition, thereby promoting its stability.
The pH
of such oral compositions of the invention is generally in the range of about
4.5 to
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about 9 or 10, preferably about 6.5 to about 7.5 or 8. The pH can be
controlled with
acid (e.g. citric acid or benzoic acid) or base (e.g. sodium hydroxide) or
buffered (as
with sodium citrate, benzoate, carbonate, or bicarbonate, disodium hydrogen
phosphate, sodium dihydrogen phosphate, etc.).
Preparation of the oral compositions of the present invention can be
carried out according to known techniques and procedures, depending upon the
particular type of vehicle employed. Where solubility is of concern, suitable
surfactants can be employed to enhance the solubility of the active
ingredients in the
selected carrier. Discussion of the preparation of oral compositions is
presented in
Harry's Cosmeticology, Seventh Edition, 1982, edited by J. B. Wilkinson and R.
J.
Moore, published by Chemical Publishing of New York, pages 609 to 617, which
is
hereby incorporated by reference in its entirety.
It will be understood that, as is conventional, the oral composition are
to be packaged according to conventional procedures. Thus a toothpaste or
dental
cream or gel dentifrice as well as a dental gel will usually be in a
collapsible tube or in
a squeeze, pump or pressurized dispenser for metering out the contents, having
a label
describing it, in substance, as a toothpaste, dental cream or the like. A
mouth rinse
will generally be in a glass or plastic bottle. Lozenges and gum will be
packaged
individually or in blister packages as is known in the art.
In a preferred use of an oral composition of the present invention, the
composition is preferably applied regularly to oral surfaces, such as every
day or
every second or third day or preferably from two to three times daily, for at
least two
weeks up to eight weeks or more up to lifetime.
Because of the properties of the flavonoids and/or terpenoids employed
in the various oral compositions of the present invention, the oral
compositions find
numerous uses for improving oral health or inhibiting the decline of oral
health. In
particular, these aspects of the present invention involve treating or
inhibiting dental
caries, gingivitis, candidiasis, denture stomatitis, and/or aphthous
ulceration, as well
as inhibiting accumulation of microorganisms on~an oral surface. Basically,
these
aspects of the present invention axe carried out by contacting an oral surface
(e.g., a
tooth, gum or other mucosal surface, tongue surface, a surface on partial or
complete
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dentures, etc.) with an effective amount of an oral composition of the present
invention under conditions effective to achieve the desired effect (i.e.,
treat or inhibit
the above conditions).
A number of oral conditions can be treated, including dental caries,
gingivitis, candidiasis, denture stomatitis, and aphthous ulcerations.
Dental caries is one of the most prevalent and significant forms of oral
disease which can lead to loss of teeth in spite of development of
therapeutics.
Caused by dental plaque formed on the surface of teeth, dental caries results
in tooth
loss as a result of organic acids, the natural metabolite of plaque bacteria
contained in
dental plaque, which decalcify dental hard tissue locally and gradually.
Although
fluoride treatments have provided some improvement in public health, there
still
exists a need to improve the prevention of dental caries. By inhibiting the
accumulation of microorganisms responsible for dental caries, such as mutans
streptococci, it is possible to inhibit or treat dental caries using the oral
compositions
of the present invention.
Bacterial plaque on tooth surfaces is also a major etiological factor in
gingivitis and periodontitis. Recently, specific oral microorganisms in plaque
have
been implicated in chronic periodontal disease. One approach to mitigate
developing
gingivitis and periodontitis is to prevent or reduce plaque formation. The
present
invention offers an improved approach for the treatment and inhibition of
gingivitis
by treating oral surfaces with an oral composition of the present invention.
Candidiasis and denture stomatitis are two conditions which are
particularly concerned with Candida invasion. Oral candidiasis is seen
frequently in
two populations, patients wearing dentures and patients who are
immunosuppressed
or have AIDS.
In the case of the AIDS patients, the oral Candida invasion is usually
severe and is one of the earliest signs of human immune deficiency virus
infection,
occurring prior to the development of opportunistic infections and full blown
AIDS
(Klein et al., 1984). Presently, there is no known explanation for this
localized oral
candidiasis, nor is it known whether the oral candidiasis itself plays a role
in the
development of systemic infections. Because of the severity of infection on
the
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mucosal surface in AIDS patients, antifungal agents are usually prescribed at
high
dose levels. Such high dosage of antifungals are generally undesirable due to
toxic
side effects. In addition, treatment failure is often observed.
In the case of the denture stomatitis patients, one of the major problems
in treating with prescription antifungals is that the disease recurs soon
after treatment
with a commercial antifungal is terminated. Investigators have found that this
recurrence is due to an inability to destroy C. albicans which adheres to and
grows on
the acrylic resin surface of the denture (Budtz-Jorgensen, 1974). Therefore,
the
denture simply reinfects the maxillary palatal mucosal surface to initiate,
maintain,
and continually aggravate the patient's oral candidiasis infection.
Thus, the oral compositions of the present invention offer an attractive
option to treat or inhibit candidiasis and denture stomatitis.
Aphthous ulcerations, commonly referred to as canker sores, are
painful oral lesions occurring on the mucous membranes of the tongue, lips,
cheek,
soft and hard palates, gingiva, floor of the mouth, or pharynx. Several
factors have
been suggested as possible causes of the disease known as aphthous stomatitis,
which
is manifested by the formation of aphthous ulcers. However, the disease is
still not
completely understood, and the factors which cause aphthous stomatitis are
still being
investigated. Regardless of their cause, the ulcers typically last from 5 to
21 days.
Often the ulcers form in groups and merge into a singular extensively
ulcerated area.
The lesions may become extremely painful, especially during periods of fatigue
or
during eating, which may become difficult if not impossible. Because of the
ability of
oral compositions of the present invention to inhibit accumulation of
microorganisms
on an oral surface, the degree of infection at the site of an aphthous ulcer
can be
reduced, thereby treating the aphthous ulcer or inhibiting its formation in
the first
place.
It is believed that the oral compositions of the present invention are
particularly well suited for use in treating or inhibiting dental caries,
gingivitis,
candidiasis, denture stomatitis, and/or aphthous ulceration, because
terpenoids and
flavonoids, particularly when used in combination, can disrupt the activity of
CA 02431044 2003-06-06
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-14-
glucosyltransferases which are in solution or bound in a solid surface as well
as
destroy microorganisms which produce the glucosyltransferases.
The oral compositions of the present invention are also well suited to
inhibit accumulation of microorganisms which promote dental caries,
gingivitis,
candidiasis, denture stomatitis, or formation of dental plaque matrix.
Exemplary
microorganisms, the accumulation of which can be inhibited, include without
limitation: lactobacilli, actinomyces, leptotrichiae, non-(3-hemolytic
streptococci,
enterococci, miscellaneous gram-positive cocci, neisseriae, diphtheroid
bacilli,
fusiform bacilli, bacteroides, spirochetes, yeasts (Candida), and combinations
thereof.
Another use of the oral compositions of the present invention concerns
the inhibition of surface-bound glucosyltransferases. It is believed that the
present
invention for the first time discloses the novel use of flavonoids to inhibit
the activity
of surface-bound glucosyltransferase, which exhibit different behavior as
compared to
soluble glucosyltransferase. This aspect of the present invention can be
achieved by
contacting a surface-bound glucosyltransferase with an effective amount of a
flavonoid or a combination of a flavonoid and a terpenoid, under conditions
effective
to inhibit the glucan-forming activity of the surface-bound
glucosyltransferase.
Glucosyltransferases which can be inhibited include, without limitation, S.
mutates
GTF B, S. mutav~s GTF C, S mutates GTF D, S. sobri~us GTF, and S. sanguihis
GTF.
EXAMPLES
The following examples are provided to illustrate embodiments of the
present invention but are by no means intended to limit its scope.
Materials & Methods
Test compounds
The compounds used in this study are classified into three groups: 1 )
flavonoids (flavonols, flavones, flavanones and dihydroflavonols); 2) cinnamic
acid
derivatives; and 3) terpenoids. The flavonols (kaempferide, kaempferol,
galangin,
isorhamnetin, rhamnetin, myricetin, fisetin, rutin), flavones (apigenin,
acacetin,
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baicalein, chrysin, luteolin, tectochrysin), flavanones (pinocembrin,
sakuranetin,
isosakuranetin), cinnamic acid derivatives (ferulic acid, p-coumaric acid,
caffeic acid)
and terpenoids (tt-farnesol, (3-caryophyllene, terpineol, syringaldehyde) were
all
obtained from Extrasynthese Co., Genay-Sedex, France. Protocatechuic acid,
vanillin, chlorhexidine (CHX), sodium fluoride, and benzoic acid were obtained
from
Sigma-Aldrich Co., Mass. The dihydroflavonols, pinobanksin, pinobanksin-7-
methyl
eter, and pinobanksin-3-acetate, were kindly provided by Prof. E. Wollenweber
(Darmdstad, Germany).
During animal studies (see Example 2 infra), a single concentration of
1.33 mM (0.035% apigenin and 0.028% tt-farnesol, w/v) was tested along with a
positive control 0.12% CHX (equivalent to 1.33 mM).
All the chemical compounds were dissolved in DMSO:ethanol (1:4,
v/v) or ethanol (99.9%, HPLC grade) just prior to carrying out the assays.
Appropriate solvent controls were always included. Table 1 summarizes all the
test
compounds used in this study and their chemical structures.
CA 02431044 2003-06-06
WO 02/47615 PCT/USO1/49032
-16-
=" -
~ L.~° ~ / x \ l ~ o
o x p
N ~ m x ~ ..x
N ~ ~~ O O
p .-~ O ~. O O _ O O
O ..,.-
I
r
'b
~ C~
O
U Q~., ~ O
"~
N
a p
a~ ~.°
0
V~ . ~ ~ ~ i O N N U ~ .~ N
..5~"~, ~ x ~bp ~ .~ ~ ~ ~ .~ ~ O M
'O ~ O ~ '~' . O ~ r~ ~ ~ d- ~ ~ M d- ,~, M l~ O
d' pO ~CS'M~~N x~~y ~ OW W ~~O ~ U
O M ~ ~ ~ ~ ~ ~ M ° O ~ O ~ O O ~ O ~' .~d .°,3
~n~~n~~~n~ ~ ~~rt~ ~n~~n ~d~'~~-~' "'' 'd
C/~ V~d'~~~1~ Md'MMI~MM d~ ~~~ MOM MMM
O
N P~
~s
'L3
OU S~ U
U cd
p
O U
m U "up '~ ...~,
~L U U p
V "d ''d S~.'
O
N ø,
N ~Nr' ~ cad ~
..*''-n .
U p ~ N
N
~_
U ~ ~ M l ~ N a ,-a
p .'d -~ n ~ p . ~ . ~ . '~~' 'd .'C~ U
S~ ~ ~ NN U~~~ .~~~ mW VU~~i O ~
cat c~ c~ m
w 4--~ 4-r .~ U> +~ e:
O O ~ ~ y~
~~ cd .~ ~ ~ U N N .~ ~ ~~' .N '~ O ~ O O O ~ ~ O cd ~ t~,
o s~, U ~ ~ a~ ~ ~ ~s o ~ ~ ~ ~ ~s o ~ ~ ~ ~ ~u U w ~
U ~ ~ pa 'U ~ E-~ ~ ~ C7 ~, ~ ~ w w a.. ~ ~, a-. a-r a, w U ss, ~ ~1 ~d
.'.,
0
0
0
v~ ~~ ~ ~ ,."U., V
N N
L7 w w w ~1 ~ p
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-17-
Bacterial strains
The bacterial strains used for the production of GTFs were: 1)
Streptococcus milleri I~SBB, which harbors the gtfB gene from S. mutans GS-5
(for
GTF B production); 2) S. milleri NHS, which contain the gtfD gene S mutans GS-
5
(for GTF D); 3) S. mutarcs WHB 410 (Wonder and Bowers, 1999), which the genes
for
GTF B, D and fructosyltransferase were deleted (for GTF C); and 4) S sanguinis
10904. For susceptibility and time-kill assays, the following bacterial
strains were
used: 1) S. mutates GS-5; 2) S muta~s UA159; and 3) S. sobrihus 6715. For
antibacterial assays, the following bacterial strains were used: 1) S. mutates
UA159;
and 2) S, sobrihus 6715. The cultures were stored at -80°C in brain
heart infusion
(BHI) or tryptic soy broth (TSB) containing 20% glycerol. The S milleri
constructs
were a kind gift from Dr. Howard I~. I~uramitsu (SUNY, Buffalo, NY) and S
mutates
UA 159 was obtained from Dr. Robert E. Marquis (Univ. of Rochester, Rochester,
NY).
GTF enzymes
All the purification procedures were carried out using buffers
containing the protease inhibitor phenylmethylsulfonylfluoride-PMSF (1 mM,
final
concentration) and NaN3 (0.02%, final concentration) as preservative. Neither
of the
reagents had any adverse effects on enzyme activity or stability.
The GTFs B, D, and Ss were obtained from culture supernatants and
purified to near homogeneity by hydroxyapatite column chromatography as
described
by Venkitaraman et al. (1995) and Vacca Smith et al. (2000). For GTF C
isolation
from S. mutans WHB 4I0 (Wonder and Bowers, 1999), cell pellets were harvested
from low molecular weight broth (2.5% tryptone, 1.5% yeast extract, 0.3%
glucose,
0.1% fructose and 0.1% sorbitol, which had been ultrafiltered through a 10-kd
molecular weight cut-off membrane) cultures of S mutaus WHB 410 (ftf gtfD- g~-
derivative of S. mutans UA 130) grown in dialysis tubing (Schilling and
Bowers,
1988). The cells were washed twice in 20 mM potassium phosphate buffer, pH 7.5
containing 1mM PMSF, and 0.02% sodium azide. The cells were then resuspended
in
30 ml of 50 mM potassium phosphate buffer, pH 7.5 containing 0.1% triton X-
100,
2M urea, 500 mM NaCI, 0.02% sodium azide and 1mM PMSF, and incubated at
25°C
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WO 02/47615 PCT/USO1/49032
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for 2h under gentle agitation. The cell suspension was centrifuged at 10,000 g
for 15
min. at 4 °C. The supernatant was carefully collected as the source of
GTF C and
dialyzed against 50 mM potassium buffer, pH 7.5 containing 1mM PMSF and 0.02%
sodium azide. The dialyzed preparation was purif ed by hydroxyapatite column
chromatography as detailed by Venkitaraman et al. (1995).
The purity of the enzyme preparations was analyzed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in a Hoefer
Mighty
Small SE245 system (Hoefer Scientific Instruments, San Francisco, CA, USA) and
silver staining (Morrisey, 1981 ). Pre-stained standards were purchased from
BioRad
Laboratories. Protein concentration was determined by the method of Lowry et
al.
(1951), with bovine serum albumin (Sigma Chemical Co., St. Louis, MO) used to
construct standard curves.
Glucosyltransferase activity was measured by the incorporation of
[14C _ glucose] from labeled sucrose (NEN Research Products, Boston, Mass.)
into
glucans (Germaine et al., 1974; Venkitaraman et al., 1995). The GTF enzyme
added
to each sample for all assays was equivalent to the amount required to
incorporate 1.0-
1.5 ~,mol of glucose over the four hours reaction (1.0-1.5 units).
Example 1 - Effect of Terpenoids and Flavonoids on GTF Activity in Solution
and Adsorbed onto Saliva-coated Hydroxyapatite Surface Assay
For solution assays, purified GTF B, C, D and Ss were mixed with a
two-fold dilution series of the test compounds (concentration ranging from 125
to 500
~,M) and incubated with [I4C-glucose]-sucrose substrate (0.2 ~,Ci/ml) (200.0
mmol/1
sucrose, 40 ~mol/1 dextran 9000, 0.02% sodium azide in adsorption buffer - 50
mM
KCI, 1.0 mM KP04, 1.0 mM CaCIZ, 0.1 mM MgCl2 - pH = 6.5) to reach a final
concentration of 100 mmol/1 sucrose (200 ~1 final volume). For the control,
the same
reaction was carried out, where ethanol:DMSO (final concentration of 7.5% and
1.25%, v/v) or ethanol (final concentration of 5%, v/v) replaced the test
agent
solutions. The samples were incubated 37°C with rocking for 4 h. After
incubation,
ice-cold ethanol (1.0 ml) was added and the samples were stored for 18 h,
4°C for
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WO 02/47615 PCT/USO1/49032
-19-
precipitation of glucans. Radiolabeled glucan was determined by scintillation
counting (Germaine et al., 1974; Venkitaraman et al., 1995).
For surface assays, the GTFs were adsorbed to hydroxyapatite beads
coated with clarified whole saliva (free of GTF activity), as detailed
elsewhere (Koo
et al., 2000c; Schilling and Bowen, 1988, Venkitaraman et al., 1995). The
saliva
coated hydroxyapatite (sHA) beads were exposed to sufficient enzyme to
saturate the
surface as determined experimentally. Following the adsorption of the enzyme,
the
beads were washed 3 times with buffer to remove the loosely bound material and
exposed to 300 ~,1 of the two-fold dilution series of test compounds (or
control) for 30
min. at the same concentrations described above. The beads were washed and
exposed
to 300 ~.1 ['øC-glucose]-sucrose substrate (100.0 mmol/1 sucrose, final
concentration).
The radiolabeled glucan formed was collected and quantified by scintillation
counting
(Germaine et al., 1974; Venkitaraman et al., 1995). All the solution and
surface assays
were done in quadruplicate in at least 3 different experiments.
The effects of the most active compounds on the activity of GTFs are
shown in the Tables 2 and 3 below. In general, flavonols and flavones reduced
the
activity of alI the enzymes tested in solution (40 to 95% inhibition) and
surface (15 to
60% inhibition) at concentration of 500 p.M. Among them, apigenin (a 5,7,4' -
trihydroxyflavone) displayed the most potent inhibition of GTFs activities.
Apigenin
inhibited 90.5 to 95% of the activity of all GTFs in solution at a
concentration as low
as 500 ~,M (135 ~,g/ml). The inhibitory effect of apigenin on surface adsorbed
enzymes was not as potent as that observed when the same enzymes were in
solution.
Nevertheless, it was an effective inhibitor (30 to 60% inhibition at a
concentration of
500 ~,M). The inhibitory effects of apigenin on GTFs are illustrated in Fig.
1.
Apigenin reduced the activity of all enzymes in solution in direct proportion
to the
amount added in the reaction test (r2 values ranging from 0.92 to 0.99). The
ICSO of
apigenin (the concentration of test compound required to inhibit the enzymatic
activity by 50%) for the GTFs in solution was between 58 (16 ~,g/ml) to 98 ~,M
(26
p,g/ml). The ICSO for surface-adsorbed enzymes were noticeably higher; the
ICso values
for GTF B and C were 478 (128 ~.g/ml) and 458 ~,M (122 ~,g/ml); those for GTF
D
and SS were >lmM. It is conceivable that an ICSO for this agent would not be
achieved
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WO 02/47615 PCT/USO1/49032
-20-
for surface-adsorbed GTFs D and Ss. The surface-adsorbed GTF B and C were
inhibited significantly more at all concentrations tested than GTF D and Ss
(p<0.05).
The flavone baicalein, and the flavonols myricetin and rhamnetin also showed
to be
effective inhibitors of GTFs in solution (70-90% at 500 ~M) and adsorbed on
saliva-
coated hydroxyapatite surface (19-40% at 500 ~.M).
For the compound with highest activity (apigenin), inhibitory curves
(concentration-activity; concentrations ranging from 62.5 ~,M to 1 mM) of all
GTFs
were plotted and ICS° values (the concentration of test compounds
required to inhibit
the enzymatic activity by 50%) were calculated from regression lines
(Copeland,
2000). For this assay, a one-way layout experimental design was used in 4x2x7
factorial scheme (enzyme x state x dose). An analysis of variance was carried
out and
qualitative treatments were compared using Tukey test at level of 5% of
significance
(p<0.05). A non-linear regression was applied in order to evaluate effects of
different
concentrations. The results axe summarized in Figures 2A-B.
CA 02431044 2003-06-06
WO 02/47615 PCT/USO1/49032
s~ r~
N
N l~
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w _ _
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CA 02431044 2003-06-06
WO 02/47615 PCT/USO1/49032
-22-
w
N N I~ ~rw
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cn~ ~ ~" ~n N
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VU r0-i~O O d- ,-,
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v~ ~
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ss,C7 ~ O O
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N
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n n r n n
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a
CA 02431044 2003-06-06
WO 02/47615 PCT/USO1/49032
- 23 -
Example 2 - Determination of Antibacterial Activity
The minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) were determined for each test compound
according
to the National Committee for Clinical Laboratory Standards guidelines
(Tentative
standard M26-T, 1992; NCCLS Publication No. M7-A5, 2000) and I~oo et al.
(2000b). The broth microdilution and macrodilution methods (in TSB) were used
for
the antibacterial tests. The starting inoculum was 5 x 105 CFU/ml and the
concentrations of test compounds ranged from 15.6 to 500 p,M (two-fold
dilutions).
The MICs and MBCs were determined in quadruplicate in at least 3 different
experiments.
The flavanones, dihydroflavonols, and some terpenoids (tt-farnesol and
[3-caryophyllene) tested in tlus study showed moderate inhibitory effects (8-
45% for
GTFs in solution and 7-24% for GTFs on surface at a concentration of 500 ~,M);
the
cinnamic acid derivatives showed negligible effects on GTF enzymes. In some
cases
the activity of GTFs was enhanced, e.g. by cinnamic acid derivatives and some
terpenoids (e.g. protocatechuic acid, terpineol).
The MIC and MBC values of the test compounds for S. mutans (GS-5
and UA 159) and S. sobrinus 6715 are shown in Table 4 below. Some of the
flavanones and dihydroflavonols, as well as tt-farnesol (terpenoid) displayed
antibacterial activity. All flavanones inhibited bacterial growth, among them
pinocembrin was the most effective with MIC values of 250 ~,M (64 pg/ml) for
all
strains tested. Pinocembrin showed bactericidal effect against S. sob~inus
6715 at 500
p,M (128 ~.g/ml). The dihydroflavonol pinobanksin-3-acetate also inhibited the
growth of S. sob~i~cus 6715 and S. mutates strains (MIC values of 500 ~.M or
157
~.g/ml). Among all test compounds, tt-farnesol was the most effective
antibacterial
agent. The MIC values were 125 ~.M (28 ~,g/ml) for S. mutans strains and 62.5
pM
(14 p.g/ml) for S sobrihus 6715. The MBC values were 500 p,M (112 p,g/ml) for
S
mutans strains and 250 ~M (56 ~.g/ml) for S sobrinus 6715. Chlorhexidine
(positive
control) yielded MIC values between 1.l-2.2 ~,M (1-2 ~Cg/ml) and MBC values of
8.9 .
~.M (8 ~,g/ml). Flavonols, flavones and cinnamic acid derivatives did not show
any
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WO 02/47615 PCT/USO1/49032
-24-
antibacterial activity at concentrations used in this assay, with exception to
baicalein
(MIC values of S00 ~,M).
Table 4: MIC and MBC values for Antibacterial Compounds Against
Mutans Streptococci Strainsa
Streptococcus Streptococcus Streptococcus
nautafas UA 159 mutans GS-5 sobri~aus 671
S
Test compounds MICa MBCa MIC MBC MIC MBC
Terpenoid
tt-Farnesol 12S S00 12S S00 62.5 2S0
Flavone
Baicalein S00 >S00 S00 >S00 S00 >S00
Flavanones
Pinocembrin 250 >S00 250 >S00 2S0 500
Sakuranetin >S00 >S00 >S00 >S00 S00 >S00
Isosakuranetin S00 >S00 S00 >S00 250
Dihydroflavonol
Pinobanksin-3-acetate S00 >S00 S00 >S00 2S0 >S00
a Values in gM.
Chlorhexidine (positive control) showed MIC values between 1.1-2.2 ~M (1-2
~g/ml) and MBC value
of 8.9 pM (8 pg/ml).
Kaempferide, kaempferol, galangin, isorhamnetin, rhamnetin, fisetin, rutin,
apigenin, acacetin,
chrysin, luteolin, tectochrysin, ferulic acid, p-coumaric acid, caffeic acid,
(3-caryophyllene, terpineol,
syringaldehyde, protocatechuic acid, vanillin, benzoic acid, pinobanksin,
pinobanksin-7-methyl eter
had MICs greater than S00 ~M.
S
For the compound with highest antibacterial activity, the time kill
studies were performed by broth macrodilution method (National Committee for
Clinical Laboratory Standards, Tentative standard M26-T, 1992). The starting
inoculum of S mutar~s GS-S and UA 159, and S sobrihus 6715 was 1-S x 106
CFU/ml. The final concentration of the antibacterial agent was 4 times MIC (or
MBC
values). Tubes containing the microorganisms and the test compound in TSB were
incubated in S% COZ at 37~C; samples were removed for viable counts at 0, 30
min.,
1, 2, 4, 8, and 24 h. Serial dilutions (10-1 to 10~) were prepared in sterile
0.9% sodium
1 S chloride solution. The diluted sample (SO p1) was plated on to tryptic soy
agar by
means of a spiral plater (Autoplate model 3000, Spiral Biotech, Inc.,
Bethesda, Md.).
The plates were incubated in S% COZ at 37 °C for 48h, when the number
of colonies
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-2S-
was determined. Killing curves were constructed by plotting loglo CFU/ml
versus time
over 24h. All the assays were done in quadruplicate on at least 3 occasions.
Bactericidal effect was defined as a >_ 3 loglo decreases in the CFU/ml from
the
original inoculum. The potential for drug carry-over to produce falsely low
viability
counts was minimized by dilution of inocula and plating of small volumes of
diluted
samples (50 ~1). In addition, no evidence of drug carry-over was detected at
the lowest
dilution used for plating (10-1).
The results of the time-kill kinetic studies are summarized in Fig. 1. tt
Farnesol at four times the MIC (or MBC) rapidly reduced the viable counts of
mutans
streptococci within 30 min. to 1h of incubation (reduction of 1 log of
CFU/ml). tt
Farnesol exerted bactericidal effects (>_ 3 log decrease in CFU/ml) on S
sobri~us 6715
and on S mutans strains between 4 - 8 h incubation. Chlorhexidine at 8.9 p,M
(MBC)
displayed bactericidal effects on mutans streptococci strains tested after 8 h
incubation.
Discussion of Examples 1 and 2
Because dental caries results from events that occur at the tooth
pellicle-plaque interface, with enzymatically active GTFs present in the
pellicle, it is
clearly desirable to determine the effects of potential inhibitors on surface-
adsorbed
GTF.
It has been demonstrated previously that propolis reduced dental caries
in desalivated rats (Koo et al., 1999). The present invention, therefore, was
directed to
the identif cation of specific compounds in propolis that can, alone and/or in
combination, inhibit growth of cariogenic bacteria and the activity of the
GTFs, which
are associated with the pathogenesis of dental caries and other oral
conditions. This
was the first step toward identifying novel inhibitors of GTF enzymes and
mutans
streptococci growth. The results obtained in the present study identified some
of the
compounds that may have been responsible for the previously reported effects
of
propolis on GTFs and bacterial growth (Koo et al., 2000a; Koo et al., 2000b;
Koo et
al., 2000c; Park et al., 1998). In general, flavonoids were the most active
compounds,
displaying distinct biological properties; flavones and flavonols were
effective GTF
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WO 02/47615 PCT/USO1/49032
-26-
inhibitors whereas flavanones and the dihydroflavonol pinobanksin-3-acetate
showed
antibacterial activity.
Apigenin, a 4', 5, 7-trihydroxyflavone, was the most effective inhibitor
of GTFs, especially GTF B and C. Most of the known GTF inhibitors tested so
far,
including currently commercially available mouthrinses, failed to inhibit
effectively
the activities of surface-adsorbed GTFs (Vacca-Smith and Bowen, 1997; blunder
and
Bowen, 1999). In contrast, apigenin greatly inhibited GTF, especially GTF B
and C,
irrespective of whether the enzyme was exposed before or after adsorption to a
surface
at concentration as low as 500 ~M. This level of inhibition has not been
observed
previously (Vacca-Smith and Bowen, 1997; blunder and Bowen, 1999). The
effective
inhibition of GTF B and C by apigenin may affect the pathogenic potential of
dental
plaque related to caries, consistent with a reduction in smooth-surface caries
observed
with mutants of mutans streptococci defective in the production of either or
both
GTFs (Yamashita et al., 1993).
Apigenin is a non-mutagenic flavonoid displaying a variety of anti-
tumor and anti-inflammatory effects (Liang et al., 1999; McVean et al., 2000).
However, the above data is believed to be the first demonstration of apigenin
as a
potent inhibitor of GTFs activity. The exact mechanism by which apigenin and
related flavonoids act to inhibit the GTFs activity is currently unknown,
although the
data reported in this study present some insights for understanding the mode
of their
inhibitory action. Without being bound by theory, it is believed that the
inhibition of
GTFs depends on the molecular structure of flavonoids and the physical state
of the
enzyme. Flavones and flavonols, which have an unsaturated double bond between
positions C2 and C3 (see Table 1), showed remarkable inhibition of GTFs
activity; in
contrast, flavanones and dihydroflavonols, which lack double bond in C2-C3,
exhibited only modest inhibitory activities. Results from previous studies
have shown
that flavones and flavonols axe the main flavonoids related to inhibition of
several
mammalian enzymes, which suggest that C2-C3 double bond may be required for
maximal inhibitory effects (Eaton et al., 1996; Wheeler and Berry, 1986). The
presence of C2-C3 double bond may provide a site for nucleophilic addition by
side
chains of amino acids in GTFs. Several amino acid residues have been
identified as
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essential for the expression of catalytic activity of GTFs, especially
aspartic acid
(Kato et al., 1992; Mooser et al., 199I). It is likely that the side chain of
aspartic acid
(CHZCOOH) may act as a nucleophile and react with flavones and flavonols,
causing
the GTF inhibition. The resistance displayed by surface-adsorbed GTF enzymes
may
be related to conformational changes the GTF undergoes during the adsorption
process, consistent with differences in physical/kinetic properties and
products
synthesized between adsorbed and soluble forms of the enzyme (Kopec et al.,
1997;
Schilling and Bowen, 1988; Venkitaraman et al., 1995).
Several compounds from propolis showed inhibition of mutans
streptococci growth. However, none of them was as potent as chlorhexidine,
which is
a clinically proven antimicrobial. Among the compounds tested, tt-farnesol was
the
most effective antibacterial agent and displayed a rapid decrease in viable
counts of
mutans streptococci. This observation is in agreement with previous reports on
antimicrobial activity of farnesol and related compounds in variety of other
taxa (Bard
et al., 1988; Dionigi et al., 1991). Terpenes such as farnesol have been
reported to
disrupt membrane function, ultimately reducing cell viability (Bard et al.,
1988). It is
noteworthy that streptococci treated with high concentrations of tt-farnesol
(> l OmM)
presented visible membrane disruption in the phase-contrast microscope;
whether the
streptococci membrane was affected at the molecular level by lower
concentrations of
tt-farnesol (e.g. 0.5 mM) needs to be further elucidated. Recently, farnesol
has been
shown to be an antifungal agent, displaying quorum-sensing molecule (QSM)
activity
(Hornby et al., 2001).
The above data supports the hypothesis that the biological activity of
propolis, such as caries prevention (Koo et al., 1999), is related to the
effects of
several compounds, as suggested by Amoros et al. (1992) and Bonhevi et al.
(1994),
rather than a single compound. Apigenin and tt-farnesol are active compounds
in
propolis. The concentrations used in this study may be readily achievable in
the
mouth through topical application. Although details of the toxicology of these
compounds were not studied here, there is no evidence in the literature that
apigenin
or tt-farnesol has any potential cellular toxicity or hemolytic effects.
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Examine 3 - Antibacterial Assays in Biofilms
To complement the data presented in Examples 1 and 2, biofilms of S.
mutans UA159 and S sob~ivcus 6715 were used for time-kill studies. Biofilms
were
formed on standard glass microscope slides in batch cultures for 5 days
(Curran et al.,
1998). Cells of mutans streptococci were grown in tryptone-yeast extract broth
with
addition of 1% (w/v) sucrose at 37°C and 5% COZ. Typically, 5-day-old
biofilms
yield approximately 109 colony forming units (CFU) per slide. The killing
assays were
performed according to Phan et al. (2000). Briefly, 5-day-old biofilms were
exposed
to test agents (1.33 mM, final concentration) in salt solution (50 mM KCI, 1
mM
MGCIz, pH 7.0) containing DMSO:ethanol (10% and 0.625%, v/v) at
25°C. At
specific intervals, the biofilms were removed, suspended in 0.89% NaCI
solution, and
subjected to sonication by a Branson Sonifier 450 (two times, each three 10-
second
pulses with 5-second intervals at 50 watts). This sonication procedure
provided the
maximum recoverable counts as determined experimentally. The homogenized
suspension was serially diluted (10' to 104) and plated on tryptic soy agar or
blood
agar by means of a spiral plater (Autoplate model 3000, Spiral Biotech, Inc.,
Bethesda, Md). The plates were incubated in 5% COz at 37 °C for 48h,
when the
number of colonies was determined. Killing curves were constructed by plotting
logo
CFU/ml versus time over 4h. All the assays were carried out in quadruplicate
on at
least 3 different occasions. Bactericidal effect was defined as a >_ 3 Iog,o
decreases in
the CFU/ml from initial viable counts, at time zero. The potential for drug
carry-over
to produce falsely low viability counts was minimized by dilution of inocula
and
plating of small volumes of diluted samples (50 ~,1). In addition, drug carry-
over was
not detected at the lowest dilution used for plating (10').
The effects of apigenin on the activity of glucosyltransferases (GTFs)
are shown in Fig. 3. Apigenin at 1.33 mM (0.035%) is a potent inhibitor of
GTFs
whether the enzyme is in solution (90-95% inhibition) or on a surface (60-
70%); the
effects of tt-farnesol on enzyme activity were negligible (10-20%).
Chlorhexidine
(CHX) showed only moderate effects (30-45% inhibition in solution and 10-20%
on
surface). In contrast, both tt-farnesol and CHX showed antibacterial activity
against S.
mutans and S sob~inus biofilms as illustrated in Fig. 4. tt-Farnesol at 1.33
mM
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(0.028%) reduced the viable counts of mutans streptococci, showing 1 loglo
decrease
in CFU/ml after 2-4h incubation. CHX at 1.33mM (0.12%) was more effective in
reducing the viability of biofilms than was tt-farnesol (2-3 loglo decrease in
CFU/mL
after same period of incubation). None of the antibacterial agents tested was
completely bactericidal against mutans streptococci biofilms. Apigenin was
devoid of
antibacterial effects against mutans streptococci.
Example 4 - Inhibition of Dental Caries in Animals
The animal experiment was performed as described previously (Bowen
et al., 1988). Seven litters of ten female Sprague Dawley rats aged 14 days
were
purchased with their dams from Charles River (Kingston, NY, USA). Dams were
infected using an actively growing overnight culture of Streptococcus sobrinus
6715.
At weaning, pups age 2I days were randomly placed into six groups and their
teeth
treated topically using a camel hair brush twice daily as follows: 1) 0.028%
tt-
Farnesol (1.33 mM); 2) 0.035% Apigenin (1.33mM); 3) vehicle control (25%
ethanol
containing 1.25% DMSO); 4) 250 ppm F; 5) tt-Farnesol + Apigenin (1.33rnM); and
6) 0.12% Chlorhexidine (1.33 mM). Each group of 11 or 12 animals was provided
with NIH diet 2000 (Keyes, 1959) (which contains 56% sucrose) and 5% sucrose
water ad li8itum. The experiment proceeded for f ve weeks, at which time the
animals
were killed by COZ asphyxiation. The lower left jaw was aseptically dissected,
suspended in 5.0 rnL of sterile saline solution, and sonicated (three 10-
second pulses
with 5-second intervals at 30 watts, Branson Sonifier 450). The suspension was
plated
on mitis salivarius agar plus streptomycin to estimate S sobrinus population
and on
blood agar to determine total cultivable flora (TCF). Dental caries was
evaluated
according to Larson's modification of Keyes' system (Larson, 1981). The
determination of caries score was blind by codification of the jaws and was
done by 1
calibrated examiner. The data were subjected to ANOVA, the Tukey-Kramer HSD
test for all pairs, using software for statistical visualization, JMP version
3.1 (SAS
Institute Inc., I989). Smooth-surface and sulcal caries scores were expressed
as
proportions of their maximum possible values (124 and 56). The level of
significance
was 5%.
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Table 5: Effects of Treatments on Percentage of Streptococcus sobrinus 6715 in
Rats
Treatments Infection by S. sobrihus, %*
1.33 mM tt-Farnesol 44.8 (8.2) a
1.33 mM Apigenin 40.6 (14.2) ab
tt-Farnesol + Apigenin (1.33mM each) 31.3 (11.3) b
250 ppm Fluoride 39.8 (13.2) ab
0.12% Chlorhexidine (1.33 mM) 34.1 (10.8) b
Control 41.5 (8.4) a6
* Means (SD): Values followed by the same superscripts are not significantly
different from each other
(p>0.05). ANOVA, comparison for all pairs using Tukey-Kramer HSD.
The rats remained in apparent good health during the 5-week
experiment. Weight gains were not significantly different among the treatment
groups
(p>0.05). The effects of the treatments on the incidence and severity of
smooth
surface caries are shown in Fig. 5. The tt-farnesol + apigenin (60% reduction,
Keyes's score: 3.3~3.7), fluoride (72%, 2.3~3.0) and chlorhexidine (75%,
2.0~1.9)
groups significantly reduced the incidence of smooth surface caries compared
to
control group (p<0.05). The smooth-surface caries severity scores were
significantly
lower in the groups treated with apigenin, apigenin+tt-farnesol, fluoride,
chlorhexidine (Ds and Dm levels) and tt-farnesol (Dm) than control (p<0.05).
The
incidence and severity of sulcal-surface caries were reduced by fluoride and
chlorhexidine treatments only (p<0.05) as shown in Fig. 6. The combination of
apigenin and tt-farnesol showed significantly better results than individual
compounds
when compared to control (p<0.05). The percentage of S sobrinus recovered from
the
jaws of the rats was calculated from the total cultivable flora and S sobrinus
population. The groups treated with tt-farnesol+apigenin or chlorhexidine
showed the
lowest levels of infection by S. sobr~inus (Table 5), although they did not
differ
statistically from control group (p>0.05).
Glucosyltransferases synthesize glucan, a capsular-like material, which
can protect microorganisms from inimical influences such as antibacterial or
antifungal agents, e.g., farnesol. It is believed that the prevention of
glucan formation
enhances the accessibility of antibacterial or antifungal agents such as
farnesol to
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contact the microorganisms which they are intended to effect, thereby
enhancing their
efficacy.
The data show clearly that apigenin (0.035%, w/v) and, to a lesser
extent, tt-farnesol (0.028 %) exhibit a cariostatic effect on smooth caries
even at low
concentrations. However, neither was as potent as chlorhexidine (0.12%, v/v)
and
fluoride (250 ppm), which axe clinically proven anti-caries agents. Apigenin
and tt-
farnesol displayed distinct biological properties. The anti-caries mechanism
of
apigenin may be related to its exceptional inhibitory effects on GTFs activity
both in
solution and adsorbed onto sHA (Fig. 3). Earlier work has showed that deletion
of
genes controlling the production of GTFs, especially Gtf B and C, resulted in
a
dramatic decline in the virulence of S mutav~s (Yamashita et al., 1992).
Apigenin
effectively inhibited the activity of Gtf B and C enzymes and also showed
cariostatic
properties in the animal based experiment (Fig. 5). This observation is
consistent with
a reduction in smooth-surface caries observed using mutants of mutans
streptococci
defective in production of either one or both GTFs (Yamashita et al., 1992).
It is likely
that apigenin affected the pathogenic potential of dental plaque related to
caries by
reducing the synthesis of extracellular glucans, because it is devoid of
antibacterial
effects on mutans streptococci.
Results from a previous study showed that tt-farnesol exhibits
bactericidal activity in vitro against planktonic cells of S. sobriuus and S.
mutates (I~oo
et al., 2001), however its effects on mutans streptococci biofilms were less
evident
(Fig. 4). It has been shown that biofilms are more resistant to antimicrobial
agents
than cells in suspension (planktonic state), as recently reviewed by Gilbert
et al.
(1997) and Lewis (2001). The modest effect of tt-farnesol (at 1.33mM) against
biofilms may explain its lack of effectiveness to reduce the percentage of S.
sobrihus
infection in rats. Nevertheless, tt-farnesol was able to reduce the severity
of smooth-
surface caxies at Ds level. Terpenes such as farnesol have been reported to
disrupt
membrane function, ultimately reducing cell viability (Bard et al., 1988).
However, a
high concentration of tt-farnesol (< 10 mM) would be needed to disrupt the
streptococci cells membrane (I~oo et al., 2001).
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The combination of apigenin and tt-farnesol was more cariostatic than
either of the compounds alone. The dynamics of the interaction of apigenin and
tt-
farnesol in the reduction of the incidence of dental caries is currently
unexplored,
although the data reported in this study present some insights towards
understanding
the mode of their inhibitory action. It appears that the inhibition of glucan
synthesis
by apigenin reduces the biomass of plaque, and also affects the susceptibility
of the
biofilm to tt-farnesol. This observation could account for the lower level of
S.
sobrinus infection in rats treated with the combination of apigenin and tt-
farnesol
compared to those treated with tt-farnesol only (p<0.05) (Table 5). Continued
research has demonstrated that apigenin, tt-farnesol, and the combination of
apigenin
and tt-farnesol inhibits formation of biofilms. Consistent with the results
reported
above, the combination of apigenin and tt-farnesol was more effective than
apigenin
or tt-farnesol alone.
The positive controls, CHX and fluoride, effectively reduced the
incidence of smooth-surface and sulcal surface caries, thereby supporting the
validity
of the selected model. The anti-plaque effect of CHX has been largely
attributed to its
antimicrobial activity and oral substantivity; it is a cationic substance that
binds to
soft and haxd tissues of the mouth, as well as to bacterial cell walls (Rolla
and Melsen,
1975; Jones, 1997). In the present study, CHX reduced the viability of mutans
streptococci biofilms (Fig. 4), although it was not bactericidal. This data
confirms the
findings in the animal experiment where lower levels of S. sobrinus infection
were
detected in the group treated with CHX. Fluoride (250 ppm) was devoid of
detectable
antimicrobial and anti-GTF activity as measured here, although it may reduce
acid
tolerance and acid production of S. muta~s (Marquis, 1990; Belli et al.,
1995).
, Nevertheless, fluoride is the most effective anti-caries agent known to date
(Rolla et
al., 1991; Clarkson et al., 2000). There is a consensus that the main effect
of fluoride
is to interfere physicochemically with caxies development by reducing
demineralization and enhancing remineralization of dental enamel (Dawes and
ten
Cate, 1990). Thus, substances, which act on virulence factors and/or
metabolism of
caxiogenic bacteria, may increase the anticariogenic effect of fluoride;
apigenin could
be such an agent.
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The above data show clearly that two natural compounds from propolis
were cariostatic in the animal model, even at low concentrations. Apigenin was
shown
to be the first natural cariostatic agent based on its ability to inhibit
GTFs; it is a
promising anti-caries compound which has a distinct mechanism of action
compared
to other clinically proven agents.
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Each of the references listed below is hereby incorporated by reference
in its entirety into the specification of this application.
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Although the invention has been described in detail for the purpose of
illustration, it is understood that such detail is solely for that purpose,
and variations
can be made therein by those skilled in the art without departing from the
spirit and
scope of the invention which is defined by the following claims.
