Note: Descriptions are shown in the official language in which they were submitted.
a
'"~ 294874
178-llo cIP
1
BACKGROUND OF THE INVENTION
The present application is a continuation-in-part
Application of U.S. Patent Application Serial No.
07/654,073, filed February 1l, 1991.
The present invention relates to methods of inhibiting
dental caries. In particular, the present invention
relates to preventing dental caries using non-antibacterial
tetracyclines.
For several decades now, there has been an increased
, awareness of the relationship between plaque formation on
tooth surfaces and dental cavities or caries. Dental
plaque is generally regarded as a film of bacteria,
bacterial polymers, salivary polymers, remnants of
epithelial cells and leukocytes. The bacteria, principally
Streptococcus mutans, is part of the naturally occurring
microflora of the oral cavity. The bacteria use natural
sugars such as sucrose and glucose included in the diet as
a nutrition source and produce cement-like polymers which
bind to the enamel tooth surface. Once bound, the bacteria
cause demineralization of the enamel by secreting acids and
ultimately enamel caries.
Although the roots of teeth lack enamel, plaque
formation may also be found below the gingival margin. The
roots of teeth contain cementin and dentin which are more
bone-like than tooth enamel. In addition, the roots are
also susceptible to breakdown by the collagenase produced
by Bacteroides c,Lng~ivalis and some other bacteria, a common
part of oral cavity microflora. Root plaque formation
ultimately leads to root caries, a leading cause of tooth
loss in adults. Root caries can be especially prevalent
when, due to periodontal disease, the gums and alveolar
bone both recede and expose the roots.
-, 2094874
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1 In the past, most efforts aimed at reducing plaque
formation and dental caries have included reducing sugar
intake, regular brushing, flossing and periodic removal of
the plaque by dental professionals.
In some cases, plaque formation on tooth surfaces may
become excessive and even pathologic. In these situations,
it is often necessary to institute prophylactic measures in
addition to those described above.' In the past, broad
spectrum antibiotics such as tetracyclines and
metronidazole have been used in the treatment of
periodontal disease to reduce oral cavity microflora, which
is the most virulent aspect of plaque and caries formation.
Although antibiotic agents are effective in reducing the
number and amount of bacteria responsible for plaque and
caries formation, extended periods of antibiotic
administration are avoided due to high incidences of side
effects. Side effects most often associated with long-term
antibacterial agent usage include intestinal disturbances,
overgrowth of yeast and fungi, and most importantly, the
development of antibiotic-resistant bacterial.
As stated above, tetracyclines are broad spectrum
antibiotics and are active against most oral cavity
microflora. The tetracycline compound exhibits the
. following general structure:
t!0 ~ , C113' N(C(!3) z.
Utl
:0N112
~u ~ vu
2094874
-3-
1 The numbering system of the ring nucleus is as follows:
~ ~ 5a 4a
a 5 4
3
9 0 11 12 1 2
12a
The tetracycline molecule is amenable to substantial
modification without losing its antibiotic properties.
Examples of modifications that may and may not be made to
the basic tetracycline structure have been reviewed by
Mitscher in the rhpmistry of Tetrac~clines, Chapter 6.
According to Mitscher, the substituents at positions 5-9 of
the tetracycline ring may be modified without complete loss
of antibiotic properties. Changes to the basic ring system
or replacement of the substituents at positions 1-4 and 10-
12, however, generally lead to synthetic tetracyclines
having substantially less or effectively no antibacterial
activity. For example, 4-dedimethylaminotetracycline is
commonly considered to be a non-antibacterial tetracycline.
Various properties of antimicrobial and non-
antimicrobial tetracyclines are known. For example, it is
. known that antimicrobial and non-antimicrobial
tetracyclines can bind to metal ions such as calcium.
. Tetracyclines are also known inhibitors of collagen
destructive enzymes such as mammalian collagenase, a
calcium dependent zinc-metalloproteinase. Collagen is a
major component of connective tissue matrices such as those
in the bone, synovium, eye, skin, tendons, gingiva,
cementum and dentin of the tooth, but not tooth surface
enamel.
2094874
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1 U.S. Patent No. 4,666,897 to Golub, et al. discloses
tetracyclines, including commercially-available
antimicrobial forms of the drug, inhibit excessive bone
resorption and collagenolytic enzyme activity. U.S. Patent
No. 4,704,383 to McNamara, et al. discloses tetracyclines
having substantially no effective antibacterial activity
inhibiting collagenolytic enzyme activity in rats.
Moreover, McNamara, et al. also disclose that ~non
antimicrobial tetracyclines reduce bore resorptiori in organ
culture.
Although some oral hygiene products such as
dentifrices have been introduced to combat dental plaque
and caries, a complete solution remains elusive.
In view of the desire to reduce caries and further in
view of the desire to avoid using antimicrobial antibiotics
to accomplish this result, it is an object of the present
invention to provide an improved method of inhibiting the
effect of caries on tooth surfaces.
It is a further object of the present invention to
provide a method of inhibiting plaque and caries formation
on tooth surfaces using non-antibacterial tetracyclines.
SUMMARY OF THE INVENTION
In accordance with the present invention, it has been
surprisingly found that tetracyclines inhibit plaque and
caries formation on tooth surfaces in a manner completely
separate from antibacterial eradication of the oral_ cavity
microflora. The method includes contacting the tooth
surfaces with an effective amount of a non-antimicrobial
tetracycline which results in the prevention of plaque and
caries formation on tooth surfaces.
2094874
4a ~ 73802-25
The present invention also provides a use of an
effective amount of a non-antimicrobial tetracycline to treat a
mammalian tooth surface to prevent root caries.
The present invention further provides a confection for
treating mammalian tooth surfaces to prevent root caries
comprising an effective amount of a non-antimicrobial tetracycline
in admixture with a suitable diluent or carrier.
The present invention yet also provides a confection for
treating mammalian tooth surfaces to prevent adhesion of root
plaque comprising an effective amount of a non-antimicrobial
tetracycline in admixture with a suitable diluent or carrier.
The present invention also provides a use of a
confection described above to treat a mammalian tooth surface to
prevent root caries or to prevent the adhesion of root plaque.
The present invention further provides a commercial
package containing as active ingredient a confection described
above together with instructions for the use thereof to treat a
mammalian tooth surface to prevent root caries or to prevent the
adhesion of root p:Laque.
'~ ~Q9487~
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1 The non-antimicrobial tetracyclines useful in the
present invention are preferably chemically modified
tetracyclines (CMT's) such as dedimethylaminotetra-
cyclines. Examples of such preferred tetracyclines include
4-dedimethylaminotetracycline, 12a-deoxydedimethylamino-
tetracycline and its derivatives, 4-dedimethylamino-5-
oxytetracycline, 6-deoxy-6-demethyl-4-dedimethylamino-
tetracycline, 7-chloro-6-demethyl-4-de.dimethylamino-
tetracycline and 6-a-deoxy-5-hydroxy-4-dedimethylamino-
tetracycline. Other suitable CMT's include, for example,
6-a-benzylthiomethylenetetracycline, the mono-N-alkylated
amide of tetracycline, 6-fluoro-6-demethyltetracycline,
tetracyclinonitrile, 4-hydroxy-4-dedimethylaminotetracy-
cline and lia-chlortetracycline.
The amount of the non-antimicrobial tetracycline used
in the methods of the present invention may be generally
described as that amount which effectively inhibits plaque
and/or caries formation on tooth surfaces. For example, a
non-antimicrobial tetracycline, may be included in
dentifrices, mouthwashes or similar oral hygiene
preparations in amounts ranging from about 10 mg% to about
100 mg%. In a preferred embodiment, the non-antimicrobial
tetracycline is included in amounts of from about 15 mg% to
about 25 mg%, with concentrations of about 20 mg% being
most preferred. When contacting tooth surfaces at these
concentrations and for time periods typical for the oral
hygiene product selected to contain the non-antimicrobial
tetracycline, the non-antibacterial tetracyclines described
herein prevent caries formation on tooth surfaces.
Naturally, the amount of the various tetracycline analogues
will vary somewhat from each other and the ranges is set
forth above are only illustrative of all possible dosage
choices. Those skilled in the art will determine optimal
concentrations for the desired non-antimicrobial
tetracycline from clinical experience in order to carry out
the present method.
209487
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As a result of the present invention, significant
improvements in oral hygiene are realized. The present
invention prevents plaque formation and reduces caries
effects on the enamel portion of tooth surfaces and also on
the root surfaces below the gingival margin. Thus, the
present invention inhibits steps in the pathologic process
of tooth decay. Moreover, the prophylaxis is achieved
without using antimicrobial agents. Thus, the oral
microflora remains intact. Antimicrobially=resistant
strains of organisms, gastrointestinal disturbances,' yeast
and fungi overgrowth which are associated. with
antimicrobial therapy are also beneficially avoided.
For a better understanding of the present invention,
together with other and further objects, references made to
the following description and its scope will be pointed out
in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a photograph showing the gum area,
gingival margin and lower incisors of a non-diabetic
control Osaka Dental University (ODU) rat.
Figure 2 is a photograph showing the gum area,
gingival margin and lower incisors of an untreated diabetic
ODU rat with extensive plaque formation on the incisors and
on the gums.
Figure 3 is a photograph showing the gum area,
gingival margin and lower incisors of a diabetic ODU rat
after having been treated with oral daily doses of the
antimicrobial agent, tetracycline hydrochloride.
'~ za94s7~
1 Figure 4 is a photograph showing the gum area,
gingival margin and lower incisors of a diabetic ODU rat
after having been treated with oral daily doses of the non-
antimicrobial tetracycline, 4-dedimethylamino-
tetracycline.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, methods for
preventing dental plaque and reducing caries are disclosed.
The method includes contacting tooth surfaces with an
effective amount of a non-antimicrobial tetracycline which
prevents plaque formation.
The non-antimicrobial tetracyclines useful in carrying
out the method of the present invention may be described as
chemically modified tetracyclines (CMT's). For purposes of
the present invention, CMT designates a tetracycline
molecule which has been modified to essentially eliminate
antimicrobial efficacy. Methods for altering and
eliminating the antimicrobial efficacy of a tetracycline
are disclosed in the ~lhemistry of Tetracyclines, Chapter 6,
Mitscher, Ed., at page 211. As pointed out by Mitscher,
modifications of the tetracycline molecule at positions 1,
2, 3, 4, 10 and 12a can lead to loss of antimicrobial
activity.
Examples of such preferable tetracyclines include
those lacking the dimethylamino side chain at position 4.
Such chemically modified tetracyclines (or CMT's) include,
for example, 4-dedimethylaminotetracycline, 4-
dedimethylamino-5-oxytetracycline, 4-dedimethylamino-7-
chlortetracycline, 4-hydroxy-4-dedimethylamino-
tetracycline, 6-demethyl-6-deoxy-4-dedimethylamino-
tetracycline, 6-a-deoxy-5-hydroxy-4-dedimethylamino-
tetracycline, and 12a-deoxydedimethyl as described by
Green, et al., J. me Chem. oc , 82, 3946-3953 (1960).
X094874
_8_
1 Further examples of tetracyclines modified for reduced
antimicrobial activity include 6-a-benzylthiomethylene
tetracycline, the mono-N-alkylated amide of tetracycline,
6-fluoro-6-demethyltetracycline, ila-chlortetracycline and
tetracyclinonitrile.
The amount of the tetracycline required to inhibit
dental caries is an amount which is effectively ~non-
antimicrobial yet is effective in inhibiting plaque and/or
caries formation. The amount of non-antimicrobial
tetracycline may also be described as a range. The highest
amount is that amount which does not cause clinically
detrimental side effects. For the purpose of the present
invention, side effects would include any untoward reaction
which would clinically warrant ceasing the tetracycline's
administration. Such side effects include, for example,
symptoms of toxicity. The lowest amount is that minimum
amount which produces the desired anti-plaque and/or anti-
caries result.
For illustrative purposes, non-antimicrobial
tetracyclines can be included in vehicles such as
dentifrices, mouthwashes, chewing gums, lozenges,
confections or other suitable dental hygiene preparations.
In such embodiments, the tetracycline may be included in an
amount of from about 10 mg% to about 100 mg%. In a
preferred embodiment, the non-antimicrobial tetracycline
may be present in amount of from about 15 mg% to about 25
mg%. In a most preferred embodiment, the non-antimicrobial
tetracycline is present in an amount of about 20 mg%. The
method of the present invention may then be carried out by
using one of the dental hygiene products described above
containing a non-antimicrobial tetracycline to contact the
tooth surfaces for a sufficient time to inhibit plaque
formation and/or dental caries.
~0948'~4
-g-
The time required for the non-antimicrobial
tetracycline to contact the tooth surface and effectively
inhibit plaque and/or caries formation is conveniently the
same amount of time as one is accustomed to for using oral
hygiene products. For example, if the non-antimicrobial
tetracycline is included in a toothpaste, normal brushing
one to three times daily is sufficient. Similarly,
contacting the teeth with an oral rinse .containing the
tetracycline far normal periods of around a minute followed
by expectorating one to three times daily would achieve the
same result.
Tests were conducted using the method of the present
invention s prevention of plaque and caries formation on
tooth surfaces. The tests demonstrate the effectiveness
and unexpected ability of non-antimicrobial tetracyclines
to prevent plaque adhesion which results in caries on tooth
surfaces without antibiotic effect on oral flora.
EXAMPLES
The following Examples serve to provide further
appreciation of the invention, but are not, in any way, to
be considered restrictive of the effective scope of the
invention.
EXAMPLE I
In this Example, the lack of antimicrobial effect of
CMT on crevicular microflora was demonstrated using a group
of six adult Osaka Dental University (ODU) rats. When
rendered diabetic, these rats exhibit large plaque
accumulations on their lower incisor teeth. Two rats were
preserved as nondiabetic controls, while the remaining four
c
-10-
rats were made diabetic by injection of 70/mg/kg of the
diabetogenic agent streptozotocin according the method set
forth, for example, by Golub et al. in Infect. Immun..~7:
1013 (1982).
The diabetic rats were then further divided into two
groups. One group was given 57 mg/kg/day of the CMT, 4-
dedimethylaminotetracycline, which was incorporated into
the rats daily food intake. The second diabeticgroup was
left untreated.
After 21 days, the rats were sacrificed and samples of
the crevicular microflora were removed from each of the
rats in all groups and transferred to a brain-heart
infusion-supplemented broth and incubated for 72 hours at
37°C.
At the end of this incubation period, the microflora
from each group of rats was compared to determine if any
changes in the microflora were attributable to 4-
dedimethylaminotetracycline. The results are summarized in
Table 1.
Table 1 shows the presence and/or absence of various
bacteria found in the microflora of the oral cavity of the
ODU rats. Changes in the makeup of the microflora found in
each group are shown below.
35
2Q948'~~~
1 FABLE 1
Non- Diabetic
Diabetic
4-dedimethyl-
Organisms Control Untreated
aminotetracycline
co i + + , ~ + .
Bacteroides + - -
Fusobacterium + + +
Proteus + -
Veillonella + + +
Leptotrichia + - -
Streptococcus -
alpha hemolytic - - -
beta hemolytic - -
gamma hemolytic + + +
Actinomyces + + +
Lactobacillus + - +
Staphylococcus - + +
Bifidobacterium + - -
Candida - C) -
+ = present
- = not present
Referring now to Table 1, the results of the isolation
and identification of the organisms show that the untreated
diabetic control animals and those treated with 4-
dedimethylaminotetracycline had essentially the same oral
flora. Moreaver, the bacterial composition and amount of
growth obtained from both the diabetic control rats and the
diabetic rats receiving 4-dedimethylaminotetracycline were
~u~4s~:~
-12-
1 essentially indistinguishable. These results, therefore,
demonstrate the lack of antibacterial activity associated
with 4-dedimethylaminotetracycline.
Thus, it can be seen that CMT's such as
dedimethylaminotetracycline have essentially no
antimicrobial effect on the crevicular microflora.
Moreover, CMT's also demonstrate essentially no microflora
altering effects. Therefore, any prevention of plaque
adhesion to tooth surfaces by CMT's cannot be attributed to
antimicrobial or microflora altering properties of the CMT.
In this Example, the ~ v vo effect of non-
antimicrobial tetracyclines on plaque accumulation on
incisors was demonstrated. Thirty-two adult male ODU rats
were distributed into four groups of eight. As was done in
Example I, one group of rats was preserved as a non-
diabetic control and the remaining rats were made diabetic
by injection with streptozotocin. One group of diabetic
rats was not treated further and designated the diabetic
control. The second group of diabetic rats was given
approximately 20 mg of the antimicrobial tetracycline
hydrochloride in their daily meal. The last group of
diabetic rats was treated by including approximately 20 mg
of CMT as part of their daily meal.
After a 3-week protocol, the rats were anesthetized
with halothane. The jaws of each rat were gently propped
open, and the lower incisor teeth were flushed with
distilled water to remove loose debris. The teeth were
then stained with a plaque-disclosing solution (erythrocin
R) to determine the presence of plaque on the incisor. The
results are set forth below and in Figs. 1-4.
~0948'~4
-13-
1 RESULTS
Referring now to Fig: 1, it can be seen that the non-
diabetic control ODU rats showed no detectable plaque
accumulation on their lower incisors at or near the
gingival margin. As can be seen in Fig. 2, the untreated
diabetic ODU rats showed large accumulations of dental
plaque on the incisors extending to and. including the
gingiva. In Figs. 3-4, it can be seen that the rats in
both the antimicrobial and non-antimicrobial tetracyaline-
treated groups showed significant reductions in plaque
accumulation on the lower incisors when compared to the
untreated diabetic group. It was also observed that the
lower incisors of the rats in the dedimethylamino-
tetracycline-treated group greatly resembled those of the
non-diabetic control rats which normally do not develop
excessive plaque formation on their tooth surfaces.
It can be seen, therefore, that non-antimicrobial
tetracyclines inhibit microbially-mediated dental plaque
accumulation on teeth ~ yivo by a mechanism independent of
antimicrobial properties of tetracyclines.
In this Example, further evidence of non-antimicrobial
tetracyclines ability to inhibit plaque formation was
demonstrated. Tiles made of polymethylmethacrylate, which
is used for dentures, and has a surface which approximates
the enamel of teeth for plaque adherence purposes were
selected. Ten tiles were incubated in test tubes with a
plaque-forming solution containing sucrose, fresh saliva,
K+, Ca++, Na+, C1' and F' ions, a buffer containing Na2 HP04
7H20 NaHC03, and a culture of oral cavity microflora
obtained from a patient at the University of New York
Dental School Clinic at Stony Brook, New York which had
been incubated for three days at 37 °C. Half of the test
X094874
-14-
1 tubes were incubated with 20 mg% or 0.02% by weight of 4-
dedimethylamino-tetracycline to demonstrate its
effectiveness against plaque formation, while the other
half were maintained as untreated controls. After the 3-
day incubation, each tile was removed from the test tube
and all non-adherent material was rinsed away with
distilled water. The tiles were then air-dried and any
bacterial plaque adhering to the tiles was stained by
dipping the tiles into a solution of Basic Fuchsin. The
amount of stain on the tile provides a direct, correlation
to plaque formation. Each tile was then destained in a
solution of 5% EDTA in 50% 2-propanol. An aliquot of the
destaining solution was then measured for light absorbence
at 550 nm in a Spect. 70 colorimeter and compared to an
aliquot from the untreated tiles. The results are set
forth below in Table 2.
SABLE 2
Spect. 70
colorimeter Absorbance
Untreated 0.513
Tiles
Treated 0.330
Tiles
(0.02% CMT)
Difference 36%
As can be seen from the above Example, the method of
the present invention provides significant advantages and
improvements in the inhibition of plaque formation on tooth
surfaces. Furthermore, by contacting tooth surfaces with
an effective amount of a non-antimicrobial tetracycline,
bacterial plaque formation can be significantly inhibited
in the oral cavity.
20948'
-15-
EXAMPLE IV
In this Example, the ~ vo effect of non
antimicrobial tetracycl'nes 'n r venting dental root ~:'~~~
caries was demonstrated.~~:e~~~a~p cif is pathogen-free ,
adult rats were distributed in four rou s. As was done irf
g P
the previous examples, one group of rats was preserved as ~l
a control group (normal control). The second group of rats
was an untreated infected control group. A third group of
rats was given 5 milligrams of the antimiarobial
tetracycline, doxycycline, in their daily meal. The.fourth
and final group was treated by including 5 milligrams of
the chemically-modified tetracycline, (CMT), 4-dedimethyl-
aminotetracycline. Prior to treatment, all of the animals
in the group were infected with ]Bacteriodes aingivalis, a
commonly-found oral bacterium responsible for caries.
After 42 days, the rats were examined for the presence
of root caries. The jaws were stained, examined and
inspected.
30
'~ 209484
-16-
1 Darker staining areas indicate greater demineralization of
the cementum, which represents root caries. The results
are set forth in Table 3, below.
TABLE 3
ging~ivalis induced root caries: Effect of doxycycline
and 4-dedimethylaminotetracycline.
Root Surfaces Root Surfaces Root Caries
as
Group with Recession with Caries % of Recession
Normal
Control 0 0 0
Untreated
Control 42 18 42.8
Doxycycline 3 0 0.0
CMT 4 0 0.0
Both the antimicrobial and non-antimicrobial
tetracyclines prevented the development of root caries.
The doxycycline eliminated the ~. g~givalis and caries by
antibacterial action. More dramatic, however, was the fact
that CMT was equally effective in preventing caries in
spite of the lack of antibacterial activity. Thus, it can
be seen that. non-antimicrobial tetracyclines not only
inhibit dental plaque accumulation on teeth, but also can
prevent dental caries. The non-antibacterial action,
therefore, affords the artisan with a valuable alternative
to antimicrobial therapy.
2094874
,~,XAMPLES V-IX
In these Examples, various oral hygiene products
containing non-antimicrobial tetracyclines are set forth.
In each of the products, the term "CMT" is used to
designate a chemically modified tetracycline such as a
dedimethylamino- tetracycline which essentially lacks
antimicrobiah activity. Each of the following illustrative
products is useful in providing a vehicle for allowing an
effective amount of the tetracycline to contact the tooth
surface and thereby inhibit plaque formation.
20
30
. . , . ;,
20948'4\'
-18-
1 EXAMPLE V
Tooth Powder
;ngredient wt %
Silica hydrogel 96.10
Zinc chloride 0.50
Sodium fluoride 0.22
Sodium gluconate . 0.27
Synthetic sweetener
(saccharin/aspartame) 0.50
Sodium methyl cocoyltaurate 1.50 .
Flavoring 0.80
C~ 0.01-0.10
EXAMPLE VI
j~o z eng~e
Ingredient
Sorbitol powder 74.50
Corn syrup 15.00
Zinc chloride 0.50
Sodium fluoride 0.22
Flavor and color 1.15
Sodium gluconate 0.30
Synthetic sweeteners 0.20
Tableting lubricant 5.00
Deionized water 3.00
0.01-0.10
35
20948'4
-19-
EXAMPLE VII
ChewiHg Gum
Ingredient wt %
Gum base 30.00
Sorbitol 48.85
Corn syrup 15.00
Flavor 1.50
Zinc chloride . 0.50
Sodium fluoride ' 0.22
Sodium gluconate 0:30
Gum tragacanth 0.50
Deionized water 3.00
0.01-0.10
EXAMPLE VIII
Dentifrice Compositio n
ingredient wt %
Glycerin 25.00
Zeo 498 (Silicone Dioxide) 21.50
HMP (Hexaphos) 6.00
Syloid 244 (synthetic silica) 3.00
Sodium Lauryl Sulfate 1.20
Flavor 1.00
Sodium Hydroxide (50% Solution) 1.00
Xanthan Gum 1.00
Sodium Benzoate 0.50
Titanium Dioxide 0.50
Sodium Saccharin 0.30
Sodium Fluoride 0.22
CMT 0.01-0.10
Deionized water to Q.S. 100
z~~~s7~
-20-
1 EXAMPLE IX
Mouthwash
Incrredient wt %
Ethyl Alcohol 15.0
Glycerol 10.0
Flavor 0.4
Sodium saccharin , 0.03
Sodium Fluoride ' 0.05
pluronic F 108 2.0
0.01-0.10
Deionized Water to Q.S. 100
Other formulations for self-treatment as well as
professional treatment can be provided by skilled artisans.
The present invention provides a highly effective and
reliable anti-plaque and anti-caries agents and treatments
which can be used without the side effects associated with
antibacterial tetracyclines.
While there have been described what are presently
believed to be the preferred embodiments of the present
invention, those skilled in the art will realize that
changes and modifications may be made thereto without
departing from the spirit of the invention, and it is
intended to claim all such changes and modifications as
fall within the true scope of the invention.
35
