Note: Descriptions are shown in the official language in which they were submitted.
QM 34589
133S3S2
ORAL HYGIENE COMPOSITION
This invention relates to oral hygiene compositions
and to methods of using such compositions to prevent or
inhibit growth of bacteria on tooth surfaces.
The prevention of the adherent deposition of
dental plaque on mammalian (particularly human) teeth
is a highly desired result. Dental plaque results when
cariogenic and other types of bacteria aggregate in
colonies on the surface of teeth and form a deposit
which adheres tenaciously to the surface. It is believed
that the deposition of plaque on the surface of a tooth
is one of the first steps in the development of dental
caries and periodontal disease.
Many attempts have been made to prevent the
deposition of plaque on tooth surfaces and to effect
removal of plaque from such surfaces. For example,
brushing, dental flossing and the use of oral irrigators
and interdental stimulators have been tried. Such
treatments are not, however, entirely successful and must
often be supplemented with periodic treatment by dental
professionals.
In our recently published European Patent
Specification No 0,182,523A, we teach that certain
pharmaceutical compositions (as therein defined)
are highly effective for preventing or significantly
reducing (a) the colonisation of tooth surfaces or
simulated tooth surfaces by cariogenic and other micro-
organisms commonly found in an oral environment, and (b)
the adherent deposition on tooth surfaces of dental
plaque resulting from such microorganisms.
The preparation of polymers for use in the
aforesaid certain pharmaceutical compositions is
described in EP 0,182,523A.
,.
`.,
~ 2 - 1335352
Chlorhexidine is a cationic antiseptic which has
been widely used by the medical profession as a topical
antibacterial agent for more than 20 years; the
preparation thereof is described in UK 705,838. It has
been reported (Loe et al, Journal of Periodontal
Research, 1970, Vol 5, pp79-83) that chlorhexidine can be
used as an antiseptic in the oral environment and that
in certain circumstances there is a tendency apparently
for chlorhexidine to stain teeth. Such staining appears
to be a property which is common to cationic antiseptics.
It has been shown (Addy et al, Journal of Periodontal
Research, volume 9, pp 134-140) that such staining is the
result of an interaction between the cationic antiseptic
and dietary components, particularly those rich in
t~nnins, e.g. coffee, tea or red wine.
We have now found that where certain surfaces, e.g.
tooth, or hydroxyapatite, are treated with a combination
of both (a) a
polymer, as hereinafter defined, which bears certain
pendant polyalkylene oxide chains, and (b) a cationic
antibacterial agent, the resulting treated tooth surfaces
surprisingly exhibit both enhanced antiadhesive and
antibacterial properties to certain oral micro-organisms,
i.e. the combination may effect control of so-called
"bio-fouling" of tooth surfaces.
Indeed, in the presence of the said polymer, equivalent
antibacterial effects can be observed where the tooth
surface is treated with a lower concentration of a
solution of the antibacterial agent. Furthermore, we
have now found, surprisingly, (i) that the aforesaid
tendency for st~;ning with chlorhexidine is at least
reduced, there is often no increase in st~i ni ~g even
where more chlorhexidine is adsorbed on the tooth surface
in the presence of the aforesaid polymer; (ii) the
antibacterial properties of certain cationic
~ 3 ~ 1335352
- antibacterial agents, e.g. chlorhexidine and alexidine,
are increased on simulated tooth surfaces if the surface
is treated sequentially with or with a a combination of,
an acidic polymer, e.g. Polymer 93W (as hereinafter
defined)and the aforesaid agent ; and (iii) where
composite restorations, e.g. Occlusion (RTM), Opalux,
Silux and Valux P50, etc, tend to be stained by cationic
antiseptics such staining is at least alleviated in the
presence of the aforesaid polymer. It will be
appreciated that reduction at least of such staining of
anterior teeth is particularly desirable.
According to the present invention there is
provided an oral hygiene composition comprising
(i) an effective amount of a cationic antibacterial
agent; and
(ii) an effective amount of at least one polymer ,as
hereinafter defined, which has one or more pendant
polyalkylene oxide groups.
As examples of cationic antibacterial agents which
may be used in the present invention may be mentioned
inter alia benzalkonium chloride, bispyridinamines, e.g. ~-
octenidine, or preferably a poly-biguanide, e.g.
alexidine, or more preferably a bis-biguanide, eg
chlorhexidine.
By "polybiguanide" we mean a compound which has a
plurality of in-chain biguanide residues of the general
formula I NH NH
Il 11
-NH-C-NH-C-NH-
or tautomers thereof. Often there are two, three or four
such in-chain residues in the antibacterial agent used in
the present invention. However, we do not exclude the
possibility that there may be sufficient to provide at
least a major portion of the repeat units of a higher
_ 4 - 1335352
molecular weight polymer, e.g of molecular weight up to
about 10,000.
It will be appreciated that where a polybiguanide
is used in the present invention it may be present as a
free base; preferably, however, it is present as a salt
thereof, e.g. acetate or hydrochloride, or more
preferably, particularly where the polybiguanide is a
bis-biguanide which has the structure shown in general
Formula II, as the di-gluconate, i.e. the di-gluconate
of 1,6-di(4-chlorophenyl-diguanido)hexane, which is known
in the art as chlorhexidine.
NH NH
Cl ~ NH-C-NH-C-NH(CH2)6NH-C-NH-C-NH ~ Cl II
NH NH
We do not exclude the possibility that where a
polybiguanide, e.g. chlorhexidine, in the form of a free
base is used in the present invention it may be in
admixture (e.g. as a salt) on the tooth surface as a salt
with an acidic polymer as hereinafter defined.
As possible explanations, without wishing to be
bound thereby, of the increased antibacterial effect of
the oral hygiene composition of the present invention, we
suggest:
(i) an increase in the quantity of chlorhexidine
adsorbed on the tooth surface; and/or
(ii) alteration in the strength of adsorption of
chlorhexidine at the tooth surface such that it is
more available to exert its antibacterial
effect at the surface; and/or
(iii) alteration in the orientation at the tooth surface
of adsorbed chlorhexidine such that the
_ 5 - 1 335352
antibacterial groups thereof are more accessible to
bacteria approaching thereto; and/or
(iv) ion-pairing between the cationic antibacterial
S agent and the acid anions, where present, of the
polymer.
In the composition according to the present
invention, multiple hydrogen-bonds between the polymer,
and the biguanide cations may contribute to the aforesaid
increase in quantity or alteration in strength of
adsorption or orientation.
Polymers of which the oral hygiene composition
according to the present invention are comprised are
preferrably acidic, by which we mean that there is at
least one carboxylic acid group appended to the polymer
backbone. However, we do not exclude the possibility that
the polymer may be amphoteric, basic or neutral, although
this not preferred.
The one or more pendant polyalkylene oxide groups
appended to the polymers of which oral hygiene
compositions according to the present invention are
comprised are preferably ethylene oxide groups.
However, we do not exclude the possibility that at least
a portion thereof may be alternative poly(lower)-
alkylene oxide groups, e.g. polypropylene.
As examples of polymers of which the oral hygiene
composition according to the present invention are
comprised may be mentioned inter alia polymers which
comprise one or more repeating units of general structure
A
1335352
' -
~ ~
(Co2H) p\
and one or more repeating units of general structure B
~~~
M
( (ZO)nR3)q
wherein X, which in the repeating units of structure A
may be the same or different, and Y, which in the
repeating units of structure B may be the same or
different, are hydrocarbyl, or substituted
hydrocarbyl residues, providing a backbone for the
polymer;
Z is -CHR1-CHR2-or-(CH2)m~; wherein, where Z is
-CHR'-CHR2-,
R1, which in the same repeating unit of structure B
(when n or ~ is 2 or more) or in different
repeating units of structure B may be the same or
different, is hydrogen or a hydrocarbyl group;
and
R2, which in the same repeating unit of structure B
(when n or ~ is 2 or more) or in different
repeating units of structure B may be the same or
different, is, hydrogen or a hydrocarbyl group;
except that R1 and R2 in a single
unit-CHR1-CHR2-O- cannot both be hydrocarbyl;
13353S2
R3, which in the same repeating unit of structure B
(when q is 2 or more) or in different repeating
units of structure B may be the same or
different, is hydrogen or a hydrocarbyl group or
an acyl group derived from an alkanoic acid
having up to five carbon atoms;
m, where present, is a number of from 2 to 10;
n is a number of from 1 to 60;
p is a number of from 1 to 4; and
q is a number of from 1 to 4;
each (CO2H) group is joined via an intermediary or
intermediaries L to the hydrocarbyl residue X, and
in cases where p is 2 to 4 may be joined by L to
the same or different carbon atoms of X;
L may be the same or different in the repeating units of
structure A and is selected from one or more direct
links and one or more groups of atoms each group
providing a chain of one or more atoms for linking
a (CO2H) group with X, except that more than two
(CO2H) groups cannot be directly linked to the same
carbon atom in X;
each ((ZO)nR3)q group is joined via an intermediary
or intermediaries M to the hydrocarbyl residue Y,
and in cases where q is 2 to 4 may be joined by M
to the same or different carbon atoms of Y;
M may be the same or different in the repeat units of
structure B and is selected from one or more direct
links and one or more groups of atoms each group
providing a chain of one or more atoms for linking
a (Z)n group with Y, except that more than two
(Z)n groups cannot be directly linked to the
same carbon atom in Y;
the ratio of the number of -CO2H groups to the number of
(ZO) groups, particularly where Z is -CH2CH2-, is
within the range of 1:20 to 20:1
- 8 - 1335352
Preferably both R1 and R2, where they are present,
are hydrogen.
Where R1 or R2 is a hydrocarbyl group, it is
preferably a lower alkyl group, more preferably methyl.
R3 is preferably a lower alkyl group, more
preferably methyl.
Where Z is -(CH~)m-, m is preferably 4; this
affords a ready preparation f~(ZO)n~ from
tetrahydrofuran.
It is to be understood that the definition of
the polymer contained in the composition (as given
above) is also intended to embrace a polymer in which
at least some of the carboxyl groups in the repeat
units of general structure A have been converted to the
corresponding salt anions CO2- (these being considered as
-CO2H group as far as the ratio of carboxyl to -ZO-
groups is concerned), the corresponding cations for
example being those of ammonium (NH+4), or alkaline earth
metals or preferably alkali metals (e.g. Na+, K+). We
do not exclude the possibility that the cation may be
derived from the cationic antibacterial agent per se;
indeed where the cationic antibacterial agent is present
as a salt of the acid polymer such that there is
substantially no free chlorhexidine in the mouth there is
a tendency for staining to be further reduced.
In general structure A, each carboxyl group is
joined to the hydrocarbyl residue X by means of an
intermediary or intermediaries (i.e. by a linking entity
or entities), this or these being denoted by L, which is
selected from one or more direct links (i.e. one or more
direct bonds) and one or more groups of atoms each group
providing a chain of one or more atoms for linking a
carboxyl group(s) with X. In cases where p is 2 to 4,
each carboxyl group may be joined by L to the same or, in
cases where L represents more than one intermediary, to
- 9 - 1335352
- the same or different carbon atoms in X, although more
than 2 carboxyl groups cannot of course be directly
linked to the same carbon atom of X (and also assuming
that in such cases X has at least 2 carbon atoms, whereas
it should be appreciated that it is within the scope of
the invention for X to have only 1 carbon atom). It will
be noted that in principle L can represent up to 4
separate intermediaries in structure A (in cases where p
is 4). L may be the same or different in the repeat
units of structure A.
In cases where L represents one or more groups of
atoms each group providing a linking chain of atoms, the
chain will normally comprise one or more carbon atoms
(which could, for example, include carbon atoms in an
aryl ring) and/or hetero atoms (particularly N and/or 0).
Examples of possible linkages provided by L are:
CH 2 CH 2 CH 2 NH CO CO
CH 2 CH CO NH NH
/ \ l l
(direct CH(CH3) CH(OH)
link or
bond)
where (apart from the direct link) the top link is to X
and the bottom link(s) is to carboxyl. It is preferred
in the present invention, however, that L is one or more
direct links, such that each carboxyl group is ~oined
directly to a carbon atom in the polymer backbone.
In the structure A, p is preferably 1 or 2, more
preferably 1 (so that L can then represent one, or at
most, two intermediaries).
- lo - 1335352
In structure B, each (Z)n R3 group is joined to
the hydrocarbyl residue Y by means of an intermediary or
intermediaries (i.e. by a linking entity or entities),
this or these being denoted by M, which is selected from
one or more direct links (i.e. one or more direct bonds)
and one or more groups of atoms each group providing a
chain of one or more atoms for linking a (Z)n R3
group(s) with Y. In cases where q is 2 to 4, each
(Z)n R3 group may be joined by M to the same or, in
cases where M represents more than one intermediary, to
the same or different carbon atoms in Y, although more
than two (Z)n R3 groups cannot of course be directly
linked to the same carbon atom of Y (and also assuming
that in such cases Y has at least 2 carbon atoms, whereas
it should be appreciated that it is within the scope of
the invention for Y to have only 1 carbon atom). M may
be the same or different in the repeat units of structure
B.
While M may represent one or more direct links, it
is preferred in the present invention that M is one or
more groups of atoms each group providing a linking chain
of atoms; such a chain will normally comprise one or more
carbon atoms (which could, for example, include carbon
atoms in an aryl ring, e.g. benzyl ether) and/or hetero
atoms (particularly N and/or 0). Particularly preferred
examples of chains provided by M are:
CO and CO
0 NH
where the top link is to Y and the bottom link is
to (Z)n R3.
1335352
In structure B, q is preferably 1 or 2, more
preferably l(so that M can then represent one, or at most
two intermediaries).
Preferably the structure A represents the repeat
unit derivable by the addition polymerisation (usually
free-radical initiated) of a polymerisable olefinically
unsaturated carboxylic acid. Examples of such acids are
maleic (or fumaric) acid, itaconic acid, the acids of
formulae
C(CH3)=CH2 CH=CH2
CO and CO
NHCH(CH3)CO2H NHCH(OH)CO2H
N-methacryloyl alanin~ N-acryloyl-hydroxy-glycine
or preferably acrylic or methacrylic acid
Preferably the structure B represents the repeat
unit derived from the polymerisation (usually
free-radical initiated) of an addition polymerisable
olefinically unsaturated ester or amide formed from the
reaction of an unsaturated carboxylic acid (or an
esterifiable or amidifiable derivative thereof such as an
acid chloride or anhydride) and a hydroxy compound of
formula HO (Z)n R3 (to form the ester) or an amine of
formula H2N (Z)n R3 (to form the amide).
Preferably the acid from which structure B is
derivable is acrylic or methacrylic acid, particularly
the latter, giving rise, where an ester or amide
derivative of methacrylic acid is used, to the following
structures respectively for B:
- 12 - 1~353~2
-C(CH3)---CH2 -C(CH3)---CH2
CO and CO
(Z)n R3, NH (Z)n R3
Preferably acidic polymers of which oral hygiene
compositions according to the present invention are
comprised have a ratio of acidic residues to pendant
polyalkylene oxide residues of about 6:1 (where each side
chain is polyethylenegylcol of molecular weight about
350, i.e. so-called PEG 350).
Polymers for use in the present invention are more
fully described in our aforesaid European Patent
Specification No 0,182,523A.
In oral hygiene compositions of the present
invention, the at least one polymer present therein is
typically at least at a concentration of about 0.05 to 30
weight % of the composition, the preferred concentration
range being from about 0.1 to 5 weight % and more
preferably 0.2 to 2 weight %.
The concentration of the at least one
antibacterial agent in oral hygiene compositions
according to the present invention is about 0.001 to 10
weight % of the composition, the preferred concentration
range being from about 0.001 to 1.0 weight % and more
preferably 0.01 to 0.1 weight %.
Preferably, the mass of the polymer is higher than
the mass of the anti-bacterial agent in oral hygiene
composition according to the present invention. However,
we do not exclude the possibility that there may be more
anti-bacterial agent than polymer present.
< -~
-' ,!1
- 13 - 1 3 353~2
The oral hygiene composition of the present
invention typically comprises only one polymer as
hereinbefore defined, although we do not exclude the
possibility that two or more such polymers may be presen~
in the composition.
The skilled man by simple experiment will be able
to formulate compositions according to the present
invention in which the ratio of antibacterial agent to
polymer is such that undesired reaction is avoided.
The oral hygiene composition of the present
invention typically comprise a pharmaceutically
acceptable vehicle which is compatible with the
antibacterial efficacy of the cationic antibacterial
agent, e.g. chlorhexidine. To maintain the efficacy of
chlorhexidine it may be necessary to adjust the
concentration thereof in a particular vehicle, a suitable
concentration may be determined by the skilled man by
experiment.
Suitable conventional pharmaceutically acceptable
vehicles that can be employed in the oral hygiene
compositions of the present invention include water,
ethanol (wherein water, or a water/ethanol mixture will
often be a major component of the vehicle); such
humectants as propylene glycol, isopropanol, glycerol and
sorbitol; such gelling agents as cellulose derivatives,
for example, hydroxypropyl and hydroxyethyl cellulose,
polyoxypropylene/polyoxyethylene block copolymers,
(so-called "Poloxamers"), for example Synperonic*PE 39/70
and PEF 87; certain gel stabilisers such as
polyvinylpyrrolidone; sweeteners such as sodium
saccharin; preservatives such as cetylpyridinium
chloride, and certain lower alkyl parahydroxy-benzoates;
surfactants such as polyoxyethylene isohexadecyl ether
(Arlasolve*200) and certain colours and flavours, on the
* Trade Mark
~., .~ .,
-. .
- 14 - 133~352
approved EEC or FD&C lists. It will be appreciated that
the aforesaid vehicle is chosen such that it does not
unduly inhibit the effectiveness of the oral hygiene
composition according to the present invention; in
particular, an anionic material, eg an anionic
cellulose derivative or an anionic Synperonic, is not
preferred.
The oral hygiene compositions of the present
invention may be in the form of any conventional
pharmaceutically acceptable oral hygiene formulation that
contains (and is compatible with) an effective amount of
a polymer and antibacterial agent as hereinbefore
defined. As examples of such formulations may be
mentioned inter alia mouthwashes, rinses, irrigating
solutions, abrasive and non-abrasive gel dentifrices,
denture cleansers, coated dental floss, coated or
impregnated toothbrush bristle (natural or synthetic),
inter-dental stimulator coatings, chewing gums, lozenges,
breath fresheners, foams and sprays.
The present invention is now illustrated by the
following Examples. The prefix "CT" to an Example number
denotes a Comparative Test.
In most of the following Examples the oral
bacterium Streptococus mutans NCTC 10449 was used as the
standard bacteria. It was grown in Brain Heart Infusion
(BHI) (ex Oxoid) in a Bioflo Model C30 Fermenter. A 750
ml pot was used cont~;n'ng 350 ml of bacterial
suspension. The bacteria were grown at 37C with a
dilution rate of 0.lh-1, an air flow of 0.24 litre/minute
and an agitation speed of 300 rpm. A sample
(approximately 20 ml) of bacterial suspension was taken
out of the fermenter for each experiment. The bacteria
- were centrifuged for 10 minutes at 4000 rpm, they were
resuspended in modified Ringer's salts solution (0.54
grams/litre NaCl; 0.02 grams/litre KC1; 0.03 grams/litre
- 15 - 1335352
CaCl 2; and 0.75 grams/litre sodium mercaptoacetate),
recentrifuged,
resuspended and diluted (10 x) in modified Ringer's salts
solutions. The approximate bacterial concentration in
the diluted salts solutions was 10 8 ml~1.
Streptococcus mitior
NCTC 7864 was grown in 100ml Brain Heat Infusion
broth in batch culture for 24 hours. The culture was then
centrifuged for 30 minutes at 3500rpm and washed twice by
resuspending the pellet in saline and centrifuging. The
bacterial suspension was then adjusted to approximately
0 7 -10 8 cells per ml.
Whole saliva was used in further examples.
Absorption surfaces
Hydroxyapatite discs were made by compressing
hydroxyapatite powder (calcium phosphate tribasic (Ca10
(OH) 2 (P4 ) 6 (ex Aldrich)) and sintering at 1100C.
The discs were re-used after heating in a furnace at
900C for 2 hours between experiments.
Application of polymers
Hydroxyapatite discs were treated for 2 minutes at
room temperature with a solution (1% w/v) of a polymer,
e.g. Polymer 93W, in a 1:1 (by volume) mixture of
industrial methylated spirits/water. The discs were
then washed by dipping and shaking 5 times in a container
of flowing water at about 15C.
Application of Anti-Bacterial Agent
Aqueous solutions of chlorhexidine and IMS
solutions of alexidine were separately adsorbed for
certain periods of time onto the surfaces of hydroxy-
apatite discs which had been treated with polymer where
appropriate (untreated discs were used in comparative
tests). The discs were then washed by dipping and
shaking 5 times into a container of flowing water.
- 16 - 133S3~2
Polymers
The polymer herein referred to as "Polymer 93W" is
an acidic polymer as described and prepared in Example 5
of our aforesaid EP.182523. (Other "acidic" polymers
hereinafter described were prepared by a similar
process). Polymer 93W comprises methacrylic acid and
PEG350MAt residues in molar ratio 6: 1.
By "PEG350MAt" we mean a polyethylene oxide of
molecular weight about 350 which has been capped with
methoxy and methacryloyl groups, i.e.
CH2=C(CH3)COO(CH2CH2O)nCH3 where n is
about 8.
PEG 150Mat, PEG1000 Mat and PEG 2000 Mat indicate
similar polyethylene oxides of molecular weights 150,
1000 and 2000 respectively.
Polymer M11 was prepared under the conditions
described in Example 15 of EP 182,523 except that a
hydroxy ended PEG was used instead of an amino ended PEG.
Loeffler's Methylene Blue
95% Ethyl alcohol (30 ml), methylene blue (0.3 g) and
water (100 ml).
Examples 1-2
These Examples demonstrate that Polymer 93W retains
its anti-adhesive properties in the presence of absorbed
chlorhexidine.
Hydroxyapatite discs were treated with a 1% w/w
solution of Polymer 93W and then with certain
antibacterial agents for set periods of time. The discs
so treated were immersed in a bacterial suspension (30
mls) in a petri-dish for 2 hours. The discs were
removed from the bacterial suspension and were washed by
dipping and shaking 5 times in a container of flowing
water. Bacteria adhering to the discs were stained using
Loeffler's Methylene Blue. The reduction in bacterial
adhesion was determined by microscopic examination.
-
- 17 -
1335352
The results are shown in Table 1.
TABLE 1
Example Antibacterial Polymer % Anti-
No Agent adhesion
CT1 0 1%93 W 99
1 C 1%93 W 99
2 A 1%93 W 99
CT1: 1% 93W was used alone
C: 1% chlorhexidine
A : 1% alexidine
From Table 1 it can be seen that chlorhexidine
and alexidine do not reduce the anti- adhesive properties
of Polymer 93W deposited on hydroxyapatite discs.
"%Anti-adhesion" ("% AA") is defined by the equation:
Area of neat surface - Area of polymer-coated
%AA = ,covered with bact. , surface covered with bact.
Area of neat surface
,covered with bacteria,
It will be appreciated that (a) where the polymer
does not decrease the area of the surface which is coated
with bacteria then:
% AA = X-X x 100 = 0
X
and (b) where the polymer prevents adhesion of bacteria
to the~ surface then:
% AA = X-O x 100 = 100
X
- 18 - 1335352
Similar results were obtained when a specimen of
material conventionally used in the preparation of a
dental prosthetic device, as hereinafter described, was
treated with polymer 93W and chlorhexidine was then
exposed to Streptococcus mitior NCTC 7864.
EXAMPLES 3-5
These Examples demonstrate that on hydroxyapatite
the antibacterial effect of chlorhexidine at certain
concentrations is increased where it is used in the
presence of Polymer 93W.
Solutions of chlorhexidine were absorbed onto
sterile hydroxyapatite discs which had been treated with
Polymer 93W. Cells of S. mutans were taken from a
fermenter and diluted 100 fold in BHI agar at 40C. The
innoculated agar was overlayed onto HAP discs.
Agar coated discs were incubated at 37C overnight.
Bacterial growth throughout the agar was assessed on a
scale of 0 (no growth) to 10 (control).
Since the surface of each hydroxyapatite disc was
brought into contact with the same number of bacteria in
each case, anti-adhesion did not contribute to the
observed result, i.e. antibacterial effect alone was
being measured. The results in Table 2 reveal that the
combination of Polymer 93W and chlorhexidine gave an
enhanced antibacterial effect compared to chlorhexidine
per se at the same applied concentration of
chlorhexidine.
In Comparative Tests CT's 2, 3, 4 and 5, the discs
were not treated with Polymer 93W. Comparative Test 2 is
a blank; in Comparative Test 2A, the disc was treated
with Polymer 93W only.
- 19 - 1335352
TABLE 2
Example Applied Treatment with Bacterial
No Chlorhexidine Polymer 93W growth
concentration
CT2 0 NO 10
CT2A 0 YES 10
CT3 1 NO 8
CT4 0.1 NO 10
CT5 0.01 NO 10
3 1 YES 0
4 0.1 YES 2
0.01 YES 4
EXAMPLES 6-9
These Examples demonstrate the combination of
anti-adhesive and antibacterial results which can be
obtained from the use of a combination of a polymer and
chlorhexidine and that such a combination provides an
improvement over the discrete components per se.
Sterile hydroxyapatite discs were treated with a 1%
w/v solution of Polymer 93W and then with solutions of
certain concentrations of chlorhexidine. The discs were
incubated in freshly collected whole saliva for 1 hour at
37C and washed by dipping and shaking 5 times into a
container of flowing water. Excess water was removed
from the surface of each disc by touching the edge
thereof with filter paper.
BHI agar cont~ning 0.04% w/v Bromo Cresol Green
(to render bacterial growth on the white hydroxy-apatite
discs visible) was pipetted at 40C onto the discs such
that a thin film of agar formed on the surface.
- 20 - 1335352
The discs were incubated at 37C overnight.
The results are shown in Table 3.
In Comparative Tests 6-10 the treatment with
Polymer 93W was omitted. In Comparative Test 11,
Polymer 93W was used, in the absence of chlorohexidine.
- 21 -
13353S2
TABLE 3
Example Concentration of Presence of Bacterial
No chlorhexidine (%) Polymer 93W growth
CT6 1 NO No Growth
6 1 YES No growth
CT7 0.1 NO Control
level 4.
7 0.1 YES No Growth
CT8 0.01 NO Control
level 5.
8 0.01 YES A few
colonies;
greater
than 99%
reduction
compared
with
control
level 5
CT9 0.001 NO Control
level 6.
9 0.001 YES 99%
reduction
compared
with
control
level 6
CT10 0 NO Thick
Growth:
control
level 7
CT11 0 YES 90%
Reduction
compared
with CT7
control
level 7
~ - 22 - 1 3 3 5 3 S 2
From Table 3 it can be seen that for certain
concentrations of chlorhexidine, e.g. 0.01 and 0.001%,
the presence of Polymer 93W increases the bactericidal
and/or bacteriostatic effect thereof. CT11 demonstrates
the reduction in bacterial growth which arises from the
anti-adhesion properties of the polymer per se.
EXAMPLES 10-20
These Examples reveal that treatment of
hydroxyapatite discs with certain polymers
(a) increases the amount of chlorhexidine absorbed
thereon and
(b) improves the retention of the absorbed
chlorhexidine through subsequent washing
treatments.
Preparation of Polymers B3 and B18
Methacryloyl chloride (0.58 moles) was added over 2
hours to a mixture of toluene (600 ml), Jeff "360" or
"2070" (0.5 moles) and 2,6-lutidine (0.56 moles) cooled
in an ice-bath. A copious white precipitate formed. The
reaction mixture was allowed to stand for 3 hours, and
the white precipitate was filtered off and washed with
toluene. The filtrate was evaporated under reduced
pressure and the residue was kept under vacuum to remove
volatiles. Products (yield 80-90%) were characterised by
infra-red and proton magnetic resonance spectroscopy.
The amino-ended products from both reactions (with
terminal butoxy or methoxy groups from "360" and "2070"
respectively) were separately converted into the
N-methacrylaloyl derivatives thereof and copolymerised
with methacrylic acid under the conditions described in
Example 11 of EP 0.182,523A.
Hydroxyapatite discs were pre-equilibrated in
double distilled water for 1 hour. The discs were
removed from the water, blotted dry and kept at room
temperature for about 30 minutes. A W reflectance scan
133S3S2
thereof was carried out. The Optical Density at 266 nm
was typically about 0.9. Any disc which had an O.D.
which was significantly different from this number was
rejected.
The acceptable discs were immersed in 1% w/w
(1:1/IMS: water) solution of polymer for 5 minutes. The
discs were removed from the polymer solution; were
washed by dipping and shaking 5 times in a container of
flowing water; were blotted dry; left for 30 minutes and
then scanned.
Each of the polymer-coated discs was immersed in
aqueous (15 ml) chlorhexidine solution (0.02% w/v) for 1
hour. They were washed as described above, allowed to
stand for 30 minutes and then scanned. They were then
placed in a flow-through (250 ml/min) washing tank (1200
ml) for 1 hour; blotted dry; allowed to stand for 30
minutes and scanned.
-- 24 --
1335352
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- 25 -
13353S2
TABLE 5
Polymer A B Molar Ratios
Backbone Side Chain
Nature Mol Wt A:B CHRlCHR 2 0:
(Source) CO 2 H
groups
73 Basic (DMAEM) PEG 350 3:1
B12 Amphoteric PEG 350 1.9:1.1:1
(MAA:DMAEM)
62 Acidic (MAA) PEG 150 3:1 1:1
86 Acidic (MAA) PEG 350 3.5:1 2.3:1
93W Acidic (MAA) PEG 350 6:1 1.3:1
66 Acidic (MAA) PEG 1000 3:1 7.7:1
B9 Acidic (MAA) PEG 1000 25:1 0.9:1
58 Acidic (MAA) PEG 2000 10:1 4.5:1
B3 Acidic (MAA) Jeff 360 6:1 1.1:1
B10 Acidic (MAA) Allyl-
PEG 350 6:1 1.3:1
B18 Acidic (MAA) Jeff2070 34:1 1.2:1
B17 Acidic (MAA) PPG 1000 17:1 1:1
Mll Acidic (MAA) PEG 350 5:1 0.9:1
DMAEM:N N-dimethyl-2-aminoethyl methacrylate.
MAA: Methacrylic acid.
MA: Maleic acid;
PEG: Polyethylene glycol.
PPG: Polypropylene glycol;
Jeff 360 : n-C~Hg(OCH2CH2)~OCH2CH(CH3)OCH2CH(CH3)NH2;
Jeff 2070: CH3OCH2CH2O(CH2CHO)nCH2CH(CH3)NH2
R
- 26 - 1335 3S2
wherein n is such that "2070" has an MW of about 2000
and R = H or CH3 in a ratio of about 7:3;
Allyl-PEG 350: Ethoxylated allyl alcohol;
Except for B10, the methacrylate or methacrylamido
derivatives of the indicated side chains were present as
general structure B;
B10: contains terminal hydroxy groups.
The aforesaid UV scanning was effected using a
Unican SP1750 Ultraviolet Spectrophotometer.
The "difference in Optical Density" results shown
in Table 4 were determined therefrom.
From Tables 4 and 5, it can be seen that the acidic
polymers significantly increased the amount of
chlorhexidine absorbed. Many of the hydroxyapatite
surfaces coated with an acidic polymer absorbed more
chlorhexidine from a 0.02% w/v solution than did the neat
hydroxyapatite surface from a 2% w/v solution of
chlorhexidine, i.e. a greater than a 100-fold
improvement was observed. The basic (Polymer 73) and
amphoteric (Polymer B12) polymers gave no improvement in
the amount of chlorhexidine adsorbed. Similarly,
polymethacrylic acid yielded no increase in chlorhexidine
adsorbed, indicating that it was the PEG (or PPG) chains,
and not the carboxyl groups, that were responsible for
the observed effect.
The results of the washing experiments showed that
after 1 hour about 23% of the initially adsorbed
chlorhexidine was still adsorbed on bare hydroxyapatite
discs, whereas the amount for polymer treated discs was
approximately 80%. After an overnight wash, the amount
of adsorbed chlorhexidine, if any, r~m~ining on the
neat HAP discs was below the detection limit; and
approximately 50-60% of the amount of chlorhexidine
- 27 - 1335~52
originally adsorbed on polymer-treated discs remained
adsorbed. Thus, polymer-treated discs adsorbed more
chlorhexidine than bare discs, and it was also less
easily washed off the surface thereof.
EXAMPLES 21-29
These Examples, in combination with Examples 10-20
and 6-9, reveal an increase in anti-bacterial properties
(at a certain chlorhexidine concentration) without the
expected increase in staining.
General Procedure
Hydroxyapatite discs were pre-equilibrated in
double- distilled water for 1 hour. The pre-equilibrated
discs were immersed in 1% w/v aqueous or IMS:water (1:1)
solution of polymer for 5 minutes. They were removed
from the polymer solution and washed by dipping and
shaking 5 times in a container of flowing water. The
washed discs were then immersed in 15 ml of an aqueous
chlorhexidine solution (of a concentration shown in Table
6) for 5 minutes. The discs were removed from the
chlorhexidine solution and immersed in 15 ml of a tea
solution for 1 hour at room temperature. The discs were
taken out of the tea solution and washed as described
above. The steps of immersion in chlorhexidine and tea
solution and washing were repeated 3 times, using fresh
chlorhexidine and tea solutions each time. After these
three cycles the discs were immersed in tea solution
overnight; they were then washed as described above,
allowed to dry for 1 hour at room temperature and the
amount of stain produced thereon was assessed.
The tea solution was prepared by adding 500 ml of
boiling water to 2 tea bags. The tea bags were removed
after 5 minutes, and the tea allowed to cool to room
temperature. The tea was filtered using standard filter
paper, and stored at 4C prior to use.
-
- 28 - 13353S2
The stained discs prepared in the General Procedure
were scanned using UV/visible reflectance
spectrophotometry as described in Examples 10-20 and
compared with tea blanks (i.e. no chlorhexidine
adsorbed). Figure 1 shows typical UV- traces which were
obtained. In Figure 1, a = bare hydroxyapatite disc; b =
tea blank; and c,d,e, = tea/chlorhexidine treatments at
0.002%, 0.02% and 0.2% concentrations of chlorhexidine
respectively.
The polymers listed in Table 6 (the chemical
composition of which are given in Table 5) were evaluated
for their effect on the stain formation of chlorhexidine
in the presence of tea. The polymers were separately
adsorbed from 1% w/v IMS/water (1:1) solutions. 0.2%,
0.2% and 0.002% w/v aqueous solutions of chlorhexidine
were used. The discs were scanned using UV/visible
reflectance spectrophotometry and the OD's at 266, 410
and 510 nm were measured. The OD's of tea blanks were
also measured at these wavelengths, and these values were
subtracted from discs treated with chlorhexidine or
polymer/ chlorhexidine combinations. Table 6 gives the
results at 510 nm. They are expressed as ratios relative
to the stain produced by a tea blank = 1Ø Similar
results were obtained at 266 nm and 410 nm. In CT 18,
the polymer was omitted, i.e. chlorhexidine per se was
used.
-
- 29 -
1335352
TABLE 6
Comparative Stain Development compared with "Natural"
build-up from exposure to tea solutions.
O.D. Ratio (compared with
natural tea stain) at 510 nm
Example Polymer at applied Chlorhexidine
No Concentrations of
0.2% 0.02% 0.002%
CT18 - 4.57 2.76 1.67
21 73 4.48 3.01 1.0
22 62 4.98 2.65 1.0
23 86 4.48 3.26 1.0
24 93W 4.98 2.79 1.0
B9 4.75 3.09 <1.0
26 B3 4.39 3.34 1.0
27 B10 4.76 3.34 <1.0
28 B18 4.20 3.12 <1.0
29 B17 5.03 2.95 <1.0
From Table 6 it can be seen that at the two higher
chlorhexidine concentrations (i.e. 0.2 and 0.02%) the
presence or nature of the polymer had no substantial
effect on the amount of stain produced on treatment of
HAP discs. At the lowest concentration (i.e. 0.002%) of
applied chlorhexidine, the majority of Examples show a
significant reduction in stain equal to or less than the
mlnlm~l levels associated with exposure of HAP discs to
the tea solutions. However, it will be appreciated from
the results in Examples 10-20 and 6-9, that, for about
the same stain as the control, the polymers had more
_ 30 - 1335352
chlorhexidine absorbed thereon and exhibited an increased
antibacterial effect.
EXAMPLES 30-31
These Examples illustrate the increase in the
quantity of chlorhexidine adsorbed onto a hydroxyapatite
disc treated with a mixture of chlorhexidine and Polymer
93W compared to treatment of the HAP disc with a
chlorhexidine solution per se.
An IMS solution (2% w/v) of Polymer 93W was mixed
with an appropriate (0.04% w/v) solution of chlorhexidine
in water, at a solution ratio of 1:1 by volume. Hydroxy-
apatite discs were allowed to stand in the mixture for 1
hour and were then washed five times with water. The
amount of chlorhexidine absorbed on the discs was
determined by UV reflectance spectrophotometrY as
described in Examples 10-20 (the Optical Density was
measured at 266 nanometers).
In Comparative Tests 20 and 21, the discs were
treated for 1 hour with 0.2 and 0.02 % solutions
respectively of chlorohexidine in a 1:1 by volume mixture
of industrial methylated spirit and water.
The results are shown in Table 7. From Table 7 it
can be seen that treatment of a HAP with the Polymer
93W/chlorhexidine mixture results in more chlorhexidine
being absorbed than from chlorhexidine solution per se.
- 31 - 1 3 35 3 S 2
TABLE 7
Example State of Concentration Increase in
No addition of Optical
of chlorhexidine Density at
composition % w/v 266 nm
Mixture 0.2 0.44
31 Mixture 0.02 0.12
CT19 Neat 0.2 0.0
chlorhexidine
solution
CT20 Neat 0.02 0.0
chlorhexidine
solution
EXAMPLES 32-33
These Examples illustrate the increased "kill" of
a Polymer 93W/chlorhexidine mixture compared with that
observed with neat chlorhexidine solution.
The discs prepared in Examples 30-31 were washed
overnight in water and subjected to an S.mutans agar
overlay experiment. They were placed in a petri-dish and
covered with BHI agar (25 mls). S mutans (100 ,ul), grown
in a fermenter (as described above) and diluted x 100 in
Ringers salt solution, was pipetted onto the agar and
evenly spread. The bacteria were grown overnight at 37C;
"lawns" of bacteria and "clear zones" free of bacteria
were noted and measured. The results are shown in Table
8. The clear zones, i.e. zones where growth did not
occur, are shown as a percentage of the area of the
disc.
- 32 - 13353~2
TABLE 8
Example Disc Concentration % Area of
No Prepared of disc where
in chlorhexidine growth did
Example % w/v not occur
No
32 30 0.2 225
33 31 0.02 64
CT21 CT19 0.2 3
CT22 CT20 0.02 0
It will be appreciated that where "% Area of disc
where growth did not occur" is more than 100, this
indicates that inhibition spread into the agar layer
beyond the perimeter of the disc.
From Table 8, it can be seen that the mixture has
an increased antibacterial activity compared with
chlorhexidine per se.
EXAMPLES 34-65
These Examples show that the combination of an
anti-adhesive compound, Polymer 93W, and chlorhexidine
reduce the amount of st~;n;ng, compared with
chlorhexidine per se, generated on a variety of surfaces
found in the oral environment. The surfaces comprised
tooth, composite restorative materials, e.g. Occlusin
and Opalux, and a methacrylate-based resin
conventionally used in the preparation of dental
133S352
prosthetic devices (hereinafter referred to for
convenience as "PR").
SPECIMENS
Residual flesh was removed with a scalpel from
freshly extracted teeth, the teeth were then tumbled for
20 minutes in 50% sodium hypochlorite solutions and
washed superficially with distilled water.
These and teeth containing restorative material were
sonicated in alcohol for 10 minutes and then dried.
Samples of DR (25 mm x 10 mm x 3 mm) and discs of
the aforementioned composite restoratives were washed in
alcohol and dried.
SOLUTIONS
a 1% and 0.5% solutions of Polymer 93W (lgm) in a
mixture of industrial methylated spirit (50 ml) and
water (50 ml).
b Solutions (0.2%, 0.02%, and 0.002%) of
chlorhexidine in water.
c Appropriate mixtures of Polymer 93W and
chlorhexidine were obtained by mixing equal volumes of
solutions from a and b to afford the concentrations shown
in the folowing Tables.
d Human saliva was obtained by taking specimens
(20 mls) from each of 6 volunteers, centrifuging for 20
minutes at 2,5000 rpm and pooling them.
e Tea solution was prepared by boiling a sample
(8g) of a commercial brand of tea in distilled water
(80 ml) for 2 minutes, cooling the product to room
temperature and filtering off the residual tea leaves.
Evaluation
Each surface was treated for 10 minutes with a
sample of the saliva. Excess saliva was washed off.
In Examples 34-49, the surface was subjected to a
first treatment for 10 minutes, superficially washed
with distilled water, subjected to the second treatment
for 10 minutes, rinsed and then immersed in the tea
_ 34 _ 1335352
solution for 1 hour; the procedure was repeated, the
sample was left in the tea solution overnight and the
whole procedure repeated every day for 5 days.
In examples 50-65 the above 5 day procedure was
repeated except that the first treatment was for 5
minutes and the second treatment was omitted, the
samples were treated with appropriate mixtures of
Polymer 93W and chlorhexidine.
The staining of the surfaces was compared visually
with the same surface treated only with water and scored
on the following scale.
Scale
O : No stain (ie Water-control taken as O, although
there was slight discolouration);
1 : Slight stain;
2 : Moderate stain;
3 : Heavy stain; and
4 : Very heavy stain.
In Tables 9, 10 and 17
OC = Occlusin
OP = Opalux
T = Tooth
PR = Prosthetic resin
A = 0.5% Polymer 93W
AA = 1% Polymer 93W
B = Water
X = 0.1% Chlorohexidine
XX = 0.2 "
Y = 0.01 "
YY = 0.02 "
Z = O . 001 "
ZZ = 0.002 ~I
W - O . 0001 "
35 - 133~352
TABLE 9
Example Surface Treatments Score
No
First or Second
Single (10 mins)
(10 mins)
CT23 OC B 0
CT24 OP B 0
CT25 T B 0
CT26 PR B 0
CT27 OC X 3
CT28 OP X 3
CT29 T X 3
CT30 PR X 3
CT31 OC AA O
CT32 OP AA O
CT33 T AA O
CT34 PR AA O
34 OC AA XX 3
'' '' YY
36 '' '' ZZ 0
CT35 '' B XX 3
CT36 '' '' YY 2
CT37 '' '' ZZ
37 OP AA XX 4
38 '' '' YY 2
39 '' '' ZZ 0
CT38 '' B XX 3
CT39 '' '' YY 2
- 36 - I 3353S2
TABLE 9 - Cont
Example Surface Treatments Score
No
First or Second
Single (10 mins)
(10 mins)
CT40 OP B ZZ
T AA XX 4
41 '' '' YY 2
42 '' '' ZZ 0
CT41 '' B XX 3
CT42 '' '' YY 2
CT43 '' '' ZZ
43 PR AA XX 4
44 '' '' YY 2
'' '' ZZ 0
CT44 '' B XX 3
CT45 '' '' YY 2
CT46 '' '' ZZ
46 OC XX AA O
47 OP '' '' 0
48 T ~ o
49 PR '' ~ o
~ 37 ~ 1335352
TABLE 10
Example Surface Treatment Score
No (mixture for 5 mins)
CT47 OC B 0
CT48 OC X 4
CT49 OC Y 3
10CT50 OC Z
CT51 OC W
CT52 OC A 0
OC A+Y 2
51 OC A+Y 0
52 OC A+Z 0
53 OC A+W 0
CT53 OP B 0
CT54 OP X 4
20CT55 OP Y 3
CT56 OP Z
CT57 OP W
CT58 OP A 0
54 OP A+X 4
OP A+Y 3
56 OP A+Z
57 OP A+W
- 38 - 133S3S2
TABLE 11
Example Surface Treatment Score
No (mixture for 5 mins)
CT59 T B 0
CT60 T X 4
CT61 T Y 3
10CT62 T Z
CT63 T w
CT64 T A O
58 T A+X 2
59 T A+Y O
T A+Z O
61 T A+W O
CT65 PR B 0
CT66 PR X 4
20CT67 PR Y 2
CT68 PR Z
CT69 PR W
CT70 PR A 0
62 PR X 2
63 PR Y 0
64 PR Z 0
PR A+W 0
From Table 9, it can be seen that at low
concentration (eg 0.002%) of chlorhexidine st~;n;ng of
dental restorative, teeth or prosthetic resin is reduced
if the surface thereof is first treated with a certain
polymer. It can be seen further (Examples 46-49) that
where the surfaces are treated with a
133~352
chlorhexidine solution and then with Polymer 93W the
staining is reduced to control levels.
Table 10 reveals the results obtained on treating
Occlusin and Opalux surfaces with mixtures of
chlorhexidine and Polymer 93W. There is a significant
reduction in the staining of Occlusin, to control levels
at chlorhexidine concentrations of 0.01% and less; a
similar trend is apparent with Opalux.
Table 11 reveals the results obtained on treating
tooth surfaces and a prosthetic resin with a mixture of
chlorhexidine and Polymer 93W. The trend in the
reduction in staining is similar to that observed with
Occlusin and Opalux.
Where a tooth with an Occlusin implant was
subjected to the above evaluation, the tooth and implant
were slightly stained to the same extent such that the
outline of the implant was further decreased.
