Note: Descriptions are shown in the official language in which they were submitted.
MICROCAPSULE COMPOSITIONS FOR TOPICAL TREATMENT
This invention relates to compositions to be used for
the delivery of benefit agents to target sites, which may be
in the mouth. In particular the invention is applicable to
the delivery of therapeutic agents active against organisms
which give rise to dental plaque.
The so-called "magic bullet" concept refers to a
therapeutic agent which is somehow bound to some entity
having an affinity for the intended target site. It is
envisaged that with such an arrangement the therapeutic
agent will be carried to its intended target and will not
display significant activity at other possible destinations
which it may have an opportunity of reaching. A particular
application of this which is frequently envisaged is the
delivery of an anti cancer drug which is also somewhat
toxic. If such a drug is formulated as a "magic bullet" it
will, so it is hoped, be delivered to the cancer against
which it is desired to act and not to other parts of the
body where the drug would merely produce undesirable side
effects.
The "magic bullet" is frequently envisaged as
comprising a microcapsule of some description as a carrier
for the therapeutic agent, with surface structures providing
a means of molecular targeting. Possible carriers include
polymeric nanocapsules and liposomes. Liposomes are small
sacs formed from certain surface active molecules, most
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commonly phospholipids, which in aqueous media arrange
themselves into a bi-layered membrane defining a microscopic
closed vesicle.
It has been proposed to use liposomes to constitute
"magic bullet" type drugs in which the therapeutic agent is
enclosed within the liposome which carries the drug to the
target site while also keeping the therapeutic agent out of
contact with alternative targets which are bypassed on the
way to the target site.
US4767615 discloses delivering therapeutic agents in
liposomes which have affinity for hydroxyapatite. This
might give selectivity as to where in the mouth receives the
therapeutic agent. However, this approach could not be
expected to improve delivery more generally, since the
amount of exposed hydroxyapatite is, in practice, likely to
be small.
We have now found that liposomes with means to attach
to specific organic surfaces can be used to give
unexpectedly effective delivery of therapeutic agents to
target sites following topical application.
The target site might be any site accessible by topical
application to the undamaged human body. It is particularly
envisaged that target sites will be in the mouth.
Such effective delivery is unexpected because the
liposome encapsulating the therapeutic agent has a much
larger bulk than the same agent when molecularly dispersed,
so that the mobility of the former in solution would be
lower, and diffusion would lead to slower delivery to the
target site. Also, if a therapeutic agent is incorporated
into a product to be used in the mouth, such as for instance
a dentifrice then the mouth would be the first part of the
human body to be eYposP~ to this product. There are no
alternative targets for the therapeutic agent to bypass on
the way.
Surprisingly we have found that when the duration of
exposure to the composition is short (which of course is a
realistic situation for a target site in the mouth) a
therapeutic agent is rendered more effective when it is
enclosed in liposomes which have means for binding it to the
desired target site.
Accordingly, the present invention provides a
composition for topical application and containing
microcapsules, particularly liposomes, which enclose an oral
benefit agent active at a target location accessible by
topical application, in the mouth, and which have on their
surface molecular structures capable of recognizing and
binding specifically to components of dental plaque.
The composition will generally include a vehicle
suitable for topical application, especially in the mouth.
This invention also relates to a method of delivering a
benefit agent to a target location, especially in the mouth,
by incorporating the agent in microcapsules as above which
are in turn incorporated into a composition for topical
application. The invention further comprises use of the
microcapsules in preparing such compositions.
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A preferred composition contains liposomes which
enclose a therapeutic agent, active against oral bacteria.
The liposomes may be prepared from those materials
which are known for the purpose; examples are given in J H
Fendler, "Membrane Mimetic Chemistry~ (Wiley-Interscience,
New York, 1982) and in J N Weinstein and J D Leserman,
Pharmac, Ther., 1984 24, 207-233. Among the materials most
commonly used are phospholipids from natural sources such as
lecithin from egg or soya, and synthetic analogues such as
L-a-dipalmitoyl phosphatidylcholine (DPPC). Charged
phospholipids such as phosphatidyl inositol are often
incorporated in liposomes to improve colloidal stability.
Techniques for preparation of liposomes are described
in G Gregoriadis, "Liposome Technology - Vol 1", (CRC Press,
1984) and in P R Cullis et al., "Liposomes - from Biophysics
to Therapeutics", Chapter 5, (Ed. M J Ostro, Marcel Dekker,
New York, 1987). The methods which we prefer are sonication
(in an ultrasonic bath) of a phospholipid dispersion and
reverse phase evaporation. Another method which may be used
is "extrusion" under pressure through very fine passages
such as provided by Nuclepore (RTM) membranes.
The techniques lead to somewhat different liposomes.
Sonication and extrusion generally lead to small vesicles,
less than 100 nm in diameter. Reverse phase evaporation
leads to larger vesicles having diameters ranging from 100
nm up to several microns.
Liposomes may act as carriers for water-soluble, oil-
soluble or microcrystalline solid materials. Incorporation
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of water-soluble material into liposomes is normally
accomplished by including the material into the aqueous
solution during liposome formation. When the liposomes are
formed, some of the solution bec ~ enclosed within the
vesicles. Oil-soluble materials may be incorporated by
including the material in the lipid mixture prior to
formation of the liposomes; such materials are normally
sequestered in the lipid membrane. Microcrystalline solid
materials may be incorporated by internal precipitation of
insoluble salts when one of the constituent ions is
encapsulated and the other is allowed to permeate through
the liposomal membrane from the external solution.
An organic material for which the liposomes have
affinity may be for example a protein, glycoprotein or
carbohydrate exposed on the surface of the oral cavity.
Possible targets include the proteins of tooth pellicle,
polysaccharides of mucosal surfaces such as gums, tongue and
cheeks, and extracellular polysaccharides of oral bacterial
species such as Streptococci, Actinomyces, Lactobacilli and
Bacteroides (which form dental plaque). Particularly
preferred targets are Streptococcus mutans or Streptococcus
sanguis.
Means for attaching liposomes to a target are suitably
at the exterior of the liposomes and may be a molecule
having strong affinity for said target, for example a
specifically binding protein, polysaccharide, glycoprotein,
lipoprotein or lipopolysaccharide. One such type of
molecule is an antibody, which might be monoclonal or
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polyclonal. Monoclonal antibodies may be generated using
the technique first described by Kohler and Milstein
(Nature, 256, 495-479, 1975). Antibodies may, alternatively
be generated by the use of recombinant DNA techniques, for
example as described in US Patents 4816397 and 4816567.
Specific binding subunits or antibody fragments may also be
used. These may be generated by enzymic digestion of intact
antibody molecules, for example using papain or pepsin, or
may be produced using the recombinant DNA techniques
described in the two US patents referred to above.
A further possibility is to use a lectin bound to the
outer surface of the liposomes. Lectins are plant-derived
proteins with binding affinity for certain sugar groups
which occur in polysaccharides and glycoproteins. Their
binding affinity tends to be less specific than that of
antibodies.
Both antibodies and lectins can be bound to liposomes
by covalent bonds. Various techniques are known for
conjugating proteins to other materials and generally entail
providing each of the two species with one of a pair of
materials which will react together. Suitable methods are
described in M J Ostro, "Liposomes - from Biophysics to
Therapeutics", Chapter 5 (Marcel Dekker, New York, 1987) and
in F J Martin et al., "Liposomes - A Practical Approach",
Chapter 4 (Ed R R C New, IRL Press, Oxford, 1990). Our
preferred technique for attaching to liposomes is to react
m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) with a
component of the phospholipid mixture before formation of
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the liposomes and to react N-succinimidyl-S-
acetylthioacetate (SATA) with the protein. After liposome
formation the residues of these substances are coupled
together in aqueous medium under mild conditions.
The use of these materials to conjugate antibodies and
lectins to liposomes has been described in F.J. Hutchinson
et al., Biochim. Biophys. Acta., 978 (1989) 17-24.
The oral benefit agent which is incorporated within the
liposomes may be for example a therapeutic agent, in
particular an anti-bacterial agent effective against
bacteria which cause dental plaque, an anti-inflammatory
agent capable of reducing gingivitis, an anti-tartar agent,
an anti-caries agent, or a tooth desensitising agent.
Alternatively, a non-therapeutic agent may be incorporated,
for example a flavour for sustained breath refreshment.
Of particular interest are antibacterial agents having
molecular weight not greater than 2000. Within this
category biphenolic compounds are of interest. A preferred
benefit agent is Triclosan, (2,4,4'-trichloro-2'-hydroxy
diphenyl ether) which is a broad spectrum antibacterial
agent active against common oral pathogens.
Preferred possibilities for forms of composition
containing the liposomes are a mouthwash and a gel. Other
possibilities include a toothpaste and a lozenge able to
dissolve in the mouth.
Proportions may vary widely. However, the benefit
agent will generally provide from 0.01 to 50% by weight of
the microcapsules which in turn will generally provide from
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0.01 to 50% by weight of the composition. The benefit agent
will generally provide from 0.001 to 10% by weight of the
composition.
The benefit agent preferably provides from 0.1 to 30~,
more especially 0.5 to 10~ by weight of the microcapsules.
The microcapsules will preferably constitute 0.1 to 10%,
more especially 1 to 5~ by weight of the composition. The
benefit agent preferably provides from 0.01 to 5%, better
0.05 to 1% by weight of the overall composition.
Example 1
Liposomes were prepared by the reverse phase
evaporation procedure referred to above incorporating
Triclosan in the lipid mixture. Some of the liposome
samples were prepared with concanavalin A (ConA) conjugated
to their surface. Details are as follows:
(a) Derivatisation of ConA with SATA 2.5~1 of a stock
solution containing 9.08mg N-succinimidyl-S-acetyl
thioacetate (SATA) in 50,ul dimethylformamide was added to
ConA solution (lOmg in 2.5ml phosphate (50mM) - EDTA (lmM)
buffer, pH7.5) at room temperature. After reaction (15min)
the derivatised protein (s-ConA) was separated from
unreacted SATA by gel filtration on a Sephadex~ G-50 column
(15 x 2cm). The s-ConA was activated by deacetylation
using, for each 2ml of protein solution, 200,ul of O.lM
hydroxylamine solution, made up in 2.5mM EDTA with
sufficient solid Na2HPO4 added to bring the pH to 7.5.
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(b) Preparation of DPPE-MBS
40mg L-a-dipalmitoylphosphatidylethanolamine (DPPE) was
dissolved in a mixture of 16ml dry chloroform, 2ml dry
methanol and 20mg dry triethylamine. 20mg maleimidobenzoyl-
N-hydroxysl~cci n; mide ester ( MBS ) was added and the reaction
mixture was stirred under nitr~gen at room temperature for
24h, after which the organic phase was washed three times
with phosphate buffered saline ( PBS, pH7.3) to remove
unreacted MBS . The DPPE-MBS derivative was recovered from
the organic phase by rotary evaporation and was stored in a
chloroform/methanol mixture (9:1 v/v) at 4~C.
(c) Preparation of liposomes
27mg dipalmitoylphosphatidylcholine ( DPPC ), 3mg
phosphatidylinositol (PI, from wheat germ) and 3mg DPPE-MBS
were dissolved in 9ml chloroform/methanol mixture (4:1 v/v).
4mg Triclosan was added to this solution when encapsulation
of the antibacterial agent was required; [14C]-DPPC and
[3H]-Triclosan were incorporated as radiotracers in some
experiments. A 3ml aliquot of this solution was placed in a
50ml round bottomed flask and the solvent removed by rotary
evaporation (60~C) to yield a thin lipid film. The film was
redispersed in 6ml 4:1 chloroform/methanol, 3ml of nitrogen-
saturated 1:10 diluted PBS was added at 60~C and the mixture
gently shaken, followed by 3min sonication under nitrogen at
50~C. The resulting homogenous emulsion was rotary
evaporated at 60~C until, after passing through an
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intermediate viscous 'gel' phase, it inverted to a water-
continuous system. The aqueous liposomal dispersion was
then purged with nitrogen for a further 15min at 50~C to
remove traces of organic solvent and was kept at that
temperature for another 15min to allow annealing to occur.
Liposome dispersions thus prepared were passed through
a Sephadex G-50 gel filtration column (30x2cm) pre-
equilibrated with PBS. The fractions collected were
analysed for phospholipid and Triclosan by scintillation
counting. Particle sizes of the liposomes were determined
by photon correlation spectroscopy, following the method
described in Hutchinson et al., Biochim. Biophys, Acta, 978
(1989) 17-24.
(d) Conjugation of ConA to liposomes
Some samples were prepared with concanavalin A covalently
coupled to the surface of the liposomes. This was
accomplished simply by mixing and equilibrating aliquots of
the dispersion with the deacetylated SATA derivative of ConA
Z0 in appropriate proportions at room temperature for 2 hours
or at 4~C overnight. After conjugation the reaction mixture
was applied to a Sepharose 4B column to separate the
proteoliposomes from unreacted protein.
(e) Assessment of efficacy
The antibacterial efficacy of Triclosan delivered in
targeted liposomes was assessed using a bacterial regrowth
assay. The oral bacterium Strep. sanguis (CR2b) was grown
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for 18h at 37~C in medium containing 10% BHI, 0.3% yeast
extract and 1% sucrose. The bacteria were harvested by
centrifugation (2000rpm, 5min), washed 3 times with sterile
PBS and resuspended in PBS to give an optical density at
550nm of 0.5. lOO,ul of suspension (containing 7.2 x 105
cells by viable cell counting) was pipetted into a
microtitre plate well and left overnight to adsorb. After
incubation the well was washed twice with 300~ul sterile PBS,
and the plate was blotted dry; the number of cells remaining
was 1.4 x 105. Non-specific binding sites were then blocked
by treatment for 30min at 20~C with a sterile solution of
0.02% w/v casein in PBS, after which the well was washed 3
times with PBS and the plate blotted dry.
lOO,ul of test solution containing either lO,ug free
Triclosan or liposomes with or without lO,ug Triclosan, all
in PBS containing 10% ethanol (Triclosan is virtually
insoluble in PBS alone; this level of ethanol does not kill
the bacteria), was added to the well and allowed to adsorb
for 2h at 37~C. The plate was then washed 3 times with
sterile PBS, lOO,ul growth medium added (10~ BHI plus 0. 3%
yeast extract and 1% sucrose), sealed and incubated
statically in a candle jar for 18h at 37~C. After this
incubation period, the plate was read using a Dynatech MR610
plate reader and the extent of continuing bacterial growth
determined from the measured optical density at 630nm.
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(f) Results
Treatment solution% inhibition of growth*
Triclosan in solution 75
Triclosan in liposomes
5 without ConA 12
Triclosan in liposomes
with ConA 38
* relative to PBS alone
Liposomes without ConA were 1.096 x 10-6 moles lipid
per ml, including 23.97% Triclosan by weight, and had weight
average diameter 251nm.
Liposomes with ConA were 9.61 x 10-7 moles lipid per
ml, including 13.67% Triclosan by weight, had weight average
diameter 213nm and weight average number of 814 ConA
molecules per liposome.
Free Triclosan was lO,ug in 100~1 of PBS containing 10~
ethanol. Total Triclosan in liposome systems was lO~g for
liposomes with ConA and 20~g for liposomes without ConA.
It can be seen that with the long (2 hour) contact
time, delivery in liposomes reduced the effectiveness of the
Triclosan. Liposomes conjugated to concanavalin A were
better than liposomes without this lectin at their surface,
but not as effective as Triclosan alone.
Example 2
The procedures of Example 1 were repeated using similar
liposomes, but with a slightly lower level of Triclosan and
with the time of exposure to the treatment solutions reduced
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from 2h to lmin, to more realistically simulate use of a
product intended to give topical application in the mouth.
This was carried out using PBS as in Example 1 for
some samples, and PBS plus human saliva (lOO,ul of 1:1
mixture of saliva with sterile PBS) for others to give a
closer representation of oral conditions. Comparative
experiments again used Triclosan in solution in PBS
containing 10% ethanol.
Results % inhibition of growth
Treatment solutionincubatedwithout saliva
with saliva
15 Triclosan in solution 3 8
Triclosan in liposomes
with concanavalin A26 20
Liposomes had weight average diameter 339nm and weight
average number of 1467 molecules of concanavalin A per
liposome.
All treatment systems contained 5~ug Triclosan in 100~1
PBS with 10% ethanol.
It can be seen that when the period of exposure to the
treatment solution is short (a realistic situation), the
liposomes greatly increase the antibacterial action compared
with Triclosan in free solution.
