Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS FOR GLUCAN PREPARATION
AND THERAPEUTIC USES OF GLUCAN
FIELD OF INVENTION
S The present invention relates to a process for the extraction of a naturally
occurring
carbohydrate (glucan) from microorganisms as well as the glucan produced by
this process.
The invention also relates to novel therapeutic uses of glucan.
BACKGROUND TO THE INVENTION
Glucan is a generic term referring to an oligo- or polysaccharide composed
predominantly
or wholly of the monosaccharide D-glucose. Glucans are widely distributed in
nature with
many thousands of forms possible as a result of the highly variable manner in
which the
individual glucose units can be joined (glucosidic linkages) as well as the
overall steric
shape of the parent molecule.
The glucan referred to in this invention typically is a linear chain of
multiple
glucopyranose units with a variable number of side-branches of relatively
short length.
The glucosidic linkages are predominantly (not less than 90%) [3-1,3 type with
a lower
number (not greater than 10%) of (3-1,6 type linkages; the (3-1.3 linkages
form the bulk of
the backbone of the molecule, while the ~i-1,6 linkages occur predominantly in
the
side-branches. The chemical name of this form of glucan is poly-(1,3)-(3-D-
alucopyranosyl-(1,6)-~i-D-glucopyranose. Glucan is a well described molecule.
This form of glucan is found principally in the cell wall of most fungi
(including yeasts and
moulds) and in some bacteria. Glucan, in combination with other
polysaccharides such as
mannan and chitin, is responsible for the shape and mechanical strength of the
cell wall.
The glucan typically accounts for approximately 40% to 50% of the weight of
the cell wall
in these cells.
The chemical structure of fungal cell wall glucan has been studied in detail
by
Bacon et al (1969) and Manners et al (1973).
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Fungal cell wall glucans have long been used in industry, particularly the
food industry,
usually in a semi-purified form. Their uses have included use as stabilizers,
binders,
thickeners and surface active materials.
It also has been known for some forty years that fungal cell wall glucans are
biologically
active, exerting a number of effects on the reticuloendothelial and immune
systems of
animals. The outstanding biological effect in this regard is their abiliy to
stimulate non
specifically the activity of the body's primary defence cells - the macrophage
and the
neutrophil. This is thought to be due to receptors to (3-1,3 glucan displayed
on the surface -
of these cells (Czop and Austen, 1985). The interaction between glucan and its
receptor
producing such stimulatory effects as enhanced phagocytosis (Riggi and Di
Luzio, 1961),
increased cell size (Patchen and Lotzova, 1980), enhanced cell proliferation
(Deimann and
Fahimi, 1979). enhanced adherence and chemotactic activity (Niskanen et al,
1978), and
production of a wide range of cytokines and leukotrienes (Sherwood et al.
1986, I987).
The aforementioned biological responses to fungal cell wall glucan have been
reported to
result in a number of clinical effects including: enhanced- resistance to
infections with
fungi (Williams et al, 1978), bacteria (Williams et al, 1983), viruses
(Williams and
Di Luzio, 1985), protozoa (Cook et al, 1979) following systemic application:
enhanced
antitumour activity following systemic application(Williams et al. 1985) or
intralesional
application (Mansell et al, 1975); and enhanced immune responsiveness
following systemic
application (Maeda and Chihara, 1973). It will be readily seen that these
clinical effects
are highly beneficial and important and represent an opportunity to develop
novel
pharmaceutics based on fungal cell wall glucans, such pharmaceutics having
potentially
wide application in both veterinary and human medicine.
Of the various fungal cell wall glucans tested, that from the yeast
Saccharomyces
cerevisiae has proven to be acceptable in terms of efficacy and safety as an
immune '
stimulant in animals and humans. Hereinafter this will be referred c~ ac
.s~~~hnrnmvrpc
cerevisiae ("Sc")-glucan. Predominantly or wholly (3-1,3 glucans from other
fungi,
bacteria or plants from the Graminaceae family have been shown to be
immunostimulatory
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in animals but compared to Sc-glucan either are not as potent or if they do
have
comparable or greater potency then that is usually associated with a higher
level of
undesirable side-effects.
Sc-glucan has been shown to be biologically active as an immune stimulant in
animals in
various forms. These include (a) a large molecular weight (typically greater
than 3 x 106
d), water-insoluble, microparticulate form, or (b) smaller molecular weight
(typically less
than X00,000 d) forms which are dispersible or soluble in water. Water-
solubility is
described as being achieved either through cleavage of the large
microparticulate glucan
form to smaller molecules using processes such as enzymatic digestion or
vigorous pH
adjustments, or by complexing to salts such as amines, sulphates and
phosphates. The
principal advantage of the smaller, water-soluble form vs the larger
microparticulate form
is that it is safer when Given by parenteral routes of administration such as
intravenously.
Also, it is likely that the smaller sized molecules are more bio-available on
a molar basis.
To date it has neither been technically possible nor economically feasible to
synthesise
glucan on a commercial basis. Thus preparation of commercial quantities of (3-
I,3 glucan
for therapeutic uses requires that it be extracted from fungi, bacteria, algae
or cereal
grams.
DESCRIPTION OF THE PRIOR ART
A number of different processes are described for the preparation of Sc-alucan
for
pharmaceutical use. A common feature of these different processes is the
extraction of
microparticulate glucan as the primary step; the glucan is either then used in
the final
therapeutic formulation in that microparticulate form or is further processed
to a smaller
molecular weight material ("soluble glucan") by modification of its chemical
and/or spatial
structures.
(l) MicroDarticulate ln~an
The extraction of Sc-alucan from whole yeast cells depends on the fact that
the bulk of the
cell wall glucan is insoluble in water, strong alkali, acid and organic
solvents whereas all
other cell wall components are soluble in one or more of these solutions.
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The essential principles of extraction of Sc-glucan are (i) lysis of the yeast
cell to allow the
intact cell walls to be separated from the less dense cytoplasmic contents,
and (ii)
subsequent or concomitant dissolution of unwanted wall components such as
other
carbohydrates (glycogen, mannan, glucosamine), lipids and proteins using
various .
combinations of water, alkali, acid and organic solvents. It is preferred in
such processes
that the three-dimensional matrix structure of the cell wall remains unaltered
and intact as a
cell wall skeleton (also known as a "cell sac"), comprised predominantly of (3-
(1,3)(1,6)-
glucan. The cell wall skeletons characteristically are spherical, hollow
structures of
approximately 4 to 20 a diameter and with a molecular weight of between
approximately
1,000,000 to 3,000,000 daltons and they are -insoluble in water. This end-
product is
termed microparticulate Sc-glucan.
A number of methods of extraction of microparticulate Sc-glucan are known,
although all
are essentially variations of a common method. The described methods entail
the
following steps.
1. Contact of whole yeast cells with strong alkali solution (pH 12 to 14).
This effects
lysis of the cells and dissolution of most of the non-glucan components except
lipids. This step is uniformly rigorous in all described processes. The
contact
usually is repeated two to three times using fresh batches of alkali and heat
also
usually is applied to speed the reaction time.
2. The cells then are exposed to acid (pH 1 to 5) with heat to effect
dissolution of
certain residual non-glucan components and to effect some hydrolysis of the
glycosidic linkages, principally the (3-1,6 linkages in the side brances and
to a
minor extent ~3-1,3 linkages in the glucan backbone side-branches. The rigour
of
this step varies considerably between the known processes of relatively mild
acid
treatment where the conformational changes are minimal and many of the side- ,
branches are retained. through to extensive acid treatment where little or no
side-
branches remain and which permits hydration of the helical glucan coils during
w
subsequent steps to convert to a water-soluble form.
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3. Contact of the cell residue with alcohol and heat with or without
additional
subsequent exposure to solvents, particularly ether or petroleum ether to
effect
removal of lipids.
N 5 See, for example, Hassid et al (1941), Manners (1973) et al, Di Luzio
(1979), and US
Patent Nos 4810694 and 4992540.
Prior art methods for the production of microparticulate glucan may be
regarded as
disadvantageous in one or more respects. These include poor yield (such as
less than about
5% w/w), low purity (such as less than about 90% purity), extended processing
time,
significant waste production, and high cost.
(ii) Soluble glue
Microparticulate Sc-glucan is water insoluble due to the tightly bound triple
helical
carbohydrate coils which resist hydration.
There are t~~o principal purposes to seek to solubilize Sc-glucan. The first
reason is the
risk of microembolization associated with the injection of microparticulate
glucan by
intravenous or other parenteral routes. The second reason is that a reduction
in molecular
weight of the Sc-glucan might reasonably be expected to be associated with
increased
biological efficacy due to greater bioavailability of the glucan molecules.
Solubilization of microparticulate glucan can be achieved in various ways.
One way is to expose the glucan to a specific enzyme, (3-1,3-glucosidase which
cuts the
long linear chain into shorter lengths. The disadvantage of this method is
that the enzymic
digestion process is difficult to control and can result in excessive
hydrolysis of the glucan
molecule to monosaccharides or oligosaccharides which lack immunostimulatory
activity.
Another way is to attach charged groups such as phosphate (US Patent Nos
4,739,046;
4,761,402), sulphate (Williams et al, 1991) and amine (US Patent No 4,707,471)
which
permit hydration of the molecule. Both phosphorylated (US Patent No 4,761,042)
and
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sulphated (Williams et al, 1991) Sc-glucans retain their immunostimulatory
activity and
are highly water soluble. A disadvantage of these methods is that of an
additional step of
complexity in processing operations, which may add considerably to overall
manufacturing
cost.
A third approach to solubilization is by sequential alkali/acid/alkali
hydrolysis. This was
first demonstrated by Bacon et al (1969) who showed that microparticulate Sc-
gluten
extracted in the traditional manner by repeated NaOH exposures followed by an
acid wash,
almost completely dissolved when subsequently exposed to 3 % NaOH at
75°C.
~ The resultant gluten is of widely
heterogenous molecular weight with a high polydispersity index associated with
the
presence of gluten molecules varying in size from approximateh' 5.000 d up to
approximately 800,000 d. That patent application describes further
purification by
diafiltration of the hydrolyzed gluten to isolate gluten molecules of defined
molecular
weight from the heterogenous molecular weight species produced, and the use of
various
resins to remove contaminating proteinaceous and lipid components.
The present im~ention insofar as it is concerned with processes for the
production of
gluten, whether in microparticulate or non-particulate form ("soluble"). seeks
to overcome
one or more of the problems/deficiencies of prior art processes for the
production of
gluten.
In addition, as described hereinafrer, this invention is also concerned with
novel
therapeutic uses of gluten, whether produced by the method herein. or other
methods
known in the prior art.
SUMMARY OF THE INVENTION
In accordance with a first aspect of this invention there is provided a
process for
production of p-(1.3)(i,6) gluten from a gluten containing cellular source
which comprises
the steps of:
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(a) extracting glucan containirig cells with alkali and heat in order to
remove alkali
soluble components;
(b) acid extracting the cells obtained from step (a) with an acid and heat to
form a
suspension;
(c) extracting the suspension obtained from step (b) or recovered hydrolyzed
cells with
an organic solvent which is non-miscible with water and which has a density
greater than that of water and separating the resultant aqueous phase, solvent
containing phase and interface so that substantially only the aqueous phase
comprising glucan particulate material suspended in water remains;
wherein the extraction with said organic solvent provides separation of glucan
subgroups comprising branched (3-(1,3)(1,6)-glucan, and essentially unbranched
(3-
(1,3) glucan which is associated with residual non-glucan contaminents; and
(d) drying the glucan material from step (c) to give particulate glucan.
In order to produce a soluble glucan, step (d) of the above process is omitted
and the pH of
the solvent extracted aqueous phase comprising glucan particulate material is
raised from
an acidic pH, to a basic pH so as to effect solubilization of the glucan
particles. This step
is carried out at a temperature below about 60°C, preferably between
about 2°C to about
25°C. more preferably between about 2°C to about 8°C, for
a time sufficient to achieve
solubilization of the glucan particles. Alternatively, soluble glucan may be
prepared by
suspending the particulate glucan of step (d) in an aqueous alkali solution so
as to effect
solubilization of the glucan particles. Temperate conditions are set out
above.
The pH of the solubilized glucan may then be adjusted as required to give a
pharmaceutical
product.
In another aspect this invention is directed to the use of glucan for the
manufacture of a
medicament for the treatment of skin ulceration or bone fracture or the
enhancement of
fixation of implanted orthopaedic devices, or the prevention/treatment of
ultraviolet light
induced skin damage.
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In a further aspect this invention is concerned with a method for the
treatment of skin
ulceration or bone fracture or the enhancement of fixation of implanted
orthopaedic
devices, or the prevention/treatment of ultraviolet light induced skin damage,
which
comprises administering to a subject glucan in association with one or more
pharmaceutically or veterinarily acceptable carriers or excipients.
In another aspect this invention is concerned with an agent for the treatment
of skin
ulceration or bone fracture or the enhancement of fixation of implanted
orthopaedic
devices, or for the prevention/treatment of ultraviolet light induced skin
damage which
comprises glucan optionally in associate with one or more pharmaceutically
acceptable
carriers or excipients.
DETAILED DESCRIPTION OF THE INVENTION
The process described in detail hereafter sets out the production of (3-
(1,3)(1,6) glucan
from a cellular glucan source, which is suitable for a variety of
pharmaceutical purposes.
In a first aspect the invention is concerned with a process for the production
of glucan from
a glucan containing cellular source. This process comprises the steps of:
(a) extracting glucan containing cells with alkali and heat, in order to
remove alkali
soluble components;
(b) acid extracting the cells of step (a) with an acid and heat to form a
suspension;
(c) extracting the suspension obtained of step (b) or recovered hydrolyzed
cells with an
organic solvent which is non-miscible with water and which has a density
greater
than that of water and separating the resultant aqueous phase, solvent
containing
phase and interface so that substantially only the aqueous phase comprising
glucan
particulate material remains;
wherein the extraction with said organic solvent provides separation of glucan
subgroups comprising branched (3-(1,3)(1,6)-glucan, and essentially unbranched
(3-
(1,3) glucan which is associated with residual non-glucan contaminents: and
(d) drying the glucan material from step (c) to give particulate glucan.
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While yeast cells generally and the yeast strain Saccharomyces cerevisiae in
particular are
the preferred source of the glucan according to this invention, any other
cells such as fungi
or bacteria containing glucan with the properties described herein may be
used. A wide
4
range of other yeast and fungal strains can be used in the present process and
the following
types are included by way of example: Sclerotium spp, Shizophyllum spp, Pichia
spp,
Hansenula spp, Candida spp, Saccharomyces spp, Torulopsis spp.
In the case of Saccharomyces cerevisiae the yeast may be grown specifically
for the
purpose of extraction of Sc-glucan or may be from a commercial source such as
yeast
manufactured for the baking industry or spent yeast from the brewing
industr~~.
The first step according to the process of the present invention involves
treatment of the
yeast cells with alkali and heat to effect cytolysis and hydrolysis of the
cytoplasmic
components and predominant cell wall components including mannan, chitin
(glucosamine), proteins and glycogen. This treatment (which may also be
referred to as
extraction or hydrolysis) releases non-glucan components into the aqueous
phase so that
they might readily be separated by a process such as centrifugation from the
intact cell
walls comprising largely glucan. The extent of non-glucan component removal
can be
readily assessed by standard analytical techniques, such as those described in
United Patent
No 4992540.
The alkali extraction step may be carried out in aqueous hydroxide of from
about 2 % to
about 6% concentration (w/v), such as between 3% and 4% (w/v). Sodium
hydroxide or
potassium hydroxide find particular application because of their availability
and relatively
low cost. However. any other strong alkali solution which has suitable
solubility
characteristics, for example, calcium hydroxide or lithium hydroxide, can be
used. The
yeast is left in contact with the alkali for a time sufficient to remove
alkali soluble non-
glucan components. Non-glucan components are removed more rapidly at higher
temperatures. The digestion may be carried out at temperatures of from about
50°C to
about 120°C, requiring exposure times to the alkali of between fifteen
minutes and sixteen
hours. During alkali exposure, the process of cytolysis and dissolution of non-
glucan
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components may be facilitated by vigorous mixing of the yeast suspension using
appropriate methods such as by example a stirring apparatus or an emulsifying
pump.
Repeat exposure of the yeast cells to fresh batches of alkali solution assists
in removing
non-glucan material, particularly protein, from the disrupted yeast cells. The
number of
alkali treatments is not limiting on the invention. However, the process
should be repeated
until it is apparent that the cells have been lysed and the majority of non-
glucan alkali
soluble components extracted. This can be confirmed by visual or chemical
analysis (such
as by gas chromatography/mass spectrometry). Treatments using low strengths of
hydroxide solution and low temperatures of alkali exposure generally may
require
increased numbers of separate alkali exposures. By way of example, alkali
treatment may
be repeated from one to six times.
In one embodiment of the present invention in relation to the alkali digestion
phase, dried
commercial Saccharo~rryces cerevisiae is suspended to 10 % w/v in sodium
hydroxide at a
strength of between 3 % and 4 % and at temperatures of between 80°C and
100°C. It has
been found that three r alkali treatments are typically required for a high
purity product.
Following each separate alkali exposure, the disrupted yeast cells and the
supernatant
solution are separated by any method which is known to this-art including, for
example,
filtration, centrifugation or chromatography. These separation techniques are
referred to
by way of example only and are not limiting to tre process of the present
invention.
The next step in the process involves the exposure of the alkali-insoluble
cell wall sacs to
acid, generally at a pH from about 2.0 to 6, preferably between 3.5 to 4.5.
This procedure
dissolves some residual contaminants such as mannan and chitin. However, the
principal
reason for this step is to induce conformational alterations to the glucan
molecule. The
principal alteration is a reduction in the number of (3-1,6 side-branches
(Table 1). In native
cell wall Sc-glucan, the proportions of glycosidic linkages is approximately
90% (3-1,3 and
10% (3-1,6. Acid hydrolysis removes the ~3-1,6 side-branches with the degree
of hydrolysis
being related directly to the vigour of the acid treatment; strong acid
treatment (low pH
and high temperature, such as pH less than 2 and temperatures above about
100°C) can
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effectively remove all side-branches whereas less vigorous treatment will
leave (3-1,6
linkages in the proportions of between approximately 1 ~ and 8 % .
Effect of acid exposure (phosphoric acid, ph 4.5, 100°C 30 minutes) on
the chemical
composition of alkali insoluble Sc-glucan as measured by gas chromatography-
mass
spectroscopy.
Pre-acidPost-acid
Mannan (96 w/w monoslccharides)0.5 0
.
(i-glycosidic linkages
(mot %a):
1,3 54.2 94.4
1,4 7.1 0
1,3.4 0.7 0.2
t,2,3 2.2 0.5
1.3.6 5.6 ?.2
1.6 9.7 0
1,4,6 0.8 0
1.2.3,4 1.5 0
1.3.4,6 1.9 0
1.2.3,6 0.4 0
Terminal-glc 6.4 2.9
glucitol hexaacetate 10.8 0
It is known in the art that the degree of branching of p-1,3-glucan molecules
has an
important influence on biological function. For example, it is known that
highly branched
glucans such as lentinan induce pro-inflammatory effects in addition to
immunostimulatory
effects and that the pro-inflammatory effects may be associated with adverse
clinical side-
effects; unbranched Sc-giucans such as those described in US Patent Nos
4,739,046,
4,761,402 and 4,7707,471 or Sc-glucan with reduced branching
are known to avoid or to greatly diminish pro-inflammatory
effects and therefore be more desirable therapeutic agents clinically.
Hitherto, however. the
structure/function relationship in terms of immunostimulatory capacity and
promotion of
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tissue repair in particular has not been defined. The inventors have defined
the optimal
degree of branching by comparing the efficacy of differently branched glucan
preparations
in an animal wound healing model. For example, a full-thickness surgical skin
incision
may be made in experimental animals such as laboratory rats. Glucan is applied
to the
wound immediately following wounding and the wound then allowed to heal. Seven
days ,
later the degree of healing is tested by determining the amount of force
required to separate
the apposing wound edges (referred to as 'wound breaking strength'). The
results of this
experiment are summarised in Table 2. It can be seen that where the degree of
branching is
measured in terms of the proportion of ~i-1,3:/3-1,6 linkages, both a low
proportion
(90 % :10 % ) as for native glucan and a high proportion ( 100 % :0 % ) are
less effective in the
promotion of dermal wound repair than moderately-branched (98 % :2 %o or 96 %
:4 % )
glucan.
Tahle 2
Tensile strength of rat skin wounds (day +7) following application of micro-
particulate Sc-
glucans with different ratios of (3-1,3 to ~3-1,6 glycosidic linkages.
Treatment r~ ~ -1.3:p -1.6 link~esWound tensile stren
tg h l
~l
,
mean (SD)
No glucan 16 - 202 (37)
Glucan 8 90% : 10% 252 (45)
Glucan 12 96% : 4% 358 (49)
Glucan 9 98% : 2% 339-- (38)
Glucan 10 100% : 0% 285 (52)
1 mg of glucan was applied at time of operation in oily base to 5 cm long full-
thickness
incisional wound.
The nature of the acid used in the acid exposure step is generally
unimportant. Preferably,
the acid is employed to provide a pH of the resultant yeast suspension from
about pH 2.0
to about 6.0, more preferably from about pH 3.5 to about 4.5. Suitable acids
include
hydrochloric, acetic, formic and phosphoric acids.
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The process of acid hydrolysis is aided by heating.
The extent of acid treatment, namely pH, temperature -and time depends on the
degree of
' (3-1,6 content sought in the glucan product. In order to produce a glucan
product generally
containing from 2% to 4% (3-1,6 linkages, the pH of the solution is selected
to be in the
range of about 2 to about 6, temperature is generally between about
50°C and about
100°C, and the time of reaction from about fifteen minutes to about
sixteen hours. The
extent of ~3-1,6 linkages in the hydrolyzed glucan can be readily determined
by standard
analytical techniques such as nuclear magnetic resonance (NMR) analysis.
Following the acid exposure stage, the yeast cells predominantly are in the
form of isolated
cell wall sacs.
In prior art methods of Sc-glucan preparation it has been proposed to expose
acid extracted
IS glucan containing cells (cell sacs) with alcohol, petroleum ether or
diethyl ether, to
selectively dissolve remaining non-glucan components. In contrast, it has been
found by
the inventors that extracting the acidified glucan containing cells with an
organic solvent
which is non-miscible with water, that is, has a density greater than I g/cm3,
is particularly
and unexpectedly advantageous. Specifically, a single extraction step with
such a solvent
provides a fine discrimination between glucan and non-glucan components, and
allows
ready separation of glucan subgroups comprising branched glucan containing
both (3-1.3
and (3-1,6 linkages (which partitions into the aqueous phase) and which is
essentially free
of non-glucan components (Table 3), and glucan comprising essentially
unbranced (3-1,3
linkages only and which is associated with residual non-glucan membrane
components such
as chitin and protein (which partitions at the interface between the aqueous
and organic
phase).
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ahle
Effect of chloroform extraction on the chemical composition of alkali/acid
treated Sc-
glucan.
Chemical
composition
(%
w/v)
GlucanMannan ProteinChitinGlycogenLipids
Prechloroform
treatment 85.5 0.5 1.4 2.1 4.3 5.6
Postchlorofonn
treatment 98.5 < 0.1 0.3 0.2 0.4 -
The branched (3-(I ,3)(1,6) glucan subgroup which partitions into the aqueous
phase may
contain minor or trace amounts of unbranched (3-1,3 glucan (less than about
5%, generally
less than about 2 % , more specifically less than about 0.5 % (w/w)) and trace
amounts of
non-glucan contaminents. It may thus be regarded as essentially branched /3-
(1,3)(1,6)
glucan which is free of other glucan and non-glucan components. The unbranched
~i-(1,3)
glucan subgroup which is associated with non-glucan contaminents and which
partitions
into the interface between the aqueous phase and organic phase can be readily
removed. It
may contain very minor or trace amounts of branched (3-(1,3)(1,6) glucan
(generally less
than about 1.3 9~ (w/w)) and hence is considered to be essentially unbranched.
Unbranched ~3-(1,3) glucan may comprise up to 20% of total glucan content
(w/w)
following alkali/acid/solvent treatment, the remainder comprising branched (3-
(1,3)(1,6)
glucan.
Branched (3-(1,3)(1,6) glucan is the most potent biologically active form of
Qlucan in terms
of wound healing as shown in Table 4.
a~ to 4
Tensile strength of rat skin wounds (day +7) following application of Sc-
glucans recovered
from either the aqueous or interface phase following chloroform extraction.
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Treatment n Post-chloroform phase Wound tensile strength fQl
mean (SD)
No glucan 12 - 185 (21 )
Glucan 14 Aqueous 345 (57)
Glucan 8 Interface 267 (59)
Thus it can be readily appreciated, particularly in terms of e~cacy of
promotion of dermal
wound healing and the production of pure glucan molecules, that there is much
potential
therapeutic benefit in separating the two glucan sub-groups by chloroform
extraction
(representative of solvents having a density greater than 1).
Solvents which may be used include chloroform (8 = 1.48 g/cm~),
methylchloroform
(8 = 1.33), tetrachloroethane (a --- 1.5953 g/cm3), dichloromethane (S =
1.325). and
carbon tetrachloride (8 = 595 g/cm3). Preferably the solvent is volatile to
allow ease of
removal of any residual. Chloroform is particularly preferred.
For convenience of description the description hereafter will refer to the use
of the
preferred solvent, chloroform. The invention is not so limited, and any
solvent having the
requisite density may be used in the invention.
The chloroform extraction may be performed in the following manner. The
acidified
aqueous suspension containing microparticulate glucan may be reacted directly
with
chloroform in the approximate ratio of chloroform:aqueous cell suspension of
between
1:10 and 5:1, preferably 1:4. The yeast cells may comprise (by volume) between
about
1 % and about 90 % of the aqueous suspension, such as between about 30 % and
50 90 . It
has been found that the process of extraction with chloroform is not
facilitated by heat and
preferably is carried out at room temperature. The chloroform and aqueous
phases are
mixed vigorously using standard methods including, for example, stirring
apparatuses or
' 30 an emulsifying pump so as to effect good contact between the chloroform
micelles and the
yeast cells. The duration of mixing is a function of the volume of the
suspension and the
stirring or mixing capacity of the stirring or mixing apparatus. An example by
way of
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illustration is that an emulsifying pump with a pumping capacity of 100 L per
minute
would be required to mix a suspension volume of 500 L for about ten minutes.
A notable feature of the chloroform extraction step is that the yeast material
changes nature
both in colour (converting from a light-gray colour to a white colour) and in
form
(converting from a material with typical cellular characteristics (cell sacs)
in suspension to
a flocculent particulate material). The bleaching and flocculating effects
observed as a
result of contact with chloroform (and other solvents having the requisite
density referred
to above), have not been observed with other organic solvents which have a
density less
than 1 g/cm3. Solvents which have been tested in this regard include acetone,
diethyl
ether, petroleum ether, methylene dichloride. ethyl acetate, ethanol. methanol
and butanol.
Following chloroform exposure and mixing such as between about five and ten
minutes,
the suspension is allowed to settle and quickly separates into three distinct
phases - a-lower
organic phase, an upper aqueous phase, and an interface between those two
phases which
is coloured gray. The three phases are well differentiated and readily
separated. The
organic phase is slightly opaque and contains lipids but no glucan. The
aqueous phase
contains glucan particles suspended in water. The interface contains a mixture
of glucan,
protein, and chitin and lipids. When analyzed by NMR, the glucan in the
aqueous phase
contains a mixture of (3-1,3 and (3-1.6 gIycosidic linkages in the approximate
ratio of 95%
to 98 % :2 % to 5 % respectively. The glucan in the interface phase contains
predominantly
unbranched (3-1,3 glycosidic linkages (generally 98 to 100% (3-1,3:0% to 2% (3-
1,6.
Effective separation of branched (3-1,3 glucan unbranched glucan and non-
glucan
contaminants is achieved.
This separation of glucan panicles based on their level of non-glucan
contaminants has
been found only with solvents having the density mentioned above, and not with
other
commonly available organic solvents having a density less than 1 g/cm3.
Without being
bound by any particular theory the fine discrimination in separating glucan
species as
exemplified by chloroform, may be due to the combination of lipophilic nature
of the
solvents and their specific density. This may allow differential separation by
weight of cell
wall glucan molecules which are associated with other ~ carbohydrates and non-
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- 17-
carbohydrates. The glucan and non-glucan molecules in this interface phase can
be
separated subsequently by evaporation of the chloroform followed by contact of
the residue
with ether and ethanol to effect dissolution of the non-glucan component,
leaving
essentially unbranched [3-1,3 glucan.
The aqueous glucan suspension collected following the specific solvent
exposure step may
be boiled briefly to effect complete removal of any residual solvent and the
glucan particles
then dried by standard methods including, for example, freeze-drying, heating,
air-drying
or spray-drying. The final product is a slightly off white, flocculent powder
comprising
particles of Sc-glucan with a diameter typically of between about 1 a up to 10
a with a
median diameter of about 3 a (such particles may be referred to as
microparticulate
glucan). The powder may be milled using standard procedures (hammer milling or
ball
milling) to give particles of desired size.
The separation of predominanly branched and uncontaminated glucan, from
relatively
unbranched glucan associated with non glucan components, is not achieved where
glucan
particles are reacted with alcohol prior to reaction with a solvent have
density greater than
1, such as chloroform. This is an unexpected finding.
Prior art description of the use of organic solvents to remove lipids from
particulate glucan
preparations failed to appreciate the discriminating effects of solvents
having a density
greater than 1 in separating predominantly branched. uncontaminated glucan
from
predominantly unbranched contaminated glucan. This invention may thus be
regarded as a
selection which confers substantial advantage as discussed above.
The microparticulate Sc-glucan produced by this process can be used as a
therapeutic in
this form. Some examples of use are application for repair of tissues such as
skin and bone
anti bowel where the microparticulate Sc-glucan is applied in formulations
such as a
powder or cream or lotion or can be used in wound dressings such as bandages
or
hydrocolloid dressings. Conventional topical formulations may be utilized as
are well
known in the art and described hereafter.
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The process of the invention described above gives rise to a high purity
product, having a
highly potent bioactivity (as it may comprise glucan having only (3-1,3 and (3-
1,6 linkages)
which is achieved with short processing time, and high yield. Table 5
demonstrates this by
comparing glucan produced according to this invention with glucan prepared
according to
the procedures of Hassid et al (1941), Di Luzio et al (1979), Manners et al
(1973), and .
Jamas (United States Patent No 4992540).
Table 5
Comparison of four standard methods of extraction of microparticulate Sc-
glucan.
Component
Processing levels
Time lucan (
ethod (days) Yield %
w/w)
GlucanMannanGlycogenProteinChitin
Hassid 8 7.8 91.7 0.4 4.5 2.9 0.4
et al
Di Luzio 12 2.0 98.1 0.3 0.5 0.7 0.2
et al
Manners 18 12.1 73,8 2.0 9.8 8.6 5.8
et al
Jamas et 2 7.4 94.6 0.3 3.1 0.8 1.1
al
The present
invention 2 7.7 98.5 < 0.1 0.4 I 0.3 0.2
The process of this invention also provides for the conversion of particulate
glucan to
glucan molecules of smaller molecular weight in the form of a solution.
dispersion or
colloid or gel which would be suitable for pharmaceutical, such as parenteral
use. Such
material may show enhanced bioactivity through the greater availability of
glucan ligands
for cytophilic glucan receptors. These glucan preparations may be regarded as
providing
glucan in a soluble form, where glucan particles dissolve in the aqueous phase
to give a
visually clear solution, or are otherwise hydrated to the extent that they
form a dispersion
or colloid, or are in the form of a gel. For convenience, these forms may be
referred to as
soluble ~lucan.
In the prior art it has been proposed to convert particulate glucan to soluble
glucan using
rigorous heat treatement (generally at 75°C or greater) in the presence
of alkali (Bacon et
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al 1969). In another proposal, the particulate glucan was treated with strong
acid (90%
formic acid) prior to exposure to alkali and heat. These approaches suffer
from a number
of disadvantages which include the production of heterogenous glucan products
of wide
- ~ polydispersity which are unsuitable for pharmaceutical use without size
fractionation,
relative inconvenience, high cost, and production of waste materials.
It has been found by the inventors that the glucan purified as described above
is readily
solubilised in alkali at low temperatures (particularly between about
2°C and about 8°C).
In the present invention, solvent extraction of acid treated cell wall sacs
with a solvent
which has a density greater than 1, where glucan partitioning takes place with
subsequent
separation and isolation of branched glucans, enables solubilisation in alkali
at low
temperatures. It is otherwise not possible to produce soluble glucan having
the properties
described hereafter.
In order to produce soluble glucan, step (d) of the process described above
may be omitted
and the pH of the solvent extracted aqueous phase comprising glucan
particulate material
may be raised from an acidic pH to a basic pH so as to effect solubilization
of the glucan
particles. This step is carried out at a temperature below 60°C,
preferably from about 2°C
to about 25°C, more preferably from about 2°C to about
8°C for a time sufficient to
achieve solubilization of the glucan particles. Alternatively, soluble glucan
may be
prepared from glucan of step (d) of the above process by reacting the
particlate glucan with
an aqueous alkali solution so as to effect solubilization of the glucan.
particles.
Temperature conditions are again below 60°C, as specified above.
An unexpected consequence of the present invention is that after alkali
solubilisation a
glucan material having a small polydispersity index (generally less than about
5, more
particularly less than about 3) results. This is highly desirable for
pharmaceutical agents.
Furthermore, no additional size fractionation steps are required. This is
contrary to prior
art teachings as set out above.
In one embodiment, microparticulate glucan isolated as described above may be
suspended
in NaOH solution at a strength of between about 2 % and 10 % (pH between pH IO
and pH
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14.5) but preferably 5 % ; the suspension contains between about 0.1 and about
30 % (w/w)
glucan, such as 5 % . A particular feature of this reaction step as discussed
above, is that
contrary to the known art it does not require prior exposure to strong acid or
applied heat
or vigorous agitation; the reaction is found to occur most advantageously at
low
temperatures (preferably between 2°C to 8°C) and with little or
no mixing; the reaction ,
time is generally between about one and twenty four hours, such as two hours.
Between
about 90% to 99% of the glucan particles are converted (through alkaline
hydrolysis) to
suspended small molecular weight molecules over the reaction time. At the
conclusion of
the reaction the undissolved particles are removed by standard methods such
as, for
example, centrifugation or filtration and the pH of the suspension adjusted
the addition of
Hcl (say from pH 8 to pH 10). This soluble glucan may be used as a
pharmaceutical
product. The glucan solution may then be adjusted to isotonicity by standard
methods such
as dialysis or ultrafiltration.
The glucan material produced by this method has a molecular weight range
between
approximately 60,000 to 250,000 with a mean of about 140,000 daltons, with a
mean
polydispersity index of about 2.4. Between approximately 70 % and 85 % of the
glucan
molecules are within 15 % of the mean molecular weight and it is found that
this result is
highly reproducible with different batches. This low polydispersity index
indicates
relatively high homogeneity. It is thus entirely suitable for use as a
pharmaceutical. It is
found that this material has high biological potency, as measured, for
example, in the
promotion of tissue repair. In a rat dermal wound repair model, this material
is
approximately five times as efficacious as microparticulate Sc-glucan when
compared on an
equivalent molar basis (Table 6).
Tensile strength of rat skin wounds (day +7) following application of a single
topical dose of 1
mg micro-particulate vs soluble Sc-glucan with 96% ((3-1,3) and 4% ((3-1,6)
linkages. '
Treatment n Wound tensile stren,~~th~_l
mean (SD)
No glucan 12 196 (23)
Micro-particulate glucan 14 356 (47)
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Soluble glucan 8 432 (69)
In that experiment the glucans were administered in a lipophilic cream base.
but it would
- be anticipated that this material could, be used as a topical therapeutic in
a variety of
formulations or could be injected as a parenteral therapeutic.
In a strongly alkaline solution, the soluble glucan molecules occur
principally as triple
helices but with little or no polymerisation of independent helical
structures. The effect of
lowering the pH of the glucan solution is to predispose the glucan molecules
to
polymerisation leading to gel formation. At a pH below approximately 9.0 there
is
progressive polymerisation of adjacent helical structures. It is observed that
the degree of
polymerisation of the glucan molecules is related directly to the
concentration of the glucan
solution. Where the glucan solution is to be diluted and dispersed in a
carrier vehicle and
it is desirable to minimise the degree of polymerisation, the concentration of
the glucan
IS solution is generally less than 10 mg/mL, and preferably no greater than 5
mglmL prior to
adjustement of the pH from a strongly alkaline state (around pH 13). In other
instances it
may be desirable to have the final glucan solution as a gel and this is
achieved if the
concentration of the glucan solution prior to pH adjustment is greater than 10
mg/mL (10%
w/w) and preferably greater than 15 mg/mL (15% w/w). for example up to about
30%
w/w. It is found that this gel state is a convenient form for topical
application. requiring
little or no additional formulation.
It can be seen that the present manufacturing process represents a significant
advance over
the current state of the art in this field. Compared to other known
manufacturing
processes, the present process yields an end-product which has greater purity,
is
manufactured in a shorter time. has greater efficiency of yield, produces a
glucan molecule
of distinctive chemical structure, and produces a product of desired
homogeneity without
the necessity of elaborate and expensive separation techniques.
It readily would be appreciated that these advantages lead to considerable
cost savings,
with the availability of a less expensive material thus allowing wider
application of
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Sc-glucan as a therapeutic in both veterinary and human medicine than is
currently
available.
The applications for which the microparticulate Sc-glucan produced by the
process of the
present invention are suitable include those applications in particular where
the risk of
direct entry of the material to the bloodstream is minimal and these include
by way of
example oral application, topical application, incradermal injection,
intramuscular injection,
subcutaneous injection, intraperitoneal injection, intrathecal injection,
intralesional
injection, intratendon injection, intraligament injection, intraarticuiar
injection, and
application to fracture sites of bones and cartilage. The therapeutic purposes
include by
way of example (a) enhancement of wound repair processes in the aforementioned
tissues,
(b) enhancement of resistance to infection from bacterial, fungal, viral and
protozoal
organisms in the aforementioned tissues, and (c) enhanced local immune
responsiveness to
carcinoQenesis.
The applications for which the small molecular weight Sc-glucan produced by
the process
of the present invention are suitable include by way of example although not
being limited
to those listed above for microparticulate Sc-glucan: indeed in these
situations the use of
solubie Sc-glucan may be preferred to that of microparticulate Sc-glucan
because of various
practical considerations such as ease of administration or the benefit of
administration in a
liquid form or because of the greater bioavailability of this form. However,
small
molecular weight Sc-glucan has particular indication for those situations
where penetration
ef intact tissues (su_l: as traps-epidermal penetcatieri ef intact shin ) is
desired or where
entry of the material to the bioodstream may occur inadvertently.
The Sc-glucans produced by the processes of the present invention can be
presented in
formulations commonly used in the pharmaceutical and cosmetic industries
including, for
example ointments, gels, suspension, emulsions, creams, lotions, powders and
aqueous
solutions. Glucan may be formulated with one or chore carriers or excipients
as are well
3C~ mown in the pharmaceutical are (see. for example. nerrtingtons
~ixarrnccr~,ic~~'~~~a~..-
;;%ah Laiticn, r~tiaci: P~~.h!ishin~ Comnarit . vas~on PA. ~c ~:~sei. e. ~,'
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Examples of carriers and excipient substances are organic or inorganic
substances which
are suitable for enteral (for example, oral or rectal), parenteral (for
example, intravenous
injection) or local (for example, topical, dermal, ophthalmic or nasal)
administration and
which do not react with the glucan, for example, water or aqueous isotonic
saline solution,
lower alcohols, vegetable oils, benzyl alcohols, polyethylene glycols,
glycerol triacetate
and other fatty acid glycerides, gelatin, soya lecithin, carbohydrates such as
lactose or
starch, magnesium stearate, talc, cellulose and vaseline.
Formulations may include one ore more preservatives, stabilizers and/or
wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffer substances,
colourants,
flavourings and/or perfumes.
Glucan may be formulated into sustained release matrices which liberate glucan
over time
providing what may be regarded as a depot effect. Glucan in the form of a gel,
as
produced according to an embodiment of the aforementioned process, may be
directly used
as a topical pharmaceutical product or - formulated with appropriate carriers
and/or
excipients.
In a further embodiment, this invention is directed to a glucan composition
which consists
essentially of branched (3-(1,3)(1,6)-glucan, and which is free or essentially
free of
unbranched (3-(1,3) glucan and non-glucan components. Reference to
"essentially free" is
to be understood to refer to less than about 2% unbranched (3-(1,3) glucan.
more
specifically less than about 0.5% unbranched X3(1,3) glucan.
These glucan formulations may comprise glucan in microparticulate form,
soluble form or
as a gel, optionally formulated or in association with one or more
pharmaceutically
acceptable carrier or excipients as herein described.
Glucan containing formulations or compositions for therapeutic purposes may
contain from
about 0.01 % to about 30% (w/w), such as from about 0.1 % to about 5 % , more
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particularly from about 0.2 % to about 1 % , even more particularly from about
0.25 % to
about 0.5% (w/w). These amounts may be regarded as therapeutically effective
amounts.
It has surprisingly been found by the inventors that Sc-glucan, whether
produced according
to this invention or by prior art processes may be used in a range of hitherto
unsuspected
and undescribed therapeutic applications. These applications include the
treatment of
ulceration or bone fracture, or the prevention/treatment of ultraviolet light
induced skin
damage.
In a further aspect this invention is directed to the use of glucan for the
manufacture of a
medicament for the treatment of skin ulceration or bone fracture, or the
implantation/fixation of orthopaedic devices, or prevention/treatment of
ultraviolet light
induced skin damage.
In a further aspect this invention is concerned with the method for the
treatment of skin
ulceration or bone fracture, or the implanation/fixation of orthopaedic
devices, or
prevention/treatment of ultraviolet light induced skin damage, which comprises
administering to a subject glucan in association with one or more
pharmaceutically or
veterinarily acceptable carriers or excipients.
In a still further aspect of this invention, there is provided an agent for
the treatment of
dermal skin ulceration, the enhancement of repair of bone and connective
tissue, or the
implanation/fixation of orthopaedic devices, or the prevention/treatment of
ultraviolet light
induced skin damage, which agent comprises gIucan in association with one or
more
pharmaceutically or veterinarily acceptable carriers or excipients.
In these novel therapeutic uses of glucan, an effective amount of glucan is
utilised. What
constitutes an effective amount will depend on the particular condition being
treated, mode
of and form of administration, and like factors. Generally, a composition or
medicament
will contain glucan in an amount from about 0.05% (w/w) to about 30% (w/w),
such as
0.1 to 5 % (w/w), more particularly from about 0.3 % to about 1 % (w/w), even
more
particularly from about 0.25 % to about 0.5 % (w/w).
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A particularly advantageous therapeutic application for glucan (such as
microparticulate,
soluble or gel forms manufactured by any of the aforementioned methods, or
produced by
prior art methods) according to the present invention is in the treatment of
dermal
ulceration. It is known that ~i-1,3-glucan will promote healing in full-
thickness, surgically-
created skin wounds in animals and humans with no dysfunctional healing. That
is, the
topically- or parenterally-applied glucan is able to accelerate the healing
response in
superficial wounds with normal healing mechanisms. It generally is assumed
that glucan
achieves this through activation of wound macrophages. Macrophages are
critical cells in
the healing process. producing a range of cytokines and growth factors which
initiate the
various components of the healing cascade - viz. fibroplasia. collagen
production,
angiogenesis, epithelialisation and collagen cross-linking. The macrophage
plays a key
modulatory role in this process, both initiating the process and helping to
ensure that the
process proceeds in a co-ordinated and integrated manner. It is assumed that a
primary
effect of the glucan is to produce a temporal acceleration of the healing
cascade.
Dermal ulcers typically are chronic wounds which have a quite different set of
physiological properties operating within the wound, compared to acute
surgical wounds.
Whereas the physiology of the healing process is well described for acute
surgical wounds,
it is ill defined for chronic ulcers. Ulcers typically show poor to negli;ible
healing because
of either constant irritation or pressure (such as decubitus ulcers or
pressure sores) or
restricted blood supply (such as in individuals with arterial ischaemia or
venous
thrombosis) or infection (such as 'tropical' ulcers) or nerve damage
('neurotrophic' ulcers)
or diabetes. Ulcers have varying pathologies, and the underlying causes. where
known,
may be quite distinct. Various types of ulcers which may be treated according
to this
invention include those associated with physical trauma (radiation, thermal
burns,
decubitus, insect bites), impaired blood flow (arterial, venous), infection
(bone, pyogenic,
synergistic gangrene, syphilis, tuberculosis, tropical diseases, fungal
diseases), neoplasia
(primary skin tumour, metastases, leukemia) and neurotrophic lesions, (spinal
cord lesions,
peripheral neuropathies).
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Ulcers associated with dysfunctional healing vary greatly in severity. from
superficial
wounds extending into the dermis and having a surface area of approximately 1-
2 cmz up
to wounds extending through dermis, subcutaneous tissue and muscle and forming
depressions and cavities with volumes of approximately 500 cm3. The larger
ulcers in
particular can be debilitating and restrictive and require intensive and
expensive therapy to
manage. Control of wound sepsis, regular wound debridement. regular dressings,
'
hypostatic drainage and corrective surgery are just some of the standard
current therapies.
However, currently available 'best-practice' wound management therapies are
not
uniformly successful, take considerable lengths of time to produce beneficial
results, are
associated with poor rates of patient compliance, generally are expensive. and
are
associated with a high incidence of ulcer recurrence. It has been noted by
Margolis (J.
I7ermatological Surgery (1995) 21(2) 145-148) that: "a paucity of data exists
that
adequately addresses the efficacy of any topical agent for the treatment of
pressure ulcers" .
It can be seen therefore that in view of the high incidence of ulcers in the
community and
the cost to the community of treatment, there is an urgent need to develop
improved
therapies. Ideally, such a therapy should have a high rate of success, be
convenient to use
and produce a clinic response quickly in order to facilitate patient
compliance, and
preferably be inexpensive.
A particular difficulty in devising a uniformly successful therapy which may
be an
improvement on current treatment modalities is the non-unifomity of the
different types of
ulcers where both the underlying aetiologic disease processes and the
pathophysiology
within the wounds show considerable variation. Confounding this variability.
is the
general poor understanding of which of the different components of the healing
response is
dysfunctional and therefore contributing the primary pathology of the
dysfunctional healing
response. So that, successful antagonism of dysfunction of any particular part
of the
healing cascade in one ulcer type may not necessarily be successful in another
ulcer type.
In particular. there is no evidence that local wound immune suppression or
macrophage
dysfunction are key pathological features or that enhancement of local immune
mechanisms
within such ulcers would result in enhanced healing responses as is seen in
uncomplicated
surgical skin wounds with no dysfunctional healing responses.
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Thus it was entirely unexpected to fmd that topical application of glucan to
decubitus,
venous stasis and arterial ischaemic ulcers induced rapid and potent healing
responses in
those wounds. This was unexpected (a) because the primary causative factor of
these ulcer
types is impaired. blood supply and there is no evidence to suggest that this
would be
responsive to antagonism by an immune stimulant, and (b) because even where it
might be
possible to promote the healing response, the impaired vasculature to the
wound could be
expected to impede the healing response as is observed with current treatment
modalities.
The beneficial effect of glucan in these ulcer types is even more remarkable
given that a
complete healing response can be achieved in the absence of other supportive
therapies
such as sepsis control, hypostatic drainage and correction of the primary
cause.
The treatment of decubitus ulcers and venostasis ulcers are particularly
preferred according
to this invention, although the invention is not limited to the treatment of
these ulcer
conditions.
Decubitus ulcers arise through multiple mechanisms. They are a disastrous
complication
of immobilization. They may result from shearing forces on the skin, blunt
injury to the
skin, drugs and prolonged pressure which robs tissue of its blood supply.
Imitative or
contaminated injections and prolonged contact with moisture. urine and faeces
also play a
prominent role. Diminished blood circulation of the skin is also a substantial
risk factor.
The ulcers vary in depth and often extend from skin to a bony pressure point.
Treatment
is difficult and usually prolonged. Surgical techniques are at present the
most important
means of achieving optimal healing.
Approximately half of venous ulcers are associated with incompetent
perforating veins in
the region of the ankle, and constitute a long temn problem in many immobile
patients.
Ulceration is rarely a manifestation of primary varicose veins but is
virtually always
_ associated with incompetence of the popliteal venous valve. Venostasis
ulcers are most
often just proximal or distal to the medial malleolus (bony ankle joint) and
often develop at
sites of minor trauma or skin infections. Scarring and secondary infection all
impair
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healing and make recurrences common if healing does occur. The natural history
of
venous ulceration is cyclic healing and recurrence.
In the case of decubitus ulcers, the glucan preferentially is applied in the
form of a powder
(microparticulate glucan) or in a cream or ointment base (microparticulate,
soluble or gel
forms of glucan). Application is generally daily and may continue for a time
period
su~cient for ulcer healing, such as seven to twenty eight days. It is observed
that the
response to the glucan therapy is apparent clinically within 2-3 days with
evidence of fresh
granulation and epithelial growth. The length of time required to heal ulcers
will vary
according to a number of factors such as ulcer size, degree of wound sepsis
and host
nutritional state. Typically wound volume is reduced by 50 % within 2-3 weeks
with
complete or near-complete wound closure effected by 4-b weeks after
commencement of
glucan therapy. It is noteworthy that most of the decubitus ulcers in which
glucan effects a
healing response have been refractory to standard therapy including a wide
range of topical
preparations and wound dressings for periods up to 7 years.
In a similar manner, application of microparticulate, soluble or gel forms of
glucan to
venostasis and arterial ischaemic ulcers promotes ulcer healing. As with the
decubitus
ulcers, treatment of these ulcers with glucan leads to a clinical response in
the wound
within 2-3 days following the start of glucan therapy with such evidence of
healing as the
appearance of fresh granulation tissue and less detritus leading to a cleaner
appearance in
the wound. Glucan in the form of a powder, cream, lotion, ointment or gel may
be
topically applied to the ulcer site daily until healing occurs. The chronic
nature of the
underlying vascular disorder in these cases means that the predisposition to
form such
ulcers remains with the patient. It may be necessary therefore to continue
glucan therapy
on a long term basis to prevent recurrence.
It can be seen therefore that it is an entirely unexpected observation that
glucan is able to
promote the healing processes in skin ulcers where the individual components
of the -
healing process are essentially normal but are unable to antagonize the
dysfunctional cause
such as inadequate blood supply, inadequate venous drainage, excessive tissue
oedema,
infection, constant pressure or other diverse causes.
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It is observed that application of glucan to ulcers as described above
produces a high rate
of therapeutic response. Skin ulcers which either are unresponsive or poorly
responsive to
conventional wound management practice, typically respond within several days
to
treatment with ~ glucan leading in a high proportion of cases to complete
healing within
several weeks of treatment. It is found that the glucan is effective in the
treatment of
ulcers when applied locally to the wound in various forms such as a powder,
gel, cream,
or dressing such as a gauze bandage or colloidal material, or any other
composition
generally known to those skilled in the art of pharmaceutical formulation.
In a related aspect the treatment of ulcers which respond to conventional
therapies (such as
normal dressings and ointments) may be accelerated with glucan administration.
Another unsuspected therapeutic application for glucan (such as,
microparticulate, soluble
or gel forms manufactured by any of the aforementioned methods, or other
processes
known in the art) according to the present invention is in the treatment of
bone fracture.
The repair of fractured bone characteristically is accomplished by a repair
process which
basically is in common with that observed in soft tissues such as skin but
which differs in
some important aspects. In bone, an important early step in the repair process
is the
formation of a fibrous structure known as a callus which bridges the fractured
site
providing a framework for the repair process and assuring a degree of
immobilization of
the fracture site. In due course the callus becomes mineralized, providing
continuity with
the uninjured bone and undergoes a degree of remodelling to approximate the
original
shape of the bone. According to this aspect of the invention the application
of glucan to
the site of injury enhances the rate of repair of injured bone thus
facilitating fracture
treatment. It is observed that the effect of such treatment is earlier
induction of the callus
formation and earlier organization of the connective tissue within that callus
to provide a
strong fibrous matrix. The result of this is the establishment of an
immobilizing callus at
an earlier time with the important clinical effect of reducing the risk of
dissociation of the
fractured edges of the bone. This is a highly desirable effect in both animals
and humans
because any disruption to the fracture site can predispose to delayed healing.
Disruption at
the fracture site remains a problem, even where methods of physical
immobilization
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through such mechanical means as rigid splints (such as casts, bandages,
etc.). or implants
(such as pins, screws, etc) are used. While it is found that the process of
mineralization is
not appreciably enhanced by the glucan treatment, it is found that the effect
of glucan in _
accelerating the callus phase has the effect of reducing the overall time to
complete
mineralization.
The glucan preferably is applied directly to the site of bone injury in a form
which will
maximize the retention of the glucan at the site of the fracture. SIow release
formulations
are well known in the art and are preferably used in these applications. It is
found that the
viscous gel formed by the embodiment ldisclosed in this invention whereby a
highly
alkaline soluble glucan solution at a concentration of greater than 15 mg/mL
(from about
l5mg/m1 to about SOOmg/ml, more preferably from about l5mg/ml to about
30mg/ml) is
adjusted to pH 7.5 (Example 4) is a preferred form. This form is sufficiently
viscous and
non-miscible with blood and tissue fluids to remain at the site of application
for periods up
to 48 hours. An additional advantage of this gel form is that it is
sufficiently tractable to be
able to be injected through fine gauge needles. In this form, the glucan can
be administered
by injection to fracture sites where the fracture is reduced without the need
for surgical
exposure of the bone. Alternatively, the gel can be administered to the
fracture site during
open surgical reduction of fractures.
The potential usefulness of glucan treatment for human bone fractures has been
evidenced
in an animal model by the inventors. The rat is a standard model used in
exnerimPnrai
medicine for bone fracture research and generally is regarded as directly
predictive of
human therapy (Bak et. al. 1992). In this animal model the inventors have
established that
injection of 2 mL of 15 mg/mL soluble glucan in a gel form at the site of a
fractured femur
resulted in accelerated healing when compared with non-treated fractures as
evidenced by
increased tensile strength of the partially healed bones at 12 days (Example
10).
It can thus be readily envisaged that glucan, being non-toxic and
physiologically -
acceptable, may find wide application in fracture treatment in human and
animal medicine.
For example. a single bolus injection or application of glucan at the site of
fracture will
promote healing and increase tensile strength of the healed bone.
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A further unexpected therapeutic benefit is that glucan enhances the fixture
of devices such
as pins, screws, artificial joints and prostheses fixed or implanted into or
onto bone. It is
observed that the application of glucan (such as by local application of a
powder or gel, or
by injection) at the site of fixation of the device enhances significantly the
local
inflammatory process which occurs in response to the contact of the device
with bone and
generally is an integral part of the strength of the bond between the bone and
the device.
A particular therapeutic indication for glucan (either microparticulate or
soluble forms
manufactured by any of the aforementioned methods or by prior art methods)
according to
the present invention is in the treatment of injured connective tissues such
as tendons and
ligaments which has not previously been described or suggested. Such tissues
are typically
densely fibrous because they are subjected to relatively high stress loads.
These injuries
include by way of example but are not limited to (a) acute or chronic
inflammation
associated with over use or strain or trauma, such conditions typically being
associated
with sporting injuries or the syndrome known as Repetitive Strain Injury or
excessive or
abnormal stress, and (b) surgery, in particular where the tissue is dissected
or transected. It
is known that injuries of this kind in such tissues typically are slow to
heal, due in part to
the relative difficulty of totally resting the injured tissue because of their
load bearing
functions, but due largely to the characteristically lower level of activity
of all aspects of
the tissue healing cascade compared to that which is seen in soft tissues. An
important
cause of this comparatively lower level of tissue repair activity in tendons
and ligaments is
a more limited blood supply compared to most soft tissues. It is found that
application of
glucan to the injured tendon or ligament either at the time of acute injury
such as following
surgery or external trauma, or with chronic injury such as chronic
inflammation will
promote both the rate of onset and the level of the healing response in these
tissues, leading ,
in the case of surgery to earlier return of normal strength and function and
in the case of
inflammation to earlier resolution of the inflammatory process. The glucan may
be
directly injected into the injured tendon or ligament. Although it has been
described that
glucan is a potent enhancer of wound repair in dermal tissue in healthy
tissues, it is not
apparent from that knowledge that glucan has the ability to effect enhanced
resolution of
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chronic inflammatory processes or of enhancing repair processes in tissues
with limited
blood supply or where the normal rate of repair is known to be relatively
slow.
A further unsuspected therapeutic indication of glucan is the
prevention/treatment of
ultraviolet light-induced skin damage which results from exposure to the sun.
It is well described that ultraviolet light exposure causes damage to skin,
particularly Long
term exposure to sunlight. This is particularly the case with Caucasians who
have light
skin colouration which predisposes them to photo-ageing and development of
certain types
of skin cancers. Both of these problems are prominent within most Western
communities.
The detrimental effects of sunlight are due primarily to its ultraviolet light
spectrum (UV-A
and UV-B). UV-B acts principally within the epidermis and rarely penetrates
deeper than
the uppermost layers of the dermis, while the longer wave-length UV-A
penetrates through
the dermal layers. The major detrimental effect of ultraviolet light is damage
to proteins,
particularly DNA and RNA where it results in dimer formation. Most of these
dimers are
repaired within several hours although a small number are either not repaired
or are mis
repaired and the accumulation of these mis-repairs over a lifetime is thought
to be a major
contributing factor to the development of skin carcinogenesis in chronically
sun-exposed
individuals.
The two principal outcomes of this damage to proteins in the skin is acute
cell damage and
mutagenicity. Cell damage is evidenced clinically in the acute phase by the
symptoms
referred to generally as 'sun-burn' which include erythema (reddening) and
oedema and in
the long-term phase by symptoms referred to generally as 'photo-ageing' which
include
skin thickening and wrinkling; mutagenicity is evidenced by skin cancer
development. A
further effect of ultraviolet light which is not clinically apparent is immune
depression.
Skin has a rich network of immune cells that are equally sensitive to the
detrimental effects
of ultraviolet light as are other skin cells and exposure to ultraviolet light
leads to
temporary dysfunction of these cells. This dysfunction is repaired generally
within 2-3
days but in this period the skin shows reduced immune capacity such as antigen-
presentation. With repeated ultraviolet light exposure such as might be
expected in
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individuals with a lifetime exposure to sunlight, the sun-exposed skin has
chronically
reduced immune function. It is likely that this predisposes to the development
of skin
cancer through reduced immune surveillance capacity within skin. However, the
relative
contributions that each of the different effects of ultraviolet light (viz.
immune depression,
chronic dermal and epidermal cell injury, mutagenicity) has in skin cancer
development
and photo-ageing remains unknown.
It has been found surprisingly by the inventors that glucan applied topically
to skin either
following or concominant with ultraviolet light Ieads to substantial
protection of the skin
from ultraviolet light-induced skin damage.
This has been found in experiments conducted with a standard. hairless mouse
strain used
as a model to study solar damage to human skin (see, for example. Canfield et
al 1985).
In this model the mice are exposed daily for 10 weeks to a minimal erythemal
dose of
mixed ultraviolet light which simulates the toxic effects of sunlight on skin.
Each daily
exposure of ultraviolet light induces a mild erythema and oedema lasting up to
about 24
hours and which mimics in appearance a mild 'sun-burn' in humans. With
continued
irradiation treatment, this on-going damage is reflected in progressive
thickening of the
skin which histologically mimics the hyperkeratinisation and elastosis
associated with
photo-ageing in chronically sun-expose skin in humans. Pre-malignant tumours
begin to
appear within several weeks of completion of the ultraviolet light treatment
regime. Over
the ensuing 6-12 months there is progressive development of pre-malignant and
malignant
tumours, the histology and behaviour of which closely mimic the actinic
keratoses and
pre-malignant and non-melanona skin cancers that develop in humans in response
to
sunlight.
The inventors have found that soluble glucan applied to the skin daily
immediately
following ultraviolet irradiation provides significant protection from both
the acute toxic
effects (evidenced by discernibly lesser skin erythema on each morning
following the
previous day's irradiation) and the chronic photo-ageing effects (evidenced by
significantly
thinner skin). This effect is particularly unexpected given that (3-1,3-glucan
is not
previously known to protect tissues from direct cytotoxic damage and that
there is no
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existing data that either confirms or suggests that (3-1,3-glucan antagonises
the
cytochemical and histopathological lesions that are consequent to acute or
chronic
ultraviolet irradiation. The ability of glucan in this model to antagonise the
acute toxic and
chronic photo-ageing effects of ultraviolet irradiation offers a novel and
important means of
protection of human skin from the damaging effects of sunlight. .
It also has been found by the inventors that soluble glucan applied topically
to human skin
immediately following exposure to sunlight affords protection from the acute
erythemal
effects of the ultraviolet light.
It further is found in the hairless mouse model that the glucan affords
considerable
protection from the development of skin cancers (see Figure 1 hereafter). The
majority of
tumours at this early stage are benign sessile-based papiIlomas, as expected;
transformation
of a proportion of these to more malignant intermediate forms culminating in
squamous
cell carcinomas is anticipated at a later stage.
Accordingly, glucan may find wide applications in ameliorating the effects of
sunlight in
the human population. In this regard, the beneficial effect of glucan is
obtained if it is
applied either prior to, during or following sunlight exposure. To this end,
it may be
formulated into sunscreen formulations or into after-sun or in general
cosmetic
formulations such as lotions, creams and gels. The particular benefits to be
gained from the
use oi~ Sc-glucan include the following: (a) amelioration of the acute toxic
effects of
sunlight on skin ('acute sunburn'); (b) amelioration of the chronic effects of
sunlight on
skin which collectively are known an photo-ageing and include symptoms such as
hyperkeratinisation, skin thickening, elastosis and wrinkling; (c)
amelioration of the
development of sunlight-induced skin carcinogenesis.
It is to be understood that the novel therapeutic uses for glucan herein
described are not
limited to glucan produced by the processes described herein, although this
material is
preferred. Any prior glucan material such as those described by Hassid et al,
Di Luzio et
al, Manners er al and Jamas et al (United States Patent Nos 5028703, 5250436,
5082936
and 4992540) may be used. Preferably the glucan is Sc-glucan
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This invention will now be described with reference to the following non-
limiting examples
which illustrate various embodiments of the invention.
Figure 1 depicts dorsal skin fold thickness measurements in mice subject to
U.V.
irradiation over 6 weeks, which mice are treated with glucan (-0-) or -tteafed
with a non-
glucan base lotion (-o-)
EXAMPLE 1
Microparticulate glucan is prepared as follows:
A 400 g sample of commercial Saccharomyces cerevisiae in dry form is added to
four
litres of 4% w/v sodium hydroxide and heated to 100°C for one hour with
vigorous
stirring. The suspension is allowed to cool to between 45°C and
50°C before the lysed
yeast cells are separated from the alkaline hydrolysate by centrifugation at
800 g for ten
minutes. The lysed yeast cells are resuspended in a fresh batch of three
litres of 3 % w/v
sodium hydroxide and boiled for 15 minutes. Following separation by
centrifugation, the
lysed yeast cells are resuspended in a fresh batch of two litres of 3 % w/v
sodium
hydroxide and boiled for 15 minutes followed by standing at 70°C for 16
hours.
Following separation by centrifugation, the lysed yeast cells are resuspended
in water and
boiled for 10 minutes. The latter step is repeated once. Following
centrifugation, the lysed
yeast cells are resusended in a fresh aliquot of 2 L water, the pH adjusted to
4.5 by the
addition of phosphoric acid and the suspension then boiled for thirty minutes.
Five
hundred mL of chloroform then is added and the suspension subjected to
vigorous agitation
for ten minutes, following which the suspension is allowed to settle for 10
minutes in a
separating funnel. The suspension separates into three distinct phases, a
lower organic
phase, an upper aqueous phase, and an interface between these two phases which
is grey
coloured. The lower chloroform phase plus a greyish intermediate phase are
discarded,
. leaving an aqueous phase which is collected and exposed as before to a fresh
batch of 500
mL of chloroform. The final aqueous phase is collected and boiled for 10
minutes to
remove any residual chloroform and then dried using a spray-drier.
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Analysis of the aqueous phase showed that it contained only branched (3-
(1,3)(1,6) glucan
in the ratio of 95 to 98% (3-1,3 : 2 to 5% (3-1,6 linkages. The organic phase
is slightly
opaque and contains lipids but no glucan. The intemediate phase (interface)
contains _
unbranched (3-(1,3) glucan (98 to 100% (3-1,3 : 0 to 2% (3-1,6) associated
within chitin,
protein and other non-glucan contaminents. Biological tests showed that the
branched
glucan was significantly more biologically active than unbranched (3(1,3)
glucan in a
wound healing test.
The chemical composition of glucan produced according to this invention is set
forth in
Table 7.
Table 7
Chemical composition of Sc-glucan produced by the process of the present
invention.
(by weight)
Glucose' > 98
Mannanl < 0.2
Protein2 < 0.5
Glycogen3 < 0.5
Chitin' < 0.3
Lipid not detectable
Glycosidic
linkages:4
(3-1, 3 96 - 97
(~1,6 3 -4
Methods of analysis: 1= HPLC; 2= Lowry method; 3= GC-MS; 4= NMR.
It is clear from this analysis that the end-product is a branched (3(1,3)(1,6)
glucan that is
substantially pure, containing only trace amounts of impurities, and
containing about 2 to
3% ~3-1,61inkages.
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EXAMPLE 2
Microparticulate Sc-glucan is prepared as follows:
A 400 g sample of commercial Saccharomyces cerevisiae in dry form is added to
four
litres of 4% w/w sodium hydroxide and heated to 100°C for one hour with
vigorous
stirring. The suspension is allowed to cool to between 45°C and
50°C before the lysed
yeast cells are separated from the alkaline hydrolysate by centrifugation at
800 g for ten
minutes. The lysed yeast cells are resuspended in a fresh batch of three
litres of 3 % w/v
sodium hydroxide and boiled for 15 minutes. Following separation by
centrifugation, the
lysed yeast cells are resuspended in a fresh batch of two litres of 3 % w/v
sodium
hydroxide and boiled for 15 minutes followed by standing at 70°C for 16
hours.
Following separation by centrifugation, the lysed yeast cells are resuspended
in water and
boiled for 10 minutes. The latter step is repeated once. Following
centrifugation, the lysed
yeast cells are resusended in a fresh aliquot of 2 L water, the pH adjusted to
4.5 by the
addition of hydrochloric acid and the suspension then boiled for ten minutes.
Five hundred
mL of chloroform then is added and the suspension subjected to vigorous
agitation for ten
minutes, following which the suspension is allowed to settle for 10 minutes in
a separating
funnel. The lower chloroform phase plus a greyish intermediate phase are
discarded.
leaving an aqueous phase which is collected and exposed as before to a fresh
batch of 500
mL of chloroform. The final aqueous phase is collected and boiled for 10
minutes to
remove any residual chloroform and then dried using a spray-drier.
The chemical composition of glucan produced according to this invention is set
forth in
Table 8.
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Table 8
Chemical composition of Sc-glucan produced by the process of the present
invention.
% (by weight)
Glucose' > 98 -
Mannan' < 0.2
Protein2 < 0.5
Glycogen3 < 0.5
Chitin' < 0.3
Lipid4 not detectable
Glycosidic
linkages:4
~i---1,3 98 - 99
(3--1, 1 - 2
6
Methods of analysis: 1= HPLC; 2= Lowry method; 3= GC-MS; 4= NMR.
It can be seen that compared to the end-product material obtained in Example
1, this
material has has a similar degree of purity but has slightly fewer (3-1,6-
glucan linkages
indicating a lesser degree of side-branching.
EXAMPLE 3
A protocol for the preparation of minimally-polymerised, soluble Sc-alucan
according to
the present invention is as follows.
Microparticulate Sc-glucan is produced as detailed in Example 2. Ten g of this
material is
suspended in 100 mL sterile 5 % NaOH solution and stirred gently for two hours
at 5°C "
(giving a pH around pH 13). The suspension then is diluted 1:1 in sterile,
distilled water
and then filtered through a 1 a membrane to remove undissolved particulate
material. The
pH of the filtered solution then is adjusted to 10 by the addition of SM HCI
and then
dialysed against 2 L distilled water (pH 10) in a Pelicon system using a
10,000 D limiting
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membrane. The solution then can be sterilised by passage through a 0.45p.
membrane and
the pH of the solution may be adjusted as desired. The soluble glucan so
produced is
useful as a pharmaceutical product.
Gel permeation chromatography (Waters Styragel HR SE~ column; effective
molecular
weight range of 10 x 104 to 4.0 x 106 daltons) of the soluble glucan showed
the material
was essentially homogenous with a very narrow molecular weight spread, having
an
average molecular weight of 140,000 daltons and a polydispersity index of
2.564. In this
determination the solvent is DMSO and the column flow rate is 1 ml/minute.
EXAMPLE 4
A protocol for the preparation of polymerised, soluble glucan according to the
present
invention is as follows.
Microparticulate Sc-glucan is produced as detailed in Example 2. Fifteen g of
this material
is suspended in 100 mL sterile 5 % NaOH solution and stirred gently for two
hours at 5°C.
The suspension then is centrifuged at 1000g to remove undissolved particulate
material.
The pH of the solution then is adjusted to 10 by the addition of SM HCI and
then dialysed
against 2 L distilled water (pH 10) in a Pelicon system using a 10,000 D
limiting
membrane. The pH then is adjusted to 7.5 by the further addition of
hydrochloric acid
producing a viscous gel which is useful as a pharmaceutical product.
Gel permeation chromatography showed the material was essentially homogenous
with a
very narrow molecular weight spread, having an average molecular weight of
320,000
daltons and a polydispersity index of 2.2.
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EXAMPLE S
A model of delayed wound healing was developed in rats to test the ability of
microparticulate Sc-glucan to promote wound healing in dysfunctional wounds.
The
breaking strength of seven day-old skin wounds in inbred young adult
laboratory rats is
determined as outlined earlier but the rats in this case are treated with
drugs intended to
depress the healing response. This is achieved by daily treatment from the
time of
wounding with a combination of prednisone (1 mg/kg), cyclosporin A (5 mg/kg)
and
azothioprine (2 mg/kg). This triple drug therapy provides a range of
depressive effects on
macrophages, lymphocytes and vascular endothelium.
Table 9 summarizes the results of the use of Sc-glucan in this model. The
effect of the
triple drug therapy was to reduce significantly (p < 0.01) the breaking
strength of the
wound at seven days. A single application of 1 mg of microparticulate Sc-
glucan (per 5
cm linear length skin wound) produced by the process of the present invention
successfully
antagonized the depressive effect of the triple drug therapy, returning the
breaking strength
of the wound to that seen in normal immunocompetent rats.
Table 9
Effect of topical Sc-glucan therapy on the breaking strength of skin wounds in
rats with
and without drug-induced depressed wound healing.
Wound breaking
Group Drug treatmentGlucan treatmentstrength (g)
(mean)
1 None None 422
2 Yes None 275
3 Yes Yes 442
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EXAMPLE 6
Glucan Formulation
A topical preparations for human and veterinary applications were prepared
from the
following components:
TOPICAL CREAM
~i-1,3-glucan (microparticulate)BP 1 mg/g
Zinc stearate BP 3 mg/g
Cetomacrogol 1000 BP 20 mglg
Cetostearyl alcohol BP 80 mg/g
Phenoxyethanol BPC 1973 5 uL/g
Glycerol BP 60 mg/g
Arachis oil BP 40 mg/g
Purified water BP to 1
g
This formulation may be referred to as Formulation #1.
A powder for topical application was prepared from the following components:
TOPICAL POWDER
(3-1,3-glucan (microparticulate) 100 mglg
Maize corn flour BP 900 mg/g
This formulation may be referred to as Formulation #2.
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A topical cream was prepared by mixing the following components:
TOPICAL CREAM
Para~n oil 80 ml
Olive oil 60 ml
Anhydrous lanolin cetomacrogol60 g
1000
Stearic acid Cetostearyl 58 g
alcohol
Glyceryl monostearate phenoxyethanol60 g
Oleic acid glycerol 25 ml
Water 1200 ml
Triethanolamine 27 ml
Soluble glucan of Example 20 ml
3
This formulation may be referred to as Formulation #3 and provides a cream
containing 5
mg soluble glucan per g.
Formulations #1 to #3 were varied by incorporating glucan in the form of a
gel. These
may be referred to as Formulations #1A to #3A.
EXAMPLE 7
A decubitus ulcer was treated successfully in a human patient using
Formulation #l.
The patient was a ninety year old male stroke victim who had been hospitalized
for ten
years and who was essentially bed-ridden. A decubitus ulcer had developed on
the right
buttock in 1986 and persisted despite regular medical and nursing attention.
By 1988 the
ulcer had grown to a diameter of 8 cm and to a depth of 4 cm. Conventional
treatments
consisting of regular wound cleansing, application of protective dressings and
body
positioning to minimize pressure to the ulcer had failed to halt the
progressive deterioration
of the ulcer.
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Treatment with Sc-glucan was commenced and involved topical application using
Formulation #l. Daily topical treatments were carried out for one week and
then ceased.
Two weeks after treatment the ulcer was totally healed; epithelialization was
complete and
there was no visible scar formation.
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EXAMPLE 8
A patient (Mr G W) suffering from persistent leg ulcers was treated with
glucan
(Formulation #1).
The patient was a fifty three year old male who suffered a sporting injury
which included a
fractured ankle. Following this injury the ankle was reconstructed twice.
After the second
reconstruction the wound did not heal and four venostasis ulcers developed
despite the use
of systemic and topical bactericides and antibiotics.
Following five successive daily applications of the glucan containing
formulation 1 wound
healing cream of Example 5 to three of the ulcers, one originally measuring
3.8 cm x
1.9 cm completely healed in ten days; one measuring 10.2 cm x 3.8 cm was
reduced to 6.3
cm x 1.3 cm during the ten day treatment period; and a further ulcer measuring
3.8 cm x
1.9 cm was reduced to 2.5 cm x 1.2 cm. The treatment was recommenced on the
tenth
day and after two further cycles of treatment comprising cream application for
seven days
and no treatment for seven days the latter two ulcers completely healed after
four weeks.
Treatment of the fourth.ulcer (10 cm x 9 cm) involving two exposed tendons and
extensive
tissue necrosis was commenced shortly thereafter. After ten days of daily
treatment, there
was clear evidence of epithelial regrowth and granulation tissue leading to
coverage of the
exposed tendons by granulation tissue and overall reduction in wound size to 8
cm x 7 cm.
The patient had never observed such positive results from any previous
treatment.
EXAMPLE 9
The posterior aspect of the forearm of a six year old thoroughbred stallion
was severely
traumatized in a fight with another stallion creating a deep cavity with an
external hole
some 40 cm x 20 cm in area. Initial treatment was irrigation with disinfectant
and
antibiotic solutions but after several days the severity of the injury became
more apparent .
and appeared to be worsening. There was extensive and deep sloughing occurring
with
necrosis of deep tissues including ligament and tendons and associated muscle
masses -
some tendon remnants were present as unhealthy looking strands and the animal
could not
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bear weight. The affected area was treated at that time by topical application
of
Formulation #1 of Example 5.
There was an immediate and profound response to glucan treatment.
S
The sequence of the clinical response to treatment was as follows:
24 hr post-treatment: Necrosis lessened with reduction in suppuration.
36 hr post-treatment: Marked improvement in appearance of wound with tissue
showing
vitality.
72 hr post-treatment: Whole area filling in rapidly with ligament and tendon
remnants
being included in new tissue.
96 hr post-treatment: General appearance of good rapid healthy healing with
peripheral
epithelialization evident.
The wound ultimately completely closed after 12 days of treatment and with
minimal
scarring.
The animal at that time was weight-bearing on all legs.
EXAMPLE 10
Four adult rats (male, Wistar, inbred) had their left femurs broken under
anaesthesia using
externally-applied force. The fracture site was located by external palpation
and a 21-gauge
needle then introduced through the skin over the fracture site and positioned
between the
fractured ends of the femur. The fracture then was immobilised in the standard
way by
insertion of an intra-medullary pin through the knee joint to emerge through
the femoral
head. In two rats, 2 mL of colloidal glucan produced as per Example 4 were
injected into
the fracture site via the previously-positioned needle. In the other two rats,
2 mL of saline
was injected instead of glucan.
The needle then was withdrawn and the rats allowed to recover from the
anaesthetic.
Twelve days later the rats were killed, the intra-medullary pins removed and
the fractured
femurs isolated for visualisation of the fracture site and determination of
the strength of the
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healing response. In the two control (saline) rats, the fracture site was
contained within a
rudimentary callus and was able to be displaced readily by torsion of the
upper and lower
femoral shafts. In the two glucan-treated rats, the callus was further
advanced, being
firmer and considerably greater force was required to displace the fractured
ends of femur.
It was concluded that the effect of the glucan had been to accelerate callus
formation,
leading to a firmed bond of the fracture site at 12 days post-fracture.
EXAMPLE 11
A 50 year-old Caucasian male exposed an area of skin approximately 4 cm x 12
cm on the
inner aspect of both forearms to direct sunlight for a period of 40 minutes.
Both areas were
exposed under identical conditions and both forearms had similar levels of
skin
pigmentation. Each exposed area was divided into 4 equal patches (4 cm x 3 cm)
which
were delineated by indelible ink. On each forearm, 1 gm of sun-cream (SPF 10)
was
applied to one of the end patches prior to sun-exposure; the remaining patches
were
untreated at this time. Two hours following sun-exposure Sc-glucan
(Formulation #3 from
Example 6) was applied to the second patches, the third patch was left
untreated, and 2gm
of Formulation #3 (Example 6) base without Sc-glucan applied to the fourth
patch. The
order of treatment was reversed on each forearm.
The skin patches were examined 24 hours following sun-exposure and the degree
of
redness assessed visually by scoring 0, +, + + ; + + + and + + + + . The
results were as
follows:
untreated + + + + -
SPF 10 +
cream base only + + + +
glucan + cream base + +
The glucan effected considerable reduction of skin redness. Hence, glucan
ameliorated the
clinical response to sun damage.
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EXAMPLE 12
Albino Skh:HR-1 hairless mice were irradiated daily with U.V. light for a
period of 12
weeks. After each daily irradiation, mice were painted with glucan cream of
(Formulation
#3), cream base alone or untreated. Results are shown in Table 10.
Table 1~
Mean no. of pre-malignant (papillomas, hyperkeratoses, kerato-acanthomas) and
malignant
(carcinomas) in albino Skh:HR-1 hairless mice, following 12 weeks ultraviolet
irradiation
painted with 0.1 ml of either cream base lotion or Sc-glucan (7mg/day) and
cream base
each day.
Mean no. skin tumours per mouse
Weeks 11 14 17 19 21
Treatment no. mice
Cream base only 20 0 1.7~2.7 4.7~3.9 7.6~3.9 13.3~8.3
Sc-glucan + cream base 20 0.05~0.2 0.05~0.2 0.95~1.9 1.7~2.6 4.6~4.7*
*p = 0.004
Mice irradiated by UV painted with neither Sc-glucan and cream base, or cream
base
alone, registered as many tumours as mice painted with cream base alone (data
not shown).
EXAMPLE 13
Mice are exposed daily for ten weeks to a minimal erythemal dose of U.V. light
which
stimulates the toxic effects of sunlight on skin. Each daily exposure of U.V.
light induces
a mild erythema and oedema lasting up to 25 hours which mimmics a mild 'sun-
burn' in
' humans. Mice were either treated with Formulation #3 after U.V. light
exposure (group
1) or treated with base lotion containing no glucan (group 2). At six weeks
notable skin
thickening (and consequential wrinkling) was observed for group 2 mice. Mice
of group 1
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were largely protected from these effects. Erythema was not observed in group
1 mice
over the treatment period. Figure 1 depicts the results obtained in one test.
After 6
weeks, glucan treated mice (-7-) showed appreciably less skin fold thickness
than untreated
mice (-o-).
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