Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS COMPRISING PHYTOSTEROL, PHYTOSTANOL OR
MIXTURES OF BOTH HAVING ENHANCED SOLUBILITY AND
DISPERSABILITY AND INCORPORATION THEREOF INTO FOODS,
BEVERAGES, PHARMACEUTICALS, NUTRACEUTICALS AND THE LIKE
FIELD OF THE INVENTION
This present invention relates to the field of phytosterol-based compositions
suitable
for incorporation into foods, pharmaceuticals, nutraceuticals and the like and
to
methods of making the same.
BACKGROUND OF THE INVENTION
While recent advances in science and technology are helping to improve quality
and
add years to human life, the prevention of atherosclerosis, the underlying
cause of
cardiovascular disease ("CVD") has not been sufficiently addressed. Research
to
date suggest that cholesterol may play a role in atherosclerosis by forming
atherosclerotic plaques in blood vessels, ultimately cutting off blood supply
to the heart
muscle or alternatively to the brain or limbs, depending on the location of
the plaque in
the arterial tree (1,2). Overviews have indicated that a 1 % reduction in a
person's iotai
serum cholesterol yields a 2% reduction in risk of a coronary artery event
(3).
Statistically, a 10% decrease in average serum cholesterol (e.g. from 6.0
mmoI/L to
5.3 mmol/L) may result in the prevention of 100,000 deaths in the United
States
annually (4).
Sterols are naturally occurring triterpenoids that perform many critical
cellular
functions. Phytosterols such as campesterol, stigmasterol and beta-sitosterol
in
plants, ergosterol in fungi and cholesterol in animals are each primary
components of
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cellular and sub-cellular membranes in their respective cell types. The
dietary source
of phytosterols in humans comes from plant materials i.e. vegetables and plant
oils.
The estimated daily phytosterol content in the conventional western-type diet
is
approximately 60-80 milligrams in contrast to a vegetarian diet which would
provide
about 500 milligrams per day.
Phytosterols have received a great deal of attention due to their ability to
decrease
serum cholesterol levels when fed to a number of mammalian species, including
humans. While the precise mechanism of action remains largely unknown, the
relationship between cholesterol and phytosterols is apparently due in part to
the
similarities between the respective chemical structures (the differences
occurring in
the side chains of the molecules). It is assumed that phytosterols displace
cholesterol
from the micellar phase and thereby reduce its absorption.
Over forty years ago, Eli Lilly marketed a sterol preparation from tall oil
and later from
soybean oil called CyteIlinTM which was found to lower serum cholesterol by
about 9%
according to one report (5). Various subsequent researchers have explored the
effects of sitosterol preparations on plasma lipid and lipoprotein
concentrations (6) and
the effects of sitosterol and campesterol from soybean and tall oil sources on
serum
cholesterols (7). A composition of phytosterols which has been found to be
highly
effective in lowering serum cholesterol is disclosed in PCT/CA95/00555 and
comprises no more than 70% by weight beta-sitosterol, at least 10% by weight
campesterol and stigmastanol. It is hypothesized in this patent application
(which has
already issued to patent in some countries) that there may be some form of
synergy
between the constituent phytosterols.
Given that phytosterols in various combinations have been proven to have wide
clinical and dietary applications in lowering total and low density
lipoprotein
cholesterol, the key problem now facing researchers in this field is the
adaptation of
the phytosterol delivery system. Studies have investigated how the form (for
example,
crystalline, suspension, granular) in which the phytosterols are dosed impacts
on their
ability to lower serum cholesterol levels. Phytosterols are highly
hydrophobic, do not
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dissolve to any significant degree in the micellar phase in the digestive
tract and
therefore are not capable of efficiently blocking cholesterol absorption. Oils
and fats
are capable to a limited but not satisfactory degree of dissolving free
phytosterols.
Since only solubilized phytosterols inhibit the absorption of cholesterol,
this "delivery"
problem must be adequately addressed.
Early research focused on grinding or milling the phytosterols in order to
enhance their
solubility (US Patent Serial Nos: 3,881,005 and 4,195,084 both to Eli Lilly).
In addition,
researchers have looked to the esterification of phytosterols in order to
enhance their
solubility in delivery systems. German Patent 2035069/January 28, 1971
(analogous
to US Patent Serial No. 3,751,569) describes the addition of phytosterol fatty
acid
esters to cooking oil. The esterification is carried out between a free sterol
and a fatty
acid anhydride, with perchloric acid as the catalyst. The significant drawback
to this
process, along with others, is the use of non-food grade catalysts and
reagents.
US Patent Serial No. 4,588,717 to David E. Mitchell Medical Research Institute
describes a vitamin supplement which comprises a fatty acid ester of a
phytosterol,
wherein the fatty acid forming the ester has from about 18 to 20 carbon atoms
in the
main carbon chain.
US Patent No. 5,270,041 to Marigen S.A. teaches the use of small amounts of
sterols,
their fatty acid esters and glucosides for the treatment of tumours. The
method of
preparation of these compositions involving the use of hazardous chemical
reagents
effectively precludes their use in foods or as dietary additives.
Other research has demonstrated that phytostanols, the 5 alpha saturated
derivatives
of phytosterols, are more effective as therapeutic agents in lowering serum
cholesterol
on a molecular weight basis than phytosterols (8). Similarly, in a further
comparison,
sitosterols infused into the GI tract resulted in a 50% reduction in serum
cholesterol as
opposed to an 85% reduction when sitostanols were infused (9). The advantages
of
stanols over sterols with respect to inhibition of cholesterol absorption from
the GI tract
are two-fold. Firstly, stanols are more chemically stable than their
unsaturated
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counterparts in heat and air due to the absence of carbon-carbon bonds in the
former.
Secondly, stanols are more effective at lowering serum cholesterol on a
molecular
weight basis than their unsaturated counterparts.
US Patent Serial No. 5,502,045 to Raision Tehtaat Oy AB (hereinafter the
"Raision
Patent") describes the preparation of a beta-sitostanol fatty acid ester
mixture
prepared by interesterifying beta-sitostanol with a fatty acid ester
containing from 2 to
22 carbon atoms in the presence of an interesterification catalyst. This
process
renders the sitostanol appreciably more soluble in fats and oils.
South African Patent Application 967616 also to Raision Tehtaat Oy AB
(hereinafter
the "SA Raision Patent") describes a similar composition to that in the
Raision Patent
but which further contains at least 10% campestanol obtained by hydrogenation
of the
phytosterol mixture.
US Patent No.5,244,887 to Straub discloses a method of making a food additive
composition which comprises dissolving a stanol (sitostanol; clionastanol;
22,23-
dihydrobrassicastanol; campestanol and mixtures thereof) with an edible
solubilizing
agent, an anti-oxidant and a carrier or dispersant.
Although the Raision Patent and the Raision SA Patent both attempt to produce
a
phytostanol delivery system which is stable and effective, there are
significant
problems with the long-term stability of these esterified products due to the
ultimate
oxidation of the unsaturated fatty acid moiety.
It is an object of the present invention to obviate or mitigate the above
disadvantages.
SUMMARY OF THE INVENTION
The present invention provides a composition suitable for use alone or for
incorporation into foods, beverages, pharmaceuticals, nutraceuticals and the
like
which comprises one or more phytosterols, phytostanols or mixtures of both
treated to
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enhance the solubility and dispersability thereof.
The present invention further comprises foods, beverages, pharmaceuticals,
nutraceuticals and the like which comprise a composition of one or more
phytosterols,
phytostanols or mixtures of both treated to enhance the solubility and
dispersability
thereof. These "formulations" include, but are not limited to, the treated
composition
incorporated into edible oils and fat-based foods (such as margarines, butter,
mayonnaise, dressing, shortenings, and cheeses), and formed into suspensions,
emulsions, microemulsions, liposomes, niosomes and general hydrated lipid
phases.
The composition additionally may be incorporated into numerous pharmaceutical
dosage forms as described in detail below.
The present invention further comprises the use of a composition which
comprises
one or more phytosterols, phytostanols or mixtures of both, treated to enhance
the
solubility and dispersability thereof, to lower serum cholesterol in animals,
including
humans.
The present invention further comprises methods of making a composition
suitable for
incorporation into foods, beverages, pharmaceuticals, nutraceuticals and the
like
which comprises enhancing the solubility and dispersability of one or more
phytosterols, phytostanols or mixtures of both by any of the techniques
described in
more detail hereinbelow.
The composition of the present invention, which comprises one or more
phytosterols,
phytostanols or mixtures of both treated to enhance the solubility and
dispersability
thereof, has marked advantages over the phytosterol/stanol compositions
previously
known and described in the art. The composition of the present invention is
more
soluble and dispersable in both lipid-based and aqueous systems which is
critically
important due to the fact that only solubilized phytosterols and phytostanols
inhibit the
absorption of cholesterol in the digestive tract. Furthermore, the techniques
described
herein to enhance the solubility and dispersability of phytosterol
compositions and
phytostanol compositions (or mixtures thereof) facilitate the incorporation of
these
compositions into foods, beverages, nutraceuticals and pharmaceuticals.
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PREFERRED EMBODIMENTS OF THE INVENTION
According to one aspect of the present invention, there is provided a
composition
suitable for incorporation into foods, beverages, pharmaceuticals,
nutraceuticals and
the like which comprises one or more phytosterols, phytostanols or mixtures of
both
treated to enhance the solubility and dispersability thereof.
As used herein, the term "phytosterol" includes all phytosterols without
limitation, for
example: sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol,
chalinosterol, poriferasterol, clionasterol and all natural or synthesized
forms and
derivatives thereof, including isomers. The term "phytostanol" includes all
saturated or
hydrogenated phytosterols and all natural or synthesized forms and derivatives
thereof, including isomers. It is to be understood that modifications to the
phytosterols
and phytostanols i.e. to include side chains also falls within the purview of
this
invention. It is also to be understood that this invention is not limited to
any particular
combination of phytosterols and/or phytostanols forming a composition. In
other
words, any phytosterol or phytostanol alone or in combination with other
phytosterols
and phytostanols in varying ratios as required depending on the nature of the
ultimate
formulation may be treated to enhance the solubility and dispersability as
described in
the present invention. For example, the composition described in
PCT/CA95/00555
which comprises no more than 70% by weight beta-sitosterol, at least 10% by
weight
campesterol and stigmastanol may be treated by the techniques of the present
invention to yield a stable and favourably soluble product for incorporation
into foods,
beverages, pharmaceuticals and the the like.
The phytosterols for use in this invention may be procured from a variety of
natural
sources. For example, they may be obtained from the processing of plant oils
(including aquatic plants) such as corn oil and other vegetable oils), wheat
germ oil,
soy extract, rice extract, rice bran, rapeseed oil, sesame oil and fish oil.
Without
limiting the generality of the foregoing, it is to be understood that there
are other
sources of phytosterols such as marine animals from which the composition of
the
present invention may be prepared. US Patent Serial No. 4,420,427 teaches the
preparation of sterols from vegetable oil sludge using solvents such as
methanol.
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Alternatively, phytosterols may be obtained from tall oil pitch or soap, by-
products of
the forestry practise as described in PCT/CA95/00555, incorporated herein by
reference.
There are numerous techniques of enhancing the solubility and dispersability
of
phytosterols and/or phytostanols which may successfully be used within the
scope of
the present invention. These techniques improve the effectiveness of the
phytosterols
and/or phytostanols in lowering serum cholesterol and ensure even distribution
of the
phytosterols and/or phytostanols throughout the food, beverage, pharmaceutical
nutraceutical and the like to which they are added. Such enhancement may be
achieved by a number of suitable mans such as, for eample, solubilizing or
dispersing
the phytosterol or phytostanol composition (or mixture thereof) to form
emulsions,
solutions and dispersions or self-emulsifying systems; reducing the particle
size by
mechanical grinding (milling, micronisation etc...), lyophilizing, spray
drying, controlled
precipitating or a combination thereof; forming solid dispersions,
suspensions,
hydrated lipid systems; forming inclusion complexations with cyclodextrins;
and
forming hydrotopes and formulations with bile salts and their derivatives.
Prior to the solubility/dispersability enhancement techniques of the present
invention, it
is preferred that the phytosterols and/or phytostanols be isolated from the
source and
formed into a solid powder through precipitation, filtration and drying, spray
drying,
lyophilization or by other conventional work-up techniques. This powder form
may
then be physically modified as described below to enhance the solubility and
dispersability of the phytosterol and/or phytostanol in the chosen delivery
medium.
Each of the tecnniques which may be used in accordance with the present
invention
are described below.
Emulsions
Emulsions are finely divided or colloidal dispersions comprising two
immiscible
phases, e.g. oil and water, one of which (the internal or discontinuous phase)
is
dispersed as droplets within the other (external or discontinuous phase). Thus
an oil-
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in-water emulsion consists of oil as the internal phase, dispersed water as
the external
phase, the water-in-oil emulsion being the opposite.
A wide variety of emulsified systems may be formed which comprise the
composition
of the present invention including standard emulsions, microemulsions and
those
which are self-emulsifying (emulsify on exposure to agitated aqueous fluids
such as
gastric or intestinal fluids).
Generally, emulsions may include oil and water phases, emulsifiers, emulsion
stabilizers and optionally preservatives, flavouring agents, pH adjusters and
buffers,
chelating agents, antifoam agents, tonicity adjusters and anti-oxidants.
Suitable
emulsifiers
(wherein bracketed numerals refer to the preferred HLB values) include:
anionic
surfactants such as alcohol ether sulfates, alkyl sulfates (30-40), soaps (12-
20) and
sulfosuccinates; cationic surfactants such as quaternary ammonium compounds;
zwitterionic surfactants such as alkyl betaine derivatives; amphoteric
surfactants such
as fatty amine sulfates, difatty alkyl triethanolamine derivatives (16-17);
and nonionic
surfactants such as the polyglycol ether derivatives of aliphatic or
cycloaliphatic
alcohols, saturated fatty acids and alkyphenols, water-soluble polyethyleneoxy
adducts onto polypropylene glycol and alkyl polypropylene glycol, nonylphenol
polyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene
oxide
adducts, tributylphenoxy-polyethoxyethanol, polyethylene glycol, octylphenoxy-
polyethoxyethanol, lanolin alcohols, polyoxyethylated (POE) alkyl phenols, POE
fatty
amides, POE fatty alcohol ethers, POE fatty amines, POE fatty esters,
poloxamers (7-
19), POE glycol monoethers (13-16), polysorbates and sorbitan esters. This
list is not
intended to be exhaustive as other emulsifiers are equally suitable.
Appropriate emulsion stabilizers include, but are not limited to, lyophilic
colloids such
as polysaccharides (e.g. acacia, agar, alginic acid, carrageenin, guar gum,
karaya
gum, tragacanth xanthan gum), amphoterics (e.g. gelatin) and synthetic or semi-
synthetic polymers (e.g. carbomer resins, cellulose ethers, carboxymethyl
chitin,
polyethylene glycol-n (ethylene oxide polymer H(OCH2CH2)nOH); finely divided
solids including clays (e.g. attapulgite, bentonite, hectorite, kaolin,
magnesium
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aluminum silicate and montmorillonite), microcrystalline cellulose oxides and
hydroxides (e.g. aluminum hydroxide. magnesium hydroxide and silica); and
cybotactic promoters/gellants including amino acids, peptides, proteins
lecithin and
other phospholipids and poloxamers.
Suitable anti-oxidants for use in the formation of emulsions include:
chelating agents
such as citric acid, EDTA, phenylalanine, phosphoric acid, tartaric acid and
tryptophane; preferentially oxidized compounds such as ascorbic acid, sodium
bisulfate
and sodium sulfite; water soluble chain terminators such as thiols and lipid
soluble
chain terminators such as alkly gallates, ascorbyl palmitate, t-butyl
hydroquinone,
butylated hydroxyanisole, butylated hydroxytoluene, hydroquinone,
nordihydroguaiaretic acid and alpha-tocopherol. Suitable preservatives, pH
adjustment agents, and buffers, chelating agents, osmotic agents, colours and
flavouring agents are discussed hereinbelow under "Supensions", but are
equally
applicable with respect to the formation of emulsions.
The general preparation of emulsions is as follows: the two phases (oil and
water) are
separately heated to an appropriate temperature (the same in both cases,
generally 5-
10°C above the melting point of the highest melting ingredients in the
case of a solid
or semi-solid oil, or where the oil phase is liquid, a suitable temperature as
determined
by routine experimentation). Water-soluble components are dissolved in the
aqueous
(water) phase and oil-soluble components are dissolved in the oil phase. To
create an
oil-in water emulsion, the oil phase is vigorously mixed into the aqueous
phase to
create a suitable dispersion and the product is allowed to cool at a
controlled rate with
stirring. A water-in-oil emulsion is formed in the opposite fashion i.e. the
water phase
is added to the oil phase. When hydrophillic colloids are a part of the system
as
emulsion stabilizers, a phase inversion technique may be employed whereby the
colloid is mixed into the oil phase rather than the aqueous phase, prior to
addition to
the aqueous phase. In using the oil-based composition of the present
invention, which
is semi-solid, it is preferred to add the composition to the oil phase prior
to heating.
Microemulsions, characterized by a particle size at least an order of
magnitude smaller
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(10-100 nm) than standard emulsions and defined as "a system of water, oil and
amphiphile which is a single optically isotropic and thermodynamically stable
liquid"
(14), may also be formed comprising the composition of the present invention.
In a
preferred form, the microemulsion comprises a surfactant or surfactant
mixture, a co-
surfactant,(usually a short chain alcohol) the oil-based composition of the
present
invention, water and optionally other additives.
This system has several advantages as a delivery system for the phytosterols
or
phytostanols or mixtures thereof having relatively high lipophilicity.
Firstly,
microemulsions tend to be created spontaneously, that is, without the degree
of
vigorous mixing required to form standard emulsions. From a commercial
perspective,
this simplifies the manufacturing process. Secondly, microemulsions may be
sterilized
using microfiltration techniques without breaking the microstructure due to
the small
diameter of the microdroplets. Thirdly, microemulsions are highly
thermodynamically
stable. Fourthly, microemulsions possess high solubilizing power which is
particularly
important as they allow for an increased solubilization of the poorly
hydrosoluble
phytosterols and phytostanols.
Surfactant or surfactant mixtures which are suitable for use in the formation
of
microemulsions can be anionic, cationic, amphoteric or non-ionic and possess
HLB
(hydrophile-lipophile balance) values within the range of 1-20, more
preferably in the
ranges 2-6 and 8-17. Especially preferred agents are non-ionic surfactants,
selected
from the group consisting of polyglycol ether derivatives of aliphatic or
cycloaliphatic
alcohols, saturated fatty acids and alkyphenols, water-soluble polyethyleneoxy
adducts onto polypropylene glycol and alky polypropylene glycol, nonylphenol
polyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene
oxide
adducts, tributylphenoxy-polyethoxyethanol, polyethylene glycol, octylphenoxy-
polyethoxyethanol, lanolin alcohols, polyoxyethylated (POE) alkyl phenols, POE
fatty
amides, POE fatty alcohol ethers, POE fatty amines, POE fatty esters,
poloxamers (7-
19), POE glycol monoethers (13-16), polysorbates and sorbitan esters.
There are many methods known and used by those skilled in the art for making
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microemulsions. In a preferred method of forming microemulsions of the present
invention, a surfactant, a co-surfactant and the phytosterol, phytostanols or
mixtures
thereof (pre-dissolved in a suitable proportion of an appropriate oil) is
mixed and then
titrated with water until a system of desired transparency is obtained.
In a further preferred embodiment, the formation of microemulsions may be
achieved
by mixing the phytosterols or phytostanols or mixtures thereof with
hydrotropic agents
and food-grade surfactants (refer to 11 ).
Solutions and Dispersions
Phytosterols or phytostanols or mixtures thereof may be dissolved or dispersed
in a
suitable oil vehicle and used in this form, for example, in general food
usage, in
basting meats and fish, and for incorporation into animal feeds.
Suitable solubilizing agents include all food grade oils such as plant oils,
marine oils
such as fish oil and vegetable oils, monoglycerides, diglycerides,
triglycerides,
tocopherols and the like, and mixtures thereof.
Self-Emulsifying Systems
Phytosterols or phytostanols or mixtures thereof may be mixed with appropriate
excipients, for example, surfactants, emulsion stabilizers (described above)
and the
like, heated (if necessary) and cooled to form a semi-solid product capable of
forming
a spontaneous emulsion on mixing with water. This semi-solid product may be
used
in numerous other forms such as filler material in two-piece hard or soft
gelatin
capsules, or may be adapted for use in other delivery systems.
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Reducing Particle Size
Many techniques of particle size reduction are suitable for use within the
present
invention including, inter alia, dry milling, micropulverization, fluid energy
grinding,
controlled precipitation, lyophilisation and spray-drying. Each of these
techniques is
well known in the art and will not be discussed in any detail other than to
provide
reference to 12 and 13, the former showing preferred processes of spray-drying
and
the latter summarizing the other techniques listed above.
It has been found that reducing the particle size to under 500um and most
preferably
under 20um allows suitable dispersability/solubility of the composition in the
carriers
and dosage forms described further below.
Solid Dispersions
An alternative means of increasing the solubility/dispersability of
phytosterols,
phytostanols or mixtures thereof involves the use of solid dispersion systems.
These
dispersions may include molecular solutions (eutectics), physical dispersions
or a
combination of both.
For example, solid dispersions may typically be prepared by utilizing water-
soluble
polymers as carriers. Without limitation, these carriers may include, either
alone or in
combination: solid grade polyethylene glycols (PEG's), with or without the
addition of
liquid grade PEG's; polyvinylpyrrolidones or their co-polymers with vinyl
acetate and
cellulose ethers and esters. Other excipients, such as additional members of
the
glycol family e.g. propylene glycol, polyols,e.g. glycerol etc.. may also be
included in
the dispersions.
Solid dispersions may be prepared by a number of ways which are familiar to
those in
the art. These include, without limitation, the following methods:
(a) fusing the ingredients, followed by controlled cooling to allow
solidification and
subsequent mechanical grinding to produce a suitable powder. Alternatively,
the
molten (fused) dispersion may be sprayed into a stream of cooled air in a
spray
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drier to form solid particles (prilling) or passed through an extruder and
spheroniser to form solid masses of a controlled particle size. In a further
alternative, the molten dispersion is filled directly into two-piece hard
gelating
capsules;
(b) dissolving the ingredients in a suitable solvent system (organic, mixed
organic,
organic-aqueous) and then removing the solvents e.g. by evaporating at
atmospheric pressure or in vacuo, spray drying, lyophilizing and the like; or,
in a
variation of the foregoing, and
(c) dissolving the ingredients in a suitable solvent system, subsequently
precipitating
them from solution by the use of an immiscible solvent in which the
ingredients
have little or no solubility, filtration, removing the solvent, drying and
optionally
grinding to provide a suitable powder form.
Other commercially available agents for enhancing solubility of the
phytosterols or
phytostanols or mixtures thereof through the formation of solid dispersions
are
considered to fall within the purview of this invention. For example, the
commercial
excipient marketed under the trade-mark GelucireTM by Gattefosse comprising
saturated polyglycolised glycerides may readily be used herein.
Suspensions
Suspensions, which may be used to enhance the solubility and/or dispersability
of the
phytosterols, phytostanols or mixtures thereof, comprise a solid, perhaps
finely
divided, internal phase dispersed in an oily or aqueous external phase (the
vehicle). In
addition, the solid internal phase may be added to an emulsion as described
above
during its' formation to produce a delivery system having properties common to
both
suspensions and emulsions.
Numerous excipients, which are commonly used in the art, may be suitable for
producing a suspension within the scope of the present invention. Typically, a
suspension comprises an oily or aqueous vehicle, the dispersed (suspended)
internal
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phase, dispersing and/or wetting agents (surfactants), pH adjustment
agents/buffers,
chelating agents, antioxidants, agents to adjust ionic strength (osmotic
agents)
colours, flavours, substances to stabilize the suspension and increase
viscosity
(suspending agents ) and preservatives.
Appropriate vehicles include, but are not limited to: water, oils, alcohols,
polyols, other
edible or food grade compounds in which the phytosterol composition is
partially or not
soluble and mixtures thereof. Appropriate dispersing agents include, but are
not
limited to: lecithin; phospholipids; nonionic surfactants such as polysorbate
65,
octoxynol-9, nonoxynol-10, polysorbate 60, polysorbate 80, polysorbate 40,
poloxamer
235, polysorbate 20 and poloxamer 188; anionic surfactants such as sodium
lauryl
sulfate and docusate sodium; fatty acids, salts of fatty acids, other fatty
acid esters,
and mixtures thereof.
Agents/buffers for pH adjustment include citric acid and its salts, tartaric
acid and its
salts, phosphoric acid and its salts, acetic acid and its salts, hydrochloric
acid, sodium
hydroxide and sodium bicarbonate. Suitable chelating agents include edetates
(disodium, calcium disodium and the like), citric acid and tartaric acid.
Suitable
antioxidants include ascorbic acid and its salts, ascorbyl palmitate,
tocopherols
(especially alpha-tocopherol), butylated hydroxytoluene, butylated
hydroxyanisole,
sodium bisulfate and metabisulfite. Suitable osmotic agents include
monovalent,
divalent and trivalent electrolytes, monosaccharides and disaccharides.
Suitable
preservatives include parabens (Me, Et, Pr, Bu), sorbic acid, thimerosal,
quaternary
ammonium salts, benzyl alcohol, benzoic acid, chorhexidine gluconate and
phenylethanol. Colours and flavours may be added as desired and may be
selected
from all nature, natural-identical and synthetic varieties.
Hydrated Lipid Systems
In a further embodiment of the present invention, the
solubility/dispersability of
phytosterols, phytostanols or mixtures thereof may be enhanced by the
formation of
phospholipid systems such as liposomes and other hydrated lipid phases, by
physical
inclusion. This inclusion refers to the entrapment of molecules without
forming a
covalent bond and is widely used to improve the solubility and subsequent
dissolution
CA 02334449 2000-12-05
of active ingredients.
Hydrated lipid systems, including liposomes, can be prepared using a variety
of lipid
and lipid mixtures, including phospholipids such as phosphatidylcholine
(lecithin),
phosphodiglyceride and sphingolipids, glycolipids, cholesterol and the like.
The lipids
may preferably be used in combination with a charge bearing substances such as
charge-bearing phospholipids, fatty acids, and potassium and sodium salts
thereof in
order to stabilize the resultant lipid systems. A typical process of forming
liposomes is
as follows:
1 ) dispersion of lipid or lipids and the phytosterols or phytostanols or
mixtures
thereof in an organic solvent (such as chloroform, dichloromethane, ether,
ethanol or other alcohol, or a combination thereof). A charged species may be
added to reduce subsequent aggregation during liposome formation.
Antioxidants (such as ascorbyl palmitate, alpha-tocopherol, butylated
hydroxytoluene and butylated hydroxyanisole) may also be added to protect
any unsaturated lipids, if present;
2) filtration of the mixture to remove minor insoluble components;
3) removal of solvents under conditions (pressure, temperature) to ensure no
phase separation of the components occur;
4) hydration of the "dry" lipid mixture by exposure to an aqueous medium
containing dissolved solutes, including buffer salts, chelating agents,
cryoprotectorants and the like; and
5) reduction of liposome particle size and modification of the state of
lamellarity by
means of suitable techniques such as homogenization, extrusion etc..
Any procedure for generating and loading hydrated lipid with active
ingredients, known
to those skilled in the art, may be employed within the scope of this
invention. For
example, suitable processes for the preparation of liposomes are described in
references 14 and 15, both of which are incorporated herein by reference.
Variations
on these processes are described in US Patent Serial No. 5,096,629 which is
also
incorporated herein by reference.
CA 02334449 2000-12-05
16
US Patent Serial No. 4,508,703 (also incorporated herein by reference)
describes a
method of preparing liposomes by dissolving the amphiphillic lipidic
constituent and
the hydrophobic constituent to form a solution and thereafter atomizing the
solution in
a flow of gas to produce a pulverent mixture.
Cyclodextrin Complexes
Cyclodextrins are a class of cyclic oligosaccharide molecules comprising
glucopyranose sub-units and having a toroidal cylindrical spatial
configuration.
Commonly available members of this group comprise molecules containing six
(alpha-
cyclodextrin), seven (beta-cyclodextrin) and eight (gamma-cyclodextrin)
glucopyranose
molecules, with the polar (hydrophilic) hydroxyl groups oriented to the
outside of the
structure and the apolar (lipophilic) skeletal carbons and ethereal oxygens
lining the
interior cavity of the toroid. This cavity is capable of accomodating
(hosting) the
lipophilic moiety of an active ingredient (the guest molecule, here the
phytosterol or
phytostanol or mixture of both) by bonding in a non-covalent manner to form an
inclusion complex.
The external hydroxyl substituents of the cyclodextrin molecule may be
modified to
form derivatives having improved solubility in aqueous media along with other
desired
enhancements, such as lowered toxicity, etc.. Examples of such derivatives
are:
alkylated derivatives such as 2,6-dimethyl-beta-cclodextrin; hydroxyalkylated
derivatives such as hydroxypropyl-beta-cyclodextrin; branched derivatives such
as
diglucosly-beta-cyclodextrin; sulfoalkyl derivatives such as sulfobutylether-
beta-
cyclodextrin; and carboxymethylated derivatives such as carboxymethyl-beta-
cyclodextrin. Other types of chemical modifications, known to those in the
art, are also
included within the scope of this invention.
The cyclodextrin complex often confers properties of improved solubility,
dispersability,
stability (chemical, physical and microbiological), bioavailability and
decreased toxicity
on the guest molecule (here, the phytosterols or phytostanols or mixtures
thereof).
There are a number of ways known in the art to produce a cyclodextrin complex.
CA 02334449 2000-12-05
17
Complexes may be produced, for example, by using the following basic methods:
stirring the phytotsterol, phytostanol or mixture thereof into an aqueous or
mixed
aqueous-organic solution of the cyclodextrin, with or without heating;
kneading,
slurrying or mixing the cyclodextrin and the phytotsterol, phytostanol or
mixture thereof
in a suitable device with the addition of an appropriate quantity of aqueous,
organic or
mixed aqueous-organic liquid, with or without heating; or by physical
admixture the
cylcodextrin and the phytotsterol, phytostanol or mixture thereof using a
suitable
mixing device. Isolation of the inclusion complex so formed may be achieved by
co-
precipitation, filtration and drying; extrusion/spheronisation and drying;
subdivision of
the moist mass and drying; spray drying; lyophilization or by other suitable
techniques
depending on the process used to form the cyclodextrin complex. A further
optional
step of mechanically grinding the isolated solid complex may be employed.
These cyclodextrin/phytosterol complexes enhance the solubility and
dissolution rate
and increase the stability of the phytosterols or phytostanols or mixtures
thereof. For a
review of cyclodextrin complexation, please refer to 16.
Complexation with Bile Salts
Bile acids, their salts and conjugated derivatives, suitably formulated, may
be used to
solubilize phytosterols, phytostanols or mixtures thereof, thereby improving
the
solubility and dispersion characteristics of these compositions. Examples of
suitable
bile acids include: cholic acid, chenodeoxycholic acid, deoxycholic acid,
dehydrocholic
acid, and lithocholic acid. Examples of suitable bile salts include: sodium
cholate,
sodium deoxycholate and their other salt forms. Examples of suitable
conjugated bile
acids include: glycochenodeoxycholic acid, glycholic acid,
taurochenodeoxycholic acid,
taurocholic acid, taurodeoxycholic acid and their salts.
A suitable system for solubilizing phytosterols or phytostanols or mixtures
thereof
consists of the sterol or stanol component plus one or more bile acids, salts
or
conjugated bile acids. Further materials may be added to produce formulations
having
additional solubilization capacity. These materials include, but are not
limited to:
phospholipids, glycolipids and monoglycerides. These ingredients may be
formulated
CA 02334449 2000-12-05
18
either in the solid phase or by the use of suitable solvents or carrier
vehicles, with
appropriate isolation and, optionally, particle size reduction using
techniques described
hereinabove.
Since bile acids and their derivatives have an unpleasant taste and may be
irritating to
the mucous membranes of the stomach and upper regions of the gastro-intestinal
tract, a suitable enteric coating may be applied to the solid formulation
particulates,
using techniques known to those skilled in the art. Typical enteric coatings
include,
inter alias cellulose acetate phthalate, cellulose acetate trimellitiate,
hydroxyproplmethylcellulose phthalate, hydroxyproplmethylcellulose acetate
succinate, poly (vinylaceate phthalate), acrylate polymers and their
derivatives (e.g.
appropriate members of the Eudragit series), ethylcellulose or combinations
thereof.
Additional excipients may be added to the coating formulation to modify
membrane
functionality or to aid in the coating process (e.g. surfactants,
plasticisers, channeling
agents, permeability modifiers and the like). Coating formulation vehicles may
comprise aqueous or organic systems, or mixtures of both.
Hydrotopic Complexation
Compounds which are capable of opening up the water structure associated with
hydrophobic (lipophilic) and other molecules are referred to as hydrotopes.
These
compounds may be used to enhance the aqueous solubility of poorly water-
soluble
substances such as phytosterols, phytostanols and their esters. Examples of
hydrotopes include, inter alia, sodium benzoate, sodium hydroxybenzoates,
sodium
salicylate, nicotinamide, sodium nicotinate, sodium gentisate, gentisic acid
ethanolamide, sodium toluates, sodium aminobenzoates, sodium anthranilate,
sodium
butylmonoglycolsulfate, resorcinol and the like.
Complex formation, which is non-covalent in nature, may be achieved by mixing
appropriate ratios of the phytosterols or phytostanols or mixtures thereof and
the
hydrotope or mixtures thereof in a suitable liquid vehicle, which may be
aqueous,
organic or a combination of both. Additional excipients such as surfactants,
polyol,
disaccharides etc.. may be added to facilitate complexation or to aid in
dispersability.
CA 02334449 2000-12-05
19
The resultant complex is isolated as a dry powder by any process known in the
art (co-
precipitation and drying, evaporation of the liquid vehicle, spray drying,
lyophilization
etc..). Particle size may be reduced by any standard technique such as those
described previously herein, if desired. The resultant hydrotope complex may
be used
without further modification or may be compounded into a variety of other
formulations
or vehicles as required.
Methods of Use:
Any phytosterol or phytostanol or mixture thereof, treated as described herein
to form
a composition of enhanced solubility/dispersability, may be used as an
effective agent
to lower serum cholesterol in animals, particularly humans. It is to be
understood,
however, that this composition is equally suited for administration to other
animals, for
example, in the form of veterinary medicines and animal foods. There are
numerous
modes or "vehicles" of delivery of this composition, accordingly, this
invention is not
intended to be limited to the following delivery examples.
1 ) Pharmaceutical Dosage Forms:
It is contemplated within the scope of the present invention that the
composition of the
present invention may be incorporated into various conventional pharmaceutical
preparations and dosage forms such as tablets (plain and coated) for use
orally,
bucally or lingually, capsules (hard and soft, gelatin, with or without
additional
coatings) powders, granules (including effervescent granules), pellets,
microparticulates, solutions (such as micellar, syrups, elixirs and drops),
lozenges,
pastilles, ampuls, emulsions, microemulsions, ointments, creams,
suppositories, gels,
and transdermal patches, modified release dosage forms together with customary
excipients and/or diluents and stabilizers.
The composition of the present invention, adapted into the appropriate dosage
form as
described above may be administered to animals, including humans, orally, by
injection (intra-venously, subcutaneously, intra-peritoneally, intra-dermally
or intra-
muscularly), topically or in other ways. Although the precise mechanism of
action is
unclear, the composition of the present invention, administered intra-
venously, lowers
CA 02334449 2000-12-05
serum cholesterol. It is believed that the phytosterol composition may have,
in addition
to the role as an inhibitor of cholesterol absorption in the intestine, a
systemic effect on
cholesterol homeostasis through bile acid synthesis, enterocycte and biliary
cholesterol excretion, bile acid excretion and changes in enzyme kinetics and
cholesterol transport between various compartments within the body
(PCT/CA97/00474 which was published on January 15, 1998). See also paper to
Peter Jones (under publication).
2) Foods/Beveraaes/Nutraceuticals:
In another form of the present invention, the composition of the present
invention may
be incorporated into foods, beverages and nutraceuticals, including, without
limitation,
the following:
1 ) Dairy Products --such as cheeses, butter, milk and other dairy beverages,
spreads and dairy mixes, ice cream and yoghurt;
2) Fat-Based Products--such as margarines, spreads, mayonnaise, shortenings,
cooking and frying oils and dressings;
3) Cereal-Based Products--comprising grains (for example, bread and pastas)
whether these goods are cooked, baked or otherwise processed;
4) Confectionaries--such as chocolate. candies, chewing gum, desserts, non-
dairy
toppings (for example Cool WhipTM), sorbets, icings and other fillings;
5) Beverages-- whether alcoholic or non-alcoholic and including colas and
other soft
drinks, juices, dietary supplement and meal replacement drinks such as those
sold under the trade-marks BoostTM and EnsureTM; and
6) Miscellaneous Products--including eggs, processed foods such as soups, pre-
prepared pasta sauces, pre-formed meals and the like.
CA 02334449 2000-12-05
21
The composition of the present invention may be incorporated directly and
without
further modification into the food, nutraceutical or beverage by techniques
such as
mixing, infusion, injection, blending, immersion, spraying and kneading.
Alternatively,
the composition may be applied directly onto a food or into a beverage by the
consumer prior to ingestion. These are simple and economical modes of
delivery.
There are some preferred techniques of enhancing solubility and dispersabitity
which
may work in concert with certain food manufacturing and/or processing methods.
Solubility and dispersability of any phytosterol or phytostanol mixture may be
enhanced via the formation of emulsions and microemulsions which may readily
be
incorporated into margarines, butter, spreads, mayonnaise, dressings, yoghurt
and the
like. Patents covering the preparation of margarines and yellow spreads
include: US
Patent Serial Nos: 5,118,522; 5,536,523; 5,409,727; 5,346,716; 5,472,728; and
5,532,020, all of which are incorporated herein by reference.
EXAMPLES
Example 1: Cyclodextrin Complex
An aqueous ethanolic vehicle was prepared by mixing water and ethanol in the
ratios
of water (9 parts - 1 part) to ethanol (1 part - 9 parts) and the temperature
was
adjusted to 20-50°C. 2-dydroxypropyl-beta-cylcodextrin was dissolved in
the mixture
to give a concentration of 10-50% w/v, with stirring. A slight calculated
excess of a
phytosterol composition comprising beta-sitosterol, campesterol and
stigmastanol was
added in fine powder form to the mixture and the vessel sealed. The mixture
was
stirred for 2-48 hours under a maintained reaction temperature. The resultant
mixture
was filtered and the filtrate allowed to attain an appropriate temperature.
The complex
was then isolated by spraying drying at 39-90°C over an appropriate
time cycle and
yielded a free-flowing powder of small and regular particle size.
Example 2: Cyclodextrin Complex with Silicon Dioxide
The mixture was prepared in accordance with the protocol outlined in Example 1
up to
and including the stage of filtration. In this example, 0.1-1 % w/w of
colloidal silicone
CA 02334449 2000-12-05
22
dioxide (based on the calculated solids content of the filtrate) was added to
the filtrate
while stirring. The complex was then isolated by spray drying at 30-
90°C over an
appropriate time cycle and yielded a free-flowing powder of small and regular
particles
size.
Example 3: Comalexation with Bile Salts
An aqueous ethanolic vehicle was prepared by mixing water and ethanol in the
ratios
of water (9 parts - 1 part) to ethanol (1 part - 9 parts) and the temperature
was
adjusted to 20-50°C. Calculated amounts of sodium cholate and sodium
taurocholate
were dissolved to give a combined concentration of 30-60% w/v, the ratios of
sodium
cholate to sodium taurocholate were between 1 part - 9 parts to 9 parts - 1
part. A
slight calculated excess of a phytosterol composition comprising beta-
sitosterol,
campesterol and stigmastanol was added in fine powder form, the vessel sealed,
and
the mixture gently stirred for 2-24 hours. The mixture was filtered and a
calculated
quantity of soybean lecithin (0.5-30% w/w based on the solids content of
filtrate) was
added with gentle stirring. Stirring was continued for a period up to 4 hours
and the
mixture allowed to attain a suitable temperature. The mixture was spray dried
at 30-
90°C over a suitable time cycle to yield an isolated solid complex as a
free flowing
powder of small and irregular particle size.
An enteric coating solution was prepared comprising a mixture of EudragitTM l_
100/
Eudragit T"" S 100 (enteric film formers) 6+1.2% w/w composite concentration,
triethylcitrate (plasticiser) 0.6+1.12% w/w, talc (anti-tack agent) 3+0.6%
w/w, water
(vehicle) 5+1 % w/w, isopropyl alcohol (vehicle) to 100% w/w. the powder,
prepared as
described above, was spray coated using equipment and methodology known in
this
field, to a percentage weight increase (based upon input weight of powder)
sufficient
to ensure an effective enteric barrier. The resultant coated powder product
was then
collected.
Example 4: Complexation with Bile Salts and Silicone Dioxide
The mixture was prepared in accordance with the protocol outlined in Example 3
up to
and including the addition of soybean lecithin, stirring and cooling. In this
example,
CA 02334449 2000-12-05
23
0.1-1 % w/w of colloidal silicone dioxide (based on the calculated solids
content of the
filtrate) was added to the filtrate while stirring. The mixture was spray
dried at 30-90°C
over a suitable time cycle to yield an isolated solid complex as a free
flowing powder of
small and irregular particle size. The enteric coating solution was then
prepared and
applied as described in Example 3.
Example 5: Complexation with Bile Salts I Sub-coatinct with Water Soluble Film
The mixture was prepared in accordance with the protocol outlined in Example 3
up to
and including the step of spray drying to form the free flowing powder. Prior
to the
application of the enteric membrane, an initial sub-coating of a water-soluble
film
former, (here, hydroxypropylmethylcellulose formulated in a hydroalcoholic
vehicle
along with a plasticiser and ant-tack agent) was applied to the powder.
Example 6: Hydrotropic Complexation
Gentisic acid anhydride (10-40% w/v) was dissolved in appropriate volume of
water,
containing 5-20% v/v ethanol at 20-60°C. A calculate slight excess of a
phytosterol
composition comprising beta-sitosterol, campesterol and stigmastanol in fine
powder
form was added, the vessel sealed and the mixture stirred vigorously for 2-24
hours.
The mixture was then filtered and the filtrate allowed to attain a suitable
temperature.
The mixture was spray dried at 30-90°C over a suitable time cycle and
a solid
hydrotropic complex isolated in free-flowing powder form, of small and regular
particle
size.
Example 7: Hydrotropic Complexation
The mixture was prepared in accordance with the protocol outlined in Example 6
up to
and including the stage of filtration. In this example, 0.1-1 % w/w of
colloidal silicone
dioxide (based on the calculated solids content of the filtrate) was added to
the filtrate
while stirring. The complex was then isolated by spray drying at 30-
90°C over an
appropriate time cycle and a solid hydrotropic complex isolated in free-
flowing powder
form, of small and regular particle size.
Unless otherwise stated for all examples hereinafter, FCP-3P2 Batch FM-P2-63
CA 02334449 2000-12-05
24
(composition: campestanol, 19.16%; sitostanol, 76.99%; campesterol, 0.13%;
beta-
CA 02334449 2000-12-05
sitosterol, 0.07%) was used in formulation work. Content uniformity data was
referenced to the total phytostanol content of the batch, i.e. 96.15%.
Example 8: Solutions and Dispersions (Oil-based)
In order to determine the lipophilicity of FCP-3P2 the compound was evaluated
in a
selection of test systems. Solubility in fixed oils, the octanol / water
partition coefficient
and solubility in pH 5 aqueous buffer were chosen as relevant characteristics.
Knowledge of these parameters would also act as guidance in future formulation
effo rts.
FCP-3P2 was represented by Batch FM-P2-48 (composition: campestanol, 20.03%;
sitostanol, 75.12%, campesterol, 3.19%).
Solubility in Fixed Oils
This was determined by adding 500mg of test compound to 5mL of each oil and
equilibrating by vortexing (VWR Multi-Tube Vortexer, setting 2) at 21 C for 16
hours, in
20mL closed glass scintillation vials. The vials were then centrifuged at 4000
rpm for 5
minutes and independently sampled for analysis by gas chromatography (GC-FID),
using a cholestane internal standard. Results are presented in Table 1.
Table 7
Fixed Oil FM-P2-48: TP
Canola 16.39
Corn 18.06
Olive 22.97
Peanut 19.08
Sesame 16.69
Key: TP = Total Phytosterols
(campestanol + sitostanol
+ beta-sitosterol),
concentration in m /mL.
CA 02334449 2000-12-05
26
The solubility of FM-P2-48 in selected fixed oils ranges from 16.39-22.97
mg/mL
Octanol / Water Partition Coefficient
The octanol / water partition coefficient was assessed by dissolving 5mg of
test
compound in 5mL of 1-octanol (oil phase), adding 15mL of pH 5.0 phosphate
buffer
and equilibrating by vortexing (VWR Multi-Tube Vortexer, setting 2) at 21 C
for 30
seconds, followed by static storage at 21 C for 16 hours, in 20mL closed glass
scintillation vials. It was observed that the two phases were completely
transparent
and no filtration step was necessary prior to analysis. The vials were then
independently sampled for analysis from the aqueous and octanol phases.
Analysis
was by GC-Mass Spectrometry.
Data for FCP-3P2 was not absolute, due to some technical difficulties with the
analytical method, but again indicated that aqueous solubility was minimal and
that the
compound was essentially confined to the octanol phase. A previous experiment
to
assess the partition coefficient of this compound, using a related but
modified
experimental procedure and GC-FID detection also supported these observations.
Thus, it was not possible to determine formal partition coefficient values for
the test
compound, but it is clear that it is essentially lipophilic in nature.
Aaueous Solubilit
This was evaluated by adding 15mg of test compound to 15mL of pH 5.0 phosphate
buffer and equilibrating by vortexing (VWR Multi-Tube Vortexer, setting 2) at
21 C for
16 hours, in 20mL closed glass scintillation vials. Samples were withdrawn
from the
vials, filtered 0.2 microns and analysed by GC-FID, using a cholestane
internal
standard.
Results indicated that FCP-3P2 possesses negligible solubility in the aqueous
buffer
(below limit of quantitation of method, ie. less than 5ng/mL total
phytosterols).
The above data indicates that FCP-3P2 is substantially lipophilic in
character, having
negligible solubility in simple aqueous media. In this respect, the data is
consistent
with comparable testing on steroids, which bear some significant structural
similarities
to the sterols and stanols.
CA 02334449 2000-12-05
27
The formulation of an oil-based solution of the active represents a feasible
delivery
system. If the quantity of FCP-3P2 exceeds it's solubility in the oily
vehicle, a
combination solution / dispersion will result. In this event, the active
particle size
distribution may be reduced, if desired, by homogenisation, e.g. using a high-
shear
device such as the Microfluidics Microfluidizer Model M-110Y or large-scale
equivalent.
Example 9: Emulsions (Macroemulsions)
A 10% w/v solution of FCP-3P2 was prepared by adding 5.062g of material to
45.248g
of soybean oil and heating to 63 C, to give a clear solution. 10mL of this
solution was
taken and 0.748g of Span 60 [polyoxyethylene-(20)-sorbitan monostearate]
dissolved
in it. This constituted the oil phase. In this case, the surfactant has a
Hydrophile-
Lipophile Balance (HLB) value of 4.7 +/- 1Ø
Tween 40 [polyoxyethylene-(20)-sorbitan monopalmitateJ, 0.750g, was dissolved
in
15mL of water, to provide the aqueous phase. Tween 40 has an HLB value of 15.6
+/-
1Ø
Both oil and aqueous phases were individually heated to 70 C, combined and
vigorously mixed using a Polytron Model PCV II mixer, on the high speed
setting, for 1
minute. The product was left to cool to ambient temperature.
This gave an oil in water emulsion, with an oil (dispersed) phase of 40% in an
aqueous
continuous phase, containing a dual surfactant system having an overall HLB of
10.0
+/- 1.0 and an active loading of ca 4% w/v (in the oil phase).
Analytical assessment of the emulsion included visual examination for phase
separation over 5 days, optical microscopic evaluation of oil droplet size, pH
measurement and FCP-3P2 content uniformity determination.
Phase Separation Assessment
15mL of emulsion was poured into a graduated centrifuge tube, which was
subsequently sealed. Daily visual inspection over 5 days indicated no phase
separation. Continued observation up to 25 days showed no separation of the
two
phases.
CA 02334449 2000-12-05
28
pH
The measured pH of the system was 6.63.
Oil Phase Droplet Size
This parameter was evaluated using an optical microscope equipped with a
calibrated
eyepiece. Sample preparation involved diluting 1 part of emulsion with 2 parts
of water
and examining a 15uL quantity on a microscope slide, under a cover slip, at
400x
magnification. Phase contrast and cross-polarization conditions were utilised.
The
dispersed oil phase consisted of droplets ranging from ca 2.5-20 microns and
no
evidence of FCP-3P2 crystallisation was observed.
FCP-3P2 Content Uniformity Determination
This was assessed on 6 samples, removed from the bulk according to a pre-
determined sample plan. Each sample (0.5mL) was extracted by vortexing for 10
minutes with dichloromethane (DCM, 5mL), followed by centrifugation at 4000
rpm for
2 minutes to separate the two phases. The analytical sample was withdrawn from
the
DCM layer and assayed by GC-FID, using a cholestane internal standard. Results
are
reported in Table 2.
Table 2
Sam le # TP
1 35.78
2 32.60
3 31.24
4 36.07
36.64
6 35.20
Mean 34.59 (93.2% of theoretical
Standard Deviation value)
Theoretical Content (of test 2.16
sample) 37.11
Key: TP = Total Phytostanols
(campestanol + sitostanol),
concentration in
m /mL.
Content uniformity is acceptable (34.59 +/- 2.16 mg/mL) and indicates
satisfactory
emulsion homogeneity. Recovery (93.2%) is a little low and this is probably
due to
pipetting errors.
CA 02334449 2000-12-05
29
Thus, this dosage delivery system has successfully enhanced both FCP-3P2
solubility
and dispersibility.
Example 10: Self-Emulsifyin4 Systems and Microemulsions
A self-emulsifying drug delivery system (SEDDS) is one that readily undergoes
emulsification in aqueous media under low or modest shear (agitation)
conditions.
Elevated temperature is not necessarily required. This ideally translates to a
spontaneous in vivo emulsification and subsequent dispersion following oral
administration. Furthermore, it is desirable for the SEDDS to form a
microemulsion on
exposure to aqueous media, thereby affording an additional enhancement of
dispersibility and an increased surface area for absorption (smaller droplet
size
distribution than a macroemulsion).
A SEDDS may be utilised in a variety of ways. For example, it is suited to
filling into a
soft gelatin capsule (softgel), or other suitable dosage form, for oral
administration, or
it may be further processed into a microemulsion prior to administration.
One example of a SEDDS and it's subsequent compounding into a microemulsion is
noted below.
Capmul MCM (a proprietary blend of medium chain glycerides), 5.30g, and Tween
80
[polyoxyethylene-(20)-sorbitan monooleate], 4.70g, were blended at ambient
temperature, to form a transparent, completely-miscible, solution. FCP-3P2,
0.10g,
was added and dissolved by sonicating for 10 minutes. A completely-transparent
solution resulted. In this SEDDS formulation, Capmul MCM constitutes the oil
phase
and Tween 80 is a high-HLB (15.0 +/- 1.0) surfactant emulsifier.
Addition of 90mL of 0.9% w/v aqueous NaCI (ambient temperature, 5mL aliquots,
gentle stirring-magnetic stir bar) affords a slightly opalescent microemulsion
system.
Analytical assessment techniques were as per Macroemulsions.
Phase Separation Assessment
Daily visual assessment over 5 days indicated no phase separation.
pH
30
The measured pH of the system was 5.72.
CA 02334449 2000-12-05
31
Oil Phase Droalet Size
Dispersed phase droplets were below the level of visual detection (contrast
with
observation under Macroemulsions) and no evidence of FCP-3P2 crystallisation
was
noted.
FCP-3P2 Content Uniformity Determination
Results are reported in Table 3.
Table 3
Sample # TP
1 0.303
_ 0.372
2
3 0.417
4 0.400
0.404
6 0.415
Mean 0.385 (84.1 % of theoretical
Standard Deviation value)
Theoretical Content (of test 0.043
sample) 0.458
Ke : TP = Total Ph ostanols
cam estanol+ sitostanol ,
concentration in m /mL.
Content uniformity is acceptable (0.385 +/- 0.043 mg/mL) and indicates
satisfactory
emulsion homogeneity. The mean recovery (84.1 %) is a little low and is
probably due
to a combination of pipetting errors and the low concentration of active in
the
formulation.
Both of these delivery systems demonstrated improvements in FCP-3P2 solubility
and
dispersibility.
Example 11: Solid Dispersions
A cosolvent mixture, consisting of 125mL chloroform and 125mL ethanol, was
prepared in a 500mL round-bottom flaskØ49g FCP-3P2 and 2.007g Benecel (a
grade
of hydroxypropylmethylcellulose) were added and the mixture was stirred at
ambient
temperature until a clear solution resulted. Solvent was removed by rotary
evaporation
under vacuum at 40 C and the resultant film was vacuum dried for a period at
ambient
CA 02334449 2000-12-05
32
temperature, following which the temperature was increased to 45 C and drying
continued to achieve a total residual solvent level of less than 200 ppm (GC-
headspace analysis). The dried film was cooled to ambient temperature and
carefully
scraped from the flask wall. This yielded a flaky powder, consisting of a
hydrophilic
cellulose matrix in which fine particles of FCP-3P2 were embedded.
Analytical assessment of product included: optical microscopic evaluation of
FCP-3P2
particle size, differential scanning calorimetry (DSC), aqueous dispersibility
testing,
measurement of FCP-3P2 levels and X-ray diffraction (XRD) evaluation.
FCP-3P2 Particle Size
FCP-3P2 did not form a complete molecular dispersion in the Benecel matrix,
following evaporation of the solvent vehicle. The complex film was visibly
opaque and
optical microscopic examination revealed rounded particulates, ranging from 20-
200
microns in size, within the matrix.
Untreated FCP-3P2 generally exhibits a rod-like crystal habit and a
significantly larger
overall mean particle size and distribution. Modification of the
crystallisation process
(crystal habit and size) by polymeric and surfactant materials has been
documented in
the literature, so this observation is not unexpected.
Since the hydrophobic (lipophilic) FCP-3P2 in this preparation was of a
markedly
smaller overall particle size than the original material and is embedded in a
hydrophilic
water-soluble matrix, some improvement in aqueous dispersibility might
reasonable be
anticipated.
DSC Evaluation
DSC is widely employed to assess specific thermal properties of single
materials and
formulated systems. Examples would include determination of melting point and
melting behaviour, identification of polymorphic forms, differentiation
between
amorphous and crystalline forms of a material and, in this case, evaluation of
potential
solid dispersion formation. Whilst characteristic melting endotherms for the
individual
components of a solid dispersion should be readily identifiable, conversion to
a true
solid dispersion would be expected to cause significant changes in their DSC
thermograms. In the case of an active material, substantial modification or
complete
CA 02334449 2000-12-05
33
elimination of the specific melting endotherm(s) for that substance are
commonly
observed.
DSC scans were run on FCP-3P2, Benecel and the FCP-3P2/Benecel formulation,
using a Dupont Model 910S Differential Scanning Calorimeter, calibrated
against an
indium standard, with helium gas purging. A scan rate of 10 C/minute, over a
temperature range of 20-200 C, was utilised. Sample sizes varied from 3.98-
4.60mg
and powders were run in crimped aluminium cups.
FCP-3P2 typically shows one major melting endotherm, with a peak value of
approximately 143.3 C. Benecel exhibited no significant endo- or exo-therms
over the
test temperature range. The FCP-3P2/Benecel formulation showed a single
endotherm at 142.6 C, corresponding to free FCP-3P2 and the area under the
endotherm curve equated to the loading level of the FCP-3P2 in the matrix.
Thus, we
may say that a true molecular dispersion has not formed between these two
substances.
Agueous Disaersibility Testing
A model system was established in an attempt to ascertain whether specific
formulation approaches could yield potential improvements in the
dispersibility of FCP-
3P2 in the gastric environment. For this purpose, a USP dissolution apparatus,
equipped with paddles (Apparatus II) and domed vessels was employed. A paddle
speed of 50 rpm and a dispersal medium comprising 300mL of 0.1 N aqueous HCI
at
37 C, were chosen as being reasonable test conditions. Stirring rate and
medium
volume were selected by experimentation to give efficient mixing without
turbulence. A
60 minute overall assessment period was set.
Untreated FCP-3P2 is hydrophobic in nature. Material (50mg) added to the
surface of
the stirred test medium, did not wet and persisted as floating particles for
up to 60
minutes, when the test was terminated.
The FCP-3P2/Benecel formulation sample was prepared by gently grinding in a
mortar
and passage through a 25 mesh sieve. Sieved material (100mg) was taken and
added
to the surface of the stirred test medium. Over the course of the 60 minute
test period,
material was observed to hydrate and commence dispersal into the medium.
Whilst
the dispersal process was not completed within this time, the test medium
became
CA 02334449 2000-12-05
34
noticeably opalescent. Optical microscopic examination of samples withdrawn
from
the bulk medium confirmed the presence of small particulates, as noted under
4.1.
Based upon this data, it would appear that the aqueous dispersibility of FCP-
3P2 has
been enhanced by this formulation approach. Wettability may be improved upon
in
future experiments, using a number of potential means. For example, addition
of a
suitable surfactant to the formulation, pre-suspension of the formulation in a
quantity of
an appropriate water-miscible liquid vehicle, substitution of spray drying for
rotary
evaporation as a process for isolating a more uniform dried product, etc.
FCP-3P2 Content Uniformity Determination
FCP-3P2 was assayed by GC-FID, using a cholestane internal standard.
The theoretical recovery of FCP-3P2 from the formulation should be 19.6% w/w.
A random sample of powder yielded an assay result of 18.8% w/w (95.9% of
theory).
This is an acceptable value, when allowances for sample processing and
analytical
variances are taken into account.
XRD Evaluation
The x-ray diffraction pattern of a substance can be used to evaluate it's
internal
structure and gives useful information as to whether a material is amorphous
or
crystalline in nature. The technique can also be used to demonstrate the
influence of
added substances on the pre-existing internal molecular arrangement of a
particular
material. As such, it constitutes a complimentary procedure to DSC
investigations and
was employed for this purpose in the current experimental work.
Material scans were conducted using a Rigaku Model D/MAX-2MB high resolution
wide angle x-ray diffractometer. A sample size of approximately 2mL was
required and
material was scanned over a 2theta range of 5.0 to 40.0 degrees.
Scans were run on the individual components of the formulation, a physical
mixture of
the two components and the test solid dispersion.
FCP-3P2 showed a characteristic pattern that indicated a reasonable degree of
crystallinity. Benecel also demonstrated a characteristic pattern, with little
evidence of
crystallinity. The physical mixture produced a composite pattern, containing
elements
of the individual components. FCP-3P2/Benecel solid dispersion showed a
pattern
CA 02334449 2000-12-05
which was quite similar to that of the physical mixture, but in addition
appeared to
indicate a reduced degree of FCP-3P2 crystallinity.
Thus, we may reasonably propose that the FCP-3P2 particulates in the Benecel
matrix
are present in a mixture of the amorphous and crystalline states, but that a
true
molecular dispersion has not formed. This observation is in agreement with the
DSC
results.
Application of this approach, or a modification thereof, should promote FCP-
3P2
dispersibility in aqueous media.
Example 12: Susaensions
An example of an aqueous suspension is noted below.
Carbopol 971 P (a proprietary grade of Carbomer 941 USNF), 0.51 g, was added
to
water, 75mL, and mixed to create a smooth lump-free suspension.
Water, 10mL, glycerol, 5mL, and Tween 80 [polyoxyethylene-(20)- sorbitan
monooleate], 0.014g, were blended together. FCP-3P2, 10g, was dispersed in
this by
vigorous mixing with a Polytron Model PCV II mixer (medium setting), to form a
smooth, fully-wetted, dispersion concentrate.
The Carbopol and FCP-3P2 dispersions were mixed together and disodium edetate,
0.10g, was dissolved in the blend. 18% w/v aqueous KOH was carefully added to
the
dispersion, with mixing, until the pH stabilised at a value of 6Ø This step
is required to
partially neutralise the acidic Carbopol groups and establish a gel structure
within the
dispersion. Finally, the product was made to volume (100mL) with water and
thoroughly mixed, to give a smooth suspension containing 100mg/mL FCP-3P2.
Analytical evaluation included: visual assessment of sedimentation, optical
microscopic appearance, pH check and FCP-3P2 content uniformity determination.
Test methods were as per Emulsions, unless otherwise stated.
Visual Assessment of Sedimentation.
Method as per Emulsions, Phase Separation Assessment.
Daily visual inspection over 5 days indicated that the suspension separated
into two
layers, from day 1 onwards. It readily re-suspended on gentle manual shaking
CA 02334449 2000-12-05
36
following the day 5 observation point, i.e. it is a flocculated system. Such
systems tend
to maintain
CA 02334449 2000-12-05
37
their re-suspendability characteristics more readily than formulations in
which the
dispersed phase is de-flocculated and ultimately tends to agglomeration and
caking.
Optical Microscopic Appearance
This characteristic was evaluated using an optical microscope equipped with a
calibrated eyepiece. The suspension was examined in the undiluted form, by
placing a
suitable quantity on a microscope slide and fitting a cover slip, at 400x
magnification
and ranged from approximately 2.5 to 25 microns in particle size (major axis).
pH
The measured pH of the system was 6.0 (initial) and 5.9 at the 5 day test
point of the
sedimentation test.
FCP-3P2 Content Uniformi~ Determination
Results are presented in Table 4.
Table 4
Sam le # TP
1 0.730
2 0.792
3 0.890
4 0.790
0.750
6 0.761
Mean 0.785 (85.8% of theoretical
Standard Deviation value)
Theoretical Content (of test 0.056
sample) 0.9153
Ke : TP = Total Ph ostanols
cam estanol+ sitostanol ,
concentration in m /mL.
Content uniformity is acceptable (0.785 +/- 0.056 mg/mL) and indicates
satisfactory
homogeneity of the dispersed active. The mean recovery (85.8%) is a little low
and
this may be due to pipetting errors and/or recovery problems.
Thus, this approach has enhanced FCP-3P2 dispersibility in aqueous systems.
CA 02334449 2000-12-05
38
Example 13: Hydrated Lipid Systems
A liposomal dispersion was evaluated as an example of a hydrated lipid system.
A solution was prepared containing the following materials: phospholipids-
dimyristoylphosphatidylcholine, 2.8328, dimyristoylphosphatidylglycerol,
1.3928; FCP-
3P2, 0.7828; dichloromethane, to 50mL. Sonication of the mixture for 30
minutes at 40
C yielded a slightly opalescent solution, which was subsequently clarified by
passage
through a 0.5 micron filter.
The solution was transferred to a 125mL round bottom flask and solvent removed
by
rotary evaporation under vacuum at 30-40 C, 35 rpm flask speed, to give a
coherent
thin film covering a substantial portion of the interior surface of the flask.
Continued
ambient temperature vacuum drying of the resultant film yielded a product with
a
residual solvent level of less than 200ppm dichloromethane (GC-headspace
analysis).
50mL of a 5% w/v aqueous glucose solution was added to the flask and the thin
film
was hydrated by rotating for 1 hour at 40 C and 60 rpm flask speed (no
vacuum). A
temperature of 40 C is well above the critical temperature of the
phospholipids (Tc =
23 C) and ensures that the membranes of the liposomal vesicles remain in a
suitably
fluid state to facilitate further processing. Hydration was completed by
gentle stirring
(magnetic stir bar) for 1 hour, at a temperature above the Tc value. This
created a
dispersion of large multilamellar vesicles (LMVs). The particle size and
lamellarity of
the LMVs was reduced by passing 250 uL quantities of dispersion (at 40 C)
through an
Avanti Mini Extruder, fitted with a 0.08 micron filter, for a total of 11
passes, to give a
dispersion of small unilamellar vesicles (SUVs).
Analytical assessment of the formulation included: liposomal particle size
evaluation,
pH measurement and FCP-3P2 content uniformity determination.
Testing methodologies were as per Emulsions, unless otherwise stated.
Liposomal Particle Size Assessment
The SUV dispersion was examined under 400x magnification and in the phase
contrast mode. A uniform dispersion of discrete liposomes having diameters of
less
than 1 micron was observed.
CA 02334449 2000-12-05
39
The measured pH of the system was 6.45.
FCP-3P2 Content Uniformity Determination
Initial attempts at performing FCP-3P2 content uniformity testing of the
liposomal
dispersion did not meet with success. Solubilisation of the liposomes with
surfactant
agents (0.5% Tween 80 and 0.5% sodium lauryl sulfate, used individually)
appeared to
lyse the liposomes, but the existing analytical procedure failed to extract
FCP-3P2
from the solubilised solutions. This may indicate that the active is tightly
associated
with one or more of the phospholipids and further studies will be required to
investigate
the matter.
This formulation approach has enhanced both the solubility and dispersibility
of the
active in aqueous media.
Example 14: Cyclodextrin Complexation
This formulation approach was investigated by application of the paste
formation
method.
2-Hydroxypropyl-beta-cyclodextrin (2-HPBC), 7.30g, and FCP-3P2, 0.888, were
thoroughly blended together in a glass vessel. 2 mL of a 30% v/v aqueous
ethanol
solution was added slowly, with mixing, to form a smooth thick paste and the
vessel
was loosely closed. The product was placed at 115 C for 2 days, to facilitate
inclusion
complex formation and subsequent evaporation of residual solvents. This
yielded a dry
friable mass, which was removed to ambient temperature and the vessel tightly
sealed.
Analytical evaluation of the product included: DSC assessment, XRD evaluation,
aqueous dispersibility testing and FCP-3P2 content uniformity determination.
Testing
methods were as per Solid Dispersions, unless otherwise stated.
DSC Evaluation
DSC scans were run on the individual components and the test formulation.
Sample
sizes varied from 3.74-4.67mg. The test temperature range was 20-350 C.
CA 02334449 2000-12-05
2-HPBC showed no significant thermal events over the temperature range 20-300
C,
but a modest progressive rise in the thermogram was noted, followed by a sharp
melting from approximately 305 C onwards. The FCP-3P2/2-HPBC formulation
showed one broad exotherm with a peak at 214.6 C and no melting endotherm for
the active (a peak at 143.3 C is typical for pure FCP-3P2). A complex melting
endotherm was noted at approximately 245 C, probably due to a lowered melting
point for the 2-HPBC.
Thus, it is clear that an inclusion complex has formed between the two
substances.
XRD Evaluation
Scans were run on the individual components of the formulation and the
complex. 2-
HPBC showed no evidence of crystallinity and appeared to be amorphous in
nature.
The characteristic FCP-3P2 peaks were absent in the scan of the complex,
which,
again, seemed to be amorphous in structure.
This data supports the DSC observations and indicates the formation of an
inclusion
complex between FCP-3P2 and 2-HPBC.
Aaueous Dispersibilit Tj~ estin
The FCP-3P2/2-HPB sample was prepared by gentle grinding in a mortar and
easily
reduced to a fine powder.
At 5 minutes, most of the material had wetted and entered solution as a fine
suspension. By 60 minutes, all material was uniformly suspended in solution,
with only
a trace of solids remaining at the surface.
Complexation with 2-HPBC has facilitated dispersibility of FCP-3P2 in aqueous
media.
7.4. FCP-3P2 Content Uniformity Determination
Results are reported in Table 5.
CA 02334449 2000-12-05
41
Table 5
Sam le # TP __
1 0.423 _
_ 0.343
2
3 0.403
4 0.371
0.391
6 0.451
Mean 0.397 (34.6% of theoretical
Standard Deviation value)
Theoretical Content (of test 0.038
sample) 1.146
Key: TP = Total Phytostanols
(campestanol + sitostanol),
concentration in
m /mL.
Reviewing Table 5 data, the mean recovery is very low, at 34.6% of
theoretical. Since
the sample preparation procedure involves extraction into DCM prior to
analysis, two
potential explanations suggest themselves. FCP-3P2 is adequately soluble in
DCM,
whilst 2-HPBC has a negligible solubility in this solvent. Under these
circumstances, it
is possible that the data recorded represents free FCP-3P2 that has not formed
an
inclusion complex with 2-HPBC. By inference, 64.4% of active should be present
as
the complex. An alternative possibility is that the extraction process has
only recovered
a percentage of FCP-3P2 from the inclusion complex. DCM is a lipophilic
solvent
which possesses a reasonable degree of polarity. Therefore, one would expect
it to
show some affinity for included FCP-3P2. The DSC and XRD data clearly indicate
inclusion complex formation between the two substances and the former shows no
melting endotherm for uncomplexed FCP-3P2. Therefore, it is felt that the
latter
explanation is the most likely to be correct. Considered in this light, the
content
uniformity data tends to suggest a homogeneous product with respect to FCP-3P2
distribution.
This formulation approach has enhanced the dispersibility of the active in
aqueous
media.
Example 15 Complexation with Bile Salts
Sodium deoxycholate (SDC) was chosen as a typical example of a human bile
salt.
CA 02334449 2000-12-05
42
A cosolvent mixture, consisting of 100mL ethanol and 50mL DCM, was added to a
500mL round-bottom flask. FCP-3P2, 1.027g, and SDC, 4.0088, were added and the
flask manually swirled, to give a clear solution at ambient temperature.
Solvent was
removed by rotary evaporation under vacuum at 40-50 C, to yield a white powder
mass. Further vacuum drying at ambient temperature reduced residual solvent to
workable levels (DCM less than 200 ppm, ethanol greater than 200 ppm- limit
testing
employed; GC-headspace analysis). The product was free of residual solvent
odour.
Analytical investigation of product included: DSC assessment, XRD evaluation,
aqueous dispersibility testing and FCP-3P2 content uniformity determination.
Testing
methods were as per Solid Dispersions, unless otherwise stated.
DSC Evaluation
DSC scans were run on the individual components and the test formulation.
Sample
sizes varied from 3.54-3.59mg.
SDC showed no significant thermal event over the test temperature range.
The thermogram for the test formulation showed two consecutive melting
endotherms,
peaking at 136.3 C and 141.8 C, separated by a small exothermic peak. FCP-3P2
typically has a melting endotherm peaking at 143.3 C. Thus, these thermal
events are
probably due to the active. One possible explanation is that FCP-3P2 may exist
in a
metastable state in the formulation, initially melting at 136.3 C,
recrystallising to the
stable form and then re-melting at 141.8 C. It was noted that the area under
the
141.8 C endotherm corresponded to the quantity of active contained in the
formulation. This indicates that FCP-3P2 has not formed a molecular dispersion
with SDC.
XRD Evaluation
Scans were run on the individual components and the test formulation.
SDC showed a pattern that is indicative of a non-crystalline material.
The test formulation scan was essentially a composite of the patterns for the
two
components and the degree of crystallinity of the FCP-3P2 has been little
affected by
it's combination with SDC. These observations support the DSC data.
CA 02334449 2000-12-05
43
Agueous Dispersibilit Tag
The test formulation presented as a fine powder and was used as received.
In this case, pH 5 phosphate buffer was used as the test medium, since the
bile acid is
present as it's sodium salt, which possesses a significantly greater aqueous
solubility than the parent acid. Contact with 0.1 N HCI, the medium initially
chosen for
dispersibility testing, would cause conversion to the acid form and
substantially
inhibit dispersion of the complex. This consideration necessitates the design
of a
dosage form which releases it's contents upon reaching the upper regions of
the
small intestine and is protected from exposure to the acidic gastric fluid,
i.e. an
enteric coated system. Since bile salts are known to cause gastric irritation
and
emesis upon oral administration, these would be further reasons to prevent
premature release in the stomach.
At 5 minutes, the test formulation had substantially wetted and appeared as a
fine
particulate dispersion in the test medium, with a small proportion of material
floating at
the surface. By 60 minutes, only a trace of material was present at the
surface, the
majority being present as a uniform particulate suspension.
This formulation has demonstrated an enhanced dispersibility in aqueous media
(pH
5.0 and above).
FCP-3P2 Content Uniformity Determination.
Results are reported in Table 6.
Table 6
Sam le # TP
__ -
1 _ 1.575
2 1.759
3 1.938
4 1.750
1.586
1.507
Mean 1.686 (91.2% of theoretical
Standard Deviation value)
Theoretical content (of test 0.159
sample) 1.848
Key: TP = Total Phytostanols
(campestanol + sitostanol),
concentration in
m /mL.
CA 02334449 2000-12-05
44
Content uniformity is acceptable(1.686 +/- 0.159 mg/mL) and indicates
satisfactory
FCP-3P2 homogeneity. The mean recovery (91.2%) is a little low and this is
probably
due to pipetting errors.
This formulation approach has improved FCP-3P2 dispersibility and wettability
in
aqueous media. Since the bile salts perform a solubilising function in vivo
(they are
surfactants), it is also possible that oral bioavailability will be enhanced.
Example 16: Hydrotropic Complexation
Sodium gentisate (SG) was selected as a model hydrotrope.
FCP-3P2, 2.066g, and SG, 4.242g, were added to 200mL of ethanol and the
mixture
was sonicated at 60 C to achieve complete dissolution. The resultant solution
was
transferred to a 250mL round bottom flask and solvent removed by vacuum rotary
drying at 60 C, to give a damp mass. Further vacuum drying at ambient
temperature
yielded a dry powder mass.
Analytical testing included: DCS assessment, XRD evaluation, aqueous
dispersibility
testing and FCP-3P2 content uniformity determination. Test methods were as per
Solid Dispersions, unless otherwise stated.
DSC Evaluation
DSC scans were run on the individual components and the test formulation.
The SG thermogram showed two melting endotherms, peaking at 89.7 C and 169.6
C.
The test formulation thermogram showed a similar pattern to that for the SDC
(Bile
Salt) complex, consisting of a minor melting endotherm peaking at
approximately
135 C, followed by a small exotherm, leading to a sharp endotherm peaking at
141.5
C (FCP-3P2 typically shows a sharp melting endotherm at 143.3 C). A similar
explanation is proposed for this behaviour (refer to section on Bile Salts
Complexation). The two melting endotherms for SG were eliminated, indicating a
potential modification of it's crystalline structure).
It would seem from this data that FCP-3P2 has not formed a molecular
dispersion in
SG.
CA 02334449 2000-12-05
XRD Evaluation
Determinations were made on the individual components and the test
formulation.
FCP-3P2 and SG both demonstrate well-defined peak patterns, suggesting a
significant crystalline component to their structures.
The test formulation showed little evidence of crystallinity, indicating that
some definite
physical interaction has occurred between the two substances. This observation
is
consistent with the DSC evaluation data.
Agueous Dispersibilit Tea
Again, pH 5.0 phosphate buffer was substituted for 0.1 N HCI, since the sodium
salt of
gentisic acid was present in the formulation.
Material initially floats on the surface of the test medium, with some
particulates
dispersing into the bulk liquid. A visible film forms at the surface,
indicating some
dissolution of the SG carrier. At 60 minutes, there was a slight increase in
the level of
dispersed particles, but the majority of the material remained at the surface
and the
SG film persisted.
This approach has not been successful in improving either the solubility or
dispersibility of FCP-3P2 in aqueous media.
FCP-3P2 Content Uniformity Determination
Results are reported in Table 7.
Table 7
Sam le # TP
.. _
__.
1 1.622
. _. _..
2 1.922
3 1.896
4 1.740
5 1.811
1.882
Mean 1.812 (121.1 % of theoretical
Standard Deviation value)
Theoretical Content (of test 0.114
sample) 1.496
Key: TP = Total Phytostanols
(campestanol + sitostanol),
concentration is in
m /mL.
CA 02334449 2000-12-05
46
The apparent mean recovery (121.1 %) is high and this will require further
investigation. Matrix interference effects are one possible explanation.
Content uniformity is acceptable (1.812 +/- 0.114mg/mL) when taken on the
basis of
relative comparison and indicates satisfactory homogeneity of the dispersed
active.
This formulation approach has not significantly enhanced the solubility or
dispersibility
of FCP-3P2 in aqueous media (pH 5.0 and above).
Example 17: Yogurt
Phytrol~ which consists of campesterol, campestanol, ~-sitosterol and
sitostanol was
mixed with nonfat milk powder in the ratio of 1:7 to 1:8. About 6 L of milk
mix was
prepared from whole milk, skimmed milk and phytrol containing milk powder.
Milk was
standardized to 0.75 - 1 % fat, 12 - 13% solids and 0.5-1 % phytrol using the
Pearsons
Square method (Hyde, K.A. and Rothwell, J., 1973, In Ice Cream, Churchill
Livingstone Ltd., London, U.K.). Milk mix was permitted to remain at room
temperature
for 30 minutes to re-hydrate powder milk and than it was homogenized using a
high
sheer batch mixer (Ultra-Turrax T50 equipped with the dispersing element S50N,
IKA
Works Inc., Wilmington, NC, USA). Other devices such as a single-stage
homogeniser, a two-stage homogeniser or a high-pressure microfluidizer may
alternatively be used for homogenization of the milk mix. Next, milk mix was
pasteurized at 69oC (156oF) for 30 minutes (batch/vat), cooled to 44oC and
hold at
this temperature for up to 15 minutes.
About 3% by weight of active yogurt culture containing Lactobacillus
bulgaricus and
Streptococcus thermophilus in the ratio 1:1 were carefully introduced into
warm milk
mix. After gentle mixing, the inoculated milk was distributed into 125 g-
containers
filling to near top. The containers were thermally sealed with aluminum leads
and
placed in incubator (44°C) equipped with good uniform air circulator
and temperature
controller. Filled containers were permitted to remain at 44°C for 3-5
hours, until a
firm, smooth gel was formed. During incubation, pH was monitored periodically.
When pH reached about 4.5, yogurt was withdrawn from the incubator, chilled
quickly and stored at 4°C.
CA 02334449 2000-12-05
47
Example 18: Bread
Breads containing 0.6% and 1.2% of Phytrol were prepared using bread maker
(Black
& Decker, Model # B2005). Phytrol~ consisted of campesterol campestanol, ~i-
sitosterol and sitostanol was mixed with multipurpose flour (1 % and 2%, w/w)
using
Hobart mixer (Model N50). Alternatively, Phytrol was mixed with milk using a
high-
pressure microfluidizer. Subsequently, all other ingredients were mixed in
proportions
indicated below.
In redients 0.6% Phytrol 1.2% Ph rol
~
Milk 334.0_ 0 334.00
Salt 7.50 7.50
Su ar 7.10 7.10
Crisco 12.00 12.00
Flour 535.00 535.00
Ph rol 5.42 10.84
Yeast 2.80 2.80
Ingredients were combined in the baking pan of bread maker. Preparation of
dough
and baking was conducting according the manufacturing instructions.
Example 19: Cereal Bar
Cereal bars of total weight 20g, and 40g that contained 3%, and 1.5% of
Phytrol,
respectively, were prepared. Phytrol~ consisted of campesterol, campestanol, ~-
sitosterol and sitostanol was dissolved in partially hydrogenated vegetable
oil in
elevated temperature (40-80oC). The oil/Phytrol blend was cooled to 30oC and
emulsified using a high sheer batch mixer (Ultra-Turrax T50 equipped with the
dispersing element S50N, IKA Works Inc., Wilmington, NC, USA). Subsequently,
two
oil blends (9.4% and 18.8% of Phytrol) were further emulsified using a high-
pressure
microfluidizer at 20,000 PSI.
CA 02334449 2000-12-05
48
Cereal bars were produced by combining binder (40%), water (5%) and edible
particles (55%). Below two typical examples of binder used for making a cereal
bar.
Sucrose containing binder
Phytrol (9.4% or 18.8%) containing oil 40%
Sucrose 22%
Water 28%
Sodium Caseinate 5%
Lecithin 2%
Glycerin 3%
Glucose containing binder
Phytrol (9.4% or 18.8%) containing oil 40%
Glucose syrup 50%
Sodium Caseinate 5%
Lecithin 2%
Glycerin 3%
Sucrose in water /glucose syrup was heated to 100oC while Phytrol containing
fat was
liquefied at 40-80oC. Hot sugar solution was placed in the bowl (Hobart mixer,
Model
N50) and fat was added followed by adding all remaining binder ingredients.
All
ingredients were thoroughly and vigorously mixed. After cooling down to 40oC,
edible
particles are added while thorough, non-vigorous mixing was carried out.
Following
edible particles were typically incorporated into the cereal bars.
Edible particles
Rolled oats 20-40%
Crisped rice 10-20%
Puffed barley 10-20%
Dried apple 10-20%
dices
Shredded coconut5-10%
Raisins 5-10%
Various nuts 5-10%
After mixing was completed, mixed material was placed in the forming mold and
pressed with a roller. After removal from the mold, it was cut into ready to
eat various
sizes cereal bars.
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49
Example 20: Spread
Light margarine (60% fat) containing 6% of Phytrol was produced in batches of
5-
10kg. Phytrol~ consisted of campesterol, campestanol, ~-sitosterol and
sitostanol was
combined with other fat ingredients and heated to 80-85oC until Phytrol was
fully
dissolved. Clear fat solution was placed in the feeding tank (20L), cooled to
40-45 oC
and stirred using (Ultra-Turrax T50 equipped with the dispersing element S50N,
IKA
Works Inc., Wilmington, NC, USA). Next, the water fraction (40%) was added and
temperature was adjusted to 60 oC. The blend was submitted into a votator and
processed at 8-10oC. The composition of margarine is describe above.
In redient Wt%
Water Phase
_
__ 39.0
Water
Salt 1.0
Potassium sorbate 0.001
Oil Phase
So bean oil 38.025
Palm kernel oil 15.0
Ph rol 6.0
Monoldi I cerides 0.6
Lecithin 0.15
Flavor 0.075
Beta-carotene 0.15
Example 21: Chocolate
Milk chocolate containing 6% of Phytrol was produced in batches of 20-50kg.
Phytrol~
consisted of campesterol, campestanol, ~-sitosterol and sitostanol was mixed
with
soybean oil using a high sheer batch mixer (Ultra-Turrax T50 equipped with the
dispersing element S50N, IKA Works Inc., Wilmington, NC, USA). The blend (20%
Phytrol) was subsequently emulsified using a high-pressure microfluidizer at
20,000
PSI. Chocolate was composed of an outer shell (42 wt%, no Phytrol) and a
center
(69%, Phytrol). Chocolate outer shell was made by mixing sugar (45%), whole
milk
powder (20%), cocoa butter (23%), cocoa mass (12%), soy lecithin (0.3%) and
pure
vanilla (0.1 %) in a heating tank. All ingredients were melted, tempered and
deposited
into molds. Center was prepare my mixing sugar, cocoa butter, whole milk
powder,
cocoa mass, soy lecithin and pure vanilla in the proportions as for outer
shell. The mix
CA 02334449 2000-12-05
was melted and tempered. Consequently, Phytrol/soybean oil blend was mixed
with
chocolate in the 1:1 ratio and deposited into molds previously filled with
chocolate
without Phytrol. Chocolate pieces were than cooled, wrapped and packed into
the
boxes. Using the molding system, 10-12 g chocolate pieces were produced.
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51
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