Note: Descriptions are shown in the official language in which they were submitted.
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_1-
Title: Oil Bodies as Topical Delivery Vehicles for Active Agents
FIE1~D OF THE INVENTION
The present invention relates to novel compositions and methods for the
delivery of active agent to animals including humans. The compositions
comprise oil bodies
5 and an active agent. The compositions are particularly useful for the
topical delivery of
the active agents.
BACKGROUND OF THE INVENTION
Active ingredients such as pharmaceutical actives can be administered to the
body via a number of routes including ingestion, injection, and topical
application.
10 Regardless of the route of delivery, the active ingredient must overcome
certain barriers
such as biological membranes, for example the stratum corneum in the skin,
before the agent
can exert the desired biological effect at ifs target site in the body.
Various techniques
have been investigated for the potential to enhance transport and delivery of
active agents
to their target site and numerous systems that facilitate the delivery of
active agents are
15 known to the prior art.
Among the most extensively evaluated delivery systems for actives are those
systems permitting delivexy of active agents into the skin. Active ingredients
exerting a
biological effect on the skin include actives capable of mitigating wrinkles,
reducing
hyperpigmentation, treating UV damaged skin cells and the like. Approaches to
20 facilitate topical delivery of these active agents include the use of
physical forces such as
ultrasound and electricity as well as the use of chemical delivery systems to
enhance
penetration of the active ingredient into the skin (see: Chen, L.-H. and
Chien, Y.W.
Enhancement of skin penetration. In: Novel Cosmetic Delivery Systems. Marcel
Dekker,
Inc., New York, pp 51-69.).
25 Chemical delivery vehicles can be divided into three broad types of
delivery
vehicles: vesicular, porous polymeric and particulate delivery vehicles.
Vesicular
delivery vehicles include, liposomes, niosomes, and transfersomes. Liposomes
are lipid
vesicles typically from about 30 to 100 nm in diameter, composed of one or
more Iipid
bilayers surrounding an aqueous interior. The traditional liposome is
constructed using
30 phopholipids like phosphatidyl choline and have more recently been
constructed using
single-chain amphiphiles or nonionic surfactants (niosomes). In general,
liposomes are
manufactured in a four-step process. The first step involves the mixing of
amphiphilic
molecules in organic solvents, the stirring of the mixture, the separation of
the solvent by
the addition of water to ~ cause the detachment of phospholipid bilayers and
the
35 homogenization to mechanically form particles of a specified size and
homogeneity.
Transfersomes are special lipid aggregates formed by the mixture of
amphiphilic
substances and active ingredients and subjecting them to filtration,
ultrasonication, stirring,
agitating or any other mechanical fragmentation (EP 0 475 160 B1).
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Porous particulate delivery vehicles include web-like systems and porous-
sphere systems. In the web-like system, an "empty" polymer can have an active
ingredient
loaded onto it or a nanoparticle can be preloaded with an active ingredient.
In the porous-
sphere system a porous membrane surrounds a solid nanoparticle. These systems
are formed
5 using cross-linked polymers, for example substituted acrylates (Nacht S.
1995. Cosmetics &
Toiletries 1I0: 25-30).
The final type of delivery vehicle is the particulate delivery system.
Examples of particulate delivery vehicles include microcapsules, beads and
microspheres.
Microcapsules are analogous to the shell of an egg. They have multilayer
construction with
10 multiple cores containing the active. The classic microcapsule is
constructed using gelatin,
cellulose-type polymers or synthetic polymexs. Beads and microspheres are
small solid
partices (fox example nylon). The active ingredient is adsorbed onto the
particle for later
delivery (Rornanowski P and Schueller R. 1999. Stability Testing of Cosmetic
Products. In:
Novel Cosmetic Delivery Systems. Marcel Dekker, Inc., New York, pp li5-130).
15 However despite considerable efforts to develop delivery systems for active
ingredients, the available delivery vehicles are frequently inefficient. There
are two
important reasons for the limited effectiveness of the currently available
delivery
technology. First, the delivery systems known to the prior art display a lack
of
permeability through the biological barriers. Second, the applied active
ingredient even
ZO if it is capable of permeating the biological barrier, does not migrate to
the active site.
Thus the effectiveness of delivery is compromised due to distribution of the
active
ingredient to non-target tissues or cells. Additionally this may result in
undesirable side-
effects, for example cosmetic actives may cause irritating reactions.
Furthermore, the
active ingredient may be metabolized prior to reaching the target. In order to
compensate
25 for the dilution of the active agent, higher doses than otherwise are
desirable must be
administered.
In the seeds of oilseed plants, which include economically important crops
such
as rapeseed and sunflower, the oil fraction is stored in discrete subcellular
structures
variously known to the art as oil bodies, oleosomes, lipid bodies or
spherosomes (Huang,
30 1992, Ann. Rev. Plant Mol. Biol. 43: 177-200). Besides a mixture of oils,
also referred to in
the chemical art as triacylglycerides, oil bodies comprise phospholipids and a
number of
associated proteins collectively termed oil body proteins. From a structural
point of view,
oil bodies are substantially spherical structures which encompass a matrix
comprising a
mixture of lipids encapsulated by a phospholipid monolayer and oil body
proteins. The
35 predominant protein present in the oil body is known as oleosin {Huang,
1992, Ann. Rev.
Plant Mol. Biol. 43: I77-200). Intact oil bodies have been isolated previously
from a
variety of oil seed crops, see for example: Jacks et al., JAOCS, 67: 353-361
and Huang, 1992,
Ann. Rev. Plant Mol. Biol. 43: 177-200. In general, the objective of this
experimental work
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has been to elucidate the in vivo structure and/or function of oil bodies in
the seed. In order
to obtain plant oils for industrially relevant applications, for example for
use in cosmetics
or detergents, seeds are generally crushed, pressed and subsequently refined.
Since the
objective of these processes is to obtain pure plant oil, the oil bodies in
the course of the
production process lose their structural integrity.
United States patents 5,683,740 and 5,613,583 disclose emulsions comprising
lipid vesicles that have been prepared from crushed oleagenous plant seeds. In
the course
of the crushing process, oil bodies substantially lose their structural
integrity. Accordingly,
these patents disclose that in the crushing process, 70% to 90% of the seed
oil is released in
10 the form of free oil. Thus the emulsions which are the subject matter of
these patents are
prepared from crushed seeds from which a substantial amount of free oil has
been released
while the structural integrity of the oil bodies is substantially lost. In
addition, the
emulsions disclosed in both of these patents are prepared from relatively
crude seed
extracts and comprise numerous endogenous seed components including
glycosylated and non-
15 glycosylated non-oil body seed proteins. It is a disadvantage of the
emulsions to which
these patents relate that they comprise contaminating seed components
imparting a
variety of undesirable properties, which may include allergenicity and
undesirable odor,
flavor, color and organoleptic characteristics, to the emulsions. Due to the
presence of seed
contaminants, the emulsions disclosed in these patents have limited
applications.
20 There is need in the art to provide improved delivery vehicles and methods
by
which active agents can be effectively topically delivered to living
organisms.
SUMMARY OF THE INVENTION
The present invention relates to the use of oil bodies as improved topical
delivery vehicles for active agents. The present inventors have shown that by
delivering
25 an active agent in an oil body formulation the absorption ar penetration of
the active agent
is enhanced. Further, certain active agents that are generally irritable to
the skin have
reduced irritability when delivered in an oil body formulation as compared to
other
formulations such as safflower oil or liposomes or when administered alone.
The present invention provides a composition for the improved delivery to the
30 skin of an active agent to a living organism wherein the composition
comprises:
(1} an active agent; and
(2} oil bodies.
The present invention further provides a use of a composition comprising an
active agent and oil bodies to deliver an active agent to a living organism.
The present
35 invention also includes a use of oil bodies to prepare a medicament or
composition to deliver
an active agent to a living organism.
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The present invention further provides a method of preparing a composition for
the delivery of an active agent which comprises formulating active agent in
the presence of
oil bodies to prepare a composition.
The present invention provides a method for the delivery of an active agent to
a
living organism comprising administering an effective amount of a composition
comprising
the active agent and oil bodies to the skin of an organism in need thereof.
The active agents that may be used in accordance with the present invention
including without limitation cosmeceuticals, cosmetically active agents and
dermatological pharmaceuticals. The active agent is topically administered to
the living
organism.
In a preferred embodiment, the active agent is a cosmetically active agent
such
as hydroquinone, salicylic acid, and tretinoin, which are formulated in fine
presence of oil
bodies, applied topically and delivered percutaneously to the skin.
Preferably, the
absorption or penetration of the cosmetically active agent in the oil body
formulation is
15 enhanced and the irritability is reduced as compared to the penetration or
absorption and
irritability of the active agent in the absence of the oil bodies.
Accordingly, the
compositions of the invention may be used in improved topical formulations to
beautify the
skin or to treat skin conditions.
In another embodiment of the invention, the active agent is a protein which is
produced recombinantly on the surface of the oil body. The oil bodies are then
formulated
and administered to the living organism.
Additional advantages and features of the present invention will become
apparent after consideration of the accompanying drawings and the following
detailed
description of the invention.
BRIEF DESCRI~'TTON OF THE DRAWINGS
Figure 1 is a Coomassie blue stained gel of washed oil body preparations
obtained from white mustard, oilseed rape (Brassica napes), soybean, peanut,
squash, flax,
sunflower, safflower and maize.
Figure 2 is a Western Blot analysis of human skin fractions wherein the
stratum
30 corneum was removed prior to the application of oil bodies in a Franz
assay. Western
analysis of epidermal fractions (50 wg /lane) from 3 individuals (lanes 3
through 5) and
dermal fractions (45 ~g /lane) from the same 3 individuals (lanes 6 through 8)
are compared
to controls (50 wg epidermal extract spiked with 0.2 ~.g of canola ail body
protein (lane 1)
and 50 ~.g control intract skin (lane 2)). The anti-oleosin antibody used in
this experiment
was a polyclonally-derived antibody to an canola oil body.
Figure 3 is a Western Blot analysis of human skin fractions wherein the
stratum
corneum was removed after the application of oil bodies and the completion of
the Franz
assay. Western analysis of epidermal fractions (50 ~.g /lane) from 3
individuals (lanes 2
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through 4) and dermal fractions (50 ~,g /lane, 45 ~.g /lane, 40 ~,g /lane
respectively) from the
same 3 individuals (lanes 5 through 7) are compared to controls (50 ~g
epidermal extract
spiked with 0.2 ~g of canola oil body protein (lane 1) and 50 ~.g /lane
control intract skin
(lane 2)). The anti-oleosin antibody used in this experiment was a
polyclonally-derived
5 antibody to an canola oil body.
Figure 4 is a histogram representing the cumulative results of 7 strips in a
skin-
stripping assay for formulations containing salicyclic acid, hydroquinone and
tretinoin.
Compared are the oil body formulations containing the active ingredient or a
simple
emulsion placebo containing the active ingredient. The cumulative percentage
recovery of
10 active ingredient obtained from 7 strips is indicated.
DETAILED DESCRIPTION OF THE INVENTION
A. Compositions
As hereinbefore mentioned, the present invention provides improved
compositions for the enhanced delivery of active agents to living organisms.
The
15 compositions comprise an active agent in an oil body formulation.
The present inventors have unexpectedly discovered that oil bodies have many
features that render them especially suitable as versatile delivery vehicles
of active
compounds. Importantly, the inventors have shown that by delivering an active
agent in an
oil body formulation the absorption or penetration of the active agent to the
skin is
20 enhanced. Further, certain active agents that are generally irritable to
the skin have
shown to have reduced irritability when delivered in an oil body formulation
as compared
to other formulations such as safflower oil or liposomes. Therefore, the oil
bodies are
extremely mild when administered to the skin and beneficial in that they mask
the
irritating effect of known cosmetic active ingredients, such as salicylic acid
and
25 hydroquinone: In addition, the creamy texture of the oil body compositions
renders these
compositions especially suitable for the preparation of delivery vehicles that
are
dermatologically acceptable. Furthermore, a wide variety of active ingredients
can easily
be formulated using oil bodies and oil bodies can be prepared on a large scale
using known
seed milling equipment combined with a simple aqueous extraction process. Oil
bodies are
30 preferably obtained from plant sources which have GRAS (Generally Regarded
As Safe)
status. The oil body based formulations therefore can be safely used to
topically deliver
active agents.
Accordingly, in one aspect, the present invention provides a composition for
the
topical delivery of an active agent which comprises:
35 (1) an active agent; and
(2) oil bodies.
The term "active agent" or "agent" as used herein means any agent that one
would like to administer to a living organism. The term includes without
limitation any
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agents capable of mediating an improvement or benefit to the physical
appearance,
health, fitness or performance of the surface area of the body. In preferred
embodiments of
the invention the active agent is a cosmetically active agent, a
cosmeceutical, or a
dermatologically active pharmaceutical active agent. The active agent may be
obtained
from any suitable source or synthesized chemically.
The terms "cosmetically active agent" or "cosmeceutical" are used
interchangeably throughout this application and denote ingredients that have a
desirable
biological effect on external or superficial surfaces of a living organism,
preferably a
human, more preferably the skin, hair, teeth and nails of the human. The
cosmeceutical
10 may be used to beautify the skin or to treat skin conditions, diseases or
abnormalities. For
example, the cosmetically active agent may be vitamin A, C or E, alpha hydroxy
acids,
such as citric, glycolic, lactic and tartaric acid, exfoliating agents such as
salicylic acid,
bleaching agents such as hydroquinone, anti-aging compounds and sunscreens
such as octyl
methoxycinnamate {Parsol MCX}, 3-benzophenone (Uvinul M40) and butylmethoxydi-
15 benzoylmethane (Parsol 1789}, or a compound that is capable of exerting an
immunostimulatory effect such as a (3-glucan. Cosmetically active agents also
include
enzymes for example papain, bromelin, ficin, lipases and proteases all of
which have been
used in the preparation of cosmeceuticals.
The terms "dermatologically active agent" or "dermatological agent" are used
20 interchangeably throughout this application and refer to all compounds
regulated as drugs,
for example antibiotics, fungicides, antiviral agents, anti-inflammatory
agents used to
treat skin conditions or diseases.
Further examples of active agents that may be employed in accordance with
the present invention, include but are not limited to amino acids, anticancer
agents
25 (carboplatin}, antimetabolite methotrexate (MTX), azidothymidine, ceramide,
corticosteroids (for example halicinonide, tiramcinolone acetonide,
betamethasone
valerate, etc.), cyclosporin, dextran, disinfectants, dithranol, econazole,
estradiol,
fibronectin, glial-derived neurotrophic factor (GDNF}, glucocorticoids, hair
growth
stimulants, herbal drugs, lectins, local anesthetics, methotrexate,
minioxidil, moisturizing
30 agents, placenta hydrolysate, phospholipase C, pregnenolone, progesterone,
prostaglandin,
retinoids (vitamin A acid, isotretinoin), rubefacients, skin protecting agents
(gelating
hydrolysate, embryonic extract, aloe extract, vanillin), steroid hormones,
terconazole,
testosterone, tetracaine, thymus extract, tretinoin, triamcinolone acetonide
(TRMA),
tyrosine and its derivatives, trimcinolone acetonide, and urea.
35 The active agent may be linked to the oil bodies, either covalently or non-
covalently. In embodiments of the instant invention where the active agent is
a protein or
peptide, one particularly advantageous way in which the biologically active
ingredient
may be included in the oil body preparation is through construction of oleosin
gene fusions as
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detailed in WO 96/21029 which is incorporated herein by reference.
Accordingly, the
present invention provides a composition for the delivery of a polypeptide of
interest
wherein the polypeptide is linked to a sufficient portion of an oleosin to
provide targeting
of the polypeptide to an oil body. Isolation of the oil body fraction results
in recovery of
5 the active agent attached to the oil bodies. In principle any desired
protein or peptide may
be produced using this technology.
Examples of therapeutic proteins may be used in accordance with the present
invention include, but are not limited to enzymes such as papain, bromeiin,
ficin, lipases,
collagenase, elastase and proteases, and proteins such as thioredoxin,
collagen, elastin, or
10 active fragments or dexivatives of any of these proteins.
It is to be clearly understood that the particular active agent is not of
critical
importance and may be as desired. Accordingly it is to be clearly understood
that in
accordance with the present invention in principle the oil body preparation
may be applied
as a topical delivery vehicle for any active agent.
15 The term "living organism" refers to any living organism which includes all
members of animal kingdom. In preferred embodiments, the living organism is a
vertebrate,
more preferably, a human.
The term "oil bodies" as used herein means a substantially intact discrete
subcellular oil or wax storage organelle. The oil bodies may be obtained from
any cell
20 containing oil bodies or oiI body-like organelles. This includes animal
cells, plant cells,
fungal cells, yeast cells (Leber, R. et al., 1994, Yeast 10: 1421-1428),
bacterial cells (Pieper-
Piirst et al., 1994, J. Bacteriol. 176: 4328 - 4337) and algae cells
(Roessler, P.G., 1988, J.
Phycol. (London) 24: 394-400). In preferred embodiments of the invention the
oil bodies are
obtained from a plant cell, which includes cells from pollens, spores, seed
and vegetative
25 plant organs in which oil bodies or oil body-like organelles are present
(Huang, 1992, Ann.
Rev. Plant Physiol. 43: 177-200). More preferably, the oil body preparation of
the subject
invention is obtained from a plant seed and most preferably from the group of
plant species
comprising: rapeseed (Brassica spp.), soybean (Glycine max), sunflower
(Helianthus
annuus}, oil palm (Elaeis gacineeis), cottonseed (Gossypium spp.), groundnut
(Arachis
30 hypogaea), coconut {Cocas nucifera}, castor (Ricinus communis), safflower
(Carthamus
tinctorius), mustard (Brassica spp. and Sinapis alba), coriander (Coriandrum
sativum),
squash (Cucurbita maxima), linseed/flax (Linum usitatissimum), Brazil nut
(Berthotlefia
excetsa), jojoba {Simrnondsia chinensis), maize (Zea mays); crambe (Crambe
abyssinica)
and eruca (Eruca sativa). In order to obtain oil bodies, plants are grown and
allowed to set
35 seed using agricultural cultivation practises well known to a person
skilled in the art. After
harvesting the seed and if desired removal of material such as stones or seed
hulls
(dehulling), by for example sieving or rinsing, and optionally drying of the
seed, the seeds
are subsequently processed by mechanical pressing, grinding or crushing. A
liquid phase
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may be added prior to or while grinding the seeds. This is known as wet
milling.
Preferably the liquid is water although organic solvents such as ethanol may
also be used.
Wet milling in oil extraction processes has been reported for seeds from a
variety of plant
species including: mustard (Aguilar et al 1991, Journal of Texture studies
22:59-84), soybean
5 (US Patent 3,971,856; Cater et al., 1974, J. Am. Oil Chern. Soc. 51:137-
141), peanut (US
Patent 4,025,658; US Patent 4,362,759), cottonseed (Lawhon et al., 1977, J.
Am. Oil, Chem.
Soc. 54:75-80) and coconut (Kumar et al., 1995, INFORM 6 (11):1217-1240). It
may also be
advantageous to imbibe the seeds for a time period from about fifteen minutes
to about two
days in a liquid phase prior grinding. Imbibing may soften the cell walls and
facilitate the
10 grinding process. Imbibition for longer time periods may mimic the
germination process and
result in certain advantageous alterations in the composition of the seed
constituents. In
another embodiment, the liquid phase is added after the seeds are ground. This
is known as
dry milling. Preferably the added liquid phase is water.
The seeds are preferably ground using a colloid mill, such as the MZ130 (Fryma
15 Inc.). Besides colloid mills, other milling and grinding equipment capable
of processing
industrial scale quantities of seed may also be employed in the here described
invention
including: flaking rolls, disk mills, colloid mills, pin mills, orbital mills,
IKA mills and
industrial scale homogenizers. The selection of the mill may depend on the
seed
throughput requirements as well as on the source of the seed which is
employed. It is of
20 importance that seed oil bodies remain substantially intact during the
grinding process.
The term "substantially intact" as used herein means that the oil bodies have
not released
greater than 50% (v/v) of their total seed content in the form of free oil.
Preferably,
grinding of the seed therefore in the release of less than about 50% (v/v) of
the total seed
oil content, more preferably less than about 20% (v/v} and most preferably
less than about
25 10% (v/v). Accordingly, in a preferred embodiment the present invention
provides a
composition for the unproved delivery of an active agent comprising (a) an
active agent and
(b) substantially intact oil bodies.
Therefore, any operating conditions commonly employed in oil seed processW g,
which tend to disrupt oil bodies are unsuitable for use in the process of the
subject invention.
30 Milling temperatures are preferably between 10°C and 90°C and
more preferably between
26°C and 30°C, while the pH is preferably maintained between 2.0
and 10.
Solid contaminants, such as seed hulls, fibrous material, undissolved
carbohydrates and proteins and other insoluble contaminants, are removed from
the crushed
seed fraction. Separation of solid contaminants, may be accomplished using a
decantation
35 centrifuge, such as a HASCO 200 2-phase decantation centrifuge or a NX310B
(Alpha
Laval). Depending on the seed throughput requirements, the capacity of the
decantation
centrifuge may be varied by using other models of decantation centrifuges,
such as 3-phase
decanters. Operating conditions vary depending on the particular centrifuge
which is
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employed and must be adjusted so that insoluble contaminating materials
sediment and
remain sedimented upon decantation. A partial separation of the oii body phase
and liquid
phase may be observed under these conditions.
Following the removal of insoluble contaminants, the oil body phase is
5 separated from the aqueous phase. In a preferred embodiment of the invention
a tubular
bowl centrifuge is employed. In other embodiments, hydrocyclones, disc stack
centrifuges, or
settling of phases under natural gravitation or any other gravity based
separation method
may be employed. It is also possible to separate the oil body fraction from
the aqueous
phase employing size exclusion methods, such as membrane ultrafiltration and
crossfiow
10 microfiltration. In preferred embodiments the tubular bowl centrifuge is a
Sharples model
AS-16 {Alpha Laval) or a AS-46 Sharples (Alpha Laval). A critical parameter is
the size
of the ring dam used to operate the centrifuge. Ring dams are removable rings
with a
central circular opening varying, in the case of the AS-16, from 28 to 36 mrn
and regulate the
separation of the aqueous phase from the oil body phase thus governing the
purity of the
15 oil body fraction which is obtained. In preferred embodiments, a ring dam
size of 29 or 30
mm is employed when using the AS-16. The exact ring dam size employed depends
on the
type of oil seed which is used as well as on the desired final consistency of
the oil body
preparation. The efficiency of separation is further affected by the flow
rate. Where the
AS-16 is used flow rates are typically between 750-1000 ml/min (ring dam size
29) or
20 between 400-600 ml/rnin (ring dam size 30) and temperatures are preferably
maintained
between 26°C and 30°C. Depending on the model centrifuge used,
flow rates and ring dam
sizes must be adjusted so that an optimal separation of the oil body fraction
from the
aqueous phase is achieved. These adjustments will be readily apparent to a
skilled
artisan.
25 Separation of solids and separation of the aqueous phase from the oil body
fraction may also be carried out concomitantly using a gravity based
separation method
such as 3-phase tubular bowl.centrifuge or a decanter or a hydrocyclone or a
size exclusion
based separation method.
The compositions obtained at this stage in the process, generally are
relatively
30 crude and comprise numerous seed proteins, which includes glycosylated and
non-
glycosylated proteins and other contaminants such as glucosinilates or
breakdown products
thereof.
In preferred embodiments of the present invention, significant amount of seed
contaminants are removed. To accomplish removal of contaminating seed
material, the oil
35 body preparation obtained upon separation from the aqueous phase is washed
at least once
by resuspending the oil body fraction and centrifuging the resuspended
fraction. This
process yields what, for the purpose of this application, is referred to as a
washed oil body
preparation. The number of washes will generally depend on the desired purity
of the oil
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body fraction. Preferably the washed oil bodies contain less than about 75%
(w/v) of ali
endogenously present non-oil body seed proteins, more preferably less than
about 50% {w/v)
of non-oil body seed proteins and most preferably less than about 10% (w/v) of
all
endogenously present non-oil body seed proteins. Accordingly, in another
embodiment, the
5 present invention provides a composition for the improved delivery of an
active agent
comprising {a) an active agent; and (h) washed oil bodies, more preferably,
substantially
intact washed oil bodies.
Depending on the washing conditions which are employed, an essentially pure
oil body preparation may be obtained. In such a preparation the only proteins
present
would be oil body proteins. In order to wash the oil body fraction, tubular
bowl centrifuges
or other centrifuges such hydrocyclones or disc stack centrifuges may be used.
Washing of
oil bodies may be performed using water, buffer systems, for example, sodium
chloride in
concentrations between 0.01 M and at least 2 M, 0.1 M sodium carbonate at high
pH {11-12),
low salt buffer, such as 50 mM Tris-HCl pH 7.5, organic solvents, detergents
or any other
15 liquid phase. In preferred embodiments the washes are performed at high pH
(11-12). The
liquid phase used for washing as well as the washing conditions, such as the
pH and
temperature, may be varied depending on the type of seed which is used.
Washing at a
number of different pH's between pH 2 and pH 11-12 may be beneficial as this
will allow
the step-wise removal of contaminants, in particular proteins. Washing
conditions are
20 selected such that the washing step results in the removal of a significant
amount of
contaminants without compromising the structural integrity of the oil bodies.
In
embodiments where more than one washing step is carried out, washing
conditions may
vary for different washing steps. SDS gel electrophoresis or other analytical
techniques
may conveniently be used to monitor the removal of seed proteins and other
contaminants
25 upon washing of the oiI bodies. It is not necessary to remove au of the
aqueous phase
between washing steps and the final washed oil body preparation may be
suspended in
water, a buffer system, for example, 50 mM Tris-HCl pH 7.5, or any other
liquid phase and
if so desired the pH may be adjusted to any pH between pH 2 and pH 10. The oil
bodies
may be preserved by heat treatment for example by pasteurization in a constant
30 temperature water bath at approximately 65°C for 20 minutes. The
pasteurization
temperature preferably ranges between 50°C and 90°C and the time
for pasteurization
preferably ranges between 15 seconds to 35 minutes.
The process to manufacture the oil body preparation may be performed in batch
operations or in a continuous flow process. Particularly when tubular bowl
centrifuges are
35 used, a system of pumps generating a continuous flow. The pumps may be for
example a 1
inch M2 Wilden air operated double diaphragm pumps or hydraulic or peristaltic
pumps
may be employed. In order to maintain a supply of homogenous consistency to
the
decantation centrifuge and to the tubular bowl centrifuge, homogenizers, such
as an IICA
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homogenizer may be added between the separation steps. In-line hornogenizers
may also be
added in between various centrifuges or size exclusion based separation
equipment
employed to wash the oil body preparations. Ring dam sizes, buffer
compositions,
temperature and pH may differ in each washing step from the ring dam size
employed in
5 the first separation step.
B. Properties of Oil bodies
When viewed under fine electron microscope, the oil bodies that are obtained
are found to be more or less spherically shaped structures (see: Example
Murphy, D. J. and
Cummins L, 1989, Phytochemistry, 28: 2063-2069; Jacks, T. J. et al., 1990,
JAOCS, 67: 353-
10 361). Typical sizes of oil bodies vary between 0.4 um for and 1.5 pro
(Murphy, D. J. and
Cummins L, 1989, Phytochemistry, 28: 20b3-2069). When analyzed using a Malvern
Size
Analyzer, it was found that oil bodies in a washed oil body preparation
isolated from
rapeseed were symmetrically and unimodally distributed around 1 Vim. Using a
Malvern
Size Analyzer a washed oil body preparation could be clearly distinguished
from
15 commercially obtainable oil-in-water emulsions including soymilk,
mayonnaise (Kraft
Real Mayonnaise) and two coconut milk preparations (Tosco, Aroy-D). The exact
size and
density of the oiI bodies depends at least in part on the precise
protein/phospholipid/triacylglyceride composition which is present.
The oil bodies present in the washed oil body preparations of the present
20 invention are resistant to exposure to strong acids and bases, including
prolonged exposure to
acidic conditions at least as low as pH 2 and alkaline conditions at least as
high as pH 10.
When exposed to pH 12, a slight loss of oil was observed, indicating a loss of
integrity of
the oil body structure. In addition, extraction with various organic
solutions, including
methanol, ethanol, hexane, isopropyl alcohol and ethyl acetate, does not or
only slightly
25 compromise the integrity of the oil bodies present in the washed oil body
preparation. The
oil bodies present in the washed oil body preparation were also found to
withstand mixing
with the anionic detergent, sodium dodecyl sulfate (SDS), the cationic,
detergent
hexadecyl trimethyl bromide and Tween-80, a non-ionic detergent. Boiling of
the washed
oil body preparation in the presence of SDS was found to result at least
partly in
30 disintegration of the oil body structure. The oil bodies present in the
washed oil body
preparation are stable when maintained for 2 hrs up to at least 100°C.
A slow freeze and
thaw of washed oil body preparations resulted in a change in their physical
appearance
characterized by the formation of clumps as opposed to a homogeneous emulsion.
Oil body
clumping following a freeze-thaw could also be prevented to a large degree by
either a)
35 flash freezing in liquid nitrogen instead of slow freezing at -20°C
or b) adding glycerol in
excess of 5% (v/v) to the oil body preparation prior to freezing. The
resistance to relatively
harsh chemical and physical conditions, is a unique characteristic of the oil
bodies present
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in the washed oil body preparation of the subject invention and makes oil
bodies uniquely
suited as delivery vehicles.
For many applications, it is also considered desirable that a purer, better
defined oil body preparation is obtained, as this allows more control over the
formulation
5 process of the final emulsion. In order for the washed oil body preparation
to be included in
a diverse set of final preparations it is desirable that volatiles are kept to
a minimum and
the colour is preferably light or white. Washing of the oil body preparation
results in a
lighter coloured preparation. In addition, a substantial amount of volatiles
is removed.
Also removed by washing are compounds which promote the growth of
microorganisms as it
10 was observed that a washed oil body preparation had a longer shelf life
than an unwashed
preparation. Other compounds which are removed by washing include anti-
nutritional
glucosinilates and/or breakdown products thereof and fibrous material. When
heat treated
to 60°C or 80°C, it was observed that larger quantities of water
remained absorbed by the
washed oil body preparation when compared with an unwashed preparation: Upon
cooling
15 down to room temperature and centrifugation, it was observed that the
washed oil body
preparation remained stable, while phase separation occurred in .the unwashed
preparation. Given the enhanced stability of washed oil bodies, they are
preferred where
the formulation process involves the application of heat. When heated to
40°C, the
washed oil body preparation was able to absorb a larger quantity of
exogenously added
20 water without resulting in phase separation. Thus in the formulation of
aqueous emulsions,
washed oil bodies may be preferred. The capacity to absorb exogenously added
oils was
also compared between a preparation of washed oil bodies and an unwashed
preparation.
Larger amounts of exogenous oil could be added to the washed oil body
preparation before
an unstable emulsion was formed. This is advantageous in applications where
exogenous
25 oils or waxes axe added in the formulation process such as in topical
applications. When
viscosity was compared between a washed oiI body preparation and an unwashed
preparation it was found that the washed preparation was more viscous. A more
viscous
preparation of oil bodies is desirable as this eliminates the need for the
addition of
thickening agents in the formulation process.
30 The above observations were made using oil body preparations obtained from
rapeseed and prepared as detailed in Example 2 of the present application. It
is believed
that resistance to relatively harsh chemical and physical conditions will be a
characteristic of the oil bodies present in the washed oil preparation of the
subject
invention regaxdless of the source of the oil bodies. However one or more of
the
35 hereinbefore documented properties for rapeseed oil bodies may vary
depending on the
living cell, plant species or the genetic line from which the washed oil
bodies preparation
is obtained. Nevertheless it is to be clearly understood that the subject
invention is drawn
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to an oil body preparation which may be obtained from any living cell
comprising oil
bodies.
In embodiments of the present invention where the oil bodies are obtained from
non-seed cells, the oiI body preparation is isolated following similar
procedures as outlined
above. In embodiments of the invention where the oil bodies are isolated from
softer
tissues, for example the rnesocarp tissue of olives, the techniques applied to
break open the
cell may vary somewhat from those used to break harder seeds. For example,
pressure-
based techniques rnay be preferred over crushing techniques. The methodology
to isolate oil
bodies on a small scale has been reported for isolation of oil bodies from
mesocarp tissues in
10 olive (Oleo europaea} and avocado (Persea americana) (Ross et al., Plant
Science, 1993, 93:
203-210) and from microspore-derived embryos of rapeseed (Brassica napus)
(Holbrook et
al., Plant Physiol., 1991, 97: 1051-1058).
In embodiments of the invention where oil bodies are obtained from non-plant
cells, the oil body preparation is isolated following similar procedures as
outlined above.
The methodology to isolate oil bodies from yeast has been documented (Ting et
al., 1997,
Journal of Biol. Chem. 272: 3699-3706).
The chemical and physical properties of the oil fraction may be varied in at
least two ways. Firstly, different plant species contain oil bodies with
different oil
compositions. For example, coconut is rich in lauric oils (C12}, while erucic
acid oils (C22)
20 are abundantly present in some Brassica spp. Secondly, the relative amounts
of oils may be
modified within a particular plant species by applying breeding and genetic
engineering
techniques known to the skilled artisan. Both of these techniques aim at
altering the
relative activities of enzymes controlling the metabolic pathways involved in
oil
synthesis. Through the application of these techniques, seeds with a
sophisticated set of
different oils are obtainable. For example, breeding efforts have resulted in
the
development of a rapeseed with a low erucic acid content (Canola) (Bestor, T.
H., 1994,
L7ev. Genet. 15: 458} and plant lines with oils with alterations in the
position and number of
double bonds, variation in fatty acid chain length and the introduction of
desirable
functional groups have been generated through genetic engineering (Topfer et
al., 1995,
30 Science, 268: 681-685). Using similar approaches a person skilled in the
art will be able to
further expand on the presently available sources of oil bodies. Variant oiI
compositions
will result in variant physical and chemical properties of the oil bodies.
Thus by selecting
oilseeds or mixtures thereof from different species or plant lines as a source
for oii bodies, a
broad repertoire of oil body preparations with different textures and
viscosities may be
35 acquired.
In one embodiment of the present invention, the oil bodies are obtained from
oil
seeds. The presence of intact oil bodies in the emulsion and the described
characteristics of
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these oil bodies clearly distinguish the subject emulsion formulation from
other materials
which may be prepared from plant seeds.
C. Method of Preparing Composition
In another aspect, the pxesent invention provides a method of preparing a
5 composition for the delivery of an active agent which comprises formulating
the active
agent in the presence of oil bodies to prepare a composition.
The term "formulating" as herein used means any process that results in
contacting oil bodies with the active agent.
The active agent may be incorporated into the oil body preparation in any
10 desired manner. The active agent may be added as a solution, suspension, a
gel or solid.
The active agent may upon formulation become associated with the oil bodies,
remain
suspended in solution, or form a suspension in which the oil bodies are
dispersed. The
active agent rnay also penetrate the phaspholipid monolayer surrounding the
oil body or
the triacylglyceride matrix. The active agent may be linked to the oil bodies
in a non
15 covalent manner, in which case the active agent may be a protein as well as
any other
molecule. Methodologies for non-covalently linking active molecules to oil
bodies are
further detailed in US Patent 5,856,452 and WO 98/27115 both of which are
incorporated
by reference herein.
When the active agent is a protein, it may be prepared as a.recombinant fusion
20 protein with an oil body protein or oleosin as described in WO 96/21029
which is
incorporated herein by reference. Accordingly, the present invention provides
a method of
preparing a composition for the delivery of an active agent comprising:
(a) introducing into a host cell a chimeric DNA sequence comprising:
(1) a first DNA sequence capable of regulating the
25 transcription in said host cell of
(2) a second DNA sequence, wherein said second sequence
encodes a recombinant fusion polypeptide and comprises a DNA
sequence encoding a sufficient portion of an oleosin protein to
provide targeting of the recombinant fusion polypeptide to an oil
30 body phase, linked in frame to (ii) a DNA sequence encoding the
active agent; and
(3} a third DNA sequence encoding a termination region
functional in said host cell;
(b) growing said host cell to produce said recombinant fusion
35 polypeptide
(c) separating the oil body fraction of said host cell from the cellular
components to prepare oil bodies and the active agent.
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The oil bodies containing the active agent may be formulated to prepare a
composition.
The amount of active agent that will be employed will be that amount that
will be necessary to deliver a desirable biological effect at the site of
delivery. In
particular, an effective amount depends, inter alia upon the particular active
agent, the
route of administration, and in the case of pharmaceutically active agents the
severity of
the condition or disorder under treatment and other factors. In general, the
concentration of
the active agent in the delivery system can vary from as little as 0.001% up
to 50% by
weight of the composition. More typically the active concentration is between
about 0.01%
and 10% by weight of the composition. Cell culture assays and animal model may
be used to
assist in determining doses appropriate for human if appropriate. Skilled
artisans will be
able to adjust the quantity of active in the composition.
A variety of additional ingredients may be included in the final composition
that is administered to the living organism. In preferred embodiments these
ingredients
are added to formulate a dermatologically acceptable formulation for topical
skin
application. For example, water may be added either directly or through
moisture
associated with the therapeutically active agent. The final amount of water is
not
critical. Generally, the final delivered formulations will contain at least 1%
of water and
up to 99°/° water by weight. Usually mixing will be required to
provide an adequate
suspension and it may be necessary to apply heat or pressure or to change the
pH.
The amount of the oil bodies and active agent in the final administered
composition may vary considerably and can vary from as little as 0.1% to
99.9%. More
typically however the oil bodies and active agent will comprise between 5% and
95% of
the final administered composition.
In other embodiments an oil or a wax will be an additianai ingredient. Oils or
waxes may partition to the triacyl glyceride matrix of the oiI bodies. Where
oils or waxes
comprise the added ingredient, the oil bodies may remain suspended in the
lipophilic
phase or double emulsions may be formed.
Generally, the compositions will be treated such that contamination by
bacteria, fungi, mycoplasmas, viruses and the like or undesired chemical
reactions, such as
oxidative reactions are prevented thus allowing the preparation of a stable
final product
with a shelf-life accepetable for the final composition. In preferred
embodiments this is
accomplished by the addition of preservatives, for example sodium
metabisulfite, Glydant
Plus, Phenonip, methylparaben propylparaben, Germall 115, Germaben II, phytic
acid, ar
other chemical additives, by irradiation, for example by ionizing radiation
such as cobalt-
60 or cesium-137 irradiation or by ultraviolet irradiation or by heat
treatment for example
by pasteurization in a constant temperature water bath at approximately
65°C for 20
minutes. The pasteurization temperature preferably ranges between 50 and
90°C and the
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time for pasteurization preferably ranges between 15 seconds to 35 minutes.
Oxidative
reactions may be prevented by the addition of anti-oxidants such as butylated
hydroXytoluene (BHT) or butyiated hydroxyanisol (BHA) or other anti-oxidants.
The physical stability of the compositions may be further enhanced if desired
by the addition of an emulsifier such as for example Arlacel. Typically,
emulsion
stabilizers are added in small amounts (less than 2% by weight).
The final compositions may be in solid or in liquid form or of any other
desired
viscosity. The viscosity of the emulsion may be modified using a viscosity
modifier such as
cetyl alcohol. The emulsion may be thickened using gelling agents such as
cellulose and
10 derivatives, Carbopol and derivatives, carob, carregeenans and derivatives,
xanthane gum,
sclerane gum, long chain alkanolamides, bentone and derivatives, Kaolin USP,
Veegum
Ultra, Green Clay, Bentonite NFBC, typically present in concentrations less
than 2% by
weight. The composition may also form a coating or film.
The compositions may further comprise surfactants to wet, foam, penetrate,
15 emulsify, solubilize and or disperse the cosmetically active agent. For
example anionic
surfactants such as sodium coconut manoglyceride sulphonate, cationic
surfactants, such as
lauryl trimethyl ammonium chloride, cetyl pyridiniurn chloride and
trimethylammonium
bromide, nonionic surfactants iuncluding pluronics, and polyethylene oxide
condensates of
alkyl phenols, and zwitterionic surfactants such as derivatives of aliphatic
quaternary
20 ammonium, phosmomium and sulphoniurn compounds may all be added as
required.
Chelating agents, capable of binding metal ions, such as tartaric acid, EDTA,
citric acid,
alkali metal citrates, pyrophosphate salts or anionic polymeric
polycarboxylates may be
also included in the final formulation as desired.
The compositions of the present invention may further comprise additional
25 hydrocarbon compounds such as plant, animal, mineral or synthetic oils or
waxes or mixes
thereof. They comprise paraffin, petrolatum, perhydrosqualene, arara oil,
almond oil,
calphyllum ail, avocado oil, sesame oil, castor oil, jojoba oil, olive oil, or
cereal germ oil.
Esters may be included such as esters of lanolic acid, oleic acid, lauric
acid, stearic acid,
myristic acid. It is also possible to include alcohols for example, oleoyl
alcohol, linoleyl
30 alcohol or linolenyl alcohol, isostearyl alcohol or octyl dodecanol,
alcohol or polyalcohol.
Further hydrocarbons which may be included are octanoates, decanoates,
ricinoleates,
caprylic/capric triglycerides or C10 to C22 fatty acid triglycerides. The
addition of these
agents may result in the formation of double emulsions. Hydrogenated oils,
which are solid
at 25°C, such as hydrogenated castor oil, palm oil or coconut oil, or
hydrogenated tallow;
35 mono- di- tri- or sucroglycerides; lanolins; and fatty acids which are
solid at 25°C may also
be included in the topically applied formulations of the present invention.
Among the
waxes which may be included are animal waxes such as beeswax; plant waxes such
as
carnauba wax, candelilla wax, ouricurry wax; Japan wax or waxes from cork
fibres or sugar
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cane; mineral waxes, for example paraffin wax, lignite wax, microcrystalline
waxes or
ozokerites and syntheiac waxes. Pigments may be included and may be white or
coloured,
inorganic or organic and/or paerlescent. These pigments comprise titanium
dioxide, zinc
oxide, ziriconium dioxide, black, yellow, red and brown iron oxides, cexium
dioxide,
5 chromium oxide, ferric blue, carbon black, barium, strontium, calcium and
aluminum lakes
and mica coated with titanium oxide or with bismuth oxide.
Moisturizing agents which may be included in topically applied compositions
axe for example mineral oil and urea. Antioxidants such as the naturally
occurring
tocopherols and polyphenols, or butylated hydroxytoluene and hydroxyanisole
may also be
10 also added.
While the final formulations or compositions may vary considerably in
composition and contain numerous additional ingredients, in general the final
compositions
for topical applications may be formulated in accordance with methods used and
known by
those skilled in the art of formulating cosmetic and dermatological
formulations.
15 D. Uses
The present invention includes all uses of the compositions of the invention
to
deliver active agents to the skin of an organism in need thereof. Accordingly,
the present
invention provides a use of a composition comprising an active agent and oil
bodies to
deliver an active agent to a living organism. The present invention also
includes a use of a
20 composition comprising oil bodies and an active agent to prepare a
medicament to deliver
an active agent to a living organism.
The present invention fuxther provides a method for the delivery of an active
agent to a living organism comprising administering an effective amount of a
composition
comprising the active agent and oil bodies to the skin of an organism in need
thereof.
25 The term "living organism" as used herein includes all members of the
animal
kingdom. Preferably the living organism is a vertebrate, more preferably a
mammal, most
preferably a human.
The term "effective amount" as used herein means an amount effective, at
dosages and for periods of time necessary to achieve the desired results.
30 The composition is preferably administered topically which includes,
without
limitation, administration to the skin, hair, teeth and nails.
Topical administration includes administration to the skin, including human or
animal skin. The compositions of the present invention may be used to beautify
the skin and
to treat the skin for example hyperpigmented or hypopigmented skin, age spots
and other
35 - skin changes associated with aging such as wrinkles, blotches and atrophy
or elastotic skin
changes characterized by leathery changes associated with intrinsic aging or
skin damage
caused by extrinsic factors such as sunlight radiation, X-ray radiation, air
pollution, wind,
cold, dampness, heat, smoke and cigarette smoking. Additional skin conditions
which may
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be treated with the delivery vehicles of the present invention include but are
not limited to
acne keratoses, paixnar or plantar hyperkeratosis, psoriasis, eczema,
seborrheic eczema,
pruritus, ichthyosis, Darier's disease, lichen simplex chronicus, inflammatory
dermatoses,
basal cell carcinoma, squamous cell carcinoma, malignant cell carcinoma, and
AIDS related
Kaposi sarcoma.
When applied topically the active ingredient is delivered percutaneously.
The term "percutaneous" as used herein refers to the delivery to the skin
without referring
to their eventual fate. Accordingly, the active ingredient may be delivered to
various
layers of the skin including the stratum corneum, the epidermis and the
dermis.
10 When it is desirable to deliver the therapeutically active ingredient to
the
epidermis or to the dermis, the active may conveniently be linked to an oil
body protein, for
example an oleosin, through covalent or non-covalent bonds and topically
applied. Based
on the observation that oil body associated proteins penetrate into the
epidermis and
dermis (examples 4 and 5), the active ingredient linked to the oil body should
be delivered
in the epidermal and dermal layers of the skin.
Upon administration of the formulation to the living organism, the active
ingredient may remain associated with the oil body and exert ifs biological
effect, for
example when a formulation is used which has been prepared by covalently or
non-
covalently linking the active ingredient to the oil body. It is also possible
that the active
20 ingredient separates from the oil body prior to exerting its biological
effect. The oil body
may also disintegrate priox to the exertion of the biological effect by the
active ingredient.
The creamy texture of the oil bodies make embodiments of the invention where
the composition is applied topically particularly preferred.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Example 1
Preparation of Oil Bodies from Oilseed rape, Soybean, Sunflower, White
Mustard, Peanut, Squash, Flax, Safflower and Maize.
Dry mature seeds obtained from Brassica napes cv Westar (oilseed rape),
30 soybean, sunflower, white mustard, peanut, squash, flax, safflower and
maize were
homogenized in five volumes of cold grinding buffer (50 mM Tris-HCI, pH 7.5,
0.4 M sucrose
and 0.5 M NaCI) using a polytron operating at high speed. The homogenate was
centrifuged at 10 x g for 30 minutes in order to remove particulate matter and
to separate oil
bodies from the aqueous phase containing the bulk of the soluble seed protein.
The oil body
35 fraction was skimmed from the surface of the supernatant with a metal
spatula and added
to one volume of grinding buffer. In order to achieve efficient washing in
subsequent steps it
was found to be necessary to thoroughly redisperse the oil bodies in the
grinding buffer.
. This was accomplished by gently homogenizing the oil bodies in grinding
buffer using a
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polytron at low speed. Using a syringe, the redispersed oil bodies were
carefully layered
underneath five volumes of cold 50 mM Tris-HCl pH 7.5 and centrifuged as
above.
Following centrifugation, the oil bodies were removed and the washing
procedure was
repeated two times. The final washed oil body preparation was resuspended in
one volume
5 of cold Tris-HCl pH 7.5, redispersed with the polytron.
The oil body samples were dissolved in SDS sample buffer and protein profiles
for the oiI body samples unique to each of the plant species were obtained
following SDS
gel electrophoresis. The results are shown in Fig. 1.
Example 2
10 The Preparation of Oil Bodies from Oilseed Rape, Sunflower and Maize on a
Large Scale.
Grinding of seeds. A total of 10 -15 kgs of dry canola seed (Brassica napus cv
Westar), sunflower (Helianthus annuus) or maize {Zeq mat's) was poured through
the
hopper of a colloid mill (Colloid Mill, MZ-130 (Fryma); capacity: 500 kg/hr},
which was
15 equipped with a MZ-120 crosswise toothed rotor/stator grinding set and top
loading
hopper. Approximately 50 - 751 water was supplied through an externally
connected hose
prior to milling. Operation of the mill was at a gap setting of 1R, chosen to
achieve a
particle size less than 100 micron at 18°C and 30°C. Following
grinding of the seeds tap
water was added to the seed slurry to a final volume of 90 litres.
20 Removal of solids. The resulting slurry, was pumped into a decantation
centrifuge {Hasco 200 2-phase decantation centrifuge maximum operating speed
6,000 rpm)
after bringing the centrifuge up to an operating speed of 3,500 rpm. Transfer
from the mill to
the decantation centrifuge at a flow rate of 360 L/hr was achieved using a 1
inch M2
Wilden air operated double diaphragm pump. In 15-20 minutes approximately 15
kg of
25 seed was decanted:
Oil body separation. Separation of the oil body fraction was achieved using a
Sharpies Tubular Bowl Centrifuge model AS-16 (Alpha Laval) equipped with a
three
phase separating bowl and removable ring dam series; capacity:150 L/hr;
ringdam: 30 mm.
Operating speed was at 15,000 rpm (13,200 x g). A Watson-Marlow (Model 70~)
peristaltic
30 pump was used to pump the decanted liquid phase (DL) into the tubular bowl
centrifuge
after bringing the centrifuge up to operating speed. This xesults in
separation of the
decanted liquid phase into a heavy phase (HP) comprising water and soluble
seed proteins
and a light phase (LP) comprising oil bodies. The oil body fraction which was
obtained
after one pass through the centrifuge is referred to as an unwashed oil body
preparation.
35 The oil body fraction was then passed through the centrifuge three more
times. Between
each pass through the centrifuge, concentrated oil bodies were mixed with
approximately
five volumes of fresh water. The entire procedure was carried out at room
temperature: The
preparations obtained following the second separation are all referred to as
the washed oil
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body preparation. Following three washes much of the contaminating soluble
protein was
removed and the oil body protein profiles obtained upon SDS gel
electrophoresis were
similar in appearance to those obtained using laboratory scale procedures
{Example 1).
Ex:~nle~
5 Comparison of Washed Oil Bodies and Lipid Vesicles with respect to Utility
as an Ingredient for the Preparation of Formulations Acceptable for Topical
Delivery to
Humans. Washed Oil bodies were prepared as described in example 2 using
safflower seed,
pasteurized and 0.1% BHT, 0.1% BHA and 0.1% Glydant plus added. Lipid vesicles
were
prepared in accordance with the specification of US Patent 5,683,740 except
that they were
10 prepared from safflower seed and pasteurized and 0.1% BHT, 0.1% BHA and
0.1% Glydant
Plus were added.
Oil bodies and lipid vesicles were compared with respect to emulsion
stability,
color changes, odor changes, viscosity, microbial growth and cosmetic
desirability
parameters. To evaluate stability, the samples were tested at 45°C,
4°C and room
15 temperature (3 months at 45°C is equivalent to approximately 2 year
shelf life at room
temperature). To evaluate emulsion stability, 150 g of each sample was
maintained at
45°C, 75 g of each sample was maintained at room temperature or at
4°C. Emulsion stability
was evaluated for emulsion separation, oil droplet separation and coalescence.
The 4°C
sample was used as the reference for comparison. Color changes were evaluated
by visual
20 inspection. Color was evaluated on the accelerated oven sample
{45°C) and the room
temperature sample and compared to the 4°C as a reference. Odor was
tested as with the
color with the 4°C sample used as a reference point. In order to
maintain consistency, the
odor was judged by two individuals who both agreed on the evaluation.
Viscosity of each
sample was measured at room temperature using a RVT Model viscometer with
Spindle E at
25 lOrpm. Microbial growth was measured on 10 g of each sample: The sample was
diluted
and 1 ml of the sample is added to 49°C Tryptic Soy Agar, swirled and
allowed to cool. The
plates were incubated at 35°C for 48 hours and a colony count was
taken. Finally, cosmetic
attributes were evaluated by 3 individuals, 2 individuals who were familiar
with oil
bodies/lipid vesicles and 1 person who was not. Cosmetic attributes include
skin
30 penetration, residue left on the skin after the sample was rubbed in,
dryness (lack of
moisture) and oiliness.
Table 1 summarizes the results for the oil bodies. The pH for the oil body
sample was constant at 6.50 throughout the test at room temperature and at
45°C. The oil
body preparation, when applied to the skin, distributed evenly on the skin,
was fast
35 penetrating and left almost no residue on the skin surface. The oil body
preparation was
also stable with respect to color, odor, viscosity and emulsion stability.
Table 2 summarizes the results for the lipid vesicles. The pH for the
lipid vesicle sample is difficult to measure because of the total separation
but was
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approximately d.8. The lipid vesicle preparation, when applied to the skin,
was very oily
and left a film residue on the skin. The lipid vesicle preparation was stable
with respect to
microbial growth but was not stable with respect to color, odor and emulsion
stability.
Thus the oil washed oil body preparation is clearly superior to lipid vesicles
5 with respect to many parameters including the following parameters, color,
odor, stability
and cosmetic parameters like penetration, residual residue, and oiliness,
which are critical
to utility as a delivery vehicle.
Example 4
Preparation of Compositions for Topical Delivery and Irritation reactions to
Oil Bodies and the Effect of Oil Bodies on Known Dermal Irritants. The
followine
formulations were used in an irritation study to test whether or not oil
bodies cause
irritation and whether or not oil bodies had an effect on known irritants used
in cosmetic
formulations (e.g. hydroquinone, alpha hydroxy acid and salicyclic acid). The
formulation
was mixed to form an emulsion with the following procedure. Phase I is the
water phase.
15 In this phase, the water in the main tank is charged. A propeller is used
to hydrate the
sodium thiosuifate, glycolic acid and/or salicyclic acid, if added to the
formulation, with
moderate agitation at room temperature. The water phase is heated to a final
temperature
of 75°C to 77°C. Phase II, the oil phase is mixed in a separate
mixing pot with moderate
agitation and then subsequently heated up to 75°C to 77°C. The
oil phase ingredients
20 include Keltrol, Arlacel 165 and Glydant Plus. The final step of
emulsification includes the
addition of the oil phase (Phase II) to the water phase (Phase I). The two
phases are
mixed under high agitation with a propeller or homogenizes for 15 minutes:
After 15
minutes of mixing the mixture is cooled slowly to 50°C. If sodium
thiosulfate and/or lactic
acid is/are added, they are added at 50°C. The agitation is decreased
as the temperature
25 decreases. At 40°C the Hydroquinone Liposomized (25% HQ) and/or
Hydroquinone is
added and when the temperature reaches about 37°C to 40°C the
safflower oil bodies are
added slowly.
Formulation A
Hydrated safflower oil bodies (0.1% 100.00%
Glydant plus, 0.1% BHT, 0.1% BHA)
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Formulation B
Distilled Water 43.85%
Keltrol 1.50%
Arlacel-165 3.00%
Glydant Plus 0.1%
Hydrated safflower oil bodies 49.00%
(0.1%
Glydant plus, 0.1% BHT, 0.1%
BHA)
Hydroquinone 2.00%
Sodium thiosulfate 0.30%
Lactic acid 0.17%
p H 4.00
Viscosity, RVT E/lOrpm (cps) 50,000
Formulation C
Distilled Water 68.04%
Keltroi 1.50%
Arlacel-165 3.00%
Glydant Plus 0.10%
Hydroquinone 2.00%
Sodium tlziosulfate 0.30%
Safflower oil 25.00%
Lactic acid 0.06%
pH 4.17
Viscosity, RVT E/lOrpm 35,000
(cps)
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Formulation D
Distilled Water 87.40%
Keltrol 1.50%
Arlacel-165 3.00%
Glydant Plus 0.10%
Hydroquinone Liposomized 8.00%
(25%)
Sodium thiosulfate 0.30%
Lactic acid 0.06%
pH 5.38
Viscosity, RVT E/l0rpm (cps)15,000
Formulation E
Distilled Water 93.10%
Keltrol 1.50%
Arlacel-165 3.00%
Glydant Plus 0.10%
Hydroquinone 2.00%
Sodium thiosulfate 0.30%
pH 4.17
Viscosity, RVT E/lOxprn 22,000
(cps)
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Formulation F
Distilled Water 46.34%
Keltroi 1.50%
Arlacel-165 3.00%
Glydant Plus 0.10%
Hydrated safflower oil bodies49.00%
(O.I%
Glydant plus, 0.1% BHT,
0.1% BHA)
Lactic acid 0.06%
pH 4.60
Viscosity, RVT E/l0rpm (cps}20,000
Formulation G
Distilled Water 41.34%
Keltrol 1.50%
Arlacel-165 3.00%
Glydant Plus 0.10%
Hydrated safflower oil bodies46.00%
(0.1%
Glydant plus, 0.1% BHT,
0.1% BHA)
Lactic acid 0.06%
Glycolic acid (AHA) 8.00%
pH 4.00
Viscosity, RVT E/l0rpm (cps)50,000
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Formulation H
Distilled Water 87.34%
Keltrol 1.50%
Arlacel-165 3.00%
Giydant Plus 0.10%
Lactic acid 0.06%
Glycolic acid (AHA) . 8.00%
pH 2.09
Viscosity, RVT E/l0rpm 7,500
{cps)
Formulation I
Distilled Water 44.34%
Keltrol 1.50%
Arlacel-165 3.00%
Giydant Plus 0.10%
Hydrated safflower oil bodies49.00%
(0.1%
Glydant plus, 0.1% BHT,
0.1% BHA)
Lactic acid 0.06%
Salicyclic acid 2.00%
pH 3.00
Viscosity, RVT E/l0rpm (cps)27500
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Formulation J
Distilled Water 93.34%
Keltrol 1.50%
Arlacei-165 3.00%
Glydant Plus 0.10%
Lactic acid 0.0b%
Salicyclic acid . 2.00%
pH 2.87
Viscosity, RVT E/l0rpm (cps) 10,000
Formulation K
Sodium Lauryl Sulfate 0.1% w/v
5 Formulation L
Saline 0.9%
In order to test the irritation reaction of oil bodies and oil bodies with
active
ingredients, a sufficient number of subjects were enrolled so that 25 test
subject completed the
study. Each test subject was tested with the 12 formulations (A to L) in a
random manner in
10 order to not introduce any bias. 0.2 mL of each formulation was applied to
the paraspinal
region of the back of each subject using a Eppendorf repeater pipette.
Formulations A
though J and L were covered by a semi-open occluded patch with adhesive
removed from
two opposing sides. Formulation K (the positive control which was known to
cause
irritation} was covered with a occluded patch using a nonwaven cotton pad
(Webril~}
15 covered by and secured on all sides by hypoallergenic tape (e.g.
BlendermTM). The
assignment of test articles to individual skin sites were rotated so that each
test article
occupies individual skin sites within the panel of test subjects, with
approximately equal
frequency, in order to eliminate any position bias. The individual test
articles were
applied to the skin for contact periods of approximately 23 hours per
application. After 23
20 hours, the subject removed the patch, bathed or showered and reported for
scoring.
Application was made every day for fourteen consecutive days on the same site
unless
reaction to any of the formulations made this inadvisable. Irritation was
scored as per
Berger and Bowman. 1984. J. Toxicology Cut. And Ocular Toxicol 1: 109-115.
Scoring was
conducted using a 100 watt incandescent blue bulb lamp. The scorer was blinded
as to the
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treatment assignments and to any previous scores. A reasonable attempt was
made to ensure
that the same individual did all of the scoring.
Scores were expressed as the Total Score for a base of 10 subjects and
classified
according to the following empirically derived categorization system. A score
of 0-33
5 indicates that the material is mild and that there was not experimental
irritation, 34-133
indicates that the material is probably mild in normal use as there was
evidence of a slight
potential for mild cumulative irritation, a score of 134-299 is indicative of
the material
being possibly mild in normal use as there was some evidence of a moderate
potential for
mild cumulative irritation, a score in the of 300-387 indicates an
experimental cumulative
20 irritant with a strong potential for mild to moderate cumulative irritation
and finally, a
score of 388-420 indicates that experimental primary irritant with evidence of
potential for
primary irritant irritation. The scores for formulations A through L are
presented in
Table 3.
Formulation K is used as the positive control with a high level of irritation
and
15 formulation L is the negative control demonstrating no experimental
irritation. The lowest
level of irritation was found in formulation A and F which are oil bodies
alone and
formulated oil bodies, respectively. This level of irritation was even lower
than the saline
control (formulation L). A substantial decrease in irritation was observed
when
hydroquinone was formulated with oil bodies (formulation B) when compared to
20 hydroquinone formulations with safflower oil (formulation C), hydroquinone
in liposomes
(formulation D) or hydroquinone formulated alone (formulation E}. Similarly
there was a
decrease in the irritation caused by salicyclic acid when the salicyclic acid
was formulated
with oil bodies (Formulation I) when compared to salicyclic acid formulated
without oil
bodies (formulation J). There was little difference with the alpha hydroxy
acid
25 formulated with and without oil bodies, in formulations G and H
respectively. This is
likely due to the fact that both formulations were occluded and this is known
to be
undesirable with AI-iA.
Thus these results indicate that oil bodies decrease the irritation caused by
active ingredients like hydroquinone (bleaching agent) and salicyclic acid
(exfolient).
30 Example 5
Detection of Oil Body-Associated Protein Penetration in Human Skin Samples
Preparation of Oil Bodies. Vllashed oil bodies were prepared aseptically from
seeds of non-transgenic canola using standard laboratory procedures.
Extraction and
washing steps were performed with sterile water. The final percentage dry
weights of the
35 oil body preparations was approximately 60%.
Franz cell methodology. Human cadaver skin from a single donor was
dermatorned to approximately 200 micro thickness. Eighteen Franz static
diffusion glass
diffusion chambers (Crown Glass Cat # FDC-100} with a magnetic stirrer mounted
on a 9-
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position Franz diffusion cell drive console with acrylic blocks, magnetic
stirrers, and
stainless steel manifolds (Crown Glass Cat #FDCD-9-LV) were used for the Franz
cell
assay. The chambers were filled with isotonic buffered saline (pH 7.4) and
equilibrated for
1 to 2 hours to a temperature of 37°C by a circulating Haake water pump
prior to applying
5 the skin specimen. Duplicate samples of two types of skin specimens were
tested:
dermatomed intact skin tape-stripped 24 hours after applying formulation and
dermatorned tape-stripped skin stripped before applying the formulation (Tape-
strip skin
with cellophane tape until "glistening" (approximately 22 stxips) or until
epidermal
separation starts to occur}. The skin sample was placed on the chamber and
sealed with an
10 O-ring. The exposed skin surface area is 1.77 cm2. 20 mg of the test
formulation (either
wild type oil bodies) was applied to the skin surface with a Gilson P100
Pipetteman-~
micropipet. A reservoir solution was collected after 1 hours and 24 hours
after application
of formulation. After 24 hours the skin surface was washed three times with
1.0 ml 2%
Oleth-20 (Croda, Inc. #9004-98-2) in water. After washing, the skin was wiped
with 3
15 sequential cotton gauze cloths. The skin was removed from the chamber. The
dermatomed
intact skin that was intended to be tape-stripped was tape-stripped. For all
of the tape-
stripped specimens {bath pre-stripped and post-stripped samples), the dermis
and
epidermis were separated by placing the sample on a 60°C hot plate for
one minute. The
epidermis and dermis tissues were collected in separate vials and
refrigerated.
20 Detection of oil body associated proteins. Tissue samples (intact skin,
stripped
skin, epidermis, dermis) were extracted with approximately 0.5 ml of SDS
extraction buffer
{2% SDS, 50 mM Tris-HCi, ph 7.5, 1 mM PMSF) by grinding with mortar and
pestle.
Reservoir samples from the Franz cells were made up to 2% SDS by addition of
1/10 volume
of a 20% SDS stock solution. All samples were heated in a boiling water bath
for 5 minutes
25 and then frozen at -20°C until required. Protein determinations were
made using the BCA
method.
Protein samples were subsequently separated through SDS PAGE and
electroblotted to PVDF membrane. A sample of 25 ug of protein from control
epidermis
samples spiked with 0.2 ug of canola oiI body protein was included on each gel
to enable
30 relative quantification of protein penetration in treated tissue samples.
Blots were probed
with either anti-oleosin (B. napus) followed by anti-rabbit IgG antibodies
conjugated to
alkaline phosphatase. Cross-reacting protein was visualized by NBT / BCIl'
assay for
alkaline phosphatase activity.
Results for the epidermal and dermal fractions wherein the stratum corneum
35 was removed with skin stripping befoxe the oil bodies were applied are
shown in Figure 2.
In this example the epidermal fractions from 3 individuals (lanes 3 through 5)
and dermal
fractions from the same 3 individuals (lanes 6 through 8) are compared to an
epidermal
extract spiked with 0.2 ~.g of canola oil body protein {lane 1) and control
intact skin (lane 2).
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The anti-oleosin antibody used in this experiment was a polyclonally-derived
antibody to
an canola oil body. As a result this anti-body will detect both oleosin
protein and any
proteins that are normally associated with canola oil bodies and were injected
when
antibodies were raised: In the epidermal fractions, oleosin as well as oil
body associated
5 proteins were detected in all individuals. In the dermis samples, oleosin
was detected and
to a lesser extent, oil bodies associated proteins were also detected. These
results indicate
that when oil bodies are applied to skin with the stratum corneum removed,
oleosin and oil
body associated proteins are able to penetrate into both the epidermis and
dermis.
Results for the epidermal and dermal fractions wherein the stratum corneum
10 was removed with skin stripping after the oil bodies were applied and the
Franz assay was
completed are shown in Figure 3. In this example the epidermal fractions from
3
individuals (lanes 2 through 4) and dermal fractions from the same 3
individuals (lanes 5
through 7) are compared to an epidermal extract spiked with 0.2 ~.g of canola
oil body
protein (lane 8) and control intact skin (lane 1). As in figure 2, the anti-
oleosin antibody
15 used in this experiment was a polyclonally-derived antibody to an canola
oil body.
Therefore this anti-body will detect both aleosin protein and any proteins
that are
normally associated with canola oil bodies. In the epidermal fractions,
oleosin and oil
body associated proteins were detected in all individuals, but with variation
between
individuals. As well, in the dermis, oleosin was detected and to a lesser
extent, oil bodies
20 associated proteins were also detected. These results indicate that when
oil bodies are
applied to skin with an intact stratum corneum, oleosin and oil body
associated proteins are
still able to penetrate into both the epidermis and dermis.
Taken together, results from figures 2 and 3 indicate that oleosin and oil
body
associated proteins are able to penetrate into both the epidermis and dermis.
As well, in
25 the presence of intact skin, oleosin and oil body associated proteins are
able to traverse
through the stratum corneum and penetrate into both the epidermis and dermis.
Exam
Preparation of Compositions for the Delivery of Cosmetic Active Agents and
Percutaneuous Absorption of Actives in Human Skin.
30 Preparation of Hydroquinone Formulations. A washed oil body preparation
was prepared from safflower as in example 2. To the washed oil body
preparation was
added: 0.1% Glydant Plus, 0.1% Butylated Hydroxyanisole (BHA) 0.1% Butylated
Hydroxytoluene) and a hydroquinone oil body formulation for use in a testing
oil body-
associated hydroquinone penetration was prepared as follows. Phase I is the
water phase.
35 In this phase, the water in the main tank is charged. A propeller is used
to hydrate the
Keltrol and Glydant plus with moderate agitation at room temperature. The
glycerin is
then added with continued mixing. The water phase is heated to a final
temperature of
76°C to 78°C. Phase II, the oil phase is mixed in a separate
mixing pot with moderate
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agitation and then subsequently heated up to 76 to 78°C. The oil phase
ingredients include;
Arlacel 165, Cetyl Alcohol, Finsolv TN, and Permethyl 101A (Isohexadecane).
The final
step of emulsification includes the addition of the oil phase (Phase II) to
the water phase
(Phase I). The two phases are mixed under high agitation with a propeller or
homogenizer
5 for 15 minutes. After 15 minutes of mixing the mixture is cooled slowly to
40°C. The
agitation is decreased as the temperature decreases. At approximately
40°C hydrated
safflower oil bodies and hydroquinone are added slowly. The mixture is allowed
to cool to
room temperature. Citric Acid is added to the formulation until the pH is 3.5
to 4Ø
Oil body-hydroquinone formulation
Purified Water 36.1%
Keltrol 0.7%
Glycerin 2.0%
Glydant Plus 0.20%
Arlacel 165 3.0%
Cetyl Alcohol 2.0%
Finsolv TN 2.0%
Permethyl 101A 2.0%
Hydrated Safflower Oil Body (0.1% 50.0%
Glydant Plus, 0.1% BHT, 0.1% BHA)
Hydroquinone 2.0%
Citric Acid to pH 3.5-4.0
A simple emulsion hydroquinone formulation for use in a testing a placebo-
associated
hydroquinone penetration was prepared as follows. Phase I is the water phase.
In this
phase, the water in the main tank is charged. A propeller is used to hydrate
the Keltrol
15 and Glydant plus with moderate agitation at room temperature. The glycerin
is then
added with continued mixing. The water phase is heated to a final temperature
of 76°C to
78°C. Phase II, the oil phase is mixed in a separate mixing pot with
moderate agitation
and then subsequently heated up to 76 to 78°C. The oil phase
ingredients include, Arlacel
165, Cetyl Alcohol, Finsolv TN, Permethyl 101A and (Isohexadecane). The final
step of
20 emulsification includes the addition of the oil phase (Phase II) to the
water phase
(Phase I). The two phases are mixed under high agitation with a propeller or
homogenizer
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for 15 minutes. After 15 minutes of mixing the mixture is cooled slowly to
40°C. The
agitation is decreased as the temperature decreases. At approximately
40°Chydroquinone
is added slowly. The mixture is allowed to cool to room temperature. Citric
Acid is added
to the formulation until the pH is 3.5 to 4Ø
Simple emulsion-hydroquinone formulation
Purified Water 87.2%
Keltrol 0.7%
Glycerin 2.0%
Glydant Plus 0.20%
Arlacel 165 3.0%
Cetyl Alcohol 2.0°/°
Finsolv TN 2.0%
Permethyl 101A 2.0%
Hydroquinone 2.0%
Citric Acid to pH 3.5-4.0
Preparation of Salicyclic Acid Formulations. A washed oil body preparation was
prepared
from safflower as in example 2. To the washed oil body preparation was added:
0.1%
10 Glydant Plus, 0.1% Butylated Hydroxyanisole (BHA) 0.1% Butylated
Hydroxytoluene)
and a salicyclic acid oil body formulation for use in a testing oil body-
associated salicyclic
penetration was prepared as follows. Phase I is the water phase. In this
phase, the water
in the main tank is charged. A propeller is used to hydrate the Keltrol and
Glydant plus
with moderate agitation at room temperature. The glycerin is then added with
continued
15 mixing. The water phase is heated to a final temperature of 76°C to
78°C. Phase II, the oil
phase is mixed in a separate mixing pot with moderate agitation and then
subsequently
heated up to 76 to 78°C. The oil phase ingredients include, Arlacel
165, Cetyl Alcohol,
Finsolv TN, and Permethyl 101A (Isohexadecane}. The final step of
emulsification
includes the addition of the oil phase (Phase II) to the water phase (Phase
I). The two
20 phases are mixed under high agitation with a propeller or homogenizer for
25 minutes.
After 15 minutes of mixing the mixture is cooled slowly to 40°C. The
agitation is decreased
as the temperature decreases. At approximately 40°C hydrated safflower
oil bodies and
salicyclic acid are added slowly. The mixture is allowed to cool to room
temperature.
Citric Acid is added to the formulation until the pH is 3.5 to 4Ø
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Oil body-salicyclic acid formulation
Purified Water 36.1%
Keltrol 0.7%
Glycerin 2.0%
Glydant Plus 0.20%
Arlacel 165 3.0%
Cetyl Alcohol 2.0%
Finsolv TN 2.0%
Permethyl 101A 2.0%
Hydrated Safflower Oil Body (0.1% 50.0%
Glydant Plus, 0.1% BHT, 0.1% BHA}
Salicyclic Acid 2.0%
Citric Acid to pH 3.5-4.0
A simple emulsion salicyclic acid formulation fox use in a testing placebo-
associated
salicyclic acid penetration was prepared as follows. Phase I is the water
phase. In this
phase, the water in the main tank is charged. A propeller is used to hydrate
the Keltrol
and Giydant plus with moderate agitation at room temperature. The glycerin is
then
added with continued mixing. The water phase is heated to a final temperature
of 76°C to
78°C. Phase II, the oil phase is mixed in a separate mixing pot with
moderate agitation
10 and then subsequently heated up to 76 to 78°C. The oiI phase
ingredients include, Arlacel
165, Cetyl Alcohol, Finsolv TN, Permethyl 201A and (Isohexadecane). The final
step of
emulsification includes the addition of the oil phase {Phase II) to the water
phase (Phase
I). The two phases are mixed under high agitation with a propeller or
homogeruzer for 15
minutes. After 15 minutes of mixing the mixture is cooled slowly to
40°C. The agitation is
15 decreased as the temperature decreases. At approximately 40°C
salicyclic acid is added
slowly. The mixture is allowed to cool to room temperature. Citric Acid is
added to the
formulation until the pH is 3.5 to 4Ø
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Simple emulsion-salicyclic acid formulation
Purified Water 85.3%
Keltrol 0.7%
Glycerin 2.0%
Glydant Plus 0.20%
Arlacel 165 3.0%
Cetyl Alcohol 2.0%
Finsolv TN 2.0%
Permethyl 101A 2.0%
Salicyclic acid 2.0%
Citric Acid to pH 3.5-4.0
Preparation of Tretinoin Formulations. A washed oil body preparation was
prepared from
safflower as in example 2. To the washed oil body preparation was added: 0.1%
Glydant
5 Plus, 0.1% Butylated Hydroxyanisole (BHA) 0.1% Butylated Hydroxytoluene
(BHT) and a
tretinoin oil body formulation for use in a testing oil body-associated
tretinoin penetration
was prepared as follows. Phase I is the water phase. In this phase, the water
in the main
tank is charged. A propeller is used to hydrate the Keltrol and Glydant plus
with
moderate agitation at room temperature. The glycerin is then added with
continued
10 mixing. The water phase is heated to a final temperature of 76°C to
78°C. Phase II, the oil
phase is mixed in a separate mixing pot with moderate agitation and then
subsequently
heated up to 76 to 78°C. The oil phase ingredients include, Arlacel
165, Cetyl Alcohol,
Finsolv TN, and Permethyl 101A (Isohexadecane). The final step of
emulsification
includes the addition of the oil phase (Phase II) to the water phase (Phase
I). The two
15 phases are mixed under high agitation with a propeller or homogenizes for
15 minutes.
After 15 minutes of mixing the mixture is cooled slowly to 40°C. The
agitation is decreased
as the temperature decreases. At approximately 40°C hydrated safflower
oil bodies and
txetinoin are added slowly. The mixture is allowed to cool to room
temperature. Citric
Acid is added to the formulation until the pH is 3.5 to 4Ø
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Oil body-tretinoin formulation
Purified Water 38.0%
Keltrol 0.7%
Glycerin 2.0%
Glydant Plus 0.20%
Arlacel 165 3.0%
Cetyl Alcohol . 2.0%
Finsolv TN 2.0%
Permethyl 201A 2.0%
Hydrated Safflower Oil Body (0.1% 50.0%
Glydant Plus, 0.1% BHT, 0.1% BHA)
Tretinoin 0.10%
Citric Acid to pH 3.5-4.0
A simple emulsion tretinoin formulation for use in a testing placebo-
associated
tretinoin penetration was prepared as follows. Phase I is the water phase. In
this phase,
5 the water in the main tank is charged. A propeller is used to hydrate the
Keltrol and
Glydant plus with moderate agitation at room temperature. The glycerin is then
added
with continued mixing. The water phase is heated to a final temperature of
76°C to 78°C.
Phase II, the oil phase is mixed in a separate mixing pot with moderate
agitation and then
subsequently heated up to 76 to 78°C. The oil phase ingredients
include, Arlacel 165, Cetyl
10 Alcohol, Finsolv TN, Permethyl 101A and (Isohexadecane}. The final step of
emulsification includes the addition of the oil phase to the water phase. The
two phases
are mixed under high agitation with a propeller or homogenizer for 15 minutes.
After 15
minutes of mixing the mixture is cooled slowly to 40°C. The agitation
is decreased as the
temperature decreases. At approximately 40°C tretinoin is added slowly.
The mixture is
15 allowed to cool to room temperature. Citric Acid is added to the
formulation until the pH
is 3.5 to 4Ø
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Simple emulsion-tretinoin formulation
Purified Water 85.3%
Keltrol 0.7%
Glycerin 2.0%
Glydant Plus 0.20%
Arlacel 165 3.0%
Cetyl Alcohol . 2.0%
Finsolv TN 2.0%
Permethyl 101A 2.0%
Tretinoin 0.10%
Citric Acid to pH 3.5-4.0
Detection of cosmetic actives in skin samples. Skin-stripping was used to
evaluate the percutaneous absorption of the therapeutic actives across human
skin by
5 removing the individual cellular layers. The formulations were applied to
the arm, which
had been cleaned and left to dry for 10 minutes. Four circles 2 cm in diameter
are marked on
the skin and forty ~tl of product was applied and exposed to open air for 3
hours. After 3
hours, a 2 cm wide semi-transparent 3M piece of tape was applied on the skin
under constant
application pressure and removed with one swift motion. The strip was placed
into a 5 ml
10 screw top tube and 1 ml of methanol was added to the tube, vortexed for one
minute and put
in a rack. The extraction was allowed to proceed for at least 15 minutes. Once
extraction
was complete, an aliquot volume was taken for injection on a HPLC Columbus
250X4.6 mrn
CB column. Concentration of the active ingredient was determined for each
strip by
comparison to the area under the curve of a known standard tested on the same
column.
15 Figure 4 is a histogram representing the cumulative results of 7 strips in
a skin-
stripping assay for formulations containing salicyclic acid, hydroquinone and
tretinoin.
Compared are the oil body formulations containing the active ingredient and a
simple
emulsion placebo containing the active ingredient. The cumulative percentage
recovery of
active ingredient obtained from 7 strips is indicated. As shown in figure 4,
all active
20 ingredients tested (salicyclic acid, hydroquinone and tretinoin) were shown
to have a
higher level of penetration (lower percent recovery) when compared to the
placebo
emulsion formulation with the same active ingredient. In particular, a higher
level of
penetration was observed for the oil body based formulation beyond the stratum
corneum.
CA 02353071 2001-05-24
WO 00130602 PCT/CA99/01138
-36-
TABLE T.
Room Temperature
Time Color Odor Stability Viscosity Microbial
(days) (cps) growth
0 Pale Very mild No 3500 +/- 500
yellow separation100
14 Pale No change No 3500 +/- 300
yellow separation100
25 Pale No change No 3500 +/- <10
yellow separation100
45C
Time Color Odor Stability Viscosity Microbial
(days) (cps) growth
0 Pale Very mild N o 3500 +/- 500
yellow separation100
14 Pale Mild No 4000 +/- <20
yellow separation100
25 Mildly Mild N o 4000 +/- <10
yellow separation100
4C
Time Color Odor Stability Viscosity Microbial
(days) (cps) growth
0 Pale Very mild N o 3500 +/- 500
yellow separation100
14 Pale Very mild N o 3500 +/- 250
yellow separation100
25 Pale Very mild No 3500 +/- <10
yellow separation100
CA 02353071 2001-05-24
WO 00/30b02 PCTlCA99/01138
-37-
TABLE 2
Room Temperature
i
Time Color Odor Stability Viscosity Microbial
(days ) (cps) growth
0 Dark Very mild SeparationApprox <20
yellow 4000
14 Dark Very mild Total Sluggish <20
yellow separation
25 Darker Very mild Total Sluggish <10
yellow separation
45C
Time Color Odor Stability Viscosity Microbial
(days) (cps) growth
0 Dark Neutral SeparationApprox. <20
yellow 4000
14 Brown Amine Total Sluggish <10
odor separation
25 Dark Fishy Total Sluggish <10
brown separation
4C
Time Color Odor Stability Viscosity Microbial
(days) (cps) growth
0 Dark Neutral SeparationApprox. <20
yellow 4000
14 Dark Neutral Total Sluggish <10
yellow separation
25 Dark Neutral Total Sluggish <10
yellow separation
CA 02353071 2001-05-24
WO 00/30602 PCT/CA99/0113$
-38-
TABLE 3
Formulation Score Classification
A 8.8 Mild material - no experimental
irritation
B 56.5 Probably mild in normal use
C 174.2 Possibly mild in normal use
D 160.7 Possibly mild in normal use
E 38.5. Possibly mild in normal use
F 5.4 Mild material - no experimental
irritation
G 377.9 Experimental cumulative irritant
H 373.2 Experimental cumulative irritant
I 156:0 Possibly mild in normal use
J 211.4 Possibly mild in normal use
K 295.1 Possibly mild in normal use
~
L 25.8 Mild material - no experimental
irritation
