Note: Descriptions are shown in the official language in which they were submitted.
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TRANSDERMAL TRANSPORT OF MOLECULES
PRIOR RELATED APPLICATION
The present application is a continuation-in-part
of pending United States patent application serial number
08/685,235 filed on July 23, 1996.
TECHNICAL FIELD
The present invention is related to a composition
and a method for topical application of a composition that
facilitates transdermal delivery of molecules. More
particularly, the present invention is related to the transdermal
delivery of molecules including macromolecules.
BACKGROUND OF THE INVENTION
Delivery of molecules to desired locations in
effective amounts has presented a continuing challenge to
health care professionals. Conventional delivery methods
include oral ~dmini.stration, ~dmini.stration by suppository or
injection, and ~dministration into the cerebrospinal fluid.
However, many of these methods involve potentially serious
side effects such as infection, hematomas, and ~l~m~ge to
nervous and muscular tissue. In addition, these conventional
methods often require the participation of a health care
professional and involve scheduling a visit and traveling to a
medical office. Other problems affect efficacious delivery of
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molecules using these methods. Molecules may be metabolized
in the liver and gastrointestinal system, or excreted through
the kidneys before reaching the desired site of action at the
desired concentration thereby necessitating ~clmini~tration of
higher concentrations of the active ingredients. Furtherrnore,
some conditions and diseases do not respond to orally
~lmini~tered molecules and formulations.
Previous attempts to ~-1mini.cter molecules through
the stratum corneum of the skin have generally been limited by
the size of the molecule since the stratum corneum provides an
effective barrier to penetration. Currently, lipophilic
molecules such as steroids and very small molecules such as
nicotine have been ~(lministered through the skin. What is
needed is a composition and method for transdermally
delivering molecules of desired size and characteristics
through the stratum corneum in close proximity to the desired
site of action so that effective concentrations may be attained
with minim~l deleterious side effects or degradation in the
gastrointestinal system.
For example, many patients with localized pain
due to arthritis, bursitis, sprain or muscle strain, bruises or
hematomas cannot tolerate conventional nonsteroidal anti-
infl~mm~tory drugs, commonly known as NSAIDS. In
addition, topical ~(lministration of conventional NSAIDS has
largely been ineffective because only a therapeutically
ineffective amount of the drug can penetrate the skin. In
addition, conditions such as acne, psoriasis and eczema are
typically refractory to topical or oral ~lmini.stration of
NSAIDS.
Bromelain is a protease composition that is
isolated from pineapple. The composition has been reported to
have anti-infl~mm~tory activity when ~lministered orally or
parenterally (see Taussig, S.J., "The mechanism of the
physiological action of Bromelain", Medical Hypotheses, 6; 99-
104, 1980). Commercially available bromelain used in the
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manufacture of pharmaceuticals is not a chemically
homogeneous substance, but the principal component is a
proteolytic enzyme that is a glycoprotein. The molecular
weight of bromelain is approximately 33,000 Daltons.
Capsaicin is an oleoresin obtained by extracting
cayenne pepper with ether. The synthetic capsaicin is trans-8-
methyl-vanillyl-6-nonenamide. Capsaicin gels have been
described in U.S. Patent No. 5,178,879 as being effective in
treating topical pain. In addition, capsaicin is available
commercially in over-the-counter compositions intended for
pain relief including lotions such as HEET, OMEG~ OIL,
SLOAN'S LINIMENT and ZOSTRIX.
Bromelain has been reported to be an anti-
infl~mnl~tory agent, an inhibitor of platelet aggregation, an
s agent that increases proteolytic and fibrinolytic activity in
blood, and a selective prostaglandin inhibitor. Bromelain has
been ~clmini.etered by injection and has been reported to be
effective after oral administration. However, because
bromelain is a relatively large molecule, it cannot be
a-lmini.stered transdermally using prior art formulations.
Accordingly, what is needed in this example is a composition
that will ef~ectively transport molecules, such as bromelain,
that are effective in treating a wide variety of infl~mm~tory
conditions by topical application of the composition.
2s Other conditions such as autoimmune diseases that
affect the connective tissue and joints could be treated by
transdermal delivery of molecules. Alleviation of the pain
associated with these conditions and inhibition of infl~mm~tory
processes would be of great benefit to these patients.
What is needed is a composition and method for
transdermally delivering molecules of desired size and
characteristics through the stratum corneum so that effective
concentrations may be attained with minim~l deleterious side
effects or degradation in the gastrointestinal system.
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BRIEF SUMMARY OF THE INVENTION
The present invention addresses the needs in the
prior art by providing a composition and method for
delivering molecules of desired size through the stratum
s corneum and into the dermis, the hypodermis, or adjacent
connective tissue and joints. The present invention also
provides a composition and method of transdermal delivery
into the bloodstream for systemic action. The present
invention is particularly useful for delivering molecules of
desired size into the germinal epithelium and nail bed. The
present invention provides novel compositions of penetrating
agents that permit transdermal delivery of molecules,
including macromolecules such as enzymes, and
immunoglobulins, after simple topical application to the skin.
This novel approach provides a method for treating a variety
of conditions without the need for injection or oral
~lmini~tration of molecules.
The present invention addresses the needs in the
prior art by providing, in one embodiment, an anti-
infl~mm~tory composition containing a proteolytic enzyme,
preferably bromelain, and capsaicin. The anti-infl~mm~tory
composition is capable of being :-~1mini.~tered transdermally to
a human or ~nim~l. The present invention is also a topical
anti-infl~mm~tory composition comprising an effective amount
2s of bromelain, an effective amount of capsaicin, and a
pharmaceutically effective penetrating agent. Preferred
penetrating agents are effective amounts of lecithin organogel,
phospholipid gels, and/or poloxamer organogel which are
optionally combined with n-decylmethyl sulfoxide. The
preferred penetration enhancers are isopropyl palmitate,
isopropyl myristate, poloxamer phospholipid gel, ethanol, n-
decylmethylsulfoxide [NDMS], and ethoxydiglycol.
In one embodiment, the present invention
provides an easy and safe method of ~clmini.~tering an effective
anti-infl~mm~tory macromolecule that, in the prior art, could
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s
only be ~-1ministered parenterally or orally. By ~mini.~tering
the composition topically, directly at the site of the
infl~mm~qtion, the present invention provides a more effective
means of treating the infl~mm~tion The present invention is
useful for treating a variety of infl~mmatory conditions which
may be painful conditions including neuralgia, myalgia,
rheumatoid arthritis, osteoarthritis, sprains, strains, bursitis,
myositis, tendonitis, carpal tunnel syndrome, chondromalacia,
eczema, infl~mm~tion due to infections by microorg~ni~m~,
o bites, acne, dermatitis, thrombi, phlebitis, hematoma, atopy,
psoriasis, and integumental pain.
The present invention provides a composition and
method for the treatment of various conditions, including but
not limited to conditions wherein the condition is pain, or
deficiencies or imbalances of the integumentary system,
immune system, endocrine system, reproductive system,
cardiovascular system, musculoskeletal system, nervous
system, digestive system, and respiratory system.
The present invention provides a composition and
method for the treatment of various conditions, including but
not limited to fungal conditions. Such fungal conditions
include, but are not limited to conditions such as onchomycosis
and other fungal diseases of the skin and scalp.
The present invention provides a composition and
method for the treatment of the effects of sun exposure on the
skin, including the condition of solar elastosis.
In addition, the present invention provides an
alternative composition and method to conventional NSAIDS
for treating infl~mm~tion. The present composition does not
have the side effects that are often seen with NSAIDS. Some
of these side effects include, but are not limited to,
somnolence, confusion, gastric upset, gastrointestinal blee~ling,
chondrocyte dysfunction and kidney damage.
In one embodiment, the composition of the
present invention comprises bromelain and capsaicin in a
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pharmaceutically acceptable penetrating composition optionally
containing n-decylmethyl sulfoxide or cholesterol. The
present invention can include other pharmaceutically
acceptable components such as gelling agents, compounding
s agents, scents and the like. In especially preferred
formulations, the composition further contains a lecithin
organogel. In other preferred formulations, the composition
further contains phospholipids such as phosphatidylcholine,
also known as lecithin, phospholipid gels, or a poloxamer
o organogel. The combination of a transdermal formulation of
bromelain and capsaicin act synergistically to reduce pain.
The composition of the present invention can also include
other pharmaceutically active agents such as antibacterial,
anticancer, antihelminthic, antifungal, antiprotozoal or
antiviral agents.
The present invention also includes methods for
topically treating inflAmm~tion due to a wide variety of causes
comprising the step of topically ~lmini~tering a therapeutically
effective amount of a composition comprising bromelain and
capsaicin in a pharm~ce~ltically acceptable vehicle. The vehicle
may include lecithin organogel, phospholipids, poloxamer
lecithin organogel, optionally combined with n-decylmethyl
sulfoxide or cholesterol, and/or ethanol.
Accordingly, it is an object of the present
invention to provide a composition and method for facilitating
transdermal transport of large molecules.
Another object of the present invention is to
provide a composition and method for facilitating transdermal
transport of molecules. Included in this category are
molecules as large as 500,000 Daltons as well as dimers,
trimers and tetramers of these molecules, although the present
invention encompasses transport of larger molecules and
constructs such as plasmids. The molecules include, but are
not limited to the following; enzymes including but not limited
to antiinfl~mm~tory enzymes, proteolytic enzymes and
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-
elastase, enzyme inhibitors, including but not limited to
protease inhibitors, coenzymes, proteins, receptors, peptides,
amino acids, hormones, hormone agonists and antagonists,
interleukins, immunoglobulins, antibodies, cytokines,
monokines, immunosuppressants, immunomodulators, drugs,
growth factors including epidermal growth factor, vit~min~,
plant extracts, lipids, plasmids, nucleic acids, including but not
limited to ribonucleic acids and deoxyribonucleic acids,
nucleotides, neurotransmitters, neurotransmitter receptor
o agonists and antagonists, mitotic inhibitors, steroids, lipids,
fatty acids, and carbohydrates.
Other molecules and compounds that may be
transported with the composition and method of the present
invention include, but are not limited to the following: skin
]s moisturizers (for example alpha hydroxyacids, etc.),
ultraviolet blockers, protease inhibitors, inhibitors of pain
transmission, vasodilators, vasoconstrictors, antibiotics,
antifungal drugs, antiprotozoal drugs, antihelminthic drugs,
antibacterial drugs, antiviral agents, and anticancer drugs,
including drugs that affect nucleotides and DNA replication,
drugs that affect transcription and/or translation, inhibitors of
signal transduction systems, and stimulators of signal
transduction systems.
It is another object of the present invention to
provide a composition and method for topically treating
conditions in patients.
Another object of the present invention is to
provide a composition and method for topically treating
painful conditions in patients.
Yet another object of the present invention to
provide a composition and method for topically treating
infl~mm~tory conditions.
It is another object of the present invention to
provide a composition and method for topically treating
arthritis.
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It is yet another object of the present invention to
provide a composition and also a method for topically ~reating
bursitis.
It is yet another object of the present invention to
provide a composition and method for treating acne, psoriasis
and eczema.
It is yet another object of the present invention to
provide a composition and method for treating flea allergy,
insect bites, dermatitis, thrombi, phlebitis, hematoma, and
atopy.
It is yet another object of the present invention to
provide a composition and method for topically treating
infl~mm~tion that is safe and effective and does not have the
side effects of conventional NSAIDS.
s Another object of the present invention to provide
a composition and method for topically treating fungal
conditions.
Yet another object of the present invention is to
provide a composition and method for treating solar elastosis.
It is a specific object of the present invention to
provide a composition and method for the topical treatment of
fungal disease of the skin and scalp.
It is another specific object of the present
invention to provide a composition and method for the topical
treatment of onchomycosis of the toenail and fingernail.
These and other objects, features and advantages
of the present invention will become apparent after a review of
the following detailed description of the disclosed
embodiments and the appended claims.
DETAILED DESCRIPTION
The present invention provides a composition
comprising a pharmaceutically effective penetrating agent, and
a method for transdermally ~1mini.~tering molecules. The
composition and method or the present invention may be used
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to treat numerous conditions by transdermally delivering
desired molecules. The term "condition" means any biological
state of a patient. Conditions may include numerous biological
states including, but not limited to, deficiencies or imbalances
s of the following systems: immune, endocrine, reproductive,
integumentary, cardiovascular, musculoskeletal, nervous,
digestive, and respiratory. Conditions include other biological
states including, but not limited to, painful conditions,
infl~mm~tory conditions, fungal conditions including fungal
o disease of the skin and scalp, and specifically onchomycosis.
Conditions may include biological states of
different tissues including, but not limited to, the following:
connective, muscle, nervous, skeletal, lymphoreticular,
cutaneous, endocrine, and exocrine. Conditions may also refer
to the need for a particular treatment with medicines, such as a
vaccination, a hormone supplementation, an anti-infl~mm~tory
regimen, an antifungal regimen, and/or pain medication.
The term "patient", as used herein, means any
human or ~nim~h The term "pharmaceutically effective
penetrating agent" means n-decylmethyl sulfoxide (NDMS),
lecithin organogel, phospholipids gels, cholesterol with or
without ethanol, ethoxy diglycol, poloxamer organogel,
poloxamer phospholipid gel and poloxamer organogels in
combination with phospholipids. The term "an effective
amount of a pharmaceutically effective penetrating agent"
means that amount of the pharmaceutically effective
penetrating agent that is capable of transdermally transporting
a molecule. More specifically, in the case of onchomycosis,
the term "an effective amount of a pharmaceutically effective
penetrating agent" means that the pharmaceutically effective
penetrating agent is capable of bringing therapeutic levels of
antifungal to the germinal epithelium as well as to the nail bed.
The term "topical administration" means
application to the surface of the skin. Topical ~dmini~tration
may take many forms including, but not limited to, gels,
. . .
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creams, sprays, rinses, ointments, salves, balms, liposomes,
time release vehicles, micelles, and skin patches or other forms
of pads.
The term "phospholipids" is used to mean water-
s insoluble biomolecules that are highly soluble in organic
solvents. Phospholipids are generally derived from glycerol
and consist of a glycerol backbone, two fatty acid chains, and a
phosphorylated alcohol. Phospholipids, either singularly, or in
combination with other phospholipids can form gels capable of
o transdermal delivery. As utilized in this invention,
phospholipids can be used individually and do not have to be
combined with other components such as lecithin organogel or
poloxamer organogel. In another embodiment of the present
invention, phospholipids may be used in combination with
lS lecithin organogel and/or poloxamer organogel.
Phospholipids may also be optionally combined with
n-decylmethyl sulfoxide (NDMS), ethoxy diglycol, cholesterol
and/or ethanol.
A preferred phospholipid for use in the present
invention is phosphatidylcholine, also known as lecithin.
Stedman's medical dictionary ~21st ed., page 879] defines
lecithin as any of a group of phospholipids which upon
hydrolysis yield two fatty acid molecules and a molecule each
of glycerophosphoric acid and choline. There are several
varieties of lecithin. Lecithin is a mixture of the diglycerides
of stearic, palmitic, and oleic acids, linked to the choline ester
of phosphoric acid. Soybean lecithin is a preferred lecithin
and may contain the following acids; palmitic, stearic,
palmitoleic, oleic, linoleic, linolenic and arachidonic. In some
lecithins both fatty acids are saturated while others contain
only unsaturated fatty acids for example, oleic, linoleic or
arachidonic. In other lecithins one fatty acid is saturated, the
other unsaturated. Lecithins are found in nervous tissue,
cardiac tissue, in egg yolks and in soy which constitutes the
most common and economical source of phosphatidylcholine.
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11
It is therefore to be understood that any reference herein to
lecithin or phosphatidylcholine is intended to include any
combination of lecithin-like phospholipid compounds as is well
known in the art. F.x~mples of other phospholipids which can
s be used in accordance with the present invention include
phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, and phosphatidic acid. A mixture of any
of the above phospholipids may be also be used in the present
invention.
o The term ~PL U R O N I c " refers to poloxamer
compounds and are sold collectively under the trademark
PLURONIC (BASF, Parsippany, NJ). PLURONIC F- 127
(PL 127) corresponds to poloxamer 407, a polyoxypropylene-
polyoxyethylene block copolymer described by Schmolka in
the Journal of Biomedical Materials Research 6:571-582, 1972.
Other PLURONICS may be used in the present invention. As
used in this application, the terms PLURONIC organogel,
poloxamer organogel, and polyoxyethylene/polyoxypropylene
organogel are synonymous. As used in this application, the
term PLURONIC phospholipid gel and poloxamer phospholipid
gel are synonymous.
The "enhanced penetration" caused by
compositions of this invention as used in topical application
with this method, means increased penetration into the skin,
and is achieved with compounds such as lecithin organogel,
poloxamer organogel, phospholipid gels or poloxamer
phospholipid gels including but not limited to
phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, and phosphatidic acid and
phosphatidylcholine optionally combined with n-decylmethyl
sul~oxide (NDMS), PLURONIC F127, ethoxy diglycol, ethanol,
or cholesterol. Enhanced penetration can be observed in many
ways known to those skilled in the art.
The present invention is a composition and
3S method for transdermally transporting molecules to treat
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conditions in patients. As defined above, these conditions
include numerous biological states. The pharmaceutically
effective penetrating agents of the present invention transport
different molecules across the stratum corneum and into the
s lower layers of the epidermis, into the dermis, the hypodermis
and into the bloodstream, lymphatic circulation, and adjacent
connective tissue and joints. The pharmaceutically effective
penetrating agents of the present invention facilitate access of
the molecules to the nervous system. In this manner, for
o example, transdermally transported molecules, such as
calcitonin gene-related peptide, inhibitors of substance P,
opiate agonists or various types of anesthesia may affect
peripheral nerve endings for modulation of pain trAn~mission
in primary sensory afferents. The pharmaceutically effective
penetrating agents of the present invention also facilitate access
of therapeutic substances, such as antifungal agents, to the
germinal level as well as to the nail bed.
The present invention is a composition and
method for topically treating conditions such as inflAmm~tion
caused by a wide variety of causes including, but not limited
to, arthritis, bursitis, phlebitis, sprains, muscle strains, bruises,
phlebitis, flea allergy, bites, phlebitis, atopy, and hematomas.
In one embodiment, the present invention is a composition
containing bromelain and capsaicin. The composition further
2s comprises lecithin organogel, and/or poloxamer organogel
and/or phospholipids optionally combined with NDMS or
cholesterol, as agents to increase the transport of molecules
across the skin.
In one embodiment, proteolytic enzymes can be
transported through the use of the present invention, including
but not limited to, bromelain, collagenase, gel~tinA~e, trypsin,
chymotrypsin, papain and elastase. In one embodiment, the
present invention comprises bromelain (PCAA, Kinghurst,
Houston, TX) at a concentration of between approximately
0.5 % and 40% by weight, with a preferred concentration of
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13
between approximately 3% and 25% by weight, with the most
preferred concentration of approximately 7.5% by weight.
The anti-infl~tnm~tory composition of the present
invention also optionally contains capsaicin. The capsaicin can
s be either naturally occurring capsaicin oleo resin (PCAA,
Kinghurst, Houston, TX) which is commonly extracted from
cayenne pepper with an organic solvent such as ether or can be
synthetic capsaicin, trans-8-methyl-vanillyl-6-nonenamide
(PCAA Kinghurst, Houston, TX). The capsaicin is present in
the transdermal anti-infl~mm~tory of the present invention at a
concentration of between approximately 0.01% and 0.5% by
weight, with a preferred concentration of between
approximately 0.1% and 0.35% by weight, with the most
preferred concentration of approximately 0.25% by weight.
Other molecules which affect pain transmission are considered
within the scope of this invention. Some of these molecules
include but are not limited to, anesthetics, analgesics,
ganglionic blockers, receptor blockers, receptor antagonists
and agonists, enzymes and enzyme inhibitors, catecholamines,
indoleamines such as histamine, serotonin and related analogs,
molecules that affect neurotransmitter reuptake mechanisms,
peptides and peptide analogs such as those related to calcitonin
gene-related peptide, substance P, bradykinin, opiate agonists
and antagonists, and steroids.
The present invention also includes a
pharmaceutically acceptable penetrating agent. A preferred
penetrating agent is NDMS used in combination with lecithin
organogel, phospholipid gels, poloxamer organogel, and
poloxamer organogel combined with phospholipid gels.
NDMS has been described as an agent that is useful in
facilitating the delivery of small molecules transdermally (see
Hoo-Kyun, C., et al. "Transdermal delivery of bioactive
peptides: The Effect of n-Decylmethyl Sulfoxide, pH, and
Inhibitors on Enkephalin Metabolism and Transport", Pharm.
3s Res., Vol. 7, No. 11, pgs. 1099-1106, 1990; Hoo-Kyun, C., et
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14
al. "Some General Influences of n-Decylmethyl Sulfoxide on
the Permeation of Drugs Across Hairless Mouse Skin", Soc.
Invest. Derm., Vol. 96, pgs. 822-826, 1991; and Smith, E.W.,
Maibach, H. eds., Percutaneous Penetration Fnh~ncers, CRC
s Press, pg. 109, 1995; which are incorporated herein by
reference). However, there is nothing in the literature known
to the inventor that would indicate that NDMS alone would be
effective in facilitating the transport of macromolecules, such
as bromelain, across the skin. NDMS must be used in
o conjunction with poloxamer organogel, phospholipids, or
lecithin organogel. Although not wanting to be bound by this
statement, it is believed that NDMS enhances perrneation of the
skin by affecting the barrier nature of the stratum corneum.
NDMS (PCAA Kinghurst, Houston, TX) is
present in the transdermal anti-infl~mm~ory composition of
the present invention at a concentration of between
approximately 0.1% and 1% by weight, with a preferred
concentration of between approximately 0.15% and 0.8% by
weight, with the most preferred concentration of
approximately 0.5% by weight. NDMS is dissolved in 10 mL
of a 75% solution of ethanol. Finally, purified water is added.
Ethanol (98%) may also be used to dissolve lecithin and then
either boiled o~f completely or partially to leave a final ethanol
concentration of 3% to 8.5%. While not wanting to be bound
by the following statement, it is believed that 3% to 8.5%
ethanol may enhance penetration.
Another preferred penetrating agent and delivery
vehicle is lecithin organogel which is a combination of lecithin,
isopropyl palmitate, or isopropyl myristate and/or ethanol and
water. Lecithin organogels have been described as vehicles
that are useful in facilitating the delivery of low molecular
weight compounds transdermally (Willim~nn, H., et al.,
"Lecithin Organogel as Matrix for Transdermal Transport of
Drugs", J. Pharm. Sci., Vol. 81, 1992, which is incorporated
herein by reference). The lecithin organogels are obtained by
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adding small amounts of water to a solution of lecithin in
organic solvents and/or ethanol. Generally, lecithin
organogels are prepared at room temperature by first
dissolving lecithin in an organic solvent such as isopropyl
palmitate or isopropyl myristate and then ~d-lin,~ enough water
while stirring to obtain the desired gel. Lecithin used in the
gel preparations of the present invention generally contain at
least 95% phosphatidylcholine.
Solvents used in the preparation of a variety of
o gels, including lecithin gels, all of which are appropriate in
practicing the present invention, are described in Scart~77ini,
et al. Journal of Physical Chemistry 92:829-833, 1988, and
Luisi, P.L. et al. Colloid and Polymer Science 268:356-374,
1990, both of which are incorporated herein by reference in
their entirety. Specifically these solvents include the
following: ethyl laurate, butyl laurate, ethyl myristate,
isopropyl myristate, isopropyl palmitate, isooctane,
cyclooctane, cyclododecane, methyl cyclohexane, tert-
butylcyclohexane, phenylcyclohexane, bicyclohexyl, 1,3,5-
triisopropylbenzene, octylbenzene, trans-decalin, (lR)-(+)-
trans-pinane, (lR)-(+)-cis-pinane, n-pentane, n-hexane, n-
heptane, n-octane, n-nonane, n-decane, n-undecane, n-
dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-
hexadecane, n-hept~(lec~ne, 2,3-dimethylbutene, l-hexene, 1,7-
octadiene, tripropyllamine, tributylamine, triisobutylamine,
trioctylamine, dibutyl ether and 2-dodecen- 1 -yl succinic
anhydride.
In addition to isopropyl palmitate and isopropyl
myristate, other solvents may be used in the practice of the
present invention. These solvents include, but are not limited
to the following: mineral spirits, kerosene, isooctane,
petroleum ether, diethyl ether, benzene, toluene, methanol,
ethanol, heptanol, methyl isobutyl ketone, cyclohexanone,
methylene chloride, choloroform, chlorodifluoromethane,
tetrahydrofuran, oleyoleate, 2-octyldodecanol, cetyl and
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16
stearyl 2-ethylhexanoate, n-octanol, ethyl laurate, isooctane,
cyclopentane, cyclohexane, and cycloheptane.
In a preferred embodiment, lecithin organogel
may be made from PHOSPHOLIPON 90 (American Lecithin
C O., Oxford, CT). In this embodiment, lecithin organogel
comprises 1:1 to 1.5:5 (weight/vol) of PHOSPHOLIPON 90 to
isopropyl palmitate. Water is added to form the desired gel.
Other penetrating agents including, but not limited to
cholesterol (2% to 100%) with a preferred range of
cholesterol to PHOSPHOLIPON 90 of 3:7 to 3:10. These
ingredients are combined with sufficient ethanol to solubilize
the mixture. Ethanol is subsequently evaporated, leaving a
complex of cholesterol:pHospHoLIpoN 90. Alternatively,
3.5% - 8% ethanol may be retained in the complex to enhance
penetration.
Willim~nn et al., Journal of Pharmaceutical
Sciences 81:871-874, 1992, examined the efficacy of lecithin
organogels for use in the transdermal delivery of drugs such as
scopolamine and broxaterol. Willim~nn et al., also observed
that lecithin organogels had no detrimental effect on skin when
compared to control samples treated with physiological saline
(see page 872, Journal of Pharmaceutical Sciences 81:871-874,
1992).
- It is to be understood that the soy lecithin of the
present invention is a preferred lecithin source and a preferred
source of phosphatidylcholine. However it is to be understood
that lecithin may be obtained from other sources. Lecithin and
may be dissolved in isopropyl palmitate or isopropyl myristate
(gm/gm) to achieve a final concentration in the composition of
from approximately 10% - 98%, with a more preferred final
concentration of from approximately 20% - 50%. Lecithin at
98% is dissolved gram per gram of isopropyl palmitate to
yield a 49% solution of lecithin in isopropyl palmitate.
Lecithins may optionally be derived from eggs, and organs
such as heart, brain, and liver, and used at concentrations of
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17
approximately 10% - 100%, with more preferred final
concentrations of from approximately 10% - 50% when
dissolved in isopropyl palmitate, isopropyl myristate or
ethanol. When PLURONIC F-127 or another poloxamer is not
s used in combination with lecithin organogel, a range of final
concentrations of lecithin in the organogel is about 0.5% -
98% and the amount of water in the composition is varied
accordingly. For example, 95% phosphatidylcholine dissolved
in equal amounts of isopropyl palmitate or isopropyl myristate
o may be diluted with 10% to 30% water to produce a final
phosphatidylcholine concentration of from 12.5% to 32.5%.
Alternatively, 100~o phosphatidylcholine may be dissolved 1:1
in isopropyl palmitate or isopropyl myristate followed by
dilution with 10% to 30% water to produce a final
lS phosphatidylcholine concentration of from 20% to 40%. In
yet another embodiment, 100% phosphatidylcholine may be
dissolved in 98% ethanol which is then boiled off before
addition of 10% to 30% water to produce a final
phosphatidylcholine concentration of 70% to 90%. The use of
PLURONIC 127 will further reduce the phosphatidylcholine
content of the gel by a factor of 3 to 4 depending on the
amount of PLURONIC 127 added to the mixture.
Another penetrating agent of the present invention
includes lecithin dissolved in isopropyl palmitate or another
solvent in combination with a final concentration of
approximately 0.1% - 45% of PLURONIC F-127 (BASF,
Parsippany, NJ), otherwise known as poloxamer 407, in a ratio
of approximately 1:2 to 1:4. A preferred final concentration
of PLURONIC F-127 is 5% to 20%. The lecithin dissolved in
isopropyl palmitate is added to 3 to 4 parts PLURONIC F-127
(for example, 20 ml lecithin in isopropyl palmitate plus 60-80
ml PLURONIC 127). Water, or any other agent known in the
art may be added to effect a gel. Other PLURONICS may be
used in the present invention. This mixture is called
PLURONIC organogel or poloxamer organogel (PLO).
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18
Other penetrating agents of the present invention
associated with lecithin are phospholipids. Phospholipids that
may be used in the present invention include, but are not
limited to the following: phosphatidylethanolamine;
s phosphatidylcholineserine; dipalmitoylphosphatidyl choline;
dimyristoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycerol
phosphocholine; 1,2-dimyristoyl-sn-gly(3)phosphoglycerol;
PHOSPHOLIPON 80TM 3-sn-phosphatidylcholine soya;
PHOSPHOLIPON 90/90 G TM 3-sn-phosphatidylcholine soya;
o PHOSPHOLIPON 90 HTM 3-sn-phosphatidylcholine soya
hydrogenated; PHOSPHOLIPON CCTM 1,2-dicaproyl-sn-
glycero (3) phosphocholine; PHOSPHOLIPON LCTM 1,2-
dilauroyl sn-glycero (3) phosphocholine; PHOSPHOLIPON
MCTM 1,2-dimyristoyl-sn-glycero (3) phosphocholine;
lS PHOSPHOLIPON PCTM dipalmitoyl-sn-glycero (3)
phosphocholine; PHOSPHOLIPON SCTM 1,2-distearoyl-sn-
glycero (3) phosphocholine; PHOSPHOLIPON MGTM 1,2-
dimyristoyl-sn-glycero (3) phosphoglycerol Na salt;
PHOSPHOLIPON PGTM 1,2-dipalmitoyl-sn-glycero (3)
phosphoglycerol Na salt; PHOSPHOLIPON SGTM 1,2-
distearoyl-sn-glycero (3) phosphoglycerol Na salt;
PHOSPHOLIPON GTM 3-(3-sn-phosphatidyl) glycerol soya;
and PHOSPHOLIPON GHTM 3-(3-sn-phosphatidyl) glycerol
soya hydrogenated. These PHOSPHOLIPON products are
available from American Lecithin Co., Oxford, Connecticut.
Phospholipid gels are made by dissolving the
desired phospholipid in any appropriate organic solvent.
Organic solvents which may be used include but are not
limited to methanol: chloroform (2:1 vol:vol) isopropyl
palmitate, isopropyl myristate or ethanol. After the
phospholipid is dissolved, 2 to 4 parts of PLURONIC 127 is
added. Water may be added in an amount required to obtain
the desired consistency. The organic solvents used for }ecithin
organogels, such as isopropyl palmitate and isopropyl
myristate and others described in the present application are
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19
also useful for preparation of gels containing natural and
synthetic phospholipids.
The composition according to the present
invention can be in the form of lotions, salves, creams,
s ointments, liposomes, micelles, sprays, gels, or ~(lmini.~tered in
a pad or patch. The desired form is lotions, ointments and
salves. Preferred embodiments include lecithin organogels,
PLURON~C organogels, and phospholipid PLURONIC gels.
A gelling agent optionally may be added to the
o formulation. Gelling agents that are suitable for use in the
present invention include, but are not limited to,
carboxycellulose, alginates, polyacrylates, bentonite, gelatin,
tragacanth, polyvinylpyrrolidone, polyvinyl alcohol, and
polyoxyethylene/polyoxypropylene block copolymers.
s A preservative, such as benzyl alcohol
(concentration range of 0.05-5.0%, with a preferred
concentration of 2.5%) or potassium sorbate, may be added to
the composition. An antioxidant such as vitamin E tocopherol,
proanthocy~nillin~, ascorbyl palmitate, ascorbic acid, or lipoic
acid may be added to lecithin organogels. Other preservatives
well known to those of ordinary skill in the art can be used in
the composition, for example, 0.1% butylated hydroxide
toluene (BHT), or 0.1% butylated hydroxide anisole (BTA).
Other preservatives well known to those of ordinary skill in
the art can be used in the composition.
Agents for improving the aroma of the
formulation, defined herein as scents, can optionally be added
to the composition. A desired aroma improving agent is honey
almond oil (PCAA). Other aroma improving agents include,
but are not limited to, avocado oil, sesame oil, castor oil, olive
oil, grapeseed oil, clove oil, groundnut oil, corn oil, lemon oil,
coconut oil, lime oil, hazelnut oil, jojoba oil, carth~ml~s oil and
wheatgerm oil. The oils can be added individually or in
combination. It is to be understood that various fragrances
3S and assorted floral scents may be optionally added to the
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composition and are commercially available (PCAA).
Stabilizers, antioxidants, preservatives, humectants, regreasing
agents, solvents or auxiliaries can be added to improve stability
and/or adhesiveness of the formulations. Cosmetic agents such
s as panthenol may also optionally be added to the formnl~tion.
The method of the present invention includes
topical ~dministration of a pharmaceutically acceptable
composition containing molecules in combination with a
penetrating agent consisting of either lecithin organogel or
o poloxamer organogel optionally combined with phospholipid
gels, any of which may be optionally combined with NDMS,
cholesterol or ethanol. It is to be understood that either
lecithin organogel, phospholipids or poloxamer organogel
and/or cholesterol may be optionally combined with NDMS to
act as penetrating agents. In one embodiment, lecithin
organogel, phospholipids and poloxamer organogel and/or
cholesterol may be used optionally in combination with NDMS
to act as penetrating agents. The composition of the present
invention can be ~dministered topically either once daily or
several times per day depending upon the nature and severity
of the condition being treated. In another embodiment
bromelain and capsaicin may be combined with NDMS and/or
lecithin organogel, phospholipids and/or poloxamer organogel
for the treatment of infl~mm~tion. The anti-infl~mm~tory
composition of the present invention can be A-lmini~tered
topically either once daily or several times per day depending
upon the nature and severity of the infl~mm~tory condition
being treated.
Preferably, the anti-infl~mm~tory composition of
the present invention is applied topically at the site of the
infl~mm~tion. For example, for osteoarthritis of the knee, the
anti-infl~mm~tory composition of the present invention is
applied topically around the knee by rubbing the composition
on the skin. Typically, the anti-infl~mm~tory composition of
the present invention is applied in widths of approximately 1.5
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cm, 2.5 cm or 3.5 cm. If the composition is applied to a
painful joint, it can be applied directly on one side of the joint
or can be evenly rubbed around the entire joint. If severe
pain exists, it is helpful to apply an equal amount of the anti-
s infl~mm~tory composition of the present invention on the
anterior and posterior surface between 1 and 4 times daily for
2 weeks. The anti-infl~rnm~tory composition of the present
invention should be applied until the pain subsides. The
amount of the composition that ig applied to the skin is not
o critical to the invention. It is important that the composition
be thoroughly rubbed into the skin.
Although not wanting to be bound by the
following hypothesis, it is believed that the composition of the
present invention causes at least a portion of a molecule, for
example bromelain, to be transdermally delivered to the site of
the infl~mm~tion. In this manner, bromelain can exert its anti-
infl~mm~tory effect at the site of infl~mm~tion. In addition,
the present invention causes more capsaicin to be delivered to
the infl~rnmAtory site thereby relieving pain presumably by
inhibiting release of substance P.
While not wanting to be bound by the following
statement, it is believed that bromelain may increase levels of
cyclic AMP which may cause a down-regulation of
interleukin-1. This reduction in interleukin-l may affect the
processing of hapten by Langerhans cells in the skin. While
not wanting to be bound by the following statement, it is
believed that reduction of interleukin- 1 can decrease
production of collagenase and prostaglandin by synovial
fibroblasts and articular chondrocytes, as well as production of
interleukin 8. It is also believed that the elevation of cyclic
AMP in psoriasis helps to stabilize the activity of keratinocytes
by down regulating colony stimulating factor and
interleukin- 1 .
A variety of other molecules may be ~lmini~tered
for transdermal transport using the composition and method of
-
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the present invention. The molecules include, but are not
limited to molecules that may be classified in the following
categories; enzymes, coenzymes, protease inhibitors, proteins,
peptides, amino acids, hormones, growth factors, interleukins,
s immunoglobulins, cytokines, monokines, drugs, vit~min.~, plant
extracts, lipids, plasmids, nucleic acids, including but not
limited to ribonucleic acids and deoxyribonucleic acids,
nucleotides, neurotransmitters, neurotransmitter agonists and
antagonists, steroids, lipids, fatty acids, carbohydrates and
o antifungal agents. As used herein, the word "molecule" is used
to describe any compound or substance, such as those
compounds or substances that fall into the categories described
in this paragraph. The word "molecule" is not limited to a
single molecule or to any number of molecules.
s The present invention and method permits the
transdermal transport of the different types of molecules
recited above for the treatment of a wide variety of conditions.
Some of these conditions include but are not limited to the
following; tendonitis, desmitis, bursitis, thrombophlebitis,
cellulitis, muscular infl~mm~tion, myalgia, arthritis including
osteoarthritis and rheumatoid arthritis, synovitis, carpal tunnel
syndrome, venomous and non-venomous insect bites,
onchomycosis of the toenail and fingernail, fungal diseases of
the skin and scalp, and joint strain and sprain. For example,
autoimmune phenomena in joints could be treated with
~ntiinfl~mm~tory agents such as polyclonal or monoclonal
antibodies designed to bind autoimmune antibodies. A variety
of immunomodulatory molecules may be transdermally
transported with the present invention including but no~
limited to interleukins, immunoglobulins, cytokines, and
monokines. The effective amount of molecule selected to treat
a particular condition will depend on the specific condition
being treated.
In another embodiment, the present invention may
be placed in the form of a skin patch or ointment for use to
CA 02262009 1999-01-22
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transdermally transport one or multiple antigens used for
immuni7~tion, thereby avoiding the use of injections and
needles. In a di~rele.lt embodiment a skin patch may be placed
on the lower back, neck, shoulder or other site for relief of
s pain. In a different embodiment, non-steroidal hormones such
as natural or recombinant pituitary hormones or hypothalamic
releasing and inhibiting factors may be transdermally
transported with the present invention to affect a variety of
conditions, including but not limited to growth, reproduction,
inflammation, metabolism, bone metabolism, electrolyte
balance, water balance, glucose homeostasis, diabetes,
production of blood cells, and hypertension.
In still another embodiment, the present invention
may be used to transport growth factors, including but not
S limited to, fibroblast growth factor, epidermal growth factor,
nerve growth factor, and growth factors that affect
granulocytes, macrophages, and reticulocytes.
It will be appreciated that other embodiments and
uses will be apparent to those skilled in the art and that the
invention is not limited to these specific illustrative examples.
Example I
Bromelain-Capsaicin Topical Composition
A bromelain-capsaicin topical composition
(Bromelain 7.5%/PLURONIC organogel) is prepared as
follows:
Bromelain Powder 7.5 g
Capsaicin 25 mg
Honey Almond Oil 2 mL
Lecithin-Isopropyl Palmitate 20 mL
20% PLURONIC Mixture 80 mL
Lecithin was prepared by dissolving 10 g of soy
lecithin granules (PCAA, Houston TX) in 10 mL of isopropyl
palmitate. 1% benzyl alcohol was added as a preservative.
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24
The mixture was stirred periodically for 24 hours until the soy
lecithin dissolved. The 20% PLURONIC mixture was prepared
by dissolving 16 g PLURONIC(~) 127 (BASF, Parsippany,
N.J.) in 80 mL of distilled water. Then PLURONIC organogel
s was made by adding the 20% PLURONIC mixture to the
lecithin-isopropyl palmitate mixture and stirring.
The Bromelain 7.5%/PLURONIC organogel was
prepared by mixing the bromelain powder with the lecithin
organogel and honey almond oil until a smooth mixture is
o prepared. The 20% PLURONIC mixture was added at 4 parts
to 1 part lecithin-isopropyl palmitate and mixed until a gel
formed. The composition was stored at room temperature.
Example II
Bromelain-Capsaicin Roll-on Formulation
A bromelain-capsaicin roll-on formulation is
prepared as follows:
Bromelain 7.5 g
~ps~icin 25 mg
n-decylmethyl sulfoxide 500 mg
Ethoxydiglycol 5 mL
Distilled water 50 mL
98% lecithin-isopropyl palmitate 20 g
PLURONIC 127 solution (10-20%) 30 mL
Honey almond oil 2 mL
The bromelain powder was stirred into water.
NDMS was added and stirred continuously for 2 hours
followed by addition of the honey almond oil. Next lecithin
organogel or poloxamer organogel was added.
-r
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Example III
Transport of Albumin Across the Stratum Corneum Using
PLURONIC Organogel
~ Bovine albumin (molecular weight 66,000
Daltons) conjugated to fluorescein isothiocyanate (FITC from
Sigma Chemical Co., St. Louis, MO) was incorporated at a
concentration of 7.5% into a mixture of PLURONIC organogel.
A rat was anesthetized with isoflurane, the back was shaved
with an oster blade, and the skin wiped with 90% ethyl
o alcohol. The albumin-FITC was applied to the skin in the
auricular area. FITC was injected intradermally in other
locations on the skin of the rats as a positive control.
Albumin-~ITC conjugate was mixed in hand cream in the
absence of PLURONIC organogel and applied to the skin of the
rat as a negative control. The hand cream employed was Rite
Aid extra strength skin care lotion and did not contain any
penetrating agents. The hand cream contained dimethicone as
the active ingredient, and also contained water, glycerin,
stearic acid, C " ~3 isoparaffin glycol stearate, petrolatum,
glyceryl stearate, triethanolamine, zinc oxide, cetyl alcohol,
potassium cetyl phosphate, carbomer 934, cetyl acetate,
acetylated lanolin alcohol, stearamide AMP, magnesium
aluminum silicate, methylparaben, and disodium EDTA.
Two hours after application of these compounds,
skin biopsies approximately 6 mm in diameter were removed,
sectioned at 4 microns (,um) in a cryostat, and placed on glass
slides. One specimen was fixed in alcohol, dehydrated through
absolute alcohol and a xylene substitute and coverslipped with
Permount. The other specimen was air dried and not
coverslipped. The slides were examined by a pathologist in an
Olympus BH-2 microscope with a reflected light fluorescent
attachment. In the albumin-FITC test condition, higher than
the background levels of fluorescence exhibited by the negative
controls were observed in the intercellular spaces of the
epidermis. Fluorescence in the epidermis of test samples was
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26
lower than the intense fluorescence in the positive controls
a~ter intradermal injection of ~ITC-albumin conjugate. These
results are consistent with FITC-labeled bovine albumin
percolating between epidermal keratinocytes in the presence of
s the PLURONIC organogel.
Example IV
Transport of Caprine Immunoglobulin Across the Stratum
Corneum Using PLURONIC Organogel
o A PLURONIC organogel was prepared (2 parts
PLURONIC 127, 2 parts lecithin-isopropyl palmitate mixture
plus 5% cholesterol) with 93% phosphatidyl choline
(Phospholipon 90, American Lecithin Co., Oxford,
Connecticut) and 10% caprine immunoglobulin (Goat-IgG,
lS approxim~te molecular weight of 150,000 Daltons) conjugated
to F~TC (SIGMA) was added to this gel. The auricular area
was shaved on both sides of two rats and the skin cleaned with
alcohol. About 0.5 mL of the PLURONIC organogel was
applied to the right auricular skin. The controls received hand
cream applied to the left auricular skin containing caprine
IgG-FlTC conJugate. Three hours later, the auricular skin was
thoroughly cleaned with alcohol and soap and biopsies were
removed. Ex~min~tion of the samples from the right auricular
skin showed fluorescence in the dermis while the controls did
not exhibit fluorescence in the dermis.
Example V
Collagenase Denaturation of Dermal Collagen in Rat Skin
Collagenase with a molecular weight of about
102,500 Daltons (PCAA, Houston, Texas) was incorporated at
10% into a gel cont~ining 40% phosphatidylcholine (Lecithin,
Sigma Chemical Co., St. Louis, MO.) and 10% PLURONIC F-
127. The collagenase and phosphatidylcholine gel was
dissolved in an equal amount of isopropyl palmitate and four
parts PLURONIC 127 (20% solution) and 2 mL of 10% calcium
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27
chloride. Two rats were shaved in the cervical region and the
gel was applied twice daily for 3 days. The ~nim~l~ were
anesthetized with isoflurane and skin biopsies were obtained at
the site of gel application and at other sites.
In one experiment, histopathological analysis of
the samples indicated intercellular edema and superficial
fragmentation. The edema and fragmentation were noted
immediately subjacent to the epithelium. The collagen bundles
appeared more widely separated and were thin, fine, and pale
o staining. Other biopsy sites untreated with gel containing
collagenase revealed normal cellular morphology and tissue
architecture.
Eighteen days following the initial biopsy new
biopsies were obtained. One specimen still demonstrated thin,
fine and pale staining collagen fibers subjacent to the
epithelium. The second experimental biopsy was normal in
appearance probably indicating that this area of the skin
underwent less severe degeneration resulting in faster collagen
regeneration. Further examination of the original biopsy
stained with Mallory trichrome revealed that one of the
specimens exhibited a sharply demarcated subepidermal area
with ~limini~hed staining intensity of collagen. The area of
~imini~hed staining was parallel to the epidermal surface. The
immediately overlying epidermis was unremarkable. The
results indicate that collagenase was transported across the
stratum corneum to the subepidermal space where it exhibited
enzymatic activity.
Example VI
Transdermal Transport of the Macromolecule Bromelain:
Demonstration of Protease Action of Bromelain
Approximately 100 mg of Eastern cottonmouth
(Agkistrodon piscivorus piscivorus) venom (SIGMA, St.
Louis, MO) was dissolved in 20 mL o~ physiological saline
solution (0.9% saline) providing a solution of 5 mg/mL. This
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28
was further diluted by adding 0.85 mL of 0.9% saline to 0.15
mL of the 5 mg/mL solution, providing 0.75 mg/mL. About
0.05 mL of this solution was injected intradermally in the
control and experimentally treated skin. Skin was
experimentally treated by applying about 0.5 mL of a 7.5%
bromelain in PLURONIC organogel to a shaved area of back
skin of a rat. Negative controls received 7.5% bromelain
mixed in hand cream (Rite aid) applied to a shaved area of skin
in the anterior lumbar region of the back. After about 45
minutes, the necrotic areas were measured with an
electrocardiogram caliper.
Five mice were treated with 0.25 mL 8%
bromelain in PLURONIC organogel and 5 mice were treated
with 0.25 mL hand cream with 8% bromelain as a control.
Copperhead venom (0.05 mL of a 0.9 mg/mL solution) was
injected intradermally to all mice under isofluorane anesthesia.
Five experimental mice survived 48 hours whereas the
controls died within 24 hours. The results demonstrate
efficacy of transdermally transported bromelain to partially
inactivate the active components of the venom.
Example VII
Transdermal Transport of the Macromolecule Bromelain:
Demonstration of Protease Action of Bromelain to Denature
2S Destructive Capabili~y of Snake Venom
Ten mice were treated with about 0.25 mL
bromelain in PLURONIC organogel (experimental) on one
location of the skin of the back. The same mice received
bromelain in hand cream (control) at another location on the
skin of the back. At each of these experimental and control
sites, approximately 0.05 mL of a solution of copperhead
venom (100 mg/ 20 mL of physiological saline. From this
solution 0.15 mL was diluted in 0.85 mL physiological saline).
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29
Table I
Size of Necrotic Regions (mm in diameter) of Control and
Experimental Cutaneous Sites
Control Experimental
11 5.5
18 10
7 5
8 4.75
21 15
22 14
16 11
19 12
24 15
~156 Total Area ~ 97.8 Total Area
The results demonstrate approximately 37% less
necrosis in experimental treatment sites showing partial
amelioration of the destructive effects of snake venom in the
presence of transdermally transported bromelain by
o PLURONIC organogel.
Example VIII
Demonstration of Transdermal Transport of a Reduced
Amount of the Macromolecule Bromelain Combined with
Cholesterol: Demonstration of Protease Action of Bromelain
to Denature Destructive Capability of Snake Venom
Potentiated by Cholesterol
The same procedure was employed as in Example
VII using cottonmouth venom with the following exceptions:
bromelain was reduced to 3~o, and 10% cholesterol was added
to the gel made from phospholipon C and PLURONIC 127
organogel. Five rats were anesthetized and prepared as
. .
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described in Example VI. The right forearm (experimental)
was treated with 0.5 mL bromelain in PLURONIC organogel
with cholesterol and the left forearm was used as a control
with 0.5 mL of hand cream (Rite aid) containing 3%
s bromelain. About 45 minutes later, all gel and cream were
removed carefully and 0.05 mL venom injected intradermally
at these experimental and control sites. Approximately 45
minutes later the necrotic areas were measured with an
electrocardiogram caliper.
Table II
Size of Necrotic Regions (mm in diameter) of Control and
Experimental Cutaneous Sites
Control Experimental
8 5
9 S
~48 Total Area ~30 Total Area
The results indicate a 38% decrease in the total
necrotic area of sites receiving bromelain application in the
presence of PLURONIC organogel and cholesterol, suggesting
that transdermal transport of reduced amounts of the protease
bromelain inhibited the extent of the venom-induced necrosis.
Example IX
Transdermal Transport of Bromelain for the Relief of Pain
Tnfl~mm~tion of the distal interph~l~ngeal joint is
a painful condition. Bromelain (approximately 7.5%) in
PLURONIC organogel was applied in a pea sized drop to
painful distal interph~l~ngeal joints in seven human volunteers
r
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31
(age range 18 to 75 years) who reported pain in these joints.
After application of the bromelain in PLURONIC-lecithin
organogel, all volunteers reported relief from the pain for a
period of about 8 to 12 hours. Complete relief from pain was
reported by 5 volunteers while 2 reported significant but not
complete relief from pain.
These results demonstrate that bromelain, a
molecule of about 33,000 Daltons molecular weight, was
effectively transported through the skin and alleviated pain,
perhaps by its action on subcutaneous nerve endings, possibly
pain afferents.
Example X
Tr~nsdermal Transport of Bromelain lnhibits Histamine-
s Induced Inflammation and Flea Antigen-Induced Inflammation
Topical application of about 0.25 mL of an 8%
solution of bromelain in PLURONIC-lecithin organogel was
followed by thoroughly rubbing it into the skin of the arm of a
human volunteer. Next, 0.05 mL of a solution of histamine (5
mg/mL) was injected intradermally into the arm at the site of
bromelain application. A second injection was ~-lmini~tered in
the forearm, a site that was not treated with bromelain. Ten
trials employing six different gels revealed a 45% reduction in
the size of the wheal produced by histamine injection at the site
of bromelain application compared to the site without
bromelain application.
In a second experiment, topical application of
about 0.25 mL of an 8% solution of bromelain in PLURONIC
organogel was followed by thoroughly rubbing it into the skin
of the arm of a human volunteer. Next, 0.05 mL of flea
antigen (Greer Labs, Lenoir, NC) diluted 1 to 100 in
physiological saline solution was injected intradermally into
the arm of a human volunteer at the site of bromelain
application. A second injection was ~(lmini~tered in the
forearm, a site that was not treated with bromelain.
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32
Table III
Effect of Transdermal Bromelain Application on Flea-antigen
Induced Wheal Formation (wheal size (diameter in mm))
Control Experimental
19.5 13
17 11
22 12
19 13
23 11
~100.5 Total Area ~60 Total Area
Wheal size was determined by rneasuring the greatest diameter
with an EKG caliper. The results indicated a 39% decrease in
the wheal size at the site of bromelain application, suggesting
o that transdermal transport of the protease bromelain inhibited
the degree of infl~mm~tion.
Example XI
Formation and Testing of Phospholipid Pluronic Organogel:
Demonstration of Transdermal Transport Using Phospholipid
Organogel
Semisynthetic phospholipids may also be utilized
- for gel preparation. A phospholipid organogel was made by
dissolving 1.94 g of Phospholipon CC ( 1,2 dicaproyl-sn
glycerol 3 - phosphocholine) (American Lecithin Co., Oxford,
CT) in about 1.94 g of isopropyl myristate, 0.5 ml ethanol, 4
ml of PLURONIC 127 (20% solution) and 4 ml of deionized
H20. Bromelain was used to evaluate protease activity against
fescue grass extract used as the allergen (0.1 ml extract + 9.9
ml H2O) (Greer Lab, Lenoir, N.C.). Bromelain (400 mg) was
added by mechanical stirring until a gel was formed
(experimental sample). Next, 400 mg of bromelain was added
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separately to a hand cream (control) to achieve the same final
concentration as the experimental sample. In order to test the
penetration capability of the phospholipid organogel compared
to the hand cream, a rat was anesthetized and the back
s carefully shaved and cleaned with 70% isopropyl alcohol.
Approximately 0.5 ml of the phospholipid organogel was
applied to the caudal aspect of the skin of the back and about
0.5 ml of the control sample was applied to the rostral lumbar
skin of the back. About 1.5 hours later the experimental
o phospholipid gel and the control cream were removed from
the skin. Approximately 0.05 ml of an aqueous 1:100 dilution
of Fescue extract (Greer Lab., Lenoir, NC) as allergen was
injected intradermally into the sites exposed to either the
experimental or control compositions. The allergen injections
s produced wheals which were measured three hours after
injection. The average size of the wheals at control sites was
40% larger than the wheals at sites treated with the
phospholipid organogel. These results demonstrate that
bromelain was transdermally transported at sites treated with
phospholipid organogel. The protease nature of the
transdermally transported bromelain partially deactivated the
allergen, resulting in reduced wheal diameter at sites treated
with phospholipid organogel compared to sites treated with
bromelain in hand cream.
Example XII
Preparation of Phospholipid Organogels
Approximately 98-99% pure phospholipids were
dissolved in isopropyl palmitate (isopropyl myristate may also
be used) with 3-5 ml 98% ethanol and allowed to stir over
night using a mechanical stirrer. Gels were made by titrating
the composition with sterile water (or saline): water was
added until the desired viscosity was achieved. Optionally,
instead of using water or saline for titration, PLURONIC 127
may also be used for making a gel. If PLURONIC 127 is
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34
used, then it is used in a proportion of 1:4, i.e., one part
phospholipid and four parts PLURONIC 127. The use of
PLURONIC 127 generally reduces the amount of
phospholipids that are utili7e~1, consequently, penetration of the
s dermis or other desired targets could be affected.
Example XIII
Treatment of Pain Associated with a Knee Injury
Severe knee injury, caused by an automobile
accident, was treated in a 68 year old woman using a
composition consisting of a PLURONIC organogel made with
lecithin, isopropyl palmitate, bromelain and capsaicin. The
knee injury caused the patient severe discomfort and pain, and
also limited flexion and extension. A PLURONIC organogel
lS cont~ining 7.5% bromelain and 0.25% capsaicin was made. A
60% lecithin powder was dissolved gram for gram with
isopropyl palmitate and 4 parts PLURONIC 127 (20% solution)
to make a gel. Following topical treatment of the knee with
this composition, the patient reported 90% reduction in pain.
Example XIV
Use of PLURONIC Lecithin Organogel for Transdermal
Delivery of Methotrexate, a Polar Molecule
Methotrexate is a highly effective drug
~clmini.ctered orally for rheumatoid arthritis, but it is not used
at the initial stages of the disease due to its toxicity.
One part of lecithin isopropyl palmitate mixture is
added to four parts of PLURONIC 127 (20% solution).
Methotrexate was added to make a 5% methotrexate gel with a
20% lecithin dissolved in equal parts of isopropyl palmitate.
0.5 ml of the gel was applied to the shaved knee of a 60 lb.
dog. Three hours later synovial fluid and blood samples were
drawn and submitted to Roche Biomedical, Burlington, VT,
for analysis. The methotrexate concentration in the blood
3s sample was less than 0.01 ~Lmol/l. The methotrexate
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concentration in the synovial sample was 0.07 ~lmol/l which
was in the low therapeutic range. The same dog was given 2.5
mg orally and 3 hrs. later blood was drawn and the
methotrexate concentration was 0.76 ~mol/l. The
s methotrexate concentration in the synovial fluid was 0.42
,umol/l .
A 120 lb dog was treated with 8 ml of
methotrexate on the knee and the synovial fluid and blood
samples were drawn 24 hrs. later. The blood level of
o methotrexate was 0.07 ,umol/l and the synovial level of
methotrexate was 0.17 ~mol/l.
Treatment Blood Synovial
Sample Fluid
Sample
0. 5 ml MTZ
gel on shaved <0.01 0.07 ~molA
knee, samples ,umol/l
drawn 3 hours
later
2.5 mg orally,
samples drawn 0.76 0.42 llmol/l
3 hours later ,umol/l
8 ml MTZ gel
on shaved 0.07 0.17 ~mol/l
knee, samples ,umol/l
drawn 24
hours later
These data suggest that rheumatoid arthritis in
children and animals could be treated more aggressively
without a concern for toxicity that precludes its use near the
onset of the disease.
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Example XV
Transdermal Transport of Collagenase with a Pluronic
Lecithin Organogel
Phospholipon 80 (American Lecithin Co., Oxford,
s CT.) was dissolved in an equal weight (gram for gram) of
isopropyl myristate and 30 ml of PLURONIC 127 (20%
solution). Deionized water (10 ml) was added to 10 ml of the
phospholipid PLURONIC mixture. As the gel was forming, 10
mg collagenase and 2 ml CaCl2 was added. A rat was treated
as previously and biopsied. Five days later the experimental
~nim~l was rebiopsied 3-5 mm on either side of the first site.
Under normal conditions a distinct basement membrane is
generally not observable at the sites of separation, but where
the basement membrane persists it is present bordering the
basal aspect of the basal epithelial cells. Collagenase has also
been successfully transported with lecithin organogel using
lecithin obtained from egg yolk. Similar results were
obtained.
Example XVI
Preparation of Lecithin Organogel
Approximately 95 % pure lecithin may be
dissolved in isopropyl palmitate or isopropyl myristate on a
weight basis of 1 g of lecithin per about 0.5 to 1.5 g of
isopropyl palmitate or isopropyl myristate and/or ethanol.
The preferred ratio of lecithin to these solvents is about 1 g to
about 0.75 g to 1 g. Next ethanol (98%) may be added while
stirring at 80~ C until the alcohol is boiled off. Water is then
added with stirring at approximately 20 to 40% with a
preferred concentration of about 30%.
A penetration enhancer of the present invention is
PLURONIC F-127 (BASF, Parsippany, NJ) which permits use
of lecithins of lesser purity than those required in formation of
lecithin organogels as taught by Willim~nn et al. PLURONIC
3s F-127 is employed at concentrations of about 0.1% to 45% in a
r ~
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37
ratio PLURONIC to lecithin of about 1:0.5 to 1:6Ø A
preferred final concentration of PLURONIC F-127 is 5% to
20% in a ratio of PLURONIC to lecithin of 1:2 to 1:4.
Lecithins of concentrations of approximately 5% to 90% are
s first dissolved in isopropyl palmitate, isopropyl myristate
and/or 98% ethanol. The addition of four parts of PLURONIC
F-127 (20% solution) to the dissolved lecithin produces a cost
effective gel. In addition, water, carboxyethyl cellulose,
carboxymethyl cellulose, other PLURONICS, and other agents
o known to one skilled in the art may be used. These mixtures
are known PLURONIC organogels or poloxamer organogels.
Example XVII
Cholesterol as a Penetration Enhancer
Water dispersible lecithin (60% concentration-
Precept 8140 Central Soya, IN) is added to an equal amount of
deionized water in a ratio of approximately 10 g lecithin to 10
g deionized water. These reagents are mechanically stirred.
Optionally, ethoxydiglycol may be added in a range of about
5% to 35% with a preferred concentration of approximately
10%. Next, cholesterol is added to the lecithin in a desired
molar proportion and heated to approximately 65~ C.
Addition of 98% ethanol to the mixture accelerates fusion of
cholesterol in the micelle and acts as a penetration enhancer.
Increasing the temperature to 80~ C will boil off excess
ethanol. A desired final concentration of ethanol, however, is
3 % to 8 % in order to enhance penetration. Once a
homogenous mixture is obtained, PLURONIC F127 (20~o
concentration) is added in a ratio of approxim~tely 3:1 to 4:1
to effect gel formation. A composition prepared by this
method may be advantageous in situations where chronic use
might provoke hypersensitivity reactions to isopropyl
palmitate, isopropyl myristate or ethanol.
Cholesterol may be used in an identical manner in
the preparation of other organogels as taught in the present
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38
invention. These organogels include lecithin organogels,
phospholipid gels, PLURONIC organogels and PLURONIC-
phospholipid gels. The use of cholesterol provides increased
stability and penetration of the gels. The relative
concentration of cholesterol to phospholipid may be
determined by one skilled in the art and would involve a
consideration of the molecules to be transported as well as the
phospholipids employed in forming the phospholipid gel.
Molar concentrations of cholesterol to phospholipid in
cholesterol phospholipid gels would be approxim~tely 0.1:1.5.
In another embodiment of cholesterol as a penetration
enhancer, approximately 7.66 g of water dispersible lecithin at
60% concentration (Precept 8140, Fort Wayne, IN) is added to
7.5 ml of deionized water and 2.5 ml of ethoxydiglycol
followed by mechanical stirring. Upon uniform dispersion of
the lecithin, 1.9 g of cholesterol (0.5 M cholesterol:l M
lecithin) is added and stirred at about 65~ C. Cholesterol
disperses and is incorporated into the micelle. Optionally,
2-10 ml of 98% ethanol may be added to accelerate this
process, however, in this case the temperature must be
increased to about 80~ C.
The excess ethanol is then boiled off so that the
final concentration of ethanol in the mixture is approximately
3 to 8 %. Next, PLURONIC 127 (20~) is added at a ratio of
approximately 3 to 4 part of PLURONIC to 1 part of the
lecithin/cholesterol mixture. After the temperature of this
mixture falls to about 25~ C, 4 g of bromelain is then
thoroughly blended into the mixture using a stirring apparatus.
A control bromelain cream is made by adding 400 mg to 9.6 g
of hand cream.
Next 1 ml of bromelain PLURONIC organogel was
applied to the medial proximal forearm and a ~imil~r amount
of the control cream was applied to the distal medial forearm.
About 1.5 hours later, 0.05 ml of a 1:100 dilution of a fescue
antigen (Greer Lab., Lenoir, NC) was injected intradermally
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39
after each site was carefully cleaned. The resulting wheals
were 30% larger in control areas when measured 3 hours after
injection. These results indicate that cholesterol may be
beneficial in transdermally transporting bromelain in the
s absence of an organic solvent since wheal sizes in experimental
areas were smaller than those observed in control areas.
Example XVIII
Transdermal Transport of Collagenase Using an Egg Yolk
o Lecithin, PLURON~C, Isopropyl Myristate Composition
Egg yolk lecithin at approximately 98%
concentration (USB Cleveland, OH) was dissolved in about 100
g of isopropyl myristate. Ten ml of this mixture was added to
about 10 ml of PLURONIC F127 (20% concentration), 20 ml of
water, 10 mg collagenase, and 1 ml CaCl2. A control sample
was prepared by dissolving collagenase in hand cream until the
same final percentage of collagenase was obtained as used in
the experimental cream. The backs of rats were shaved in the
anterior lumbar region and the experimental composition was
applied in a volume of approximately 0.5 ml twice at 12 hour
intervals. The next morning the ~nim~l~s were anesthetized and
vesicles were evident at biopsy which were not present in
controlled treated ~nim~ Histopathological analysis revealed
extensive subepidermal bulla formation at sites of application
of the experimental composition containing collagenase. In the
center of these biopsy specimens, the epidermis was separated
from the subjacent dermis by a wide clear space. Collagen was
slightly paler in appearance than the collagen subjacent to the
clear space and was also less distinct and slightly paler than the
collagen subjacent to the intact epithelia. Biopsies from
control animals were described as normal in histologic
appearance. The lesions observed in biopsies from
experimental ~nim~ls resembled those observed in bullous and
vesicular diseases caused by loss of the structural integrity of
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the basement membrane zone. These results indicate the utility
of combining phospholipids in PLURONIC gels.
It should be understood that the foregoing relates
only to preferred embodiments of the present invention and
s that numerous modifications or alterations may be made
therein without departing from the spirit and the scope of the
invention as set forth in the appended claims.
