Note: Descriptions are shown in the official language in which they were submitted.
WO 01/45645 CA 02394901 2002-05-21 PCT/US00/35319
CHITOSAN BIOPOLYMER FOR THE TOPICAL
DELIVERY OF ACTIVE AGENTS
FIELD OF THE INVENTION
This invention relates to carrier bases for the topical delivery of active
agents comprising high viscosity chitosan biopolymers. Preferred carrier bases
comprise chitosan having a molecular weight of at least 250,000 Dalton. The
invention also relates to carrier bases comprising high viscosity chitosan at
a
concentration of at least 2 weight%. The present invention further provides a
delivery system for therapeutic agents, such as retinoids, that overcomes many
of the previously known problems associated with delivery systems for
retinoids.
BACKGROUND OF THE INVENTION
A number of changes occur in skin tissue as a consequence of aging,
photodamage, and diseases, e.g., skin cancer and acne. Skin connective tissue
is comprised primarily of fibrillar collagen bundles and elastic fibers, along
with
extracellular matrix (ECM) molecules such as glycosaminoglycans (GAG),
proteoglycans, glycoproteins, peptide growth factors. Keratinocytes and
fibroblasts are the main cell types embedded within the ECM. The predominant
component of the ECM is hyaluronan (HA). HA is the primordial and simplest
of the GAGs, and the first ECM to be developed in the developing embryo. HA is
thought to be largely a product of fibroblasts.
The components of the extracellular matrix (ECM) form a highly
organized structure endowed with hydration properties, and structural proteins
such as collagen and to a lesser extent, elastin. HA is the primordial and
simplest of the GAGS, and the first ECM to be developed in the developing
embryo. HA is thought to be largely a product of fibroblasts.
A number of changes occur in the structure of skin connective tissue as
a consequence of aging or photodamage. Age-related changes include a
decrease in the number of fibroblasts, and connective tissue abnormalities
such
as ( 1) thinning of the collagen fiber bundles, (2) an increase in space
between
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
collagen fiber bundles, (3) an increase in collagen fiber bundle
disorganization
and (4) increase in depth of disorganization (Varani et al., 2000). In
addition,
the HA in the epidermal extracellular matrix has disappeared completely in
aged skin (Neudeker et al., 2000). These alterations are believed to be
largely
responsible for the thin, fragile, and finely wrinkled quality of naturally-
aged
skin. Photoaged skin is characterized by the presence of elastotic material
and
damage to the collagen bundles. Clinically, photoaged skin appears thick and
rough, with course wrinkles and mottled pigmentation (Lavker, 1995).
The alterations in skin connective tissue in skin aging and photodamage
and skin diseases seem to be mediated mainly by collagen which comprises the
bulk of the connective tissue (90% wet weight) and by hyaluronan which is the
predominant component of the extracellular matrix. In terms of quantity both
reduction in collagen synthesis and increased destruction seem to occur.
Collagen synthesis is reduced in both photoaged and naturally aged skin
(Griffiths et al., 1993; Talwar et al., 1995; Varani et al., 2000). In vivo
studies
have demonstrated decreased collagen synthesis in aged fibroblasts (Johnson et
al., 1986, Gregory et al. 1986; Mays et al., 1990; Furth, 1991) In
photodamaged
skin UV irradiation has been shown to increase production of matrix
metalloproteinases (MMP) which destroy collagen and cause tissue damage
(Fisher et al., 1996, 1997). The quality of the fiber bundle architecture
seems to
be mediated by extracellular and structural molecules such as hyaluronan.
There are many known agents that are used for the treatment of skin
diseases and defects, including, e.g., retinoids, vitamins, and alpha-hydroxy
acids. Topical application of retinoids such as All-traps retinoic acid and
retinol
has been shown to stimulate collagen synthesis in naturally aged as well as
photoaged skin (Varani et al., 2000; Griffiths et al., 1993). The active
substance
seems to be All-traps retinoic acid. However, the two retinoids All-traps
retinoic
acid and retinol are related. Indirect evidence exists that retinol transforms
into
All-traps retinoic acid in human skin (Kang et al, 1995). Retinoids appear to
affect the quantity of collagen by increasing the number of collagen-producing
fibroblasts, increasing collagen synthesis and/ or by reducing MMP levels in
skin, thereby decreasing destruction of collagen (Varani et al., 2000).
However,
retinoids do not seem capable of affecting the quality of the collagen being
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
produced as evidenced by wo change in the dermal connective tissue
abnormalities after retinoic~ trea'~ment (Varani et al., 2000). For increasing
the
quality of the collagen being produced by the retinoids there seems to be a
need
for additional molecules which play a role in tissue reorganization.
Although retinoid treatment induced measurable changes in the dermal
fibroblast population, it did not alter age-associated connective tissue
abnormalities such as correct collagen fiber deposition (Varani et al., 2000).
Thus, it would be desirable to have a carrier base that is capable of altering
these abnormalities and reverse or minimize the effects of aging or
photodamage on the skin.
Retinoids are also used to treat other skin conditions such as acne,
actinic keratosis, psoriasis, skin cancers and have been found to useful
therapeutic agents in the chemoprevention of melanoma (Stam-Postuma,1998;
Halpern, 1994; Kligman, 1998).
The incidence of melanoma is increasing in the United States at a rate of
about 2.7% annually, even as most other cancers are experiencing a decline in
incidence. Furthermore, melanoma is the seventh most commonly diagnosed
cancer in U.S. men and women. Chemoprevention is a strategy to prevent the
development of melanoma through the administration of drugs. The recognition
of dysplastic nevi as markers of melanoma risk and intermediate steps of
melanocytic tumor progression has significant implications for melanoma
chemoprevention.
The incidence of malignant melanoma of the skin, the most serious form
of skin cancer, is increasing faster than that of any other cancer in the
United
States (Koh 1991). Trends in melanoma incidence rates have continued to
increase substantially (from 1990-1996: =2.7% per year; p<0.001) while all
other cancer incidence decreased (except for non-Hodgkin's lymphoma) (Wingo
et al., 1999). Data from the Surveillance, Epidemiology, and End Results
Program Registry (SEER 1973-1994) indicates that the increasing incidence
rates of melanoma may represent a true increase in cancer rates with data also
showing an increase in advanced disease (thick tumors-2 year mortality).
(Dennis, 1999) similar to that reported in Australia (Hall et a1.,1999).
While strategies for malignant melanoma have included ( 1 ) public health
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interventions (Koh and Geller, 1998), (2) adjuvant therapies (Demierre and
Koh,
1997) and (3) immunotherapy (Curiel-Lewandrowski and Demierre, 1999),
recent research suggests chemoprevention is an important strategy for the
management of malignant melanoma (Halpern, 1994, 1998). Chemoprevention
entails the use of specific agents to block, reverse or suppress
carcinogenesis
and thereby prevent the development of primary or secondary cancers.
Melanocytic nevi, particularly dysplastic nevi confer a risk factor for the
development of melanoma, with quantitative measures correlating directly with
the magnitude of risk. (Tucker et al 1997; Grob et al., 1990; Egan et al.,
1998;
Meier et al., 1998) and a count of benign melanocytic nevi as a major
indicator
of risk for non-familial nodular and superficial spreading and nodular
melanoma (Grob et al., 1990). In a multicenter prospective case-control study
of
716 newly diagnosed melanoma patients and 1014 controls conducted by
Tucker et a1.(1997), an increased risk of melanoma was determined according to
the number of non-dysplastic and dysplastic nevi. Individuals with numerous
small nevi had a double risk of melanoma. Having additional large non-
dysplastic nevi increases the risk four-fold. Having just one dysplastic nevus
was associated with approximately a 2-fold risk, while 10 or more conferred a
12-fold risk of melanoma.
Furthermore, clinical and histopathologic features of melanoma have
suggested five steps of melanoma progression: (1) common acquired and
congenital nevi with structurally normal melanocytes, (2) dysplastic nevus
with
structural and architectural atypia, (3) early radial growth phase primary
melanoma, (4) advanced vertical growth phase primary melanoma with
competence for metastasis, and (5) metastatic melanoma (Sauter and Herlyn,
1998). The recognition of dysplastic nevi both as markers of melanoma risk
and intermediate steps of melanocytic tumor progression has significant
implication for melanoma chemoprevention.
A national chemoprevention multicenter randomized Phase II trial led by
the Eastern Cooperative Oncology Group (ECOG) is investigating the effects of
topical tretinoin (ATRA) and systemic fenretinide (4-HPR). Small pilot studies
have demonstrated a significant effect of topical tretinoin on the appearance
and histology of dysplastic nevi. Topical tretinoin is also active in the
treatment
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WO 01/45645 CA 02394901 2002-06-21 PCT/US00/35319
of inflammatory diseases (acne vulgaris), precancerous lesions (actinic
keratosis) and photodamage.
Retinoids are among the most promising chemopreventive agents with
clinical effects of retinoid chemoprevention having been demonstrated in
cancers of the head and neck, lung, cervix, ovaries and skin (Lotan, 1996;
Sankaranarayanan and Mathew, 1996, Labrecque et al., 1999). Topical
application of tretinoin (all-trans retinoic acid, ATRA) has been shown to
decrease melanocyte numbers and reduce melanocytic atypia in the treatment
of photodamaged skin (Bhawan et al., 1996) and small pilot studies have
demonstrated a significant effect of topical tretinoin on the appearance and
histology of dysplastic nevi (Halpern et al., 1994, 1998; Stam-Posthuma et
al.,
1998). In addition, in a malignant melanoma murine model, with ATRA or 9-cis-
RA treatment there was a reversible conversion of malignant melanoma into a
benign, melanocytic phenotype (Spanjaard et al., 1997; Clifford et al., 1990).
It
is well known that there are two structurally and pharmacologically distinct
families of retinoid receptors: the retinoic acid receptor (RAR) family with
subtypes a, (3, y and the retinoid X receptor (RXR) family with subtypes a,
(3, y.
ATRA binds and activates RARs, whereas the panagonist 9-cis-RA, a novel
retinoid, binds and activates all six of the retinoid receptors. Of note,
melanoma
expresses all three of the RAR subtypes (Nagpal and Chandraratna, 1996).
These data suggests that melanoma chemoprevention of persons at high risk of
developing melanoma might benefit from both ATRA and 9-cis-RA.
In presently used topical delivery systems for agents used to treat skin
ailments, one side effect is increased irritation. For example, compared to
oral
administration, topical delivery of retinoids increases the concentration of
retinoids in the dermal compartment 10- to 100-fold (Lehman et al., 1988).
However, topical tretinoin (ATRA) induces irritation in 90% of patients
(Gilchrest, 1997), and other side effects include patchy erythema, localized
swelling, xerosis, and scaling. Irritation has been attributed, in part, by an
overload of the tretinoin dependent pathways with non-physiological amounts
of exogenous tretinoin in the skin. (Siegenthaler et al., 1994). This
irritation
may be the reason for discontinuation of treatment for close to 50% of
patients
(Stam-Posthuma et al., 1998). This high incidence of irritation, leading to
poor
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WO 01/45645 CA 02394901 2002-06-21 PCT/US00/35319
compliance, can preclude its use.
The incorporation of drugs into polymeric carriers provides advantages,
e.g., preferable tissue distribution of the drug, prolonged half life,
controlled
drug release and reduction of drug toxicity. Examples of percutaneous drug
delivery systems for retinoids delivery presently on the market include ATRA
formulations containing a synthetic material, polyolprepolymer-2 (PP2) (Avita,
Penederm Inc., Foster City, CA). These retinoid formulations have been shown
to be less irntating than currently marketed ATRA formulations (Quigley and
Bucks, 1998). The addition of the synthetic polymer appeared to reduce the
percutaneous flux to about 50% of an equivalent ATRA commercial formulation
(0.025% ATRA) after 6 hours of delivery. Another synthetic polymer system
based on acrylates for retinoid delivery is described in U.S. Patents
5,145,675
and 5,955,109 in Won et al. ( 1992; 1999). However, these formulations utilize
a
non-biodegradable synthetic polymer as a carrier of the drug. High molecular
weight polymers (360,000 to 400,000 Dalton) have been shown to penetrate the
stratum corneum (Brown et al., 1999). The possibility of other polymers, such
as the synthetic polymers described above, to penetrate the skin and enter the
systemic circulation has been suggested by the authors after careful
radiolabeled analysis of the tissue distribution and accumulation in various
tissue organs of their target high molecular weight polymer after topical
application (Brown et al., 1999). Thus it would be desirable to have a topical
delivery system which is entirely biodegradable due to the likelihood of it
entering the systemic circulation and accumulating in target tissues.
In addition, there is presently no controlled topical delivery system of
retinoids for use in melanoma chemoprevention. A controlled delivery system
could make retinoid topical therapy a viable chemoprevention treatment for
melanoma. In addition, it would be useful to have a delivery system that
utilizes
a non-synthetic carrier which is biodegradable after penetrating the skin
layers.
Thus, it would be desirable to have a controlled delivery vehicle for active
agents used to treat skin ailments, which would prevent the irritation seen in
present treatments. For example such a delivery system for retinoids would
enable chronic use of topical retinoids for treating skin ailments, including
for
melanoma chemoprevention. A controlled delivery system could make tretinoin
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WO 01/45645 CA 02394901 2002-06-21 PCT/US00/35319
topical therapy a viable ch°moprevention treatment for melanoma in
individuals
with dysplastic nevi who are at nigh risk of developing melanoma.
Chitosan is a natural, biodegradable cationic polysaccharide derived by
deacetylating chitin, a natural material extracted from fungi, the
exoskeletons
of shellfish and from algae and has previously been described as a promoter of
wound healing (Balassa, 1972; Balassa, 1975). Chitosan comprises a family of
polymers with a high percentage of glucosamine (normally 70-99%) and N-
acetylated glucosamine ( 1-30%) forming a linear saccharide chain of molecular
weight from 10,000 up to about 1000,000 Dalton. Chitosan is polycationic.
Chitosan, through its cationic glucosamine groups, interacts with anionic
proteins such as keratin in the skin conferring bioadhesive characteristics.
When not deacetylated, the acetamino groups of chitosan are an interesting
target for hydrophobic interactions and contribute to some degree to its
bioadhesive characteristics. Modified chitins and chitosans have been
administered to humans in the form of dressings for wounded soft tissues and
for the controlled delivery of drugs (Muzzarelli et al, 1986; 1999;
Muzzarelli,
1993; 1996; Tokura and Azuma, 1992; Wada, 1995; Maekawa and Wada, 1990;
Mita et al., 1989). For the purpose of soft tissue healing the most relevant
characteristics of chitin-based biomaterials are their biodegradability,
biocompatibility and similarity to hyaluronan, beside their capacity to
release
glucosamine and N-acetyl-glucosamine monomers and oligomers (Muzzarelli,
1999).
Chitosan is insoluble in neutral to alkaline water and thus, it has to be
exposed to acidic conditions to render it soluble. Methods for solubilizing
chitosan include the use of a slightly acid solution (pH<6) containing acidic
acid, glycolic acid, lactic acid, or other alpha-hydroxy acids. Other methods
include producing derivatives of chitosan which obviate the need for acids to
solubilize chitosan. For example, U.S Patent No. 3,953,608 in Vanlerberghe and
Sebag describes a method of making chitosan soluble in water at pH>7 by
acylation of the chitosan using organic anhydrides. This patent describes the
use of these derivatives mainly as film formers for coloring of the skin,
deodorizing products and making antispot products. United States Patent Nos.
4,929,722 and 4,946,870 describe the use of chitosan derivatives in delivery
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WO 01/45645 CA 02394901 2002-os-21 ' PCT/US00/35319
systems for the delivery of pharmaceutical or therapeutic compositions. Patent
No. 4,929,722 describes, in particular, the method of making a chitin or
chitosan salt or covalent derivative from highly crystalline, partially
deacetylated chitin or chitosan. ThesF. ionic derivatives of chitosan called
chitosonium polymers and covalent chitosan derivatives have been made by
dispersing chitosan in an aqueous/solvent mixture. Patent No. 4,946,870
describes the use of these chitosonium polymers and covalent chitosan
derivatives. U.S. patent No. 5,300,494 describes the same delivery system to
deliver quaternary and related compounds.
It would be useful to have a delivery system that incorporates drugs,
such as retinoids, into polymeric carriers to provide advantages such as
preferable tissue distribution of the drug, prolonged half life, controlled
drug
release and reduction of drug toxicity. The use of a controlled topical
delivery
vehicle for retinoids may prevent the overload of retinoids into the systemic
circulation, which may be responsible for irritation and allow chronic use of
topical retinoids. In addition, it would be useful to have a controlled
topical
delivery system of retinoids for melanoma chemoprevention. A controlled
delivery system could make tretinoin topical therapy a viable chemoprevention
treatment for melanoma.
It would also be useful to have a controlled delivery system for the
delivery of retinoids in which the carrier of the drug promotes connective
tissue
abnormalities in the damaged tissue, in order to increase the effectiveness of
the treatment.
SUMMARY OF THE INVENTION
The present invention relates to a carrier base for the topical delivery of
an active agent comprising a high viscosity chitosan biopolymer. Preferably,
the
carrier base comprises a high viscosity chitosan having a molecular weight of
at
least about 100,000 Dalton, more preferably at least about 250,000 Dalton and
most preferably at least about 300,000 Dalton. In other preferred embodiments
the chitosan has a concentration of at least about 2 weight% . In an
especially
preferred embodiment, the carrier bases comprises a high viscosity chitosan
biopolymer having a molecular weight of at least about 300,000 Dalton and at a
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W~ 01/4$645 CA 02394901 2002-06-21 PCT/US00/35319
concentration of at least 2 weight%.
The present invention also relates to a composition for the topical
delivery of an active agent comprising a carrier base as described above and
an
active agent. Examples of active agents include pharmaceutical actives and
therapeutic actives. Preferred pharmaceutical actives are those used for the
treatment of skin diseases, e.g., retinoids, corticosteroids, non-steroidal
anti-
inflammatory drugs (NSAIDS), hormones, anti-fungal agents, anti-septic agents,
local anaesthetics, kerolytic agents, and 5-FU. Examples of useful therapeutic
actives include, but are not limited to vitamins and moisturizing agents such
as
alpha-hydroxy acids, etc. as further described below. In certain embodiments,
the compositions contain more than one active agent, thus the compositions
comprise at least one additional active agent, which can be either a
pharmaceutical active or a therapeutic active. A preferred composition
comprises the carrier, retinoids and alpha-hydroxy acid.
In certain compositions of the present invention the chitosan has a
molecular weight of at least about 300,000 Daltons. In certain of these
embodiments, the chitosan is present in a concentration greater than about
2%. These compositions are especially useful for obtaining the slow, sustained
release of the active agent.
In certain embodiments of the present invention, the chitosan has a
molecular weight of about 10,000 to about 250,000 Dalton. In certain of these
embodiments the chitosan is present in a concentration greater than about 5%,
more preferably between about 5% up to about 8%.
The invention further relates to compositions for the topical delivery of
retinoids comprising a Garner base and a retinoid, wherein the carrier base
comprises a high viscosity chitosan. Preferably, the carrier base comprises a
high viscosity chitosan having a molecular weight of at least about 100,000
Dalton, more preferably at least about 250,000 Dalton and most preferably at
least about 300,000 Dalton. In other preferred embodiments the chitosan has a
concentration of at least about 2 weight %. In an especially preferred
embodiment, the carrier bases comprises a high viscosity chitosan biopolymer
having a molecular weight of at least about 300,000 Dalton and at a
concentration of at least 2 weight %.
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The invention provides for compositions of the present invention in the
form of gels, creams and lotions. The manufacture of such gels, creams or
lotions are known in the art.
The invention further relates to a method of controlling the release of an
active agent from a carrier base, comprising as a carrier base a high
viscosity
chitosan; providing the active agent; and mixing the active agent and the
chitosan. Preferably, the carrier base comprises a high viscosity chitosan
having
a molecular weight of at least about 100,000 Dalton, more preferably at least
about 250,000 Dalton and most preferably at least about 300,000 Dalton. In
other preferred embodiments the chitosan has a concentration of at least about
2 weight % . In an especially preferred embodiment, the carrier base comprises
a high viscosity chitosan biopolymer having a molecular weight of at least
about
300,000 Dalton and at a concentration of at least 2 weight %.
In certain methods, the method further comprises the step of selecting a
concentration of chitosan depending on the molecular weight of the chitosan
provided so that a viscosity of at least about 100 cps is obtained.
In preferred methods of controlling the release of an active agent from a
carrier, the active agent comprises a pharmaceutical active, e.g., an agent
that
is used for the treatment of skin diseases. Examples of pharmaceutical actives
include, but are not limited to retinoids, such as corticosteroids, non-
steroidal
anti-inflammatory drugs (NSAIDS), hormones, antiviral, anti-histamines, anti-
fungal agents, anti-septic agents, local anaesthetics, kerolytic agents, 5-FU,
etc.
In other embodiments, the active agent comprises a therapeutic active, e.g.,
vitamins, moisturizing agents such as alpha-hydroxy acids, etc., as further
described below. In certain embodiments, the composition contains more than
one active agent, thus the compositions comprise at least one additional
active
agent, which can be either a pharmaceutical active or a therapeutic active.
The invention also relates to a method of treating skin diseases providing
to the diseased skin a composition containing a high viscosity chitosan
biopolyrner and an active agent. Preferably, the high viscosity chitosan has a
molecular weight of at least about 100,000 Dalton, more preferably at least
about 250,000 Dalton and most preferably at least about 300,000 Dalton. In
other preferred embodiments the chitosan has a concentration of at least about
WO 01/45645 cA 02394901 2002-os-21 PCT/US00/35319
2 weight % . In an especia 1y preferred embodiment, the high viscosity
chitosan
biopolymer has a molecular weight of at least about 300,000 Dalton and at a
concentration of at least 2 weight %.
Examples of skin diseases include, but are not limited to, acne,
melanoma, premature skin aging, and photodamage. In preferred embodiments
the active agent comprises a pharmaceutical active, e.g., an agent that is
used
for the treatment of skin diseases. Examples of pharmaceutical actives
include,
but are not limited to retinoids, such as corticosteroids, non-steroidal anti-
inflammatory drugs (NSAIDS), hormones, anti-viral, anti-histamines, anti-
fungal agents, anti-septic agents, local anaesthetics, kerolytic agents, 5-FU,
etc.
In other embodiments, the active agent comprises a therapeutic active, e.g.,
vitamins, moisturizing agents such as alpha-hydroxy acids, etc., as further
described below. In certain embodiments, the compositions contains more than
one active agent, thus the compositions comprises at least one additional
active
agent, which can be either a pharmaceutical active or a therapeutic active. In
certain embodiments of the present invention, the methods of treating skin
diseases comprises the compositions of the present invention, as described
herein, in conjunction with other treatments for the disease. For example, in
treating precancerous skin conditions, it may be useful to use the
compositions
of the present invention with standard treatments that use an anti-cancer
drug,
e.g., 5-FU for the treatment of actinic keratosis.
The invention further relates to compositions for the topical delivery of an
active agent comprising a chitosan biopolymer and the active agent, wherein
the
chitosan has a molecular weight of at least about 300,000 Daltons and is
present at a concentration less than about 2%, preferably less than about 1
weight %. These compositions are useful for increasing the transdermal
delivery of the active agent.
In preferred compositions of the present invention, the chitosan
biopolymer comprises a chitosan having a molecular weight of at least about
100, 000 dalton. Preferably the chitosan has a molecular weight ranging from
about 250,000 daltons to about 1000,000, more preferably about 300,000 to
about 1000,000, and most preferably from about 300,000 to about 800,000
Dalton.
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In certain embodiments the chitosan has a molecular weight from about
300,000 to about 800,000, at a concentration of at least about 2%. In other
embodiments, the chitosan has a molecular weight from about 100,000 Daltons
to about 300,000 and a concentration of at least about 5%.
In preferred methods and compositions of the present invention, the
chitosan has a degree of deacetylation of from about 70% to about 90%.
In preferred embodiments, the pharmaceutical active comprises a
retinoid. Examples of retinoids comprise retinoic acid or retinol. In
preferred
embodiments of the present invention, the retinoic acid comprises all traps
retinoic acid (ATRA).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph that shows ATRA distribution with chitosan topical
delivery.
Figure 2 shows the use of high molecular weight (HMW) chitosan to
enhance transdermal delivery.
Figure 3 shows ATRA distribution using 3% HMW chitosan.
Figure 4 is a graph showing ATRA permeation with the high molecular
weight chitosan (TD012).
Figure 5 is a graph that shows ATRA permeation of the high molecular
weight chitosan and middle molecular weight chitosan (TM761).
Figure 6 shows the stability of ATRA gels of the present invention at
20°C.
Figure 7 shows the stability of retinol creams of the present invention at
40°C.
Figure 8 shows the stability of ATRA in HMW chitosan.
Figure 9 is a graph that shows that as the chitosan concentration
increases from 1% to 3% this results in a more gradual release of retinoic
acid
from the chitosan matrix.
DETAILED DESCRIPTION OF THE INVENTION
The methods of the present invention provide a system of incorporating
active agents, e.g., pharmaceuticals, such as retinoids, into polymeric
carriers
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WO 01/4$645 CA 02394901 2002-os-21 PCT/US00/35319
to provide advantages, such as preferable tissue distribution of the drug,
prolonged half life, controlled drug release and reduction of drug toxicity.
More
particularly, the present invention relates to the use of a chitosan carrier
for the
topical delivery of an active agent, e.g., retinoids, where the sustained
release of
the drug can be altered by varying the properties of the chitosan that is used
as
a carrier base for the drug.
As used herein, the term "active agent" refers to any substance that when
introduced into the body has an affect on either the appearance of tissue to
which it is applied, or alters the way the body functions. The term
"pharmaceutical active" refers to a drug, i.e., a substance which when applied
to, or introduced into the body, alters in some way body functions, e.g.,
altering
cell processes. Examples of pharmaceutical actives include, but are not
limited
to, agents that are used for the treatment of skin diseases, e.g., retinoids,
corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDS), hormones,
anti-viral agents, anti-fungal agents, anti-septic agents, local anaesthetics,
anti-
histamines, kerolytic agents, 5-FU, etc. Other examples of such actives
include,
but are not limited to growth factors, recombinant human interleukin-2 and
DNA, RNA and oligonucleotides and the like.
The term "therapeutic active" as used herein, refers to a substance which
either alters processes within the body, or alters the cosmetic appearance of
the
tissue of interest, e.g., skin, but is not technically considered a drug.
Examples
of therapeutic actives include, but are not limited to, vitamins, e.g.,
vitamins A,
B, C, D and E, alpha-hydroxy acids, moisturizers and other additives, as
further described below.
In certain embodiments, the compositions contains more than one active
agent, thus the compositions comprises at least one additional active agent,
which can be either a pharmaceutical active or a therapeutic active. For
example, in a preferred embodiment, the compositions includes a retinoid as a
pharmaceutical active and alpha-hydroxy acid as a therapeutic active.
The invention will be discussed in relation to retinoids. However, it is to
be understood that any active agent that can be used in a topical delivery
system can be used in the compositions and methods of the present invention.
Preferably the active agent is a substance that has a molecular weight less
than
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about 300,000 Daltons. For example, preferred agents include retinoids, e.g.,
retinoic acid and retinol (Vitamin A), 5-FU, anti-fungal agents, anti-viral
agents,
anti-histamines, hormones and corticosteroids.
The term "topical" as used herein is known in that art and includes the
application of the compounds of the present invention to skin surfaces,
including mucosal surfaces, such as labial, rectal and genital mucosal
surfaces.
The term "carrier base" as used herein includes a component of the
delivery system that assists in the release of the active agent that is being
delivered. Preferred carrier bases comprise a high viscosity chitosan having a
molecular weight of at least about 100,000 Dalton, more preferably at least
about 250,000 Dalton and most preferably at least about 300,000 Dalton. In
other preferred embodiments the chitosan has a concentration of at least about
2 weight %. In an especially preferred embodiment, the carrier bases comprises
a high viscosity chitosan biopolymer having a molecular weight of at least
about
300,000 Dalton and at a concentration of at least 2 weight%.
The term "high viscosity" chitosan refers to a chitosan biopolymer having
a viscosity of at least about 100 cps. The viscosity of the chitosan solution
can
readily be determined by one of ordinary skill in the art, e.g., by the
methods
described in Li et al., Rheological Properties of aqueous suspensions of
chitin
crystallites. J Colloid Interface Sc 183:365-373, 1996. In addition, viscosity
can
be estimated according to Philipof's equation: V= ( 1 + KC)$, where V is the
viscosity in cps, K is a constant, C is the concentration expressed as a
fraction
(Form No. 198-1029-997GW, Dow Chemical Company). In certain
embodiments, the high viscosity chitosan preferably has a viscosity greater
than
at least 100 pcs, and more preferably greater than at least S00 cps. The term
"low viscosity" chitosan refers to a chitosan solution having a viscosity of
at
least about 1-30 cps. "Middle viscosity" refers to a chitosan having a
viscosity
of about 30-100 cps. Viscosity measurements reported here refer to a chitosan
solution at 1 % concentration in 1 % acetic acid measured in a Brookfield LVT
viscometer with appropriate spindle at 30 RPM, as common in the art.
The term "high concentration" as used herein, may refer to a
concentration of greater than about 2% chitosan in the solution. The term "low
concentration" refers to up to about 1% chitosan. The term "middle
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WO 01/45645 CA 02394901 2002-os-21 pCT~S00/35319
concentration" refers to between about 1 and about 2%.
The term "high molecular weight" chitosan, also referred to herein as
HMW, refers to chitosan having a molecular weight of at least about 250,000
Dalton. The term "middle molecular weight" chitosan, also referred to herein
as
MMW, refers to chitosan having a molecular weight of at least about 50,000 up
to about 250,000 Dalton. The term "low molecular weight" chitosan, also
referred to herein as LMW, refers to chitosan having a molecular weight up to
about 50,000 Dalton. In preferred embodiments, the carrier base is a chitosan
having a molecular weight of at least about 250,000 Dalton, more preferably at
least about 300,000.
The compositions and methods of the present invention rely on the
discovery of the inventors that the desired viscosity of the chitosans can be
achieved by manipulating the concentration, i.e., percentage, of different
molecular weight chitosans. For example, as shown in Table 1, a viscosity of
greater than 100,000 cps can be obtained by using 12% of a LMW chitosan, 5%
of a MMW chitosan or 3% of a HMW chitosan.
Table 1. Viscosity-concentration relationship for
different viscosity-grade Chitosans
LMW MMW HMW
Viscosity % Viscosity % Viscosity
c s) c s c s
7 1 66 1 552 1
21,263 9 151,403 5 15,862 2
~ 116,882 12 3.27 E+06 8 ~ 171,163 3~
~ ~ ~ ~
The methods and compositions of the present invention enable the
control of the active agent by varying the concentration, molecular weight
and,
therefore the viscosity of the chitosan. For example, in one embodiment of the
present invention, the use of a greater concentration of a lower molecular
weight chitosan will provide similar release rates as a higher molecular
weight
chitosan.
Retinoids, e.g., retinoic acid, are hydrophobic and highly insoluble. We
have found that delivery of retinoic acid is highly dependent on the viscosity
of
the carrier base. Thus, we have found that the higher the viscosity of the
colloidal solution of chitosan, the slower the release of the agent being
WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
delivered. For example, the retinoids in the present compositions are released
as the polymer film on the skin surface becomes hydrated. As the film
containing the drug and carrier dissolves away, new layers of the compositions
containing the drug are exposed, leading to further release of the drug to the
affected area of the skin.
The inventors have found that the chitosan-based controlled delivery
system of the present invention for delivery of retinoids enhances the
transdermal delivery of retinoids where warranted, yet prevents the overload
that results from traditional retinoid treatments and thus reduce skin
irritation.
As discussed further below, experiments using Franz diffusion cells have shown
that carrier bases of the present invention slow down the release of retinoids
which is delivered across the epidermal membrane, thus limiting the overload
of
retinoids to the dermal compartment. Thus, the compositions of the present
invention enable the slow, sustained release of the drugs, as desired.
The cumulative All-Trans-Retinoic Acid (ATRA) levels in each skin
compartment of hairless mouse skin after about 200 hrs exposure to different
chitosan formulations is shown in Figure 1. By varying the viscosity of the
chitosan from 550 cps for the 1% High Molecular Weight (HMW) chitosan
(MW~360,000 Dalton) to an estimated 3.27 million cps for the 8% Middle
Molecular Weight (MMW) chitosan (MW~120,000 Dalton) it is possible to obtain
a wide range of retinoid distributions. The cumulative percutaneous
penetration
across the skin is inversely proportional to the amount of retinoid remaining
on
the skin surface. As the amount of retinoid remaining on the skin surface
decreases from around 90% of the applied dose for the 8% MMW chitosan to
less than 30% for the 1% HMW, the percutaneous penetration of retinoid
increases from less than 10% to around 70%. Likewise, the amount of retinoids
in the skin layers increases from less than 1% for the 8% MMW to around 5%
for the 1% HMW.
Figure 2 shows the 1% HMW chitosan, containing 0.1% ATRA compared
to a control gel, containing 0.1 g ATRA. The 1% HMW chitosan contains 0.1%
ATRA (0.1 g ATRA, 0.04 g butylated hydroxytoluene (BHT), 1 g of Cremophor0
RH40, 15 g ethanol (200 proof, 1 g of Chitosan HMW, 81.8 g water, 1 g of
glacial acetic acid]. The control gel contained the following: 0.1 g ATRA,
0.04 g
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
BHT, 1 g of Cremophor~ RH40, 15 g of ethanol, 0.5g of Carbopol 940 NF, 76 g
water and 0.7 g Triethanolamine. The results show a higher percutaneous
penetration was obtained with the 1%HMW compared with the standard gel. A
full 70% of the applied retinoid dose was delivered transcutaneously with the
HMW formulation compared to around 45% with the control gel formulation. A
1% HMW chitosan formulation can be used to enhance the transdermal
penetration of retinoids to maximize the therapeutic power of retinoids.
Figure 3 shows that the 3 % HMW chitosan [containing 0.1% ATRA (0.1 g
ATRA, 0.04 g butylated hydroxytoluene (BHT), 1 g of Cremophor~ RH40, 15 g
ethanol (200 proof), 3 g of Chitosan HMW TD012, 80.8 g water, 1 g of glacial
acetic acid] compared to a standard control gel [containing the following: 0.1
g
ATRA, 0.04 g BHT, 1 g of Cremophor~ RH40, 15 g of ethanol, 0.5g of Carbopol
940 NF, 76 g water and 0.7 g Triethanolamine]. A lower percutaneous
penetration was obtained with the 3% HMW compared with the control gel. 32%
of the applied retinoid dose was delivered percutaneously with the HMW
formulation compared to 45% with the control gel formulation. A 3% HMW
chitosan formulation could be used to control release the retinoids and limit
the
potential for irritation.
Figure 4 shows the ability to release ATRA from the chitosan
formulations is highly dependent on their viscosity which range from 552 cps
for 1% HMW to 171,163 cps for the 3% HMW estimated from the Philipof's
equation: V= ( 1 + KC)8, where V is the viscosity in cps, K is a constant, C
is the
concentration expressed as a fraction. The higher the viscosity of the HMW,
the
slower the percutaneous release of ATRA over a period of 220 hours of a single
application in a Franz cell apparatus. The topical control gel consisting of
Carbopol~ 940 NF polymer displays a percutaneous ATRA delivery which lies
somewhere in between the Topical ATRA formulations ranging from 1% to 3%
HMW.
In Figure 5, the percutaneous permeation of MMW chitosan gels of high
viscosity (viscosity of 3.27 million cps for the 8% MMW estimated from the
Philipof's equation) compared to a 2.9% HMW with an estimated viscosity of
117,163 cps). The topical ATRA formulations containing the higher viscosity
chitosan display a lower percutaneous penetration through hairless mouse skin
17
WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
after 220 hours of continuous application in a Franz cell apparatus.
One of ordinary skill in the art can readily select an appropriate chitosan
component as the carrier for the compositions and methods of the present
invention, based upon the teachings described herein. For example, as
described above, one of ordinary skill in the art can use Phillipof's equation
for
predicting release rates from polymer concentrations and viscosities. As
aforesaid, a lower viscosity chitosan used at higher concentrations will
provide
similar release rates as a higher viscosity chitosan. Thus, if it is desirable
to
have a slow release of the retinoids, one would select a carrier base having a
high viscosity chitosan, e.g., a chitosan with molecular weight of at least
about
100,000 Dalton, e.g., 300,000, at a concentration of least 2 weight%. This
type
of composition is desirable to minimize the overload of retinoids which may
lead
to irritation of the skin.
Alternatively, if it is desirable to have a faster release of the retinoid,
one
would select a chitosan solution having a high molecular weight, e.g., of at
least
about 250,000, at a lower concentration, e.g.,. from about 1% to about 2%.
Such compositions are useful for increasing the transdermal release of the
active agent over a shorter period of time.
The combination of chitosan and retinoids in the compositions of the
present invention enhances the normal tissue architecture of naturally and
photoaged skin while reducing skin irritation, normally seen with retinoid
preparations.
The compositions of the present invention can be formulated into gels,
lotions, ointments or creams according to known methods. The delivery
systems can be used to form gels at concentrations greater than 2%. In
addition, these gels can be used as is or formed into creams by including an
oil
and emulsifying the mixture, by known methods. Preferred oils include
avocado oil, sea buckthorn oil, jojoba oil, etc. Other compounds can also be
added as desired to increase the effectiveness of the formulations. Examples
of
such additives may include, but are not limited to, vitamins such as A, B, C,
D,
E, K, etc., moisturizers such as alpha-hydroxy acids, etc. Other additives may
be used to improve the appearance of the formulation, e.g., odor, texture or
visual appeal. Examples of such additives include, but are not limited to,
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WO 01/45645 cA 02394901 2002-os-21 PCT/US00/35319
fragrances, coloring, enroll, ents and ingredients for the enhanced
percutaneous
absorption of various thera.peuti.c actives, such as glycerol, propylene
glycol,
oleic acid, surfactants, etc.
The delivery systems of the present invention can contain a large number
of pharmaceutical and therapeutic actives that can be applied topically either
singularly or in combination. Examples of these actives include, but are not
limited to compounds such as the following: Anti-fungal agents such as
Imidazoles, Clotrimazole, Clotrimazole/betamethasone dipropionate, Econazole,
Ketoconazole, Miconazole, Oxiconazole, Sulconazole, Allylamines, Naftifine,
Terbinafine, Polyenes, Nystatin, Nystatin/triamcinolone, Ciclopirox olamine,
Triacetin/sodium propionate/benzalkonium chloride/chloroxylenol, Tolfanate,
Undecylenic acid/zinc, undecylenate. Anti-inflammatory agents such as coal
tar, shale tar, wood tar, non-steroidal anti-inflammatory drugs (NSAIDS)
salicylic acid, salicylate esters and salts, acetylsalicylic acid, and the
like. Local
anaesthetics such as cocaine, benzocaine, tetracaine, lidocaine, bupivacaine,
their hydrochloride salts, and the like. Antibiotic agents such as bacitracin,
mupirocim, erythromycin, neomycin, clindamycin, doxycycline, trimethoprim-
sulfamethoxazole, penicillin-V, trimthoprim-sulfamethoxazole, chloramphenicol,
gentamycin, azithromycin, ciprofloxacin, ofloxacin, ceftriaxone, minocycline,
amoxicillin-clavulanate, first-generation cephalosporin, ceftriaxone, and the
like. Sulfanilamide antibacterial agents such as sulfanilamide, sulfacetamide,
sulfadiazine, sulfisoxazole, sulfamethoxazole, trimethoprim, pyrimethamine,
and the like. Antiviral agents such as Imiquamod, acyclovir, valacyclovir,
famcyclovir, penciclovir, idoxuridine, trifluridine, foscarnet, cidofovir,
interferons, IFN-a, IFN-a2b, IFN-an3, nucleoside analogues, protease
inhibitors
and the like. Antiseptic agents such as acridine dyes, alcohols, bronopol,
chlorhexidine, phenols, hexachlorophene, organic mercurials, organic
peroxides, i.e., benzoyl peroxide, quaternary ammonium compounds, and the
like. Vitamin and vitamin derivatives such as Vitamin A, retinol, retinoic
acid
(both cis and trans), alpha-tocopherol (Vitamin E), 7-dehydrocholesterol
(Vitamin D), Vitamin K, thiamine riboflavin, niacin, pyridoxine, biotin,
pantothenic acid, ascorbic acid, choline, inositol, and the like. Anti-
inflammatory corticosteroids such as progesterone, hydrocortisone, prednisone,
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
fludrocortisone, triamcinolone, dexamethasone, betamethasone, fluocinolone,
and the like. Autacoids such as prostaglandins, prostacyclin, thromboxanes,
leukotrienes, angiotensins (captopril), as well as other pharmaceutically
active
peptides such as serotonin, endorphins, vasopressin, oxytocin, and the like.
Kerolytic agents such as benzoyl peroxide, salicylic acid, trichloroacetic
acid,
and piroctone, and wart treatment compounds such as salicyclic acid,
trichloroacetic acid and lactic acid, singularly or in combination with anti-
viral
agents. Anti-alopecia agents such as niacin, nicotinate esters and salts, and
minoxidil. Sun-Protective agents such aminobenzoates, Para-aminobenzoic
acid (PABA), Ethyl-4-[bis(hydroxypropyl)-aminobenzoate, Glyceyl PABA, Amyl p-
dimethylaminobenzoate (padimate A), 2-ethylhexyl PABA (padimate O),
Cinnamates, Dietholamine p-methoxycinnamate (Parsol MCX), Salicylates, 2-
ethylhexyl salicylate, Homosalate (homomenthyl salicylate), Octyl salicylate,
Triethanolamine salicylate, Trolamine salicylate, Benzophenones,
Dioxybenzone, Sulisobenzone, Oxybenzone, Ethylhexyl, 2-cyano-3, 3-diphenyl-
acrylate (octocrylene), Lawsone and dihydroxyacetone, 2-phenylbenzimidazole-
5-sulfonic acid, Digalloyl trioleate, Red veterinary petrolatum, Titatium
dioxide,
Methyl anthranilate, Butylmethoxydibenzoyl methane (avobenzone), zinc oxide.
Other additives can also be used, e.g., moisturizing agents such as lactic
acid, pyrrolidone carboxylic acid, glycolic acid, water, glycerine, propylene
glycol, sorbitol, other alphahydroxy carboxylic acids, and various salts of
these
esters and salts, and the like and additives for the enhanced percutaneous
absorption of various pharmaceutical or therapeutic actives. Such
percutaneous enhancers include propylene glycol, glycerol, urea, diethyl
sebecate, sodium lauryl sulfate, sodium laureth sulfate, sorbitan ethoxylates,
nicotinate esters (such as hexyl nicotinate), oleic acid, pyrrolidone
carboxylate
esters, (such as dodecyl pyrrolidone carboxylate), N-methyl pyrrolidone, N,N-
diethyl-mtoluamide, dimethyl sulfoxide, decyl methyl sulfoxide, alkyl methyl
sulfoxides, N,N-dimethyl formamide, cis-11-octadecenoic acid, 1-
dodecylazacycloheptan-2-one, and 1,3-dioxacyclopentane or 1,2-
dioxacyclohexane containing at least one aliphatic group of four to eighteen
carbon atoms.
The amount of active employed will be that amount necessary to deliver a
WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
pharmaceutically or therapeutically effective amount to achieve the desired
result at the site of application. In practice, this will vary depending upon
the
particular medicament, severity of the condition as well as other factors. In
general, the concentration of the actives in the delivery systems can vary
from
as little as 0.0001 up to 5 percent or higher, by weight of the delivery
system.
For retinoids, a preferred dose is between 0.01 %-1 % for retinol and between
0.01% -0.1% for all-trans-retinoic acid.
Other adjuvant ingredients such as glycerin, propylene glycol, sorbitol,
preservatives, stearic acid, cetyl alcohol, other high molecular weight
alcohols,
surfactants, menthol, eucalyptus oil, other essential oils, fragrances,
penetration enhancers, and the like to give stable cremes, ointments, lotions,
aerosols, solutions, may also be included.
Alternatively, solutions or mixtures of the actives with the chitosan
derivatives may be prepared with or without some of the adjuvant ingredients,
and these solutions or mixtures may be fabricated into films, rods, sheets,
sponges or fibers for use as suppositories, medicated sutures, medicated
sheets, medicated bandages, patches, and the like. It is relatively easy to
process chitosan into various forms such as small particles, gel, and cotton
mesh for drug delivery applications. Such methods are known in the art.
In a preferred composition, alpha-hydroxy acid (AHA) is used to
completely dissolve the chitosan. AHA is also referred to as glycolic acid in
the
methods and examples described below. The benefit of using alpha -hydroxy
acid is two-fold. One advantage is that it helps dissolve the chitosan.
Another
advantage is that the combination of alpha-hydroxy acid and chitosan, which is
basic, raises the pH of the composition which in turn, minimizes the peeling
seen with standard alpha-hydroxy acid formulations. Neutral or mildly acidic
vehicles of alpha-hydroxy acids are actively being sought (Neudecker et al.,
2000). It is common practice to use ammonium salts to neutralize the alpha-
hydroxy acids present in most current cosmetic preparations. Ammonium salts
present in most current cosmetic preparations of alpha-hydroxy acids may
prevent hyaluronan (HA) enhancement (Neudecker et al., 2000). Chitosan,
through the presence of its amino groups on the polymer chain, can be used to
neutralize the alpha hydroxy acids. The addition of 3% HMW chitosan raises
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
the pH of an alpha hydroxy solution from 3.5 to 5.5 , thus bringing the pH of
the AHA formulation in the mildly acidic range where the action of AHA can
effect the ability to stimulate HA production rather than implement their
action
by peeling the skin and cause diffuse wound healing.
AHA is thus useful as an active agent alone, or in conjunction with
another pharmaceutical or therapeutic active.
The compositions of the present invention are stable, as is necessary for
topical treatments. ATRA gels made from the HMW chitosan at concentrations
greater than 2% are stable for at least 120 days and comparable in stability
to
the standard control gels made from Carbopol as shown in Figure 6. Lower
concentrations of chitosan may cause a reduction in the stability of the ATRA
in
the gel formulation. As shown in Figure 7, creams made from the 3% HMW are
highly stable, again as a result if the high viscosity of this type of
chitosan when
present at greater than 2% concentration. Similar results would be obtained
with the MMW chitosan present at concentration than 5% w/w. The difference
in stability is related to the addition of the surfactant Cremophor RH40 which
causes a reduction in ATRA stability compared to the HMW formulation alone.
The inventors have found that the use of a carrier base with a high-
viscosity grade chitosan, e.g., having a molecular weight of at least about
300,000 Dalton and at a concentration, e.g., of at least 2 weight % results in
a
greater stability of the retinoid preparation, over a period of months. See
Figure
8 and Example 3; below. Thus, one advantage of using a high molecular weight
chitosan in delivering an active agent, such as retinoids, is the ability to
use a
lower concentration to obtain a sufficient viscosity required for
stabilization of
the retinoids. Stability of formulations is often tested at 40 °C for a
period of
several months.
To the best of our knowledge there are presently no chitosan-based
retinoid delivery systems. For percutaneous drug delivery chitosan offers
unique advantages. For example, chitosan is used in cosmetology to make
moisturizing creams. The concentration in moisturizers and soaps varies from
0.3% to 1% chitosan. These concentrations have been experimentally tested by
the manufacturers and are well tolerated on the skin. It is also used in hair
sprays, styling gels and shampoos: its cationic nature enables a close bond to
22
WU 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
the keratin anion (Sachetto, 1986; Cleenewerck, 1994). Chitosan is a
biodegradable polymer which has advantages over a synthetic polymer, e.g.,
PP2. For example, chitosan is. completely degraded in the body. It degrades
without leaving residual matter which could build up in the tissues. As suture
material, chitosan has been shown to be completely absorbed in one to two
months so it would release the drug during the same period (Suzuki, 1995). It
is unnecessary to remove chitosan from the body after the complete release of
the drug because chitosan has good biodegradability and is completely
dissolved by enzymes such as lysozyme.
As aforesaid, the present invention provides methods for the treatment of
many skin ailments. To our knowledge there is no controlled topical delivery
system of retinoids for melanoma chemoprevention. One aspect of the present
invention is a chitosan based percutaneous delivery system for the
chemoprevention of melanoma in individuals with dysplastic nevi who are at
high risk of developing melanoma.
In addition, the combination of retinoids and a chitosan -based delivery
system takes advantage of the immunostimulating properties of chitosan for the
delivery of therapeutic actives in skin conditions that necessitate an immune
response. The compositions of the present invention utilize the property of
chitosan to initiate immune and reparative functions, either directly or
indirectly through the stimulation of macrophages in the skin tissue.
Activation and production of cytokines such as IL-1 leads to increased
angiogenesis and skin reparative functions. IL-1 and TNF- a, produced by
macrophages, stimulate fibroblasts (Chang J et a1.1986). Chitosan has been
shown to stimulate macrophage production, resulting in activation of cytokines
such as interleuken-1 (IL-1 ) and interferon gamma (IFN-y). (Chensue et al.,
1989; Shibata et al., 1997). The degree of deacetylation for immunostimulatory
activity is optimal around 70% and other degrees of deacetylation result in
the
reduction of immunostimulatory activity (Nishimura et al, 1984, 1985, 1986,
1990). A 70% deacetylated chitin has been used in combination with
petrolatum to immunostimulate the skin in the management of senile
erythroderma. (Horuchi & Otoyama, 1996). The chitin derivative is not
employed in these studies as a delivery system but rather as the active
23
WO 01/4564$ CA 02394901 2002-os-21 PCT/US00/35319
ingredient in the topical petrolatum-based formulation.
In addition, the chito-oligomers released from chitosan by the in vivo
hydrolytic action of lysozyme and N acetyl-(3-D-glucosaminidase after
penetration of chitosan into the skin may stimulate hyaluronan synthesis.
Recent evidence is found for the presence of DG42 protein (a chito-oligomer
synthase) during embryogenesis, producing chito-oligomers acting as primers in
the synthesis of hyaluronan. Overexpression of DG42 in mouse cells leads to
the synthesis of chito-oligomers, and hyaluronan synthase preparations also
contain chitin synthase activities (Varki A, 1996; Semino et al., 1996;
Bakkers
et al., 1997).
Chitosan has the potential, directly or indirectly through the formation of
hyaluronic acid, to correct this deficiency and to provide correct deposition
of
collagen fibers such as reduced space and fiber thinness, fiber
disorganization
and depth of disorganization.
Therefore the administration of retinoids via a chitosan carrier base has
the potential of enhancing both the quantity and quality of new collagen
production in skin connective tissue.
The methods of the present invention take advantage of the reparatory
effect of chitosan to stimulate fibroblasts in conjunction with the
therapeutic
effect of retinoids to obtain a synergistic effect. The increase in collagen
repair
is useful for treating conditions that which would benefit from an
immunostimulatory response, e.g., in preparations used for anti-wrinkle
products as well as for products that are used to treat photodamage and other
such skin conditions.
As aforesaid, the compositions of the present ivention are useful for
treating skin diseases. Examples of skin diseases which can be treated
include,
but are not limited to, acne, melanoma, premature skin aging, and
photodamage. In preferred embodiments the active agent comprises a
pharmaceutical active, e.g., an agent that is used for the treatment of skin
diseases. Examples of pharmaceutical actives include, but are not limited to
retinoids, such as corticosteroids, non-steroidal anti-inflammatory drugs
(NSAIDS), hormones, anti-fungal agents, anti-septic agents, local
anaesthetics,
kerolytic agents, 5-FU, etc. In other embodiments, the active agent comprises
a
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therapeutic active, e.g., vitamins, moisturizing agents such as alpha-hydroxy
acids, etc., as further described below. The amount and frequency of the
application of the delivery systems can readily be determined by one of
ordinary
skill in the art, based upon the type and severity of the ailment, as well as
the
amount of agent present in the system.
As aforesaid, in some methods of treating certain skin diseases, it may be
useful to use the compositions of the present invention in conjunction with
other treatments for the disease. For example, in treating precancerous skin
conditions, it may be useful to use the compositions of the present invention
with standard treatments that use an anti-cancer drug, e.g., 5-FU, for the
treatment of actinic keratosis.
The present invention is further illustrated by the following Examples.
The Examples are provided to aid in the understanding of the invention and are
not construed as a limitation thereof.
All examples are carried out using standard techniques, which are well
known and routine to those of skill in the art, except where otherwise
described
in detail. Routine techniques of the following examples can be carried out as
described in standard laboratory manuals.
EXAMPLES
Summary of experiments:
In the design of the topical delivery system different polymer
formulations were prepared. Table 2 shows the types of chitosan used. The
chitosan was obtained from Primex Ingredients, Avaldnes, Norway.
These formulations were then tested in in vitro assays, i.e. penetration
and recovery studies using conventional and radiolabeled retinoids and long-
term stability studies at 20 °C and 40 °C, as described below
with a Franz
diffusion cell. Human subjects are then exposed to selected formulations (in
vivo) and compared to current dermal retinoid formulation to test their
ability to
reduce irritation.
WO 01/45645 cA 02394901 2002-os-21 PCT/US00/35319
Table 2
TYPE OF
VISCOSITY1 DEGREE OF
CHITOSAN DESCRIPTION
LOT # (MpAS) DEACETYLATION2
Soluble in 1% Acetic
Acid
HMW 552 89 or 2% Glycolic Acid
0%
(TD012) . Gel at concentration
of
3%
Soluble in 1% Acetic
Acid
MMW 66 96.1% or 2% Glycolic Acid
(TM761) Gel at concentration
of
5% or hi her
Soluble in 1% Acetic
Acid
LMW 7 95 or 2% Glycolic Acid
0%
(TM615) . Slightly viscous liquid
at
concentration of 3%
Soluble in 1% Acetic
Acid
LMW 23 80 or 2% Glycolic Acid
8%
(TM816) . Slightly viscous liquid
at
concentration of 3%
Soluble in 1% Acetic
Acid
LMW 10 87 or 2% Glycolic Acid
8%
~I (TM611) . Slightly viscous liquid
at
concentration of 3%
1. The viscosity of 1% solutions in 1% acetic acid was measured on a
Brookfield LVT viscometer, 25 °C, with appropriate spindle at 30
rpm (From Primex Ingredients, Product Literature).
2. The degree of deacetylation was measured by the UV-method
(From Primex Ingredients, Product Literature).
In the following examples, sample TD012 is an example of a high
molecular weight (HMW) chitosan, TM761 is an example of a middle molecular
weight (MMW) chitosan, and TM615, TM816 and TM611 are examples of low
molecular weight (LMW) chitosans.
EXAMPLE 1: Preparation of chitosan-retinoid compositions
Gel Chitosan TD012 has a viscosity of 500 cP when dissolved with 1%
glacial acetic acid at 1% concentration. The viscosity increases as a function
of
concentration of the polymer, reaching an estimated 171,163 cps at 3%
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
concentration.
Colloidal solutions up to 3% (wt/wt) chitosan were obtained by dissolving
high molecular weight chitosm (HMW (TD012); MW 360,000 Da.itons) in 1%
glacial acetic acid at room temperature. Carrier bases up to 8% were obtained
by suspending chitosan powder of middle molecular weight (MMW (TM761); MW
120,000) (8 g in 66 g of deionized water) in water at room temperature,
raising
the temperature to 90 °C and adding 25 g of water and 1 g of glacial
acetic acid,
dropwise to chitosan to form a clear, highly viscous solution after cooling at
room temperature.
EXAMPLE 2 - In vitro skin penetration studies using radiolabeled retinoids.
Fresh hairless mouse skin samples were obtained from surgery, and
upon arnval to the lab they were stored in a freezer (-20 °C).
Immediately prior
to the permeation experiments, skin samples without subcutaneous fat were
thawed by floating on water at 22 °C for about 10-20 minutes. A 1.0 cm2
portion of the skin samples was fastened between the Franz diffusion cell's
receptor chamber and chimney top by an o-ring and a spring clamp
(PermeGear, Inc.) (Lehman et al., 1988.)
For gel sample preparation, 20 uL of radiolabeled 3H-Retinoic Acid (20
microcuries) (NEN LifeSciences, Boston, MA) were added to 1.5 grams of a
retinoic acid stock solution, comprised of 100 mg of retinoic acid in 15 grams
of
absolute ethanol (200 proof and 1 g of hydrogenated castor oil (cremophor
RH40, BASF Corporation) and were mixed with 8.5 grams of the chitosan
colloidal solution.
For the cream sample preparation, 20 uL of radiolabeled 3H-Retinoic Acid
(20 microcuries) (NEN LifeSciences, Boston, MA) were added to 0.6 g of the
retinoic acid stock solution (comprised of 100 mg of retinoic acid, 10 g of
avocado oil and 1 g of Cremophor RH40). The solution was then mixed with 1.5
g of glycerin, 0.05 g of Vitamin E, 0.1 g of Seabuckthorn Seed Extract.
Finally, 7.8 g of TD012 (2.9%) chitosan, dissolved in glycolic acid (pH 5.5)
was added homogeneously.
Approximately 200 mg of each formulation was applied to the sample
compartment (i.e. the epidermal side) of the skin sample. The dermal surface
of
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
the skin was perfused with receptor phase solution (phosphate buffered saline
containing 0.5% Volpo surfactant (Croda, Inc.). Each formulation was tested in
triplicate.
The receptor volume was sampled every 24 hours by withdrawing 500
~L. It was then mixed with scintillation fluid for scintillation counting.
At the end of the run the entire content of the reservoir compartment of
the Franz cell (5 ml) was removed and placed in a scintillation vial with 10
ml of
scintillation fluid. Any retinoid remaining on the surface of the skin (top
wash)
was extracted with 2 x 500 ~L of ethanol containing 1% glacial acetic acid and
placed in a scintillation vial containing 9 ml of scintillation fluid
(Packard)
The epidermis and dermis were digested overnight in 4 ml of tissue
solubilizer (Solvable Tissue and Gel Solubilizer -Packard Instruments) to
which
6 ml of scintillation fluid (Ultima Gold -Packard Instruments) was added and
analyzed by scintillation counting.
The permeation of all-trans retinoic acid (ATRA) across hairless mouse
skin as a function of concentration of the high viscosity chitosan TD012 and
middle viscosity TM761 is shown in figure 1. As shown in Figure l, it is
possible
to increase the percutaneous penetration from 8% to 68% ATRA percutaneous
penetration by changing the chitosan polymer from 8% TM761 (the medium
viscosity chitosan: 10 cP at 1% concentration) to 1% TD012 (high viscosity
chiosan: 552 cP at 1% concentration). As the amount of ATRA penetrating
increases, there is a concomitant decrease of ATRA on the skin surface. The
amount in the skin layers decreases from 5% to 0.5% as the amount of ATRA
penetrated decreases.
As the concentration of the high viscosity chitosan (TD012) decreases,
the amount of ATRA permeated through the skin into the Franz Cell Reservoir
compartment increases as shown in Figure 4. The ATRA release from a
standard gel made with CarbopolT"' 940 NF acrylate polymer (BF Goodrich) is
intermediary between the 1% and the 2% chitosan TD012.
These results show that it is possible to control the delivery of the
retinoid ATRA by changing the chitosan concentration, in relation to the
viscosity of the chitosan. An increase in concentration of the middle
viscosity
chitosan TM761 further reduces the permeation rate (Figure 5).
28
W~ 01/4564$ CA 02394901 2002-06-21 PCT/US00/35319
EXAMPLE 3 - Stability Testing of retinoid gels and creams
A. Preparation of gels and creams based on retinoic acid and chitosan
TD012.
For the preparation of gels and creams the high molecular weight TD012
chitosan (M.Wt 360,000 Dalton) was chosen due its slow release characteristics
for retinoic acid. We chose to use the TD012 Chitosan (2.9%) because it forms
a
highly viscous colloidal solution at room temperature and it offers a
favourable
ATRA release profile.
Preparation of retinoic acid gel.
Solution A was prepared by dissolving chitosan in a 1% glacial acetic
acid solution as follows: 2.9% Chitosan TD012, 79.98%Water in 1% Acetic Acid.
Solution B was prepared by dissolving cremophor RH40 in ethanol in an amber
container followed by BHT and retinoic acid. The amounts are as follows: 15%
Ethanol, 1% Cremophor RH40, 0.02% BHT and 0.1% Retinoic Acid. Solution B
was mixed into solution A using a 3-blade laboratory mixer.
Preparation of Retinol Cream was as follows:
Solution A: 3% Chitosan TD012
appx. 62.84%Water
2.86% Glycolic Acid (70% solution)
appx. 3.5% NaOH Solution ( l Og in 100m1 water)
to bring to pH=3.5
Solution B: 15% Glycerin
1% Cremophor RH40
0.5% Vitamin E Acetate
10% Avocado Oil
1% Sea buckthorn Seed Oil
0.1% Perfume
0.2% Retino150C
Solution A was prepared by adding glycolic acid to water. While stirring, NaOH
( l Og/ 100 ml) was added dropwise to raise the pH from 2.12 to 3.5. Then
chitosan was added and allowed to dissolve completely overnight. The final pH
was 5.3-5.5. Solution B was prepared by combining the glycerin, cremophor
RH40, vitamin E acetate, avocado oil, and sea buckthorn oil. The perfume and
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WO 01/45645 CA 02394901 2002-os-21 pCT/US00/35319
retinol 50C (50% w/w of retinol in Polysorbate 20 -BASF) were added
sequentially to obtain an homogeneous solution. Solution B was then
incorporated into Solution A using a 3-blade laboratory stirrer.
Preparation of 0.1% Retinoic Acid Cream
For the retinoic acid cream 100 mg retinoic acid was substituted for the
200 mg of retinol 50 C. The retinoic acid was initially suspended in 10 g of
avocado oil containing 1 g of cremophor RH40. The rest of procedure is similar
to the retinol cream.
Stability Testing
The stability of the retinoic acid gels was tested at both 20 °C
and 40 °C
in a water bath. Retinoid concentrations were tested by dissolving 0.2g of the
gel (or cream) in 6.7g of a 1% acetic acid in ethanol solution. The solution
was
then stirred using a magnetic stirring bar and plate until the retinoid and
chitosan had dissolved.
For the retinoic acid sample, a 100 ~L quantity was diluted 10-fold in 1%
acetic acid/ethanol solution and the absorbance measured at 351 nm using a
Pharmacia Biotech Ultrospec 2000 Spectrophotometer. For the retinol samples,
a 50 ~L quantity was diluted 20-fold in 1% acetic acid/ethanol solution and
absorbance readings at 326 nm. The stability measurement was repeated once
per week over several weeks.
Gel samples designated 87-1 consist of 0.1% ATRA in 2.9% TD012 a.s in
EXAMPLE 1; 101-1 is 0.1 % ATRA in 0.5% Carbopol 940 NF instead of 2.9
TD012 ; 109-1 is 0.1% ATRA as in EXAMPLE 1 with 3.5% TD012 instead of
2.9% TD012. Cream samples 2-3-1 consist of 1% retinol in TD012 (2.9%) as in
EXAMPLE 2. Cream sample 2-5-1 is the same as 2-3-1 without the Cremophor
component.
EXAMPLE 4 -Patch Testing in Healthy Individuals
Human studies are undertaken to evaluate the irritation potential of the
chitosan/ATRA percutaneous delivery system. 15 patients having signed an
informed consent are patch tested with commercial creams containing
W~ 01/45645 CA 02394901 2002-06-21 PCT/US00/35319
conventional ATRA and wits a cream of the present invention containing
chitosan and retinoids at an equivalent dose. The creams are prepared
according to the methods in Example 3 and as shown below. The irritant
potential of the tretinoin/chitosan delivery system on human skin is assessed
by means of patch test evaluations as follows:
For assessing irritation (Seaton, 1995), the occlusive Hill Top Chamber
patch testing system (Hill Top Research, Inc., Cincinnati, Ohio) incorporates
0.2
ml of sample.
The human evaluation involved three strengths of commercially available
tretinoin (ATRA) cream (0.01%, 0.05% and 0.1%) with two concentrations of
chitosan (1% and 3%) in the formulation.
The data is evaluated in terms of a Mean Irritation Score by evaluating
the extent of erythema, as previously described (Mills and Berger, 1998).
Statistical evaluation includes both frequency and severity of erythema seen
at
sites treated with tretinoin containing chitosan and commercially available
tretinoin using analysis of variance (ANOVA) and the paired t-test.
Patch testing of ATRA Cream
The drug product (ATRA Cream) consists of a modified retinoic acid
formulation. The control cream was obtained from Technical bulletin ME 142e
for Retinoic acid (BASF Corporation, NJ). To test the chitosan-based cream on
irritancy levels the following formulations are prepared:
Control Cream
I Luvitol~ EHO ( 1 ) 8 g
II Cremophor A 6 ( 1 ) 3.0 g
Cremophor A 25 ( 1 ) 1.5 g
Glycerol monostearate 3.0 g
Cetyl alcohol 3.0 g
Tegiloxan~ 100 (2) 0.5 g
III Butylated hydroxytoluene 0.04 g
1,2-Propylene glycol 4.0 g
Nip-Nip~ (3) 0.2 g
Germail~ (4) 0.3 g
Perfume 0.2 g
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
Water 76.2 g
Mixture II is heated to 75 °C and stir in Solution I. Mixture III is
heated until
a completely clear solution is obtained, then added to the heated Mixture I/II
and stirred until cold.
Control Cream + 0.1% ATRA
I ATRA (USP) 100 mg
Luvitol~ EHO ( 1 ) 8 g
II Cremophor A 6 (1) 3.0 g
Cremophor A 25 (1) 1.5 g
Glycerol monostearate 3.0 g
Cetyl alcohol 3.0 g
Tegiloxan~ 100 (2) 0.5 g
III Butylated hydroxytoluene 0.04 g
1,2-Propylene glycol 4.0 g
Nip-Nip~ (3) 0.2 g
Germail~ (4) 0.3 g
Perfume 0.2 g
Water 76.2 g
Mixture II is heated to 75 stir in Solution I. Mixture III
C and is heated until
a completely clear solution is ed, then added to the heated Mixture
obtain I/II
and stirred until cold.
HMW-Chitosan Cream
I Glycerol 15 g
Cremophor0 RH40 ( 1) 1 g
Vitamin E Acetate 0.5 g
Avocado Oil 10 g
Sea Buckthorn Seed Oil 1 g
Perfume 0.1 g
II Chitosan TD012 3.0 g
Glycolic Acid (70%) 2.86 g
NaOH Solution ( 10%) 3.5 g
Water 62.84 g
Mixture I is incorporated ~~ith solution II and the Mixture I/II is
homogenized to a fine consistency.
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
3% HMW-Chitosan Cream + 0.1% ATRA
I ATRA 100 mg
Glycerol 15 g
Cremophor~ RH40 (1) 1 g
Vitamin E Acetate 0.5 g
Avocado Oil 10 g
Sea Buckthorn Seed Oil 1 g
Perfume 0.1 g
15
25
II Chitosan TD012 3.0 g
Glycolic Acid (70%) 2.86 g
NaOH Solution ( 10%) 3.5 g
Water 62.84 g
Mixture I is incorporated with solution II and the Mixture I/II is
homogenized to a fine consistency.
Product Suppliers and Manufacturers
1. BASF Corporation, Ludwigshafen, Germany
2. Th. Goldschmidt AG, Essen, Germany
3. Henkel KgaA, Dusseldorf, Germany
4. Ru-Jac Inc., Upper Montclair, NJ
Clinical Experimental Design - The clinical study is performed in three parts:
Part I
Part I involves 6 human volunteers. Each volunteer receives the 6
formulations listed below. Each formulation consists of 0.2 g of test sample,
applied to the volar forearm (3 formulations on each forearm) in the form of a
patch (Hill Top Research, Inc., Cincinnati, OH). Each human subject is
evaluated at 24 hours for signs of irritancy (e.g. erythema).
Patients No. 1 to 6: Formulation (A,B,C,D as referred above)
Site 1 A (Control Cream)
Site 2 B (Control Cream + 0.1% ATRA)
Site 3 C (1% HMW-Chitosan)
Site 4 D ( 1 % HM W-Chitosan 1 % + 0.1 % ATRA)
Site 5 C (3% HMW-Chitosan)
Site 6 D (3% HMW-Chitosan) + 0.1% ATRA)
The location of each test sample is rotated for each individual according to
latin square design.
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WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
Part II
Given that the results of Part I show no irritation from the volar application
of the formulations, Part II involves 3 additional human subjects, each
subject
receiving 3 patches containing 0.2 grams of test sample to the paraspinal area
of the back to verify any irritation caused by the base alone without ATRA.
The
patch application is for 24 hours with irritancy evaluation at 30 minutes
after
patch removal and 24 hours after patch removal.
For Patients 7 to 9 Formulation (A, C as referred above
Site 1 A (Base Cream)
Site 2 C (HMW-Chitosan 1%)
Site 3 C (HMW-Chitosan 3%)
The location of each test sample is rotated for each individual according to
Latin square design.
Part III
Given that the results of Part II show no irritancy, Part III involves the
testing of 6 additional human subjects. Each participant receives 6 patches
applied to the paraspinal area on the back, including 3 patches of the control
cream and 3 patches of the 3.9% HMW-chitosan cream each containing 3
strengths of ATRA. Patches are removed after 24 hours and irritancy scored 30
minutes and 24 hours. Statistical evaluation includes ANOVA and paired t-test
to evaluate any significant difference between treatments, sites and patients.
For Patients 10 to 15 Formulations (B, C as referred above)
Site 1 B (Control Cream + 0.01% ATRA)
Site 2 B (Control Cream + 0.05% ATRA)
Site 3 B (Control Cream + 0.1% ATRA)
Site 4 D (3% HMW-Chitosan + 0.01% ATRA)
Site 5 D (3% HMW-Chitosan + 0.05% ATRA)
Site 6 D (3% HMW-Chitosan + 0.15% ATRA)
The location of each test sample is rotated for each individual according
to Latin square design.
EXAMPLE 5. CHITOSAN GELS AS DELIVERY VEHICLES FOR RETINOIC ACID
The topical carrier base consisting of high viscosity chitosan with a
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WO 01/45645 cA 02394901 2002-os-21 pCT/[JS00/35319
molecular weight of at least 300,000 Dalton and at a concentration of at least
2
weight% acts as a delivery system to control the release of retinoic acid
(RA).
Studies with [3H)retinoic acid. A high molecular weight chitosan (viscosity of
552 cP with 1% solutions in 1% acetic acid measured on a Brookfield LVT
viscometer at 25 C, appropriate spindle at 30 rpm, MWt of 360,000 Dalton). As
the chitosan concentration increases from 1% to 3% this results in a more
gradual release of retinoic acid from the chitosan matrix as shown in Figure
4.
EXAMPLE 6 - Preliminary in vitro evaluation of topical chitosan delivery
system
for retinoids
A. Skin sample preparation:
Fresh skin (female abdominal) was obtained from surgery, and upon
arrival to the lab was washed and stored with 0.1 M phosphate-buffered saline
(PBS) buffer (pH 7.4).
Subcutaneous fat was removed and the skin was rinsed in PBS, it was
then dried and stored in the freezer (-20 C).
Prior to skin splitting, full skin was thawed overnight in sterile PBS. The
split skin procedure consisted of taking a 4 x 4 cm full skin sample and
immersing it in water at 60 °C for approximately 60 sec. The epidermis
was
then carefully removed with forceps and placed on aluminum foil and stored at
-20 °C. Prior to the permeation experiment, split skin samples were
thawed by
floatation in water at 22 °C for - 20-40 minutes.
B. Vehicle Preparation
3.5% HMW-Chitosan (88.8% deacylated chitosan, 1000 cps viscosity,
800,000 MWt; Primex Ingredients SA, Avaldsnes, Norway) was dissolved in 1%
acetic acid for 24 hours prior to mixing. The retinoid/chitosan formulation
was
made up by adding concentrations of retinoids (ATRA or 9-cis-RA) ranging from
0.01% to 0.1% in a colloidal formulation containing 50% ethanol, 1% vitamin E,
8% cremophore RH40, 40% water and 1.75% HMW-chitosan
C. Franz diffusion cell setup
WO 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
All experiments used 9 mm amberized Franz diffusion cells purchased
from PermeGear Inc. (Riegelsville, PA). Amberized cells were used to limit
light
exposure to the retinoic acids. The Franz cells were clamped in series, and
water from a water bath (37 °C) was circulated through all cells. A
magnetic
stirrer was placed underneath all 3 Franz cells to ensure constant agitation
of
the fluid within the receptor compartment and hence a more homogeneous
distribution of the permeant (retirioic acid). Split skin (epidermis) samples
of
approximately 2.5 cm2 surface area were carefully placed upon the receptor
compartment (dermis side facing down). The donor cap was then placed upon
the skin and carefully clamped into place with a horseshoe clamp.
Receptor fluid (consisting of 25% ethanol and 75% PBS) was placed
within the receptor compartment. This concentration of ethanol in PBS
prevented the formation of a two-phase system (turbidity) while maintaining
the
retinoid in solution.
D. Retinoid Percutaneous Studies
A known quantity of conventional retinoid (0.01 %-0.1 %) was placed in
the donor compartment, covered with aluminum foil to prevent evaporation.
Samples (200 ~1) for spectrophotometric analysis were then removed from the
receptor port at timely intervals up to 48 hours and stored in amberized lml
Teflon-capped vials. The same quantity of receptor fluid (at 37 °C)
was then
returned to the receptor compartment to ensure a constant volume. Samples
from the vials were diluted five-fold and then quantitated via UV absorbance
using a Shimadzu UV 160U spectrophotometer. Maximum absorbance of ATRA
(all trans retinoic acid) and 9cRA (9-cis retinoic acid) was at 348.5 nm and
340
nm respectively. The cumulative amount of the applied dose which crossed the
epidermis into the receptor chamber was determined as follows: C = R*25/A,
where: C= cumulative amount, (~g/cm2); R = retinoid (fig) (from UV reading and
standard curve), 25 = dilution factor; A= Area of skin exposed to formulation
in
sample compartment (0.785 cm2).
E. Preliminary Radiolabeled ATRA percutaneous studies
Retinoid penetration through human skin was determined as follows:
36
W0 01/45645 CA 02394901 2002-os-21 PCT/US00/35319
5~1 of 3H-ATRA (NET-1117) were mixed homogenously to 500 ~1 of HMW-
Chitosan to make a 0.001 % gelling solution.consisting of 0.05 g Tretinoin, 50
ml 95% Ethanol, 3.2 g Cremophor RH-40, 1.0 g Vitamin E acetate, 50 ml 2.5%
Chitosan ( high MW Primex Superior). For the ethanol solution, the chitosan
was omitted in the formulation.
200 ~1 of this solution was then placed on the skin section within the
Franz cell. A surface wash was performed at 24 hrs. The skin was washed and
blotted and all IVR59 solution, washes and blots placed together in
scintillation
fluid. The cleaned skin was then dissolved O/N in Soluene 350 and 5m1
scintillation fluid was then added to this solution. An aliquot was removed
from
the reservoir of the Franz diffusion cell and added to the scintillation fluid
(Aquasol-II). All scintillation solutions (top wash, skin and reservoir) were
diluted 1:1000 and the radioactivity levels in these samples were counted.
F. Preliminary in vitro toxicity and irritation studies
The EpiDermT"" Skin Model (Epi-200, MatTek Corporation, Ashland, MA)
is used to obtain in vitro skin toxicity MTT and IL-1 a measurements
indicative
of skin irritation as follows: Individual human equivalent cultures are
transferred to six-well culture plates, each well containing 0.9 ml of culture
medium and placed in a humidified incubator at 37 °C, 5% C02, for 1
hour.
Prior to dosing, the medium is replaced with fresh medium. 25 ~~L of test
solution containing 0.05% ATRA with either ethanol or 1.25% IVR59 are
topically applied to the apical surface of each culture in duplicate and the
culture plate is returned to the incubator..
Culture plates are removed at 18 hrs, according to the protocol.
Deionized water is used as the negative control and 0.3% Sodium Dodecyl
Sulfate (SDS) as the positive control. The cultures are assayed for residual
mitochondria) dehydrogenase enzyme activity ("MTT assay") as an indicator of
culture viability (Osborne and Perkins, 1994). Washed cultures are incubated
for 3 hrs in a humidified chamber at 37 °C in MTT reagent (Sigma) at a
concentration of 1 mg MTT dye per 1.0 ml of incubation medium (EpiDermT""
Assay Medium). The remaining medium was saved for IL-la cytokine analysis.
At the end of the MTT dye-incubation step, cultures are washed again in
37
W~ 01/45645 CA 02394901 2002-06-21 PCT/US00/35319
PBS and 2 ml of 2-propanol was added to each culture plate to extract the
purple formazan product of the MTT dye metabolism. Extraction is performed at
room temperature for 2 hrs.
The absorbance of 200 ~L aliquots of the formazan/alcohol extracts are
measured at 570 nm. The percent viability is calculated using the following
formula: %viability = 100 x [OD(sample)/OD(negative control)].
IL-la was measured on the saved culture medium using a standard
ELISA and protocol from Cayman Chemical Corporation (Ann Arbour, MI). The
level of absorbance in the 0 pg/ml sample is subtracted from all other
standard
concentration absorbencies. A linear regression formula for the standard curve
was obtained providing the IL-la concentrations.
The invention has been described in detail with particular references to
the preferred embodiments thereof. However, it will be appreciated that
modifications and improvements within the spirit and scope of this invention
may be made by those skilled in the art upon considering the present
disclosure.
The references cited herein are incorporated by reference.
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