Note: Descriptions are shown in the official language in which they were submitted.
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1
PATCH FOR TRANSCUTANEOUS IMMUNIZATION
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of provisional U.S. Appln. No. 601276,497,
filed March 19, 2001.
FIELD OF THE INVENTION
The invention relates to a protein-in-adhesive patch for transcutaneous immu-
nization, their use to treat disease, and their manufacture.
BACKGROUND OF THE INVENTION
A variety of antigens are effectively administered by transcutaneous immuni-
zation (TCI) to induce antigen-specific immune responses. See WO 98/20734, WO
99/43350, and WO 00/61184; U.S. Patents 5,910,306 and 5,980,8.98; and U.S.
Patent Applns. 09/257,188; 09/309,881; 091311,720; 09/316,069; 091337,746; and
09/545,417. The immune response may require the use of an adjuvant (e.g., ADP-
ribosylating exotoxins). Vaccines are safe and effective when applied
epicutane-
ously, in contrast to the disadvantages associated with the use of some
adjuvants
when administered by an enteral, mucosal, transdermal or other parenteral
route
(e.g., subcutaneous, intramuscular, intraperitoneal, intraarterial,
intravenous). Skin
antigen presenting cells can be activated and antigen processed without
eliciting
undesirable immune reactions (e.g., atopy, dermatitis, eczema, psoriasis, and
other
allergic or hypersensitivity reactions). Here, we describe patches for
transcutaneous
immunization in which protein antigen is incorporated into an adhesive
component in
contact with skin of the human or animal to be immunized.
Drug-in-adhesive patches have been described, but most of them are limited
to the transdermal administration of small molecular weight drugs (e.g.,
androgens,
nicotine, nitroglycerin) to be introduced into the systemic circulation. But
the incorpo-
ration of proteins, which are much larger than the aforementioned drugs and
more
so unstable in their chemical and physical structure, into an adhesive portion
of a patch
for transcuta-neous immunization has not been described. Proteinaceous
adjuvants
and antigens are subject to denaturation and degradation. Herein, we show that
both
a patch according to the present invention and its immunogenic proteins are
mechanically and chemically stable, respectively. Moreover, biological
activity of the
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2
immunogenic protein is maintained and microbial contamination is avoided even
after prolonged storage at room temperature.
Protein-in-adhesive patches used for transcutaneous immunization, as well as
processes for making and using them, are disclosed herein. In particular, the
stability
of protein in this formulation and use of the patch in TCI are demonstrated.
Use of
one or more stabilizers may avoid protein aggregation, degradation,
denaturation, or
combinations thereof. Other advantages of the invention are discussed below or
would be apparent from the disclosure herein.
~o SUMMARY OF THE INVENTION
A protein-in-adhesive patch for transcutaneous immunization is comprised of
at least four different components: (i) backing layer; (ii) pressure-sensitive
adhesive
layer adhering to the backing layer; (iii) at least one immunologically-active
protein of
an immunogenic formulation applied to the pressure-sensitive adhesive layer
oppo-
~ site the backing layer and/or incorporated in the pressure-sensitive
adhesive layer
such that the at least one protein is in contact with adhesive; and (iv)
stabilizer which
maintains the immunological activity of the at least one protein under ambient
condi-
tions.
The backing layer may be occlusive or semi-occlusive (e.g., dressing). An
20 optional release liner may be included. A single unit may be produced by
enclosing a
patch in packaging material sufficient for storage under ambient conditions.
The pressure-sensitive adhesive layer may be comprised of at least one
aqueous-based adhesive (e.g., acrylate or silicone). The stabilizer may be a
sugar or
polymer to protect the protein: for example, a nonreducing disaccharide,
sucrose, or
25 trehalose may be used. Other excipients such as plasticizes, tackifier, and
thickener
may be included in a formulation containing adhesive, adjuvant, or antigen.
The
plasticizes may be a short-chain trialkyl citrate. The tackifier may be a
glycol and/or
succinic acid. The thickener may be a short-chain hydroxyalkyl cellulose or
starch.
The protein may have adjuvant activity, antigen activity, or both. The protein
so may be the antigen against which the immune response is induced or it may
act as
adjuvant to promote the immune response induced by a heterologous antigen. An
ADP-ribosylating exotoxin (e.g., cholera toxin, diphtheria toxin, E coli heat-
labile
enterotoxin, Pseudomonas exotoxin A, pertussis toxin), a chemokine, a
cytokine,
other known adjuvants, or derivatives thereof may be the protein. Examples of
the
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derivatives are fragments (including those that have been chemically
conjugated or
genetically fused with a portion of the wild-type adjuvant) or mutants
(including those
that are naturally occurring variants or other changes, insertions, or
deletions in the
amino acid sequence) which have adjuvant activity.
An effective amount of the protein is provided by the patch. For example, the
patch may comprise an amount of protein between 1 pg and 1 mg, 5 pg and 500
pg,
pg and 100 pg, or intermediate ranges thereof. Depending on the immunologic
activity of the protein, the effective amount of a particular protein may
vary.
Transcutaneous immunization may be used for inducing an antigen-specific
~o immune respone, treating an existing disease, or preventing a disease for
which the
subject is at risk. Hydration or penetration of the skin at the site where the
patch is
used may enhance the antigen-specific immune response or prevent unwanted
immune reactions. A possible target for activation and/or presentation of
antigen is a
dendritic cell underlying the skin.
~5~ A wet blend may be formulated containing adhesive and stabilized protein,
and then used to manufacture a patch. Protein may be applied to the surface of
an
adhesive layer, incorporated in an adhesive as a suspension or in solution, or
the
adhesive- and protein-containing formulations may be separately made and then
mixed or laminated together. Casting, coating, extrusion, laminating, and
printing
2o may be used to bring protein in contact with adhesive.
Effectiveness may be assessed by one or more clinical or laboratory criteria,
surrogate markers which are correlated to health, or morbidity or mortality
criteria.
Further aspects of the invention will be apparent to a person skilled in the
art from
the following detailed description and claims, and generalizations thereto.
DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic, partial cross-section of a protein-in-adhesive (PIA)
patch
10. A backing layer 12 and a pressure-sensitive adhesive layer 14 adhere to
each
other. The skin-side of the patch 10 is.optionally covered prior to use by a
release
liner 18. An immunogenic formulation 16 is located on the exposed side of the
patch
10 by application to the skin-side of the pressure-sensitive adhesive layer
14. It is not
necessarily shown to scale.
Fig. 2 is a schematic, partial cross-section of a protein-in-adhesive (PIA)
patch
20. A backing layer 22 and a pressure-sensitive adhesive layer 24 adhere to
each
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other. The skin-side of the patch 20 is optionally covered prior to use by a
release
liner 28. An immunogenic formulation 26 is located on the exposed side of the
patch
20 by incorporation in the pressure-sensitive adhesive layer 24. It is not
necessarily
shown to scale.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
One or more active immunogenic proteins used in transcutaneous immuni-
zation may be applied to and/or incorporated in an adhesive portion of a patch
or an
adhesive formulation per se by dispersing or solubilizing proteins with a
stabilizer
~o under nondenaturing conditions. In contrast to a syrup or other sticky
solution, the
adhesive is pressure sensitive. The protein-containing immunogenic formulation
is
stabilized against degradation and loss of adjuvant and/or antigen activity.
If not it is
not soluble in an aqueous adhesive, the stabilized protein or particles
containing the
stabilized protein may be added as a slurry or suspension. We have termed this
a
15 "protein-in-adhesive" (PIA) patch.
Formulations typically used for drug-in-adhesive (DIA) products are unaccept-
able for proteins as the solubilization of the adhesive in DIA was usually
performed in
an organic solvent which was thought to be unsuitable for unprotected
proteins. By
changing the base of the adhesive to an aqueous-based adhesive formulation and
2o incorporating proteins into the water-based adhesive formulation, the
active ingre-
dients for transcutaneous immunization can be incorporated into an adhesive
portion
of a patch or an adhesive formulation per se.
The DIA concept has been used in transdermal drug delivery, which is distin-
guished from transcutaneous immunization by several features (Glenn et al.,
Exp.
25 Opin. Invest. Drugs, 8:797-805, 1999). Thus, use of PIA formulations and
patches for
transcutaneous immunization represents a new and nonobvious invention.
The adhesive may be used for one or more of the following purposes: to keep
the patch in place on the subject, to incorporate other components of the
formulation
such as optional skin penetration enhancer chemicals or non-active components,
to
so stabilize labile components of the formulation, and other purposes known to
skilled
artisans. The use of adhesive patches for purposes of transcutaneous
immunization
provides a convenient and practical method for administration of vaccine.
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SKIN STRUCTURE AND IMMUNOBIOLOGY
Skin, the largest human organ, plays an important part in the body's defense
against invasion by infectious agents and contact with noxious substances. But
this
barrier function of the skin appears to have prevented the art from
appreciating that
transcutaneous immunization provided an effective alternative to enteral,
mucosal,
and other parenteral routes of administering vaccines. It has recently been
shown
that epicutaneous application of a vaccine targets specialized antigen
presenting
cells and induces a robust immune response.
Anatomically, skin is composed of three layers: the epidermis, the dermis, and
subcutaneous fat. Epidermis is composed of the basal, the spinous, the
granular,
and the cornified layers; the stratum corneum comprises the cornified layer
and lipid.
The principal antigen presenting cells of the skin, Langerhans cells, are
reported to
be an the mid- to upper-spinous layers of the epidermis in humans. Dermis
contains
primarily connective tissue. Blood and lymphatic vessels are confined to the
dermis
and subcutaneous fat.
The stratum corneum, a layer of dead skin cells and lipids, has traditionally
been viewed as a barrier to the hostile world, excluding organisms and noxious
substances from the viable cells below the stratum corneum. Stratum corneum
also
serves as a barrier to the loss of moisture from the skin: the relatively dry
stratum
2o corneum is reported to have 5% to 15% water content while deeper epidermal
and
dermal layers are relatively well hydrated with 85% to 90% water content. The
barrier
function of skin is reinforced by extensive crosslinking between corneocytes.
Only
recently has the secondary protection provided by antigen presenting cells
(e.g.,
Langerhans cells) been recognized. Moreover, the ability to immunize through
the
25 skin with or without penetration enhancement (i.e., transcutaneous
immunization)
using a skin-active adjuvant has only been recently described. Although
undesirable
skin reactions such as atopy and dermatitis were known in the art, recognition
of the
therapeutic advantages of transcutaneous immunization might not have been
appre-
ciated in the past because the skin was believed to provide a barrier to the
passage
so of molecules larger than about 500 daltons (Bos et al., Exp. Dermatol.,
9:165-169,
2000).
The epidermis is composed primarily of keratinocytes, but also has a signifi-
cant population (about 1 % to 3%) of immune surveillance cells called
Langerhans
cells (LC) distributed amongst the viable keratinocytes. Although LC are a
relatively
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6
small population of cells in the skin, they account, for 25% of the total skin
surface
area in humans. Langerhans cells represent an extensive, superficial network
barrier
of immune cells that make an attractive target for vaccine delivery. They are
bone
marrow derived dendritic cells that migrate to epithelial surfaces Where they
perform
immunosurveilance. Under normal circumstances, there is a baseline traffic of
LC
from the skin to the draining lymph nodes. In the face of a stimulus such as
infecting
microbes, the number of LC migrating out of the skin is greatly increased,
fulfilling
the immunosurveillance function of an antigen presenting cell. Langerhans
cells
stimulated by the danger signals created by interaction with microbes, foreign
mate-
~ o rials, or adjuvants orchestrate an effector immune response in the lymph
node
through the highly specific and amplified response created by their antigen
presen-
tation function.
A system for transcutaneous immunization (TCI) is provided which induces an
immune response (e.g., huri~oral and/or cellular effector specific for an
antigen) in a
~5~ human or animal. The delivery system provides simple, epicutaneous
application of
a formulation comprised of at least one adjuvant and one or more antigens to
the
skin of a human or animal subject (Glenn et al., J. Immunol., 161:3211-3214,
1998a;
Glenn et al., Nature, 391:851, 1998b; Glenn et al., Nature Med., 6:1403-1406,
2000;
Hammond et al., Adv. Drug Deliv. Rev., 43:45-55, 2000; Scharton-Kersten et
al.,
2o Infect. Immun., 68:5306-5313, 2000). An antigen-specific immune response is
thereby induced with or without chemical and/or physical penetration
enhancement
as long as the skin is not perforated through the dermal layer. This delivery
system
may also be used in conjunction with enteral, mucosal, or other parenteral
immuni-
zation techniques. Thus, the patch technologies described here could be used
for
25 treatment of humans and animals such as, for example, immunotherapy and
immunoprotection: therapeutically to treat existing disease, protectively to
prevent
disease, to reduce the severity and/or duration of disease, to ameliorate one
or more
symptoms of disease, or combinations thereof.
The transit pathways utilized by antigens to traverse the stratum corneum are
so unknown at this time. The stratum corneum (SC) is the principal barrier to
delivery of
drugs and antigens through the skin. Transdermal drug delivery of polar drugs
is
widely held to occur through aqueous intercellular channels formed between the
keratinocytes (Transdermal and Topical Drug Delivery Systems, Eds. Ghosh et
al.,
Buffalo Grove: Interpharm Press, 1997). Although the SC is the limiting
barrier for
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7
penetration, it is breached by hair follicles and sweat ducts. Whether
antigens pene-
trate directly through the SG or via the epidermal appendages may depend on a
host
of factors. These appendages are thought to play only a minor role in
transdermal
drug delivery (Barry et al., J. Control Rel., 6:85-97, 1987). Despite some
evidence in
mice that transcutaneous immunization using DNA may utilize hair follicles as
the
pathway for skin penetration (Fan et al., Nature Biotechnol., 17:870-872,
1999), it is
more likely that the, robust immune responses utilize more of the skin surface
area.
Because disruption of the SC barrier can be accomplished by simple hydration
of the
skin (Roberts et al., In: Pharmaceutical Skin Penetration Enhancement, Eds.
Waiters
o et al., New York: Marcel Dekker, 1993), this has been employed for
transcutaneous
immunization.
Activation of one or more of adjuvant, antigen, and antigen presenting cell
(APC) may promote the induction of the immune response. The A.PC processes the
antigen and then presents one or more epitopes to a lymphocyte. Activation may
~5 promote contact between the formulation.and the APC (e.g., Langerhans
cells, other
dendritic cells, macrophages, B lymphocytes), uptake of the formulation by the
APC,
processing of antigen and/or presentation of epitopes by the APC, migration
and/or
differentiation of the APC, interaction between the APC and the lymphocyte, or
combinations thereof. The adjuvant by itself may activate the APC. For
example, a
2o chemokine may recruit and/or activate antigen presenting cells to a site.
In particular,
the antigen presenting cell may migrate from the skin to the lymph nodes, and
then
present antigen to a lymphocyte, thereby inducing an antigen-specific immune
response. Furthermore, the formulation may directly contact a lymphocyte which
recognizes antigen, thereby inducing an antigen-specific immune response.
25 In addition to eliciting immune reactions leading to activation and/or
expansion
of antigen-specific B-cell and/or T-cell populations, including antibodies and
cytotoxic
T lymphocytes (CTL), the invention may positively and/or negatively regulate
one or
more components of the immune system by using transcutaneous immunization to
affect antigen-specific helper (Th1 and/or Th2) or delayed-type
hypersensitivity T-cell
3o subsets (TprH). The desired immune response induced is preferably systemic
or
regional (e.g., mucosal) but it is usually not undesirable immune responses
(e.g.,
atopy, dermatitis, eczema, psoriasis, and other allergic or hypersensitivity
reactions).
As seen herein, the immune responses induced are of the quantity and quality
that
provide therapeutic or prophylactic immune responses useful for treating
disease.
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8
Hydration of the intact or penetrated skin before, during, or immediately
after
epicutaneous application of the formulation is preferred and may be required
in some
or many instances. For example, hydration may increase the water content of
the
topmost layer of skin (e.g., stratum corneum or superficial epidermis layer
exposed
by penetration enhancement techniques) above 25%, 50% or 75%. Skin may be
hydrated with an aqueous solution of 10% glycerol, 70% isopropyl alcohol, and
20%
water. Addition of an occlusive dressing or use of a semi-liquid formulation
(e.g.,
cream, emulsion, gel, lotion, paste) can increase hydration of the skin. For
example,
lipid vesicles or sugars can be added to a formulation to thicken a solution
or
1o suspension. Hydration occurs with or without disruption of all or at least
a portion of
the stratum corneum at the site of application of the formulation, along with
possibly
also a portion of the epidermis, as long as the dermis is not perforated. The
intent is
for the formulation to act on skin antigen presenting cells instead of
introducing
immunologically-active components of the formulation into the systemic
circulation,
~5 although some portion of the formulation may act at distal sites.
Skin may be swabbed with an applicator (e.g., adsorbent material on a pad or
stick) containing hydration or chemical penetration agents or they may be
applied
directly to skin. For example, aqueous solutions (e.g., water, saline, other
buffers),
acetone, alcohols (e.g., isopropyl alcohol), detergents (e.g., sodium dodecyl
sulfate),
2o depilatory or keratinolytic agents (e.g., calcium hydroxide, salicylic
acid, ureas),
humectants (e.g., glycerol, other glycols), polymers (e.g., polyethylene or
propylene
glycol, polyvinyl pyrrolidone), or combinations thereof may be used or
incorporated in
the formulation. Similarly, abrading the skin (e.g., abrasives like an emery
board or
paper, sand paper, fibrous pad, pumice), removing a superficial layer of skin
(e.g.,
25 peeling or stripping with an adhesive tape), microporating the skin using
an energy
source (e.g., heat, light, sound, electrical, magnetic) or a barrier
disruption device
(e.g., blade, needle, projectile, spray, tine), or combinations thereof may
act as a
physical penetration enhancer. See WO 98/29134, WO 01 /34185, and WO
02/07813; U.S. Patents 5,445,611, 6,090,790, 6,142,939, 6,168,587, 6,312,612,
so 6,322,808 and 6,334,856 for description of microblades or microneedles, gun
or
spray injectors, and for microporation of the skin and techniques that might
be
adapted for transcutaneous immunization. The objective of chemical or physical
penetration enhancement in conjunction with TCI is to remove at least the
stratum
corneum, or a superficial or deeper epidermal layer, without perforating skin
through
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9
past the dermal layer. This is preferably accomplished with minor discomfort
at most
to the human or animal subject, and without bleeding at the site. For example,
applying the formulation to intact skin may or may not involve thermal,
optical, sonic,
or electromagnetic energy to perforate layers of the skin to below the stratum
s corneum or epidermis.
The difference between transcutaneous immunization as practiced in WO
98/20734 and 99/43350 is whether all or at least a portion of the stratum
corneum is
disrupted. The term "penetration enhancer" as used herein refers to those
chemicals
which when applied in the formulation, before application, during application,
or after
application results in such disruption. Some chemicals (e.g., alcohols) may or
may
not disrupt the stratum corneum depending on how vigorously they are applied
(e.g.,
swabbing or scrubbing with sufficient pressure). For example, including
alcohol, O/W
or W/O emulsions, lipid micelles, or lipid vesicles in the formulation may
enhance
penetration of one or more immunologically-active ingredients of the same
formu-
15 lation across intact skin without detectable disruption of the 'stratum
corneum.
Formulations which are useful for vaccination are also provided as well as
processes for their manufacture. The formulation may be in liquid or semi-
liquid form.
For example, the formulation may be provided as a liquid: cream, emulsion,
gel,
lotion, ointment, paste, solution, suspension, or other liquid forms.
Formulation may
2o be air dried, dried with elevated temperature, freeze or spray dried,
coated or
sprayed on a solid substrate and then dried, dusted on a solid substrate,
quickly
frozen and then slowly dried under vacuum, or combinations thereof to a low
moisture content. Adhesive formulations may be cured to a desired amount of
cross-
linking by suitable choice of initiator, rate accelerator or decelerator, and
terminator.
25 A "patch" refers to a product which includes a solid substrate (e.g.,
occlusive
or nonocclusive surgical dressing) as well as at least one active ingredient.
Liquid or
semi-liquid formulations may be incorporated in~a patch. Here, the patch
comprises
backing layer, pressure-sensitive adhesive layer, and immunogenic formulation.
The
solid substrate is at least the backing layer, but the adhesive and
immunogenic
so formulations may also form part of the solid substrate is they are suitably
dried and
cured. One or more active components of the immunogenic formulation may be
applied on the adhesive layer, incorporated in the adhesive layer, or
combinations
thereof. Layers may be formed, and then adhered or laminated together.
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The moisture content of the adhesive layer may be more than 0.5%, more
than 1 %, more than 2%, less than 10%, less than 5%, less than 2%, and interme-
diate ranges thereof. The patch may be a pliable, planar substrate from about
1 cm2
to about 100 cm2. An effective amount of the protein is provided by a single
patch.
For example, the patch may comprise an amount of protein between 1 pg and 1
mg,
5 pg and 500 pg, 10 pg and 100 pg, or intermediate ranges thereof. Depending
on
the immunologic activity of the protein, the effective amount of a particular
protein
may vary. The patch may be stored in a moisture-proof package (e.g., blister
pack,
foil pouch) for at least one or two years at room temperature (e.g.,
20°C to 30°C)
with an immunological activity between 85% and 115% of the patch's initial
activity.
Formulation in liquid or semi-liquid form may be applied with one or more
adjuvants and/or antigens both at the same or separate sites or simultaneously
or in
frequent, repeated applications. The patch may include a controlled-release
reservoir
or a rate-controlling matrix or membrane may be used which allows stepped
release
Of adjuvant and/or antigen. It may contain a single reservoir with adjuvant
and/or
antigen, or multiple reservoirs to separate individual antigens and adjuvants.
The
patch may include additional antigens such that application of the patch
induces an
immune response to multiple antigens. In such a case, antigens may or may not
be
derived from the same source, but they will have different chemical structures
so as
2o to induce an immune response specific for different antigens. Multiple
patches may
be applied simultaneously; a single patch may contain multiple reservoirs. For
effective treatment, multiple patches may be applied at intervals or
constantly over a
period of time; they may be applied at different times, for overlapping
periods, or
simultaneously.
2s Solids (e.g., particles of nanometer or micrometer dimensions) may also be
incorporated in the formulation. Solid forms (e.g., nanoparticles or
microparticles)
may aid in dispersion or solubilization of active ingredients; assist in
carrying the
formulation through superticial layers of the skin; provide a point of
attachment for
adjuvant, antigen, or both to a substrate that can be opsonized by antigen
presenting
so cells, or combinations thereof. Ingredients that are insoluble or poorly
soluble in an
aqueous solution may be formulated in an emulsion, lipid vesicles, or
micelles.
The formulation may be manufactured under conditions acceptable to
appropriate regulatory agencies (e.g., Food and Drug Administration) for
biologicals
and vaccines. Optionally, components like binders, buffers, colorings,
dessicants,
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11
diluents, humectants, preservatives, stabilizers, other excipients, adhesives,
plasti-
cizers, tackifiers, thickeners, patch materials, or combinations thereof may
be
included in the formulation even though they are immunologically inactive.
They
may, however, have other desirable properties or characteristics which improve
the
effectiveness of the formulation.
A single or unit dose of formulation suitable for administration is provided.
The
amount of adjuvant or antigen in the unit dose may be anywhere in a broad
range
from about 0.001 pg to about 10 mg. This range may be from about 0.1 pg to
about 1
mg; a narrower range is from about 5 pg to about 500 pg. Other suitable ranges
are
o between about 1 pg and about 10 pg, between about 10 pg and about 50 pg,
between about 50 pg and about 200 pg, and between about 1 mg and about 5 mg. A
preferred dose for a toxin is about 50 pg or 100 pg or less (e.g., from about
1 pg to
abraut 50 pg or 100 pg). The ratio between antigen and adjuvant may be about
1:1
(e.g., an ADP-ribosylating exotoxin when it is both antigen and adjuvant) but
higher
s ratios may be suitable for poor antigens (e.g., about 1:10 or less), or
lower ratios of
antigen to adjuvant may also be used (e.g., about 10:1 or more).
A formulation comprising adjuvant and antigen or polynucleotide may be
applied to skin of a human or animal subject, antigen is presented to immune
cells,
and an antigen-specific immune response is induced. This may occur before,
during,
~o or after infection by pathogen. Only antigen or polynucleotide encoding
antigen may
be required, but no additional adjuvant, if the immunogenicity of the
formulation is
sufficient to not require adjuvant activity. The formulation may include an
additional
antigen such that application of the formulation induces an immune response
against
multiple antigens (i.e., multivalent). In such a case, antigens may or may not
be
25 derived from the same source, but the antigens will have different chemical
structures so as to induce immune responses specific for the different
antigens.
Antigen-specific lymphocytes may participate in the immune response and, in
the
case of participation by B lymphocytes, antigen-specific antibodies may be
part of
the immune response. The formulations described above may include binders,
so buffers, colorings, dessicants, diluents, humectants, preservatives,
stabilizers, other
excipients, adhesives, plasticizers, tackifiers, thickeners, and patch
materials known
in the art.
The invention is used to treat a subject (e.g., a human or animal in need of
treatment such as prevention of disease, protection from effects of infection,
therapy
CA 02445486 2003-09-18
WO 02/074325 PCT/US02/08099
12
of existing disease or symptoms, or combinations thereof). Diseases other than
infection include cancer, allergy, and autoimmunity. When the antigen is
derived
from a pathogen, the treatment may vaccinate the subject against infection by
the
pathogen or against its pathogenic effects such as those caused by toxin
secretion.
The invention may be used therapeutically to treat existing disease,
protectively to
prevent disease, to reduce the severity and/or duration of disease, to
ameliorate
symptoms of disease, or combinations thereof.
The application site may be protected with anti-inflammatory corticosteroids
such as hydrocortisone, triamcinolone and mometazone or nonsteroidal anti-
inflammatory drugs (NSAID) to reduce possible local skin reaction or modulate
the
type of immune response. Similarly, anti-inflammatory steroids or NSAID may be
included in the patch material, or liquid or solid formulations; and
corticosteroids or
NSAID may be applied after immunization. IL-10, TNF-a,,other immunomodulators
may be used instead of the anti-inflammatory agents. Moreover, the formulation
may
~5 be~applied to skin overlying more than one draining lymph node field using
either
single or multiple applications. The formulation may include additional
antigens such
that application induces an immune response to multiple antigens. In such a
case,
the antigens may or may not be derived from the same source, but the antigens
will
have different chemical structures so as to induce an immune response specific
for
2o the different antigens. Multi-chambered patches could allow more effective
delivery
of multivalent vaccines as each chamber covers different antigen presenting
cells.
Thus, antigen presenting cells would encounter only one antigen (with or
without
adjuvant) and thus would eliminate antigenic competition and thereby enhancing
the
response to each individual antigen in the multivalent vaccine.
25 The formulation may be epicutaneously applied to skin to prime or boost the
immune response in conjunction with or without penetration techniques, or
other
routes of immunization. Priming by transcutaneous immunization (TCI) with
either
single or multiple applications may be followed with enteral, mucosal,
transdermal,
and/or other parenteral techniques for boosting immunization with the same or
so altered antigens. Priming by an enteral, mucosal, transdermal, andlor other
paren-
teral route with either single or multiple applications may be followed with
transcuta-
neous techniques for boosting immunization with the same or altered antigens.
It
should be noted that TCI is distinguished from conventional topical techniques
like
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13
mucosal or transdermal immunization because the former requires a mucous
membrane (e.g., lung, mouth, nose, rectum) not found in the skin and the
latter
requires perforation of the skin through the dermis. The formulation may
include
additional antigens such that application to skin induces an immune response
to
multiple antigens.
In addition to antigen and adjuvant, the formulation may comprise a vehicle.
For example, the formulation may comprise an AQUAPHOR, Freund, Ribi, or Syntex
emulsion; water-in-oil emulsions (e.g., aqueous creams, ISA-720), oil-in-water
emulsions (e.g., oily creams, ISA-51, MF59), microemulsions, anhydrous lipids
and
oil-in-water emulsions, other types of emulsions; gels, fats, waxes, oil,
silicones, and
humectants (e.g., glycerol).
Antigen may be derived from any pathogen that infects a human or animal
subject (e.g., bacterium, virus, fungus, or protozoan), allergens, and self-
antigens.
The chemical structure of the antigen may be described as one or more of carbo-
~5 hydrate, fatty acid, and protein (e.g., glycolipid, glycoprotein,
lipoprotein). Proteina-
ceous antigen is preferred. The molecular weight of the antigen may be greater
than
500 daltons, 800 daltons, 1000 daltons, 10 kilodaltons, 100 kilodaltons, or
1000
kilodaltons (including intermediate ranges thereof). Chemical or physical
penetration
enhancement may be preferred for macromolecular structures like cells, viral
2o particles, and molecules of greater than one megadalton, but techniques
like
hydration and swabbing with a solvent may be sufficient to induce immunization
across the skin. Antigen may be obtained by recombinant techniques, chemical
synthesis, or at least partial purification from a natural source. It may be a
chemical
or recombinant conjugates: for example, linkage between chemically reactive
groups
25 or protein fusion. Antigen may be provided as a live cell or virus, an
attenuated live
cell or virus, a killed cell, or an inactivated virus. Alternatively, antigen
may be at
least partially purified in cell-free form (e.g., cell or viral lysate,
membrane or other
subcellular fraction). Because most adjuvants would also have immunogenic
activity
and would be considered antigens, adjuvants would also be expected to have the
so aforementioned properties and characteristics of antigens. For example,
adjuvants
and antigens may be prepared using the same techniques (see above).
The choice of adjuvant may allow potentiation or modulation of the immune
response. Moreover, selection of a suitable adjuvant may result in the
preferential
induction of a humoral or cellular immune response, specific antibody isotypes
(e.g.,
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14
IgM, IgD, IgA1, IgA2, IgE, IgG1, IgG2, IgG3, and/or IgG4), andlor specific T-
cell
subsets (e.g., CTL, Th1, Th2 andlor TpTH). The adjuvant is preferably a
chemically
activated (e.g., proteolytically digested) or genetically activated (e.g.,
fusions,
deletion or point mutants) ADP-ribosylating exotoxin or B subunit thereof.
s An "antigen" is an active component of the formulation which is specifically
recognized by the immune system of a human or animal subject after
immunization
or vaccination. The antigen may comprise a single or multiple immunogenic
epitopes
recognized by a B-cell receptor (i.e., secreted or membrane-bound antibody) or
a T-
cell receptor. Proteinaceous epitopes recognized by T-cell receptors have
typical
lengths and conserved amino acid residues depending on whether they are bound
by major histocompatibility complex (MHC) Class I or Class II molecules on the
antigen presenting cell. In contrast, proteinaceous epitopes recognized
antibody may
be of variable length including short, extended oligopeptides and longer,
folded
polypeptides. Single amino acid differences between epitopes may be
distinguished.
The antigen may be capable of inducing an immune response against a molecule
of
a pathogen, allergenic substances, or mammalian host (e.g., autoantigens,
cancer
antigens, molecules of the immune system). For immunoregulation, that molecule
may be an allergen, autoantigen, internal image thereof, or other components
of the
immune system (e.g., B- or T-cell receptor, co-receptor or ligand thereof,
soluble
2o mediator or receptor thereof). Thus, antigen is usually identical or at
least derived
from the chemical structure of the molecule, but mimetics which are only
distantly
related to such chemical structures may also be successfully used.
An "adjuvant" is an active component of the formulation to assist in inducing
an immune response to the antigen. Adjuvant activity is the ability to
increase the
2s immune response to a heterologous antigen (i.e., antigen which is a
separate
chemical structure from the adjuvant) by inclusion of the adjuvant itself in a
formu-
lation or in combination with other components of the formulation or
particular immu-
nization techniques. As noted above, a molecule may contain both antigen and
adjuvant activities by chemically conjugating antigen and adjuvant or
genetically
so fusing coding regions of antigen and adjuvant; thus, the formulation may
contain only
one ingredient or component. Some naturally-occurring proteins such as CT and
LT
have both adjuvant and antigenic properties; some recombinant proteins are
known
to have similar properties (LeIF); some non-protein adjuvants may also induce
anti-
bodies to themselves, such as LPS or lipid A. The combination of adjuvant and
anti-
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genie qualities may be used to induce protective immune responses. For
example,
LT antibodies are protective against ETEC, LeIF immune responses are effective
in
manifestations of Leishmaniasis and LPS antibodies may be protective in
protection
against diseases caused by gram-negative organisms.
s The term "effective amount" is meant to describe that amount of adjuvant or
antigen which induces an antigen-specific immune response. A "subunit"
immunogen
or vaccine is a formulation comprised of active components (e.g., adjuvant,
antigen)
which have been isolated from other cellular or viral components of the
pathogen
(e.g., membrane or polysaccharide components like endotoxin) by recombinant
o techniques, chemical synthesis, or at least partial purification from a
natural source.
Induction of an immune response may provide treatments of a subject such
as, for example, immunoprotection, desensitization, immunosuppression,
modulation
of autoimmune disease, potentiation of cancer immunosurveillance, prophylactic
vaccination to prevent disease, and therapeutic vaccination to ameliorate
established
15 disease. A product or method "induces" when its presence or absence causes
a
statistically significant change in the immune response's magnitude and/or
kinetics;
change in the induced elements of the immune system (e.g., humoral vs.
cellular,
Th1 vs. Th2); effect on the number and/or the severity of disease symptoms;
effect
on the health and well-being of the subject (i.e., morbidity and mortality);
or combi-
2o nations thereof.
The term "draining lymph node field" as used in the invention means an ana-
tomic area over which the lymph collected is filtered through a set of defined
lymph
nodes (e.g., cervical, axillary, inguinal, epitrochelear, popliteal, those of
the abdomen
and thorax). Thus, the same draining lymph node field may be targeted by
immuni-
nation (e.g., enteral, mucosal, transcutaneous, transdermal, other
parenteral,) within
the few days required for antigen presenting cells to migrate to the lymph
nodes if
the sites and times of immunization are appropriately spaced to bring
different
components of the formulation together (e.g., two closely located patches with
either
adjuvant or antigen applied at the same time may be effective when neither
alone
so would be successful). For example, a patch delivering adjuvant by the
transcuta-
neous technique may be placed on the same arm as is injected with a
conventional
vaccine to boost its effectiveness in elderly, pediatric, or other
immunologically
compromised populations. In contrast, applying patches to different limbs may
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16
prevent an adjuvant-containing patch from boosting the effectiveness of a
patch
containing only antigen.
Without being bound to any particular theory for the operation of the
invention
but only to provide an explanation for our observations, we hypothesize that
this
transcutaneous delivery system carries antigen to cells of the immune system
Where
an immune response is induced. The antigen may pass through the normally
present
protective outer layers of the skin (i.e., stratum corneum) and induce the
immune
response directly, or through an antigen presenting cell population in the
epidermis
(e.g., macrophage, tissue macrophage, Langerhans cell, other dendritic cells,
B
lymphocyte, or Kupffer cell) that presents processed antigen to lymphocytes.
Thus,
with or without penetration enhancement techniques, the dermis is not
penetrated for
TCI as it is for subcutaneous injection or transdermal techniques. Optionally,
the
antigen may pass through the stratum corneum via a hair follicle or a skin
organelle
(e.g., sweat gland, oil gland).
~5 ~ Transcutaneous immunization with bacterial ADP-ribosylating exotoxins
(BARE) as an example, may target the epidermal Langerhans cell, known to be
among the most efficient of the antigen presenting cells (APC). Maturation of
APC
may be assessed by morphology and phenotype (e.g., expression of MHC Class II
molecules, CD83, or co-stimulatory molecules). We have found that BARE appear
to
2o activate Langerhans cells when applied epicutaneously to intact skin.
Adjuvants such
as trypsin-cleaved bARE may enhance Langerhans cell activation. Langerhans
cells
direct specific immune responses through phagocytosis of the antigens, and
migration to the lymph nodes where they act as APC to present the antigen to
lymphocytes, and thereby induce a potent antibody response. Although the skin
is
25 generally considered a barrier to pathogens, the imperfection of this
barrier is
attested to by the numerous Langerhans cells distributed throughout the
epidermis
that are designed to orchestrate the immune response against organisms
invading
through the skin. According to Udey (Clin. Exp. Immunol., 107:s6-s8, 1997):
Langerhans cells are bone-marrow derived cells that are present
so in all mammalian stratified squamous epithelia. They comprise all of
the accessory cell activity that is present in uninflamed epidermis, and
in the current paradigm are essential for the initiation and propagation
of immune responses directed against epicutaneously applied
antigens. Langerhans cells are members of a family of potent
35 accessory cells ('dendritic cells') that are widely distributed, but
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17
infrequently represented, in epithelia and solid organs as well as in
lymphoid tissue.
It is now recognized that Langerhans cells (and presumably other
dendritic cells) have a life cycle with at least two distinct stages.
s Langerhans cells that are located in epidermis constitute a regular
network of antigen-trapping 'sentinel' cells. Epidermal Langerhans cells
can ingest particulates, including microorganisms, and are efficient
processors of complex antigens. However, they express only low
levels of MHC class I and II antigens and costimulatory molecules
(/CAM-1, B7-1 and B7-2) and are poor stimulators of unprimed T cells.
After contact with antigen, some Langerhans cells become activated,
exit the epidermis and migrate to T-cell-dependent regions of regional
lymph nodes where they localize as mature dendritic cells. In the
course of exiting the epidermis and migrating to lymph nodes, antigen-
15 bearing epidermal Langerhans cells (now the 'messengers') exhibit
dramatic changes in morphology, surface phenotype and function. In
contrast to epidermal Langerhans cells, lymphoid dendritic cells are
essentially non-phagocytic and process protein antigens inefficiently,
.. but express high levels of MHC class I and class II antigens and
2o various costimulatory molecules and are the most potent stimulators of
naive T cells that have been identified."
The potent antigen presenting capability of Langerhans cells can be exploited
for transcutaneously-delivered immunogens and vaccines. An immune response
using the skin's immune system may be achieved by delivering the formulation
only
2s to Langerhans cells in the stratum corneum (i.e., the outermost layer of
the skin
consisting of cornified cells and lipids) and subsequently activating the
Langerhans
cells to take up antigen, migrate to B-cell follicles and/or T-cell dependent
regions,
and present the antigen to B and/or T lymphocytes. If antigens other that bARE
(e.g.,
toxin, colonization or virulence factor) are to be phagocytosed by Langerhans
cells,
so then these antigens could also be transported to the lymph node for
presentation to
T lymphocytes and subsequently induce an immune response specific for that
antigen. Thus, a feature of TCI is the activation of the Langerhans cell,
presumably
by BARE or derivatives thereof, chemokines, cytokines, PAMP, or other
Langerhans
cell activating substance including contact sensitizers and adjuvants.
Increasing the
s5 size of the skin population of Langerhans cells or their state of
activation would also
be expected to enhance the immune response (e.g., acetone pretreatment). In
aged
subjects or Langerhans cell-depleted skin (i.e., from UV damage), it may be
possible
to replenish the population of Langerhans cells (e.g., tretinoin
pretreatment).
Adjuvants such as bARE are known to be highly toxic when injected or given
4o systemically. Intradermal injection has also been shown to induce
persistent nodules
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18
when LT is included as the adjuvant (Guy et al., Vaccine, 17:1130-1135, 1999).
But if
placed on the surface of intact skin (i.e., epicutaneous), they are unlikely
to induce
systemic toxicity. Thus, the transcutaneous route may allow the advantage of
adjuvant effects without systemic toxicity. A similar absence of toxicity
could be
expected if the skin were penetrated only below the stratum corneum (e.g.,
near or
at the epidermis), but not through the dermis. Thus, the ability to induce
activation of
the immune system through the skin induces potent immune responses without
systemic toxicity.
The magnitude of the antibody response induced by affinity maturation and
isotype switching to predominantly IgG antibodies is generally achieved with T-
cell
help, and activation of both Th1 and Th2 pathways is suggested by the
production of
IgG1 and IgG2a. Alternatively, a large antibody response may be induced by a
thymus-independent antigen type 1 (TI-1 ) which directly activates the B
lymphocyte
or could have similar activating effects on B lymphocytes such as up-
regulation of
15 MHC Class II, CD25, CD40, B7-1/CD80, B7-21CD86, and ICAM-1 molecules.
The spectrum of commonly known skin immune responses is represented by
atopy and contact dermatitis. Contact dermatitis, a pathogenic manifestation
of
Langerhans cell activation, is directed by Langerhans cells which phagocytose
antigen, migrate to lymph nodes, present antigen, and sensitize T lymphocytes
that
2o migrate to the skin and cause the intense destructive cellular response
that occurs at
affected skin sites. Such responses are not generally known to be associated
with
antigen-specific IgG antibodies. Atopic dermatitis may utilize the Langerhans
cell in a
similar fashion, but is identified with Th2 cells and is generally associated
with high
levels of IgE antibody.
2~ On the other hand, transcutaneous immunization with bARE provides a useful
and desirable immune response. There are usually no findings typical of atopy
or
contact dermatitis given the high levels of IgG that are induced. Cholera
toxin or E.
coli heat-labile enterotoxin epicutaneously applied to skin can achieve
immunization
in the absence of lymphocyte infiltration 24, 48 and 120 hours after
immunization.
so The minor skin reactivity seen in preclinical and clinical trials were
easily treated.
This indicates that Langerhans cells engaged by transcutaneous immunization as
they "comprise all of the accessory cell activity that is present in
uninflamed epi-
dermis, and in the current paradigm are essential for the initiation and
propagation of
immune responses directed against epicutaneously applied antigens" (Udey,
1997).
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19
The uniqueness of the transcutaneous immune response here is also indicated by
both thehigh levels of antigen-specific IgG antibody and the type of antibody
produced (e.g., IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA), and generally the
absence
of antigen specific IgE antibody. Transcutaneous immunization could
conceivably
occur in tandem with skin inflammation if sufficient activation of antigen
presenting
cells and T lymphocytes were to occur in a transcutaneous response coexisting
with
atopy or contact dermatitis.
Transcutaneous targeting of Langerhans cells may also be used in tandem
with agents to deactivate all or part of their antigen presenting function,
thereby
~o modifying immunization or preventing sensitization. Techniques to modulate
Langerhans activation or other skin immune cells include, for example, the use
of
anti-inflammatory steroidal or nonsteroidal agents (NSAID); cyclosporin,
FK506,
rapamycin, cyclophosphamide, glucocorticoids, or other immunosuppressants;
interleukin-10; interleukin-1 monoclonal antibodies (mAB) or soluble receptor
~ antagonists (RA); interleukin-1 converting enzyme (ICE) inhibitors; or
depletion via
superantigens such as through Staphylococcal enterotoxin A (SEA) induced
epidermal Langerhans cell depletion. Similar compounds may be used to modify
the
innate response of Langerhans cells and induce different T-helper responses
(Th1 or
Th2) or may modulate skin inflammatory responses to decrease potential side
2o effects of the immunization. Similarly, lymphocytes may be immunosuppressed
before, during or after immunization by administering immunosuppressant
separately
or by coadministration of immunosuppressant with the formulation. For example,
it
may be possible to induce a potent systemic protective immune responses with
agents that would normally result in allergic or irritant contact
hypersensitivity but
2s adding inhibitors of ICE may alleviate adverse skin reactions.
ANTIGEN
A transcutaneous immunization system delivers agents to specialized cells
(e.g., antigen presentation cell, lymphocyte) that produce an immune response.
3o These agents as a class are called antigens. Antigen may be composed of
chemical
structures such as, for example, carbohydrate, glycolipid, glycoprotein,
lipid, lipo-
protein, phospholipid, polypeptide, conjugates thereof, or any other material
known
to induce an immune response. Antigen may be conjugated to carrier. Antigen
may
be provided as a whole organism such as, for example, a bacterium or virion;
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antigen may be obtained from an extract or lysate, either from whole cells or
membrane alone; or antigen may be chemically synthesized or produced by recom-
binant technology. Antigen may be incorporated into a formulation by
solubilization
or dispersion.
Antigen of the invention may be expressed by recombinant technology,
preferably as a fusion with an affinity or epitope tag; chemical synthesis of
an oligo-
peptide, either free or conjugated to carrier proteins, may be used to obtain
antigen
of the invention. Oligopeptides are considered a type of polypeptide.
Oligopeptide
lengths of 6 residues to 20 residues are preferred. Polypeptides may also by
~ o synthesized as branched structures. Antigenic polypeptides include, for
example,
synthetic or recombinant B-cell and T-cell epitopes, universal T-cell
epitopes, and
mixed T-cell epitopes from one organism or disease and B-cell epitopes from
another. Antigen obtained through recombinant technology or peptide synthesis,
as
well as antigen obtained from natural sources or extracts, may be purified by
the
15 antigen's physical and chemical characteristics, preferably by
fractionation or
chromatography. Recombinants may combine antigen fragments or fuse them into
chimerae. -A multivalent antigen formulation may be used to induce an immune
response to more than one antigen at the same time. Conjugates may be used to
induce an immune response to multiple antigens, to boost the immune response,
or
2o both. Transcutaneous immunization may be used to boost responses induced
initially by other routes of immunization such as by oral, nasal or other
parenteral
routes. Such oral/transcutaneous or transcutaneous/oral immunization may be
especially important to enhance mucosal immunity in diseases where mucosal
immunity correlates with protection.
Antigen may be solubilized in a buffer or water or organic solvents such as
alcohol or DMSO, or incorporated in gels, emulsions, lipid micelles or
vesicles, and
creams. Suitable buffers include, but are not limited to, phosphate buffered
saline
Ca++/Mg++ free, phosphate buffered saline, normal saline (150 mM NaCI in
water),
and Hepes or Tris buffer. Antigen not soluble in neutral buffer can be
solubilized in
so 10 mM acetic acid and then diluted to the desired volume with a neutral
buffer such
as PBS. In the case of antigen soluble only at acid pH, acetate-PBS at acid pH
may
be used as a diluent after solubilization in dilute acetic acid. Dimethyl
sulfoxide and
glycerol may be suitable nonaqueous buffers for use in the invention.
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21
A hydrophobic antigen can be solubilized in a detergent or surfactant, for
example a polypeptide containing a membrane-spanning domain. Furthermore, for
formulations containing liposomes, an antigen in a detergent solution (e.g.,
cell
membrane extract) may be mixed with lipids, and liposomes then may be formed
by
removal of the detergent by dilution, dialysis, or column chromatography.
Certain
antigens (e.g., membrane proteins) need not be soluble per se, but can be
inserted
directly into a lipid membrane (e.g., virosome), in a suspension of virion
alone, or
suspensions of microspheres or heat-inactivated bacteria which may be taken up
by
activate antigen presenting cells (e.g., opsonization). Antigens may also be
mixed
1o with a penetration enhancer as described in WO 99/43350.
Many antigens are known in the art which can be used to vaccinate human or
animal subjects and induce an immune response specific for particular
pathogens,
as well as methods of preparing antigen, determining a suitable dose of
antigen,
assaying for induction of an immune response, and treating infection by a
pathogen
~5 (e.g., bacterium, virus, fungus, or protozoan). Environmental and food
allergens, as
well as self-antigens of the mammalian host (e.g., human, animal) are examples
of
antigens that are not derived from a pathogen. Antigen used to produce
formulations
and vaccines for transcutaneous immunization may be the natural product per
se,
genetically-engineered or chemically-synthesized forms thereof, fragments
thereof,
2o fusions, or conjugates. The immune response will usually recognize only a
portion of
the antigen (e.g., one or more immunogenic epitopes).
Plotkin and Mortimer (Vaccine, 2nd Ed., Philadelphia: W.B. Saunders, 1994)
provide antigens which can be used to vaccinate humans or animals to induce an
immune response specific for particular pathogens, as well as methods of
preparing
2s antigen, determining a suitable dose of antigen, assaying for induction of
an immune
response, and treating infection by a pathogen.
Bacteria include, for example: anthrax, Campylobacter, Vibrio cholera, clostri-
dia including Clostridium difficile, Diphtheria, enterohemorrhagic E, coli,
enterotoxi-
genic E, coli, Giardia, gonococcus, Helicobacter pylori, Hemophilus influenza
B,
so Hemophilus influenza nontypeable, Legionella, meningococcus, Mycobacteria
including those organisms responsible for tuberculosis, pertussis,
pneumococcus,
salmonella, shigella, staphylococcus, Group A beta-hemolytic streptococcus,
Streptococcus B, tetanus, Borrelia burgdorfi and Yersinia. Products thereof
which
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22
may be used as antigen. Antigen includes, for example, toxins, toxoids,
subunits
thereof, or combinations thereof; virulence or colonization factors; and
products.
Viruses include, for example: adenovirus, dengue serotypes 1 to 4, ebola,
enterovirus, hanta virus, hepatitis serotypes A to E, herpes simplex virus 1
or 2,
human immunodefi-ciency virus, human papilloma virus, influenza, measles,
Norwalk, Japanese equine encephalitis, papilloma virus, parvovirus B19, polio,
rabies, respiratory syncytial virus, rotavirus, rubella, rubeola, St. Louis
encephalitis,
vaccinia, viral expression vectors containing genes coding for other antigens
such as
malaria antigens, varicella, and yellow fever. The viral products or
derivatives thereof
~ o may be used as sources for antigen.
Fungi including entities responsible for tines corporis, tines unguis,
sporotri-
chosis, aspergillosis, candida and other pathogenic fungi. The fungal products
or
derivatives thereof may be used as sources for antigen.
Protozoans include, for example: Entamoeba histolytica, Plasmodium, Leish-
~ 5 mania, and the Helminthes; Schistosomes; and products thereof. The
protozoan
products or derivatives thereof may be used as sources for antigen.
Of particular interest are pathogens that enter on or through mucosal surfaces
such as, for example, pathogenic species in the bacterial genera Actinomyces,
Aeromonas, Bacillus, Bacteroides, Bordetella, Brucella, Campylobacter,
Capnocyto-
2o phaga, Clamydia, Clostridium, Corynebacterium, Eikenella, Erysipelothrix,
Escheri-
chic, Fusobacterium, Hemophilus, Klebsiella, Legionella, Leptospira, Listeria,
Myco-
bacterium, Mycoplasma, Neisseria, Nocardia, Pasteurella, Proteus, Pseudomonas,
Rickettsia, Salmonella, Selenomonas, Shigella, Staphylococcus, Streptococcus,
Treponema, Vibrio, and Versinia; pathogenic viral strains from the groups
Adeno-
25 virus, Coronavirus, Herpesvirus, Orthomyxovirus, Picornovirus, Poxvirus,
Reovirus,
Retrovirus, Rotavirus; pathogenic fungi from the genera Aspergillus,
Blastomyces,
Candida, Coccidiodes, Cryptococcus, Histoplasma, and Phycomyces; and patho-
genic protozoans in the genera Eimeria, Entamoeba, Giardia, and Trichomonas.
Vaccination has also been used as a treatment for cancer, allergies, and auto-
so immune disease. For example, vaccination with tumor antigen (e.g., HER2,
prostate
specific antigen) may induce an immune response in the form of antibodies,
CTLs
and lymphocyte proliferation which allows the body's immune system to
recognize
and kill tumor cells. Tumor antigens useful for vaccination have been
described for
leukemia, lymphoma, and melanoma. Allergens are known for animals (e.g., bird,
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23
cat, dog, rodents), cockroaches, fleas, mites, and plant pollen (e.g.,
grasses, trees).
Vaccination with T-cell receptor or autoantigens (e.g., pancreatic islet
antigen) may
induce an immune response that halts progression of autoimmune disease.
ADJUVANT
The formulation contains an adjuvant, although a single molecule may contain
both adjuvant and antigen properties (e.g., ADP-ribosylating exotoxin).
Because
most adjuvants would also have immunogenic activity and would be considered
antigens, adjuvants would also be expected to have the aforementioned
properties
and characteristics of antigens. For example, adjuvants and antigens may be
prepared using the same techniques (see above).
Adjuvants are substances that are used to specifically or nonspecifically
potentiate an antigen-specific immune response, perhaps through activation of
antigen presenting cells (e.g., dendritic cells in various layers of the skin,
especially
Langerhans cells). See also Elson et al. (in Handbook of Mucosal Immunology,
Academic Press, 1994). Although activation may initially occur in the
epidermis or
dermis, the effects may persist as the dendritic cells migrate through the
lymph
system and the circulation. Adjuvant may be formulated and applied with or
without
antigen, but generally, activation of antigen presenting cells by adjuvant
occurs prior
2o to presentation of antigen. Alternatively, they may be separately presented
within a
short interval of time but targeting the same anatomical region (e.g., the
same
draining lymph node field).
Adjuvants include, for example, chemokines (e.g., defensins, HCC-1, HCC-4,
MCP-1, MCP-3, MCP-4, MIP-1 a, MIP-1 Vii, MIP-1 ~, MIP-3a, MIP-2, RANTES);
other
ligands of chemokine receptors (e.g., CCR1, CCR-2, CCR-5, CCR-6, CXCR-1 );
cytokines (e.g., IL-1 a, IL -2, IL-6, IL-8, IL-10, IL-12; IFN-y; TNF-a; GM-
CSF); other
protein ligands of receptors for those cytokines, heat shock proteins and
derivatives
thereof; Leishmania homologs of eIF4a and derivatives thereof; bacterial ADP-
ribosylating exotoxins and derivatives thereof (e.g., genetic mutants, A
and/or B
3o subunit-containing fragments, chemically toxoided versions); chemical
conjugates or
genetic recombinants containing bacterial ADP-ribosylating exotoxins or
derivatives
thereof; C3d tandem array; and superantigens. See also Nohria et al.
(Biotherapy,
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24
7:261-269, 1994) and Richards et al. (in Vaccine Design, Eds. Powell et al.,
Plenum
Press, 1995) for other useful adjuvants.
Adjuvant may be chosen to preferentially induce antibody or cellular
effectors,
specific antibody isotypes (e.g., IgM, lgD, lgAl, lgA2, secretory IgA, IgE,
IgG1, IgG2,
s IgG3, and/or IgG4), or specific T-cell subsets (e.g., CTL, Th1, Th2 and/or
TpTH). For
example, antigen presenting cells may present Class II-restricted antigen to
precursor CD4+ T cells, and the Th1 or Th2 pathway may be entered. T helper
cells
actively secreting cytokine are primary effector cells; they are memory cells
if they
are resting. Reactivation of memory cells produces memory effector cells. Th1
characteristically secrete IFN-y (TNF-~i and IL-2 may also be secreted) and
are
associated with "help" for cellular immunity, while Th2 characteristically
secrete IL-4
(IL-5 and IL-13 may also be secreted) and are associated with "help" for
humoral
immunity. Depending on disease pathology, adjuvants may be chosen to prefer a
Th1 response (e.g., antigen-specific cytolytic cells) vs. a Th2 response
(e.g., antigen-
~5 specific antibodies).
Most ADP-ribosylating exotoxins (BARE) are organized as A:B heterodimers
with a B subunit containing the receptor binding activity and an A subunit
containing
the ADP-ribosyltransferase activity. Exemplary bARE include cholera toxin (CT)
E.
coli heat-labile enterotoxin (LT), diphtheria toxin, Pseudomonas exotoxin A
(ETA),
2o pertussis toxin (PT), C. botulinum toxin C2, C. botulinum toxin C3, C,
limosum
exoenzyme, 8. cereus exoenzyme, Pseudomonas exotoxin S, S. aureus EDIN, and
B. sphaericus toxin. Mutant BARE, for example containing mutations of the
trypsin
cleavage site (e.g., Diclcenson et al., Infect Immun, 63:1617-1623, 1995) or
mutations affecting ADP-ribosylation (e.g., Douce etal., Infect Immun,
65:28221-
25 282218, 1997) may be used.
TCI may be accompished through the ganglioside GMT binding activity of CT,
LT, or subunits thereof (e.g., CTB or LTB). Ganglioside GMT is a ubiquitous
cell
membrane glycolipid found in all mammalian cells. When the pentameric CT B
subunit binds to the cell surface, a hydrophilic pore is formed which allows
the A
3o subunit to insert across the lipid bilayer. Other binding targets on the
APC may be
utilized (e.g., ETA binds a2-macroglobulin receptor-low density lipoprotein
receptor-
related protein). The LT B subunit binds to ganglioside GMT in addition to
other
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gangliosides and its binding activities may account for its the fact that LT
is highly
immunogenic on the skin.
TCI with bARE or B subunit-containing fragments or conjugates thereof may
repuire their ganglioside GMT binding activity. When mice were
transcutaneously
immunized with CT, CTA and CTB, CT and CTB were required for induction of an
immune response. CTA contains the ADP-ribosylating exotoxin activity but only
CT
and CTB containing the binding activity are able to induce an immune response
indicating that the B subunit was necessary and sufficient to immunize through
the
skin. We conclude that the Langerhans cells or other APC may be activated by
CTB
binding to its cell surface resulting in a transcutaneous immune response.
CT, LT, ETA and PT, despite having different cellular binding sites, are
potent
adjuvants for transcutaneous immunization, inducing IgG antibodies but not IgE
antibodies. CTB without CT can also induce IgG antibodies. Thus, both bARE and
a
derivative thereof can effectively immunize when epicutaneously applied to the
skin.
15 Native LT as an adjuvant and antigen, however, is clearly not as potent as
native CT.
But activated bARE can act as adjuvants for weakly immunogenic antigens in a
transcutaneous immunization system. Thus, therapeutic immunization with one or
more antigens could be used separately or in conjunction with
immunostimulation of
the antigen presenting cell to induce a prophylactic or therapeutic immune
response.
2o In general, toxins can be chemically inactivated to form toxoids which are
less
toxic but remain immunogenic. We envision that the transcutaneous immunization
system using toxin-based immunogens and adjuvants can achieve anti-toxin
levels
adepuate for protection against these diseases. The anti-toxin antibodies may
be
induced through immunization with the toxins, or genetically-detoxified
toxoids
25 themselves, or with toxoids and adjuvants. Genetically toxoided toxins
which have
altered ADP-ribosylating exotoxin activity or trypsin cleavage site, but not
binding
activity, are envisioned to be especially useful as nontoxic activators of
antigen
presenting cells used in transcutaneous immunization and may reduce concerns
over toxin use.
so bARE can also act as an adjuvant to induce antigen-specific CTL through
transcutaneous immunization. The bARE adjuvant may be chemically conjugated to
other antigens including, for example, carbohydrates, polypeptides,
glycolipids, and
giycoprotein antigens. Chemical conjugation with toxins, their subunits, or
toxoids
with these antigens would be expected to enhance the immune response to these
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26
antigens when applied epicutaneously. To overcome the problem of the toxicity
of
the toxins (e.g., diphtheria toxin is known to be so toxic that one molecule
can kill a
cell) and to overcome the problems of working with such potent toxins as
tetanus,
several workers have taken a recombinant approach to producing genetically-
produced toxoids. This is based on inactivating the catalytic activity of the
ADP-
ribosyl transferase by genetic deletion. These toxins retain the binding
capabilities,
but lack the toxicity, of the natural toxins. Such genetically toxoided
exotoxins would
be expected to induce a transcutaneous immune response and to act as
adjuvants.
They may provide an advantage in a transcutaneous immunization system in that
they would not create a safety concern as the toxoids would not be considered
toxic.
Activation through a technique such as trypsin cleavage, however, would be
expected to enhance the adjuvant qualities of LT through the skin which lacks
trypsin-tike enzymes. Additionally, several techniques exist to chemically
modify
toxins and can address the same problem. These techniques could be important
for
~5 certain applications, especially pediatric applications, in which ingested
toxins might
possibly elicit adverse reactions.
Adjuvant may be biochemically purified from a natural source (e.g., pCT or
pLT) or recombinantly produced (e.g., rCT or rLT). ADP-ribosylating exotoxin
may be
purified either before or after proteolysis (i.e., activation). B subunit of
the ADP-
2o ribosylating exotoxin may also be used: purified from the native enzyme
after
proteolysis or produced from a fragment of the entire coding region of the
enzyme.
The subunit of the ADP-ribosylating exotoxin may be used separately (e.g., CTB
or
LTB) or together (e.g., CTA-LTB, LTA-CTB) by chemical conjugation or genetic
fusion. A fragment of the ADP-ribosylating exotoxin which retains the ability
to bind
25 its cell membrane receptor may also be biochemically purified or
recombinantly
produced, and then used instead of the B subunit.
Point mutations (e.g., single, double, or triple amino acid substitutions),
deletions (e.g., protease recognition site), and isolated functional domains
of ADP-
ribosylating exotoxin may also be used as adjuvant. Derivatives which are less
toxic
so or have lost their ADP-ribosylation activity, but retain their adjuvant
activity have
been described. Specific mutants of E. coli heat-labile enterotoxin include LT-
K63,
LT-R72, LT (H44A), LT (R192G), LT (R192G1L211A), and LT 0192-194). Toxicity
may be assayed with the Y-1 adrenal cell assay (Clements and Finkelstein,
Infect.
Immun., 24:760-769, 1979). ADP-ribosylation may be assayed with the NAD-
CA 02445486 2003-09-18
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27
agmatine ADP-ribosyltransferase assay (Moss et al., J. Biol. Chem., 268:6383-
6387,
1993). Particular ADP-ribosylating exotoxins, derivatives thereof, and
processes for
their production and characterization are described in U.S. Patents 4,666,837;
4, 935, 364; 5, 308, 835; 5, 785, 971; 6, 019, 982; 6, 033, 673; and 6,149,
919.
An activator of Langerhans cells may also be used as an adjuvant. Examples
of such activators include proteins like chemokines, cytokines,
differentiation factors,
and growth factors (e.g., members of the TGF~i superfamily).
If an immunizing antigen has sufficient Langerhans cell activating
capabilities
then a separate adjuvant may not be required, as in the case of LT which is
both
~o antigen and adjuvant. Alternatively, such antigens can be considered not to
require
an adjuvant because they are sufficiently immunogenic. It may also be possible
to
use low concentrations of activators of Langerhans cells to induce an immune
response without inducing skin lesions.
Other techniques for enhancing activity of adjuvants may be effective, such as
~5 adding surfactants and/or phospholipids to the formulation to enhance
adjuvant
activity of ADP-ribosylating exotoxin by ADP-ribosylation factor. One or more
ADP-
ribosylation factors (ARF) may be used to enhance the adjuvanticity of bARE
(e.g.,
ARF1, ARF2, ARF3, ARF4, ARFS, ARF6, ARD1 ). Similarly, one or more ARF could
be used with an ADP-ribosylating exotoxin to enhance its adjuvant activity.
2o Undesirable properties or harmful side effects (e.g., allergic or
hypersensitive
reaction; atopy, contact dermatitis, or eczema; systemic toxicity) may be
reduced by
modification without destroying its effectiveness in transcutaneous
immunization.
Modification may involve, for example, removal of a reversible chemical
modification
(e.g., proteolysis) or encapsulation in a coating which reversibly isolates
one or more
25 components of the formulation from the immune system. For example, one or
more
components of the formulation may be encapsulated in a particle for delivery
(e.g.,
microspheres, nanoparticles) although we have shown that encapsulation in
lipid
vesicles is not required for transcutaneous immunization and appears to have a
negative effect. Phagocytosis of a particle may, by itself, enhance activation
of an
so antigen presenting cell by upregulating expression of MHC Class I and/or
Class II
molecules and/or costimulatory molecules (e.g., CD40, B7 family members like
CD80 and CD86). Alternative methods of upregulating such molecules by
activating
an antigen presenting cell are also known (see above).
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28
FORMULATION
Processes for manufacturing a pharmaceutical formulation are well known.
The components of the formulation may be combined with a pharmaceutically-
s acceptable carrier or vehicle, as well as any combination of optional
additives (e.g.,
at least one binder, buffer, coloring, dessicant, diluent, humectant,
preservative,
stabilizer, other excipient, or combinations thereof). See, generally,
Ullmann's Ency
clopedia of Industrial Chemistry, 6t" Ed. (electronic edition, 1998);
Remington's
Pharmaceutical Sciences, 22~d (Gennaro, 1990, Mack Publishing); Pharmaceutical
~ o Dosage Forms, 2"d Ed. (various editors, 1989-1998, Marcel Dekker); and
Pharma-
ceutical Dosage Forms and Drug Delivery Systems (Ansel et al., 1994, Williams
&
Wilkins).
Good manufacturing practices are known in the pharmaceutical industry and
regulated by government agencies (e.g., Food and Drug Administration). A
liquid
15 formulation may be prepared by dissolving an intended component of the
formulation
in a sufficient amount of an appropriate solvent. Generally, dispersions are
prepared
by incorporating the various components of the formulation into a vehicle
which
contains the dispersion medium. For production of a solid form from a liquid
formu-
lation, solvent may be evaporated at room temperature or in an oven. Blowing a
2o stream of nitrogen or air over the surface accelerates drying;
alternatively, vacuum
drying or freeze drying can be used.
Suitable procedures for making the various dosage forms and production of
patches are known. The size of each dose and the interval of dosing to the
subject
may be used to determine a suitable size and shape of the container,
compartment,
25 or chamber. Formulations will contain an effective amount of the active
ingredients
(e.g., at least one adjuvant and/or one or more antigens) together with
carrier or
suitable amounts of vehicle in order to provide pharmaceutically-acceptable
compositions suitable for administration to a human or animal. Formulation
that
include a vehicle may be in the form of a cream, emulsion, gel, lotion,
ointment,
so paste, solution, suspension, or other liquid forms known in the art;
especially those
that enhance skin hydration. For a patch, successive coatings of formulation
may be
applied to the substrate or several formulation-containing layers may be
laminated to
increase its capacity for active ingredients.
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29
The relative amounts of active ingredients within a dose and the dosing
schedule may be adjusted appropriately for efficacious administration to a
subject
(e.g., animal or human). This adjustment may depend on the subject's
particular
disease or condition, and whether therapy or prophylaxis is intended. To
simplify
administration of the formulation to the subject, each unit dose would contain
the
active ingredients in predetermined amounts for a single round of
immunization.
There are numerous causes of protein instability or degradation, including
hydrolysis and denaturation. In the case of denaturation, the protein's
conformation
is disturbed and the protein may unfold from its usual globular structure.
Rather than
refolding to its natural conformation, hydrophobic interaction may cause
clumping of
molecules together (i.e., aggregation) or refolding to an unnatural
conformation.
Either of these results may entail diminution or loss of antigenic or adjuvant
activity.
Stabilizers may be added to lessen or prevent such problems.
The formulation, or any intermediate in its production, may be pretreated with
protective agents (i.e., cryoprotectants and drying stabilizers) and then
subjected to
cooling rates and final temperatures that minimize ice crystal formation. By
proper
selection of cryoprotective agents and the use of preselected drying
parameters,
almost any formulation might be dried for a suitable desired end use.
It should be understood in the following discussion of optional additives like
2o binders, buffers, colorings, dessicants, diluents, humectants,
preservatives, and
stabilizers are described by their function. Thus, a particular chemical may
act as
some combination of the aforementioned. Such chemicals would be considered
immunologically-inactive because they do not directly induce an immune
response,
but it increases the response by enhancing immunological activity of the
antigen or
adjuvant: for example, by reducing modification of the antigen or adjuvant, or
dena-
turation during drying and hydrating cycles.
Stabilizers include dextrans and dextrins; glycols, alkylene glycols,
polyalkane
glycols, and polyalkylene glycols, sugars and starches, and derivatives
thereof are
suitable. Preferred additives are nonreducing sugars and polyols. In
particular,
so trehalose, hydroxymethyl or hydroxyethyl cellulose, ethylene or propylene
glycol,
trimethyl glycol, vinyl pyrrolidone, and polymers thereof may be added. Alkali
metal
salts, ammonium sulfate, magnesium chloride, and surfactants (e.g., nonionic
detergent), may stabilize proteinaceous adjuvants or antigens; optionally
adding a
carrier (e.g., agar, albumin, gelatin, glycogen, heparin), and freeze drying
may
CA 02445486 2003-09-18
WO 02/074325 PCT/US02/08099
further enhance stability. A polypeptide may also be stabilized by contacting
it with a
sugar such as, for example, a monosaccharide, disaccharide, sugar alcohol, and
mixtures thereof (e.g., arabinose, fructose, galactose, glucose, lactose,
maltose,
mannitol, mannose, sorbitol, sucrose, xylitol). Polyols may stabilize a
polypeptide,
5 and are water-miscible or water-soluble. Various other excipients may also
stabilize
polpeptides, including amino acids, fatty acids and phospholipids, metals,
reducing
agents, and metal chelating agents. The stabilizer may be between 0.1 % (w/v)
and
10% (w/v) or between 1 % (w/v) and 5% (w/v) of the adhesive formulation.
Single-dose formulations can be stabilized in poly(lactic acid) (PLA) and poly
10 (lactide-co-glycolide) (PLGA) microspheres by suitable choice of stabilizer
or other
excipients. Trehalose may be advantageously used as an additive because it is
a
nonreducing saccharide, and therefore does not cause aminocarbonyl reactions
with
substances bearing amino groups such as proteins. Although stabilizers like
high
concentrations of sugar will combat the growth of microbes like bacteria and
fungi,
~5 preservatives are typically antimicrobial agents that actively eliminate
(e.g., bacterio-
cidal) or reduce the growth of microbes (e.g., bacteriostatic). Antioxidants
may also
be used to prevent oxidation of active ingredients of the formulation.
It is conceivable that a formulation or patch that can be administered to the
subject in a dry, nonliquid (i.e., solid) form, may allow storage in
conditions that do
2o not require a cold chain. An antigen may be mixed with a heterologous
adjuvant,
placed on a dressing to form a patch, and allowed to completely dry. This dry
patch
can then be placed on skin with the dressing in direct contact with the skin
for a
period of time and be held in place covered with an occlusive backing layer
(e.g.,
plastic or wax film).
25 Patch material may be nonwoven or woven (e.g., gauze dressing). Layers
may also be laminated during processing. It may be nonocclusive or occlusive,
but
the latter is preferred for backing layers. The optional release liner
preferably does
not adsorb significant amounts of the formulation, perhaps by treating a film
with
silicone or fluorocarbon. The patch is preferably hermetically sealed for
storage {e.g.,
so foil packaging). The patch can be held onto the skin and components of the
patch
can be held together using various adhesives. One or more of the adjuvant
and/or
antigen may be applied to and/or incorporated in the adhesive portion of the
patch.
Generally, patches are planar and pliable, and they are manufactured with a
uniform
shape. Optional additives are plasticizers to maintain pliability of the
patch, tackifiers
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31
to assist in adhesion between patch and skin, and thickeners to increase the
viscosity of the formulation at least during processing.
Metal foil, cellulose, cloth (e.g., acetate, cotton, rayon), acrylic polymer,
ethyl-
enevinyl acetate copolymer, polyamide (e.g., nylon), polyester (e.g., poly-
ethylene
naphthalate, ethylene terephthalate), polyolefin (e.g., polyethylene, poly-
propylene),
polyurethane, polyvinylidene chloride (SARAN), natural or synthetic rubber,
silicone
elastomer, and combinations thereof are examples of patch materials (e.g.,
dressing,
backing layer, release liner).
The adhesive may be an aqueous-based adhesive (e.g., acrylate or silicone).
Acrylic adhesives are available from several commercial sources. Acrylic
polymers
may be a copolymer of C4-C18 aliphatic alcohol with methacrylic alkyl ester or
the
copolymer of methacrylic alkyl ester having C4-C18 alkyl, methacrylic acid,
and/or
other functional monomers. Examples of the methacrylic alkyl ester may include
butyl acrylate, isobutyl acrylate, hexyl acrylate, octyl acrylate, 2-
ethylhexyl acrylate,
iso-octyl acrylate, decyl acrylate, isodecyl acrylate, lauryl acrylate,
stearyl acrylate,
methyl methacrylate, ethyl methacrylate, butyl methacrylate, isobutyl
methacrylate,
2-ethylhexyl methacrylate, iso-octyl methacrylate, decyl methacrylate, etc.
Examples of the functional monomers may include a monomer containing
hydroxyl group, a monomer containing carboxyl group, a monomer containing
amide
2o group, a monomer containing amino group. The monomer containing hydroxyl
group
may include hydroxyalkyl methacrylate such as 2-hydroxyethyl methacrylate,
hydroxypropyl methacrylate and the like. The monomer containing carboxyl group
may include a-(3 unsaturated carboxylic acid such as acrylic acid, methacrylic
acid
and the like; malefic mono alkyl ester such as butyl malate and the like;
malefic acid;
fumaric acid; crotonic acid and the like; and anhydrous malefic acid. Examples
of the
monomer containing amide group may include alkyl methacrylamide such as acryl-
amide, dimethyl acrylamide, diethyl acrylamide and the like;
alkylethylmethylol
methacrylamide such as butoxymethyl acrylamide, ethoxymethyl acrylamide and
the
like; diacetone acrylamide; vinyl pyrrolidone; dimethyl aminoacrylate. In
addition to
so the above exemplified monomers for copolymerization, vinyl acetate,
styrene, a-
methylstyrene, vinyl chloride, acrylonitrile, ethylene, propylene, butadiene
and the
like may be employed.
Commercially available acrylic adhesives are sold under the tradenames
AROSET, DUROTAK, EUDRAGIT, GELVA, and NEOCRYL. EUDRAGIT polymers
CA 02445486 2003-09-18
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32
form a diverse family of polymers whose common feature is a polyacrylic or
poly-
methacrylic backbone that is compatible with the gastrointestinal tract and
which
have been widely used in pharmaceutical preparations, especially as coatings
for
tablets, but it has also been used as a coating for other medical devices.
EUDRAGIT
s polymers are characterized as (1 ) an anionic copolymer based on methacrylic
acid
and methylmethacrylate wherein the ratio of free carboxyl groups to the ester
groups
is approximately 1:1, (2) an anionic copolymer based on methacrylic acid and
methylmethacrylate wherein the ratio of free carboxyl groups to the ester
groups is
approximately 1:2, (3) a copolymer based on acrylic and methacrylic acid
esters with
a low content of quaternary ammonium groups wherein the molar ratio of the
ammo-
nium groups to the remaining neutral methacrylic acid esters is 1:20, and (4)
a copo-
lymer based on acrylic and methacrylic acid esters with a low content of
quarternary
ammonium groups wherein the molar ratio of the ammonium groups to the
remaining
neutral methacrylic acid esters is 1:40. The copolymers are sold under
tradenames
~5 EUDRAGIT L, EUDRAGIT S, EUDRAGIT RL, and EUDRAGIT RS. EUDRAGIT E is
a cationic copolymer based on diethylaminoethyl methacrylate and neutral metha-
crylic acid esters; EUDRAGIT NE is a neutral copolymer of polymethacrylates.
For
methacrylate or acrylate polymers, there are EUDRAGIT RS, EUDRAGIT RL, and
EUDRAGIT NE; also available are EUDRAGIT RS-100, EUDRAGIT L-90,
2o EUDRAGIT NE-30, EUDRAGIT L-100, EUDRAGIT S-100, EUDRAGIT E-100,
EUDRAGIT RL-100, EUDRAGIT RS-100, EUDRAGIT RS-30D, EUDRAGIT E-1008,
and EUDRAGIT RTM.
Furthermore, for the purpose of increasing or decreasing the water absorption
capacity of an adhesive layer, the acrylic polymer may be copolymerized with
hydro
25 philic monomer, monomer containing carboxyl group, monomer containing amide
group, monomer containing amino group, and the like. Rubbery or silicone
resins
may be employed as the adhesive resin; they may be incorporated into the
adhesive
layer with a tackifying agent or other additives.
Alternatively, the water absorption capacity of the adhesive layer can be also
so regulated by incorporating therein highly water-absorptive polymers,
polyols, and
water-absorptive inorganic materials. Examples of the highly water-absorptive
resins
may include mucopolysaccharides such as hyaluronic acid, chondroitin sulfate,
dermatan sulfate and the like; polymers having a large number of hydrophilic
groups
in the molecule such as chitin, chitin derivatives, starch and carboxy-
methylcellulose;
CA 02445486 2003-09-18
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33
and highly water-absorptive polymers such as polyacrylic, polyoxyethylene,
polyvinyl
alcohol, and polyacrylonitrile. Examples of the water-absorptive inorganic
materials,
which may incorporated into the adhesive layer to regulate its water
absorptive capa-
city, may include powdered silica, zeolite, powdered ceramics, and the like.
The plasticizes may be a trialkyl citrate such as, for example, acetyl-
tributyl
citrate (ATBC), acetyl-triethyl citrate (ATEC), and triethyl citrate (TEC).
The plasti-
cizes may be between 0.001 % (w/v) and 5% (w/v) of the adhesive formulation. A
suitable concen-tration may be empirically determined by selecting for
pliability of the
adhesive layer, and avoiding brittleness.
Exemplary tackifiers are glycols (e.g., glycerol, 1,3 butanediol, propylene
glycol, polyethylene glycol); average molecular weights of 200, 300, 400, 800,
3000,
et=c. are available for the polyakylene glycols. Succinic acid is another
tackifier. The
tackifier may be between 0.1 % (w/w) and 10% (w/w) of the adhesive
formulation. A
suitable concentration may be empirically determined by avoiding brittleness
of the
~5 adhesive layer and its pliability.
Thickeners can be added to increase the viscosity of an adhesive or immuno-
genic formulation. The thickener may be a hydroxyalkyl cellulose or starch, or
water-
soluble polymers: for example, poloxamers, polyethylene oxides and derivatives
thereof, polyethyleneimines, polyethylene glycols, and polyethylene glycol
esters.
2o But any molecule which serves to increase the viscosity of a solution may
be suitable
to improve handling of a formulation during manufacture of a patch. For
example,
hydroxyethyl or hydroxypropyl cellulose may be between 1 % (w/w) and 10% (w/w)
of
the adhesive or immunogenic formulation. The formulation as a layer may be
film
cast or extruded, and then layers may be coated or laminated during
manufacture of
25 a patch. The capacity for protein might be increased by successive coatings
or lami-
nating several thin, adhesive layers together. Alternatively, a viscous
formulation
may be spread on a substrate (e.g., backing or adhesive layer) with minimal
loss of
immunologically-active ingredients like adjuvant or antigen. Thickeners are
sold as
NATROSOL hydroxyethyl cellulose and KLUCEL hydroxypropyl cellulose.
3o Gel and emulsion systems can be incorporated into patch delivery systems,
or
be manufactured separately from the patch, or added to the patch prior to
application
to the human or animal subject. Gels or emulsions may serve the same purpose
of
facilitating manufacture by providing a viscous formulation that can be easily
manipu-
lated with minimal loss. The term "gel" refers to covalently crosslinked,
noncross-
CA 02445486 2003-09-18
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34
linked hydrogel matrices. Hydrogels can be formulated with at least one
protein with
immunologic activity for PIA patches. Additional excipients may be added to
the gel
systems that allow for the enhancement of antigen/adjuvant delivery, skin
hydration,
and protein stability. The term "emulsion" refers to formulations such as
water-in-oil
creams, oil-in-water creams, ointments, and lotions. Emulsion systems can be
either
micelle-based, lipid vesicle-based, or both micelle- and lipid vesicle-based.
Emulsion
systems can be formulated with at least one adjuvant and/or antigen as the
protein-
in-adhesive systems. Additional excipients may be added to the emulsion
systems
that allow for the enhancement of antigen/adjuvant delivery, skin hydration,
and
1o protein stability.
Formulation may be applied with a patch in contact with skin of the subject.
It
may be covered with a nonocclusive or occlusive backing layer, the latter
prevents
evaporation and traps moisture at the site of application. Such a formulation
may be
applied to single or multiple sites, to single or multiple limbs, or to a
large surface
area of skin. Other substrates that may be used are pressure-sensitive
adhesives
such as acrylics, polyisobutylenes, and silicones. The formulation may be
incorpo-
rated directly into such substrates, perhaps with the adhesive per se instead
of
adsorption to a porous pad (e.g., cotton gauze) or bilious strip (e.g.,
cellulose paper).
The adhesive and immunogenic formulations may be at least partially mixed
20 or even throroughly blended, and then adhered to the backing layer. The
immunolo-
gically-active ingredient may be dispersed or dissolved in the formulation.
Alterna-
tively the immunogenic formulation may be applied to the surface of the
adhesive
layer by coating or spreading over the adhesive using a Meyer rod, casting a
layer
and then laminating in close apposition with the adhesive using a roller,
printing on
2s the adhesive using a rotogravure, etc. Adhesive may be brought into contact
with a
release liner. Adhesive and immunogenic formulations may also be brought into
contact with microblade or microneedle arrays or tines by coating, dipping the
device
into the formulation and drying, or spraying the device with the formulation.
Polymers added to the formulation may act as a stabilizer or other excipient
of
so an active ingredient as well as reducing the concentration of the active
ingredient
that saturates a solution used to hydrate an at least partially-dried form
(i.e., dry or
semi-liquid) of the active ingredient. Such reduction occurs because the
polymer
reduces the effective free volume by filling "empty" space in the solvent. In
this way,
quantities of adjuvant/antigen can be conserved without reducing the amount of
CA 02445486 2003-09-18
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saturated solution. An important thermodynamic consideration is that an active
ingre-
dient in the saturated solution will be "driven" into regions of lower
concentration
(e.g., through the skin). For dispersal or dissolution of at least one
adjuvant and/or
one or more antigens, polymers can also stabilize the adjuvant/antigen-
activity of
5 those components of the formulation. Such polymers include ethylene or
propylene
glycol, vinyl pyrrolidone, and (3-cyclodextrin polymers and copolymers.
TRANSCUTANEOUS DELIVERY
Transcutaneous delivery of the formulation may target Langerhans cells and,
1o thus, achieve effective and efficient immunization. Cells are found in
abundance in
the skin and are efficient antigen presenting cells (APC), which can lead to T-
cell
memory and potent immune responses. Because of the presence of large numbers
of Langerhans cells in the skin, the efficiency of transcutaneous delivery may
be
related to the surface area exposed to antigen and adjuvant. In fact, the
reason that
~5 transcutaneous immunization is so efficient may be that it targets a larger
number of
these efficient antigen presenting cells than intramuscular immunization.
Immunization may be achieved using epicutaneous application of a simple
formulation of antigen and adjuvant, optionally covered by an occlusive
dressing or
using other patch technologies, to intact skin with or without chemical or
physical
2o penetration. Transcutaneous immunization according to the invention may
provide a
method whereby antigens and adjuvant can be delivered to the immune system,
especially specialized antigen presentation cells underlying the skin (e.g.,
dendritic
cells like Langerhans cells). The patch may be worn for as briefly as 30 sec;
1 min to
5 min; or less than 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 15
hours, 18
25 hours, 24 hours, or 48 hours. In contrast to transdermal patches delivering
drugs, the
release characteristics of the patch of the invention does not need to be
constant or
prolonged. It is preferred that the immunologically-active protein may be
released
quickly and quantitatively.
Moreover, transcutaneous immunization may be superior to immunization
so using hypodermic needles as more immune cells would be targeted by the use
of
several locations targeting large surface areas of skin. A therapeutically-
effective
amount of antigen sufficient to induce an immune response may be delivered
transcutaneously either at a single cutaneous location, or over an area of
skin
covering multiple draining lymph node fields (e.g., cervical, axillary,
inguinal,
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36
epitrocheiear, popliteal, those of the abdomen and thorax). Such locations
close to
numerous different lymphatic nodes at locations all over the body will provide
a more
widespread stimulus to the immune system than when a small amount of antigen
is
injected at a single location by intradermal, subcutaneous, or intramuscular
injection.
Antigen passing through or into the skin may encounter antigen presenting
cells which process the antigen in a way that induces an immune response.
Multiple
immunization sites may recruit a greater number of antigen presenting cells
and the
larger population of antigen presenting cells that were recruited would result
in
greater induction of the immune response. It is conceivable that use of the
skin may
o deliver antigen to phagocytic cells of the skin such as, for example,
dendritic cells,
Langerhans cells, macrophages, and other skin antigen presenting cells;
antigen
may also be delivered to phagocytic cells of the liver, spleen, and bone
marrow that
are known to serve as the antigen presenting cells through the blood stream or
lymphatic system.
~ 5 Langerhans cells, other dendritic cells, macrophages, or combinations
thereof
may be specifically targeted using their asialoglycoprotein receptor, mannose
receptor, Fcy receptor CD64, high-affinity receptor for IgE, or other highly
expressed
membrane proteins. A ligand or antibody specific for any of those receptors
may be
conjugated to or recombinantly produced as a protein fusion with adjuvant,
antigen,
20 or both. Furthermore, adjuvant, antigen, or both may be conjugated to or
recombinantly produced as a protein fusion with protein A or protein G to
target
surface immunoglobulin of B lymphocytes. The envisioned result would be
widespread distribution of antigen to antigen presenting cells to a degree
that is
rarely, if ever achieved, by current immunization practices.
25 A specific immune response may comprise humoral (i.e., antigen-specific
antibody) and/or cellular (i.e., antigen-specific lymphocytes such as B
lymphocytes,
CD4+ T cells, CD8+ T cells, CTL, Th1 cells, Th2 cells, and/or ToTH cells)
effector
arms. Moreover, the immune response may comprise NK cells and other leukocytes
that mediate antibody-dependent cell-mediated cytotoxicity (ADCC).
so The immune response induced by the formulation of the invention may
include the elicitation of antigen-specific antibodies and/or lymphocytes.
Antibody
can be detected by immunoassay techniques. Detection of the various antibody
isotypes (e.g., IgM, igD, IgA1, IgA2, secretory IgA, IgE, IgG1, igG2, IgG3 or
IgG4)
can be indicative of a systemic or regional immune response. Immune responses
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37
can also be detected by a neutralizing assay. Antibodies are protective
proteins
produced by B lymphocytes. They are highly specific, generally targeting one
epitope
of an antigen. Immunization may induce antibodies that neutralize biological
activity
of an allergen, cell-entry receptor, growth factor receptor, or toxin. For
example,
s inducing antibodies may treat a disease by specifically reacting with
antigen (e.g.,
cholera toxin, HER2, influenza hemagluttinin) derived from a pathogen or
cancer.
Challenge studies in a host using infection by the pathogen or administration
of toxin,
comparison of morbidity or mortality between immunized and control
populations, or
measurement of another clinical criterion (e.g., high antibody titers or
production of
~o IgA antibody-secreting cells in mucosal membranes may be used as a
surrogate
marker) can demonstrate protection against disease or therapy of existing
disease.
CTL are immune cells produced to protect against infection by a pathogen.
They are also highly specific. Immunization may induce CTL specific for the
antigen
in association with self-major histocompatibility complex antigen. CTL induced
by
immunization with the transcutaneous delivery system may kill pathogen-
infected
cells or cancers. Immunization may also produce a memory response as indicated
by boosting responses in antibodies and CTL, proliferation of lymphocyte
cultures
stimulated with the antigen, and delayed-type hypersensitivity (DTH) responses
to
intradermal skin challenge of the antigen alone.
2o The following is meant to be illustrative of the invention, but practice of
the
invention is not limited or restricted in any way by the following examples.
EXAMPLES
Stability of Lysozyme-in-Adhesive Formulation
25 Many proteins and large biomolecules exhibit thermal lability, as well as
chemical instability due to pH factors or incompatibility with a variety of
compounds.
Many adhesive systems are solvated in solvents which are detrimental to drug
stability. Also many of these adhesive polymers contain functional groups
which are
incompatible with many reactive molecules. In addition, conventional
technology
so often requires high temperatures to dry, extrude, or set the adhesive
blend. It is
therefore very difficult to formulate and process drug-in-adhesive systems for
these
compounds. The formulation and process described here allow for production of
a
protein-in-adhesive (PIA) system without thermal or chemical degradation of
these
delicate molecules. The formulation is also particularly suitable to large
molecular
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38
weight biomolecules, owing to the water-soluble characteristics of the
adhesive
polymer which allow for release of the biomolecule when exposed to water.
Lysozyme was used as a model protein for the PIA formulation. Proteins can
be chemically-labile compounds that aggregate, degrade, or otherwise denature
when subjected to heat or a variety of solvents or reactive chemical sites.
Lysozyme
and assay of its enzymatic activity makes this a good model for proteins which
needs
stabilization.
Lysozyme was applied to patches made with two aqueous-based adhesives:
lysozyme was applied to either a silicone adhesive or an acrylic adhesive on a
patch.
o Protein was extracted from the adhesive in 20 ml water for 1 hr. Lysozyme
recovery
was about 35% to 90% from silicone-adhesive patches stored at room temperature
or stored overnight at 40°C, as well as acrylic-adhesive patches stored
at room
temperature. Ground lysozyme was assayed as a positive control with a recovery
of
about 95% and activity of about 107% (bioactivity was assayed by UV
spectroscopy
~s and scan monitoring of reaction kinetics of lysozyme with Micrococcus
lysodeikticus).
Activity for the lysozyme extracted from adhesives of the patches was about
100% to
110% of original.
A methacrylate adhesive-KLUCEL thickener emulsion was prepared with a
liquid plasticizer and emulsifier using water as the primary solvent at room
tempe
2o rature. Stability of the lysozyme in the wet blend was demonstrated for
over seven
days. The wet blend was coated soon after preparation and then dried using
room
temperature air or nitrogen, blown very close to the adhesive surface. The
resulting
partially-dried protein-in-adhesive demonstrated stability for over 30 days at
room
temperature as shown by lysozyme bioactivity. It also had acceptable adhesive
and
25 wear properties.
E. coli Heat-Stable Enterotoxin (LT)-in-Adhesive Formulation
LT was obtained from Dr. John Clements of Tulane University (LTc). This
material was obtained as a dry powder lyophilized from a TRIS buffer
containing
30 200mM NaCI. This LT has never been exposed to lactose. Unless otherwise
noted,
this is the source of LT used throughout this example. LT obtained from SSVI-
Berne
(LTs). This material was provided as a dry powder lyophilized from a phosphate
buffered saline (PBS) formulation that contains 5% lactose. LTR~9~c or
LT(R192G)
mutant protein is a single amino acid residue mutant of LT: arginine at
position 192 is
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39
mutated to glutamine. PBSX, pH 7.4 is 10 mM potassium phosphate buffered
saline
with pH of 7.4; the subscript x indicates the concentration of NaCI (e.g.,
PBS2oo,
pH7.4 has 200 mM NaCI).
KLUCEL EF thickener is hydroxypropyl cellulose, a viscosity enhancer made
by Hercules, and Was prepared as a 20% (w/w) stock in water. NATROSOL 250L NF
is hydroxyethyl cellulose, a viscosity enhancer made by Hercules, and was
prepared
as a 12% (w/w) stock in water.
The adhesive fomulation is a suspension of EUDRAGIT EPO polymer (Rohm)
in water containing 37.4% nonvolatile components (NVC). The modified adhesive
o formulation is the standard adhesive formulation to which was added 6% (w/w)
glycerol and 4% (w/w) 1,3-butanediol. The final suspension contains 43.7% NVC.
These additives are included to increase the plasticity and tackiness of cured
films of
EUDRAGIT adhesive.
Adhesive formulations have been identified in which LT shows good stability
~5 and recoverability. Wet blends contain about 500 pg/gm LT, 5% disaccharide
(e.g.,
sucrose, trehalose), and 3% KLUCEL thickener. The blend is prepared by mixing
EUDRAGIT adhesive and protein solution buffered at pH 7.4 (with disaccharide
as
nonreducing sugar and KLUCEL thickener) in a mass ratio of about 1:1.
Excellent
stability at 5°C and good stability (there was some loss of recovered
protein) at room
2o temperature were observed over the course of 6 to 7 weeks.
Patches were manufactured by combining protein solutions (disaccharide
containing) in a weight ratio of about 1:1 with standard EUDRAGIT adhesive.
These
wet blends were then cast as thin films on 1012 plastic backing using an 8-mil
knife.
The films were allowed to air dry at room temperature and then covered with a
25 release liner. Patches with an area of about 1 cm2 were punched out using a
7/16-
inch diameter multi-purpose punch. Patches were placed in 5 ml glass
lyophilization
vials (with 20 mm mouth) and sealed under nitrogen. The sealed vials were
placed
on incubation and sampled at intervals.
Patches were rehydrated with ddH20, and the samples were prepared and
so analyzed according to the following procedures. EUDRAGIT adhesive is
soluble
under acidic conditions. This procedure can be used with ddH20, PBS2o, pH7.4,
or
SE-HPLC buffer (200 mM Na phosphate, pH7.2) as the rehydration buffer. Two
patches without release liner were placed into a 1.7 ml EPPENDORF centrifuge
tube. About 0.5 ml of rehydration buffer was added and the patches were able
to
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rehydrate in buffer at room temperature for several hours with occasional
manual
agitation (about every half hour). The EPPENDORF tube was centrifuged for 5
min
at 14,OOOg and 4°C. The supernatant was recovered and used for HPLC
analysis.
Reverse-phase high-performance liquid chromatography (RP-HPLC) was
5 used to detect degradation of proteins such as LT and LTR192G. Protein
subunits
were eluted from a Vydac column (Protein & Peptide C4 with 2.1 mm ID x 25 cm)
at
a rate of 0.3 ml/min using a gradient made from 0.1 % (w/v) trifluoroacetic
acid (TFA)
in ddH20 for buffer A and 0.1 % (w/v) TFA in 95% (v/v) acetonitrile for buffer
B. Both
subunits A and B of LT were resolved, along with peaks for degraded protein
and
o aggregated protein.
Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) consisted of a
14% separating gel with a 5% stacking gel. Protein samples were reduced by
boiling
samples for 5 min in buffer containing (i-mercaptoethanol. Separation of the A
and B
subunits with SDS-PAGE was used to confirm the RP-HPLC results.
15 Series 1A patches were prepared using LTs from SSVI Berne. Lyophilized
samples were reconstituted with half the recommended volume of ddH20 to give a
solution with nominally 2 mg/ml of LT and 10% lactose in PBS. Trehalose was
dissolved in this solution to a final concentration of 5% (w/v).
Series 2A patches were prepared using LTc from the Clements laboratory at
2o Tulane University. LTc was reconstituted using ddH20 and then dialyzed into
PBS~so,
pH7.4. The dialyzed LTc was concentrated using a 30,000 MW cutoff CENTRICON
unit. Trehalose was dissolved in the concentrated LTc to give a final solution
with 1.2
mg/ml LTc and 5% (w/v) trehalose in PBS~5o, pH7.4.
Series 1A patches were incubated at 40°C for one month. Series 2A
patches
2s were incubated at 40°C for two months. LTc was better stabilized by
a disaccharide:
5% (w/v) trehalose was a better stabilier for this protein than 10% (w/v)
lactose. Peak
heights in the chromatogram were lower and the ratio between the peak areas
for A
and B subunit products showed that there more degradation with lactose.
The standard EUDRAGIT adhesive was blended with other components (PBS
3o buffer with LT, with or without trehalose, and with or without KLUCEL
thickener) at a
mass ratio of about 1:1.2 for blends with KLUCEL thickener, and at a mass
ratio of
about 1:1 for blends without KLUCEL thickener. Final concentrations in the wet
blend
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41
were 3% (w/w) KLUCEL thickener, 5% (w/v) disaccharide (when present), and 410
pg/gm to 460 pg/gm LT.
Comparison of formulations containing trehalose to those without trehalose
clearly shows that the presence of trehalose dramatically increased the
stability of LT
even at elevated temperatures as high as 60°C. Even in the absence of
trehalose,
patches prepared using standard EUDRAGIT adhesive showed greater LT stability
than those prepared using modified EUDRAGIT adhesive. The purpose for
modifying
the EUDRAGIT adhesive, is to increase the tackiness and malleability of
partially-
dried films. Films of standard EUDRAGIT adhesive that included trehalose had a
o tendency to crumble and flake off the patch backing substrate. However, this
flaki-
ness dramatically decreased (or disappeared entirely) after incubation at
elevated
temperatures. The presence of KLUCEL thickener enabled the casting of
consistent
films. Films from blended formulations without KLUCEL thickener were not cast
with
any consistency.
Two blended adhesive and immunogically-active protein compositions were
studied: KLUCEL thickener with sucrose or trehalose. The modified EUDRAGIT
adhesive was blended with the other ingredients (PBS buffer with LT,
disaccharide,
and KLUCEL thickener) in a mass ratio of about 1:1.3. Final concentrations in
the
wet blend of KLUCEL thickener and disaccharide were 2.6% and 5%, respectively,
2o and about 450 pg/gm LT. Patches from each blend composition were incubated
at
5°C, room temperature, 40°C or 60°C.
Chromatograms of the elution profile from RP-HPLC for protein extracted from
each sample (i.e., KLUCEL thickener-pressure sensitive adhesive formulation
with
either sucrose or trehalose incubated at the four different temperatures) were
analyzed. Changes in peak area ratios and normalized peak areas with
incubation
time were plotted. When 5% (w/v) disaccharide was included, the stability of
LT was
enhanced relative to the conditions in which little or no disaccharide is
present, even
at 40°C. Trehalose is a better stabilizer than sucrose under these
conditions. At 5°C,
protein was stabilized by both disaccharides over the incubation time tested.
But LT
so was not stabilized at 60°C; no LT was recovered after one week at
this temperature.
Five blend compositions were studied with the modified EUDRAGIT adhesive:
KLUCEL thickener and no disaccharide, KLUCEL thickener and sucrose, KLUCEL
thickener and trehalose, NATROSOL thickener and sucrose, and NATROSOL
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42
thickener and trehalose. For patches containing KLUCEL thickener, the modified
EUDRAGIT adhesive was blended with the other components (PBS buffer with LT,
with or without disaccharide and KLUCEL thickener) at a mass ratio of about
1:1. For
patches containing NATROSOL thickener, it was blended in a mass ratio of about
s 1:1.4. Final concentrations in the wet blend of thickener and disaccharide
(if present)
were 3% and 0.4%, respectively for KLUCEL patches, and 3.5% and 0.3%,
respectively for NATROSOL patches. Final concentration of LT in the wet blend
was
about 500 ~,g/gm. Patches from each of the five blends were incubated at room
temperature, 40°C or 60°C.
o Chromatograms of the elution profile from RP-HPLC for protein extracted from
each sample (i.e., KLUCEL thickener or NATROSOL thickener-pressure sensitive
adhesive formulation incubated at the three different temperatures) were
analyzed.
Changes in peak area ratios and normalized peak areas with incubation time
were
plotted. When 5% (w/v) disaccharide was included, the stability of LT was
signifi-
s cantly enhanced relative to the conditions in which little or no
disaccharide was
present. Thickeners are used to enhance the viscosity of a formulation
component
so it can be cast as a uniform film. KLUCEL thickener is preferred to NATROSOL
thickener under these conditions, because LT appears to be more stable in the
presence of KLUCEL thickener than NATROSOL thickener. At low concentrations of
2o disaccharide (i.e., 0.3% to 0.4%), no additional stability appears to be
conferred to
LT after one week of incubation. Therefore, higher disaccharide concentrations
are
preferred.
An adhesive-protein formulation further containing about 3% (w/v) thickener
(e.g., KLUCEL hydroxypropyl cellulose) and about 5% (w/v) nonreducing sugar
(e.g.,
2~ trehalose) is preferred. A period of curing at an.elevated temperature
(40°C to 60°C)
might be used to address any problem of crumbling and flakiness of at least
partially-
dried films. It may also be possible to include glycols at concentrations low
enough
(e.g., 1 % or less of glycerol andlor 1,3-butanediol in the final wet blend
may be used
as a starting point, up to about 5%, 10% or 15%) not to destabilize the
protein but
so sufficient to confer malleability and cohesion to the partially-dried
pressure-sensitive
adhesive layer. Thin films prepared by casting and drying wet blends of
standard
EUDRAGIT adhesive and buffers containing 3% (w/v) to 5% (w/v) disaccharide
have
a tendency to flake off the backing material used for patches and do not have
much
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43
adhesive character. To improve malleability and adhesiveness, the standard
EUDRAGIT adhesive may be modified by adding glycerol (up to about 6%) and 1,3-
butanediol (up to about 4%). Addition of these plasticizers achieve the
desired effect
in terms of malleability and adhesiveness, but they may also be detrimental to
LT
stability.
Patches cast from a blend of standard EUDRAGIT adhesive and protein in a
buffer containing disaccharide resulted in very inconsistent coat weights from
patch
to patch. It was found that including about 3% (w/w) KLUCEL or NATROSOL
thickener in the final wet blend greatly increased the ability to cast
consistent coats.
o Patch compositions have been identified in which LT shows good stability and
recoverability. Wet blended formulations contain about 500 pg/gm LT, 5% (w/v)
disaccharide (e.g., sucrose, trehalose), and 3% (w/v) KLUCEL thickener. The
blend
was prepared by mixing EUDRAGIT adhesive and buffered protein solution (pH
7.4;
containing disaccharide and KLUCEL thickener) at a mass ratio of about 1:1.
There
5 was excellent stability at 5°C. There was good stability at room
temperature so we
observed over the course of 6 to 7 weeks.
Addition of a stabilizer, which was a disaccharide (e.g., sucrose, trehalose),
at
a high concentration of 5% (w/v) may protect against aggregation, degradation,
and
denaturation. This structural stability is correlated to retention of
biological activity.
2o Trehalose appears to confer slightly more stability than sucrose, but
lactose is
detrimental to LT stability in patches. Excipients such as glycerol and 1,3-
butanediol
may also be somewhat detrimental to LT stability in an at least partially-
dried patch.
The presence of lactose in the LT and LTR~s2c formulations from commercial
suppliers also has a deleterious effect on solubility. LT formulated in
lactose (such as
25 that obtained from SSVI) is very poorly soluble in lactose-free solutions.
Additionally,
lactose may chemically modify a protein as indicated by mass spectromefiry
results
showing a 14 amu difference in fragments generated from the lactose-formulated
LT
relative to the lactose-free LT adhesive formulation. .
Adding KLUCEL thickener allowed the casting of consistently uniform films
so using a knife. Increasing concentrations of disaccharide and KLUCEL
thickener
increasingly caused the film to be brittle or flaky and to lose adhesive
properties.
Modification of EUDRAGIT adhesive by including excipients like glycerol and
1,3-
butanediol (in concentrations of around 3% and 2%, respectively, in the final
wet
blend) restore the malleability and much of the adhesiveness of the film.
These
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44
additions, however, are detrimental to protein stability. Incubation at
elevated
temperature (40°C) for about a week, showed a restoration of
malleability and film
cohesiveness. This suggests that "curing" films for a short period of a few
hours or
less at elevated temperatures (40°C to 50°C) may be a viable
means of restoring film
malleability and integrity without having to add excipients harmful to protein
stability.
Freshly prepared EUDRAGIT adhesive should be used to avoid excessive cross-
linking between polymers.
LT-in-Adhesive Formulation for Transcutaneous Immunization
The following aqueous-based adhesive was used for the pressure-sensitive
adhesive layer. An acrylate adhesive is blended with acetyl-tributyl citrate
(ATBC) as
plasticizer and succinic acid as tackifier.
Table 1. Adhesive Formulation
Ingredients NonvolatileWet Weight Dry Weight
Component
NVC weight (gm)% weight
(gm)
Methacrylate Polymer100 22.8 22.0 22.8 58.8
Succinic Acid 100 1 0.96 1 2.58
ATBC 100 15 14.5 15 38.7
Water 0 65 62.6 0 0
Total 103.8 100 38.8 100
Dry weight % is the total weight of the component multiplied by % NVC divided
by
the total weight of all components multiplied by their respective % NVC. This
adhe-
sive formulation is used to make an emulsion containing protein.
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Table 2. Adjuvant-in-Adhesive Formulation
Ingredients NonvolatileWet Weight Dry Weight
Component
NVC weight (gm)% weight (gm)
1 x P B S / Lactose6. 05 23.4 38.8 1.42 13.3
Adhesive Formulation37.5 23.4 38.8 8.78 82.8
NATROSOL thickener2.5 13.5 22.4 0.338 3.18
LT protein adjuvantN/A 0.0234 0.04 0.023 0.22
Tween 20 100 0.05 0.08 0.05 0.47
Five blends were made using the adjuvant-in-adhesive formulation:
~ Blend 1 was as shown in Table 2.
~ Blend 2 included glycerol (5.4% dry weight).
5 ~ Blend 3 included 1,3 butanediol (5.4% dry weight).
~ Blend 4 substituted KLUCEL thickener for NATROSOL thickener.
~ Blends 5 and 6 were Blend 1 applied by rotogravure and laminated to pressure-
sensitive acrylate or silicone adhesive layer.
o Table 3. Adjuvant / Co-Administered Antigen-in-Adhesive Formulation
Ingredients NonvolatileWet Weight Dry Weight
Component
NVC weight (gm)% weight (gm)
1 x PBS / Lactose 6.05 15.6 38.7 0.94 13.3
EUDRAGIT EPO 37.5 15.6 38.7 5.85 82.3
NATROSOL thickener2.5 9.0 22.3 0.23 3.16
CS6 protein antigen100 0.0468 0.11 0.047 0.66
LT protein adjuvant100 0.0156 0.04 0.016 0.22
Tween 20 100 0.03 0.07 0.03 0.42
Two blends were made using the adjuvant-in-adhesive formulation:
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46
~ Blend 7 was as shown in Table 3.
~ Blend 8 was a 1:1 mixture of 26.6 mg/ml CS6 and Blend 3 applied by
rotogravure
and laminated to draw down of LT alone.
The following formulations were made:
A LT formulated in EUDRAGIT EPO adhesive / 3.2% NATROSOL thickener /
0.5% Tween
B LT formulated in EUDRAGIT EPO adhesive / 3.0% NATROSOL thickener /
5% glycerol / 0.4% Tween
o C LT formulated in EUDRAGIT EPO adhesive / 3.2% NATROSOL thickener
5% 1,3 butanediol / 0.4% Tween
D LT formulated in EUDRAGIT EPO adhesive / 3.2% KLUCEL thickener / 0.5%
Tween
E LT and CS6 formulated in EUDRAGIT EPO adhesive / 3.2% NATROSOL
~5 thickener / 0.4% Tween
Chemical stability of formulations A to E were determined by reverse phase
HPLC and physical stability was determined size exclusion HPLC. Reverse phase
chromatography separates protein according to binding affinity and allowed
detection
20 of fragments that result from protein degradation. Size exclusion
chromatography
separates protein according to passage through pores and allowed detection of
aggregates, dissociated subunits, precipitates, and unfolded polypeptide
chains.
Samples were stored at 15°C, 25°C or 40°C for one week.
Dissociation of the LT-B
subunit (a pentamer) from the LT-A subunit was only detected with formulation
B.
25 Mice were transcutaneously immunized as described previously (Scharton-
Kersten et al., Infect. Immun., 68:5306-5313, 2000). Briefly, the animals were
shaved
on the dorsum with a No. 40 clipper, which leaves no visible irritation or
changes in
the skin, and rested for 48 hr. Mice were anesthetized intramuscularly (IM) in
the
hind thigh or intraperitoneally (1P) with a ketamine/xylazine mixture during
the immu-
so nization procedure to prevent self-grooming. The exposed skin surface was
hydrated
with an aqueous solution of 10% glycerol, 70% isopropyl alcohol, and 20%
water; the
stratum corneum was at least partially disrupted with sandpaper. A 1 cm2 patch
with
pg/cm2 protein was applied epicutaneously for 24 hr with an adhesive tape
placed
CA 02445486 2003-09-18
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47
over the patch to secure it on the animal. After removal of the patch, the
animals
were extensively washed, tails down, under running tap water for about 30 sec,
patted dry, and washed again.
Induction of an antigen-specific immune response was assayed by ELISA of
antibody against LT or CS6. IMMULON-2 polystyrene plates (Dynex Laboratories)
were coated with 0.1 pg/well of antigen, incubated at room temperature
overnight,
blocked with a 0.5% casein buffer in PBS, washed, serial dilutions of specimen
applied, and the plates incubated for 2 hr at room temperature. IgG {H+L)
antibody
was detected using HRP-linked goat anti-mouse IgG (H+L) (Biorad) for 1 hr.
Bound
1 o antibody was revealed using 2,2'-azino-di (3-ethylbenzthiazoline sulphonic
acid)
substrate (ABTS; Kirkegaard and Perry) and the reaction stopped after 30 min
using
a 1 % SDS solution. Plates were read at 405 nm. Antibody titer results are
reported in
either OD (405 nm) or ELISA Units, which are defined as the inverse dilution
of the
sera that yields an optical density (OD) of 1Ø
Table 4. ELISA Results for Immunized Mice
Formulation Dose Geometric Mean of ELISA Units
A 10 pg/cm~ 3325
B 10 pg/cm2 6962
C 10 pg/cm2 4896
D 10 pg/cm2 12,707
E 10 pg/cm2 (LT) 9959
30 pg/cm~ (CS6) 614
Gauze Patch (LT)10 pg 4861
Gauze Patch (LT)10 pg 3109
Gauze (-) controlPBS 12
Gauze patches were produced by adding an LT-containing solution to a 1 cm2
Nu-Gauze backing layer. The substrate is held in place on the mouse with a
piece of
2o adhesive tape.
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48
Protein-in-Adhesive Formulation for Transcutaneous Immunization
These protein-in-adhesive formulations are intended to treat enterotoxigenic
E. coli (ETEC) by incorporating one or more ETEC subunit antigens into an
adhesive
formulation. The formula is also suitable for incorporating killed ETEC whole
cells
0104 to 10$ killed bacteria per dose) with or without LT-adjuvant. The blend
is then
cast over a sheet of occlusive (or semi-occlusive) backing as a thin film. The
formu-
lation is allowed to cure (room temperature or 40°C to 60°C)
until the film is at least
partially-dried (water content may vary between 0.5% and 5%; less than about 1
% or
~0 2% is preferred for a patch according to the invention). The cast film may
be cut from
the die-cast to the desired size and shape. The patch may then be sealed in a
light-
tight, waterproof plastic or metal foil pouch. Patches produced in this manner
may be
stored refrigerated or at ambient temperatures (e.g., 20°C to
30°C). The protein-in-
adhesive is flexible in that the multivalent vaccine blend may be varied to
incorporate
different amounts and ratios of one or multiple antigens and adjuvant. In
addition, the
patch size may be varied in order to adjust dosing. Depending upon the age of
the
individual, patch size (dose) can be varied for use in children and adults.
Protein-in-adhesive formulations are flexible and uniquely allow the vaccines
to be coated in layers. These patches are manufactured in a manner wherein
each
2o vaccine component is layered separately onto the patch backing. The
objective is to
create a multilaminar membrane in which an adhesive formulation is adhered
onto
the backing layer, a first immunogenic formulation is applied on the adhesive
formu-
lation, a second immunogenic formulation is applied on the first immunogenic
and
adhesive formulation, and the release liner is the layer most distal with
respect to the
backing layer. The advantage of this approach is that it provides flexibility
to the
formulation (i.e., a patch may be produced from the same process using
different
ratios of antigen-containing first immunogenic formulation and adjuvant-
containing
second immunogenic formulation, or where a patch is manufactured to contain
only
one or two active ingredients). The multilaminated patch also has the
advantage of
so controlling the release rates of each antigen and the adjuvant. In some
instances, it
will be desirable to have LT adjuvant released immediately in order to pre-
prime the
skin dentritic cells (e.g., Langerhans cells) prior to release of other
antigens. Then
the LT-primed Langerhans cells may more efficiently capture and process the
toxin
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49
and colonization factor antigens. Controlled delivery is a more efficient use
of the
adjuvant and antigens and will allow the doses to be further reduced.
Formulations are described that may be suitable for stabilizing proteins with
at
least adjuvant and/or antigen activity in contact with an adhesive. The
following are
intended to be examples of such formulations and are not intend to restrict
the
formulation.
Gel formulations for delivery of ETEC subunit vaccines (CS3, CS6, CFAII, ST
and
LT) and killed ETEC whole cells
~o Gels are examples of fully hydrated or wet patches. These formulations are
intended to incorporate one or more ETEC subunit antigens entrapped within a
gel
matrix. This formulation is also suitable for transcutaneous delivery of
killed ETEC
whole cells 0104 to 10$ killed bacteria per dose) with or without LT. The
vaccines
are formulated by blending a solution containing the antigens in the desired
amounts
5 and ratios with Carbomer, Pluronic, or a mixture of the two gel components
(see
below). The gel-containing immunogenic formulation is then coated on a strip
that
holds the gel in place without spilling. It is important that the material
have a low
binding capacity for the proteins in the formulation. The strip may comprise
patch
materials as described above. The strip may be a single layer or a laminate of
more
2o than one layer. Generally, the strip is substantially water impermeable and
helps to
maintain the skin in hydrated condition. The material may be any type of
polymer
that meets the required flexibility and low binding capacity for proteins.
Preferred
polymers include, but are not limited to, polyethylene, ethyl vinylacetate,
ethylvinyl
alcohol, polyesters, or Teflon. The strip of material for holding the gel is
less than 1
25 mm thick, preferably less than 0.05 mm thick, most preferably 0.001 to 0.03
mm
thick.
The gel-loaded strip may be of different sizes and shapes. It is preferred
that
the corners be rounded for ease of application. The length of the strip can
vary and
is dependent upon the intended user (i.e., children or adults). It may be from
about 2
so cm to about 12 cm, and is preferably from about 4 cm to about 9 cm. The
width of
the strip will vary but it may be from about 0.5 cm to about 4 cm. The strip
may have
shallow pockets or dimples as reservoirs for the gel. To hold in place, when
the gel-
containing formulation is coated onto the strip, the gel should fill the
reservoirs. The
shallow pockets may be about 0.4 mm across and about 0.1 mm deep. The gel-
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loaded patch is about 1 mm thick, with a preferred thickness of about 0.5 mm
or less.
The gel-loaded strip is held in place by adhering it to a pressure-sensitive
adhesive
layer with the gel surface facing away from the adhesive. The backing material
may
be occlusive or semi-occlusive (e.g., TEGADERM dressing).
5 The flexural stiffness is important since maximal contact between the gel
and
the skin must be maintained. The strip will need to conform to the contour of
the
anatomical location where the patch is applied (e.g., skin over the deltoid
muscle,
volar forearm, neck, behind the ear, or other locations). Flexural stiffness
can be
measured with a Handle-0-Meter (Thwing Albert Instruments). The flexural
stiffness
o should be less than 5 gm/cm, more preferably less than 3 gmlcm. The
relatively low
stiffness enables the strip of material to drape over the contoured surface
with little
force being exerted. The backing layer is designed to hold the patch in place,
to aid
in maintaining maximal contact between the skin and gel, and to prevent the
gel from
dehydrating during wear.
15 To prevent dehydration of the wet patch during storage and handling, it may
be placed on an inert plastic strip, which is fairly rigid. The gel surface
would be in
direct contact with the plastic strip, and the gel/plastic interface has low
peel force
making it easy to separate the gel strip from the plastic strip. The plastic
strip may be
made of polyethylene or similar material. The gel-containing patch can be
packaged
2o in a light-proof and water tight plastic or foil pouch. The pouch can be
stored at room
temperature or in a refrigerator.
The following are intended as examples of the hydrated gel formulation and
are not intended to restrict it: gels in phosphate buffered saline; 1 %
Carbomer 1342;
1.5% Carbomer 940; 1.5% Carbomer 934; 1.5% Carbomer 940, 2% sucrose, 10%
25 isopropyl alcohol, 10% glycerol; 50% Pluronic F87; and 30% Pluronic F108.
Carbomer polymers are high molecular weight, acrylic acid-based polymers
that may be cross-linked With allyl sucrose or allylpentaerythritol, and/or
modified
with C10 -C30 alkyl acrylates. These may or may or not be incorporated into a
patch
or may be delivered by other means know in the art into the skin.
so Formulations may be comprised of carbomers of different average molecular
weights. For example, the polymers may be Carbomer 1342 (e.g., 1 % Carbomer
1342, 0.6 mg/ml LT, 0.3% methylparaben, 0.1 % propylparaben, 2.5% lactose, in
1 x
PBS); Carbomer 934 (e.g., 1.5% Carbomer 934, 0.6 mg/ml LT, 0.3% methylparaben,
0.1% propylparaben, 2.5% lactose, in 1x PBS); or Carbomer 940 (e.g., 1.5%
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51
Carbomer 940, 0.6 mg/ml LT, 0.3% methylparaben, 0.1 % propylparaben, ~.5%
lactose, in 1x PBS). Each formulation can be prepared in a phosphate buffered
saline solution and contain LT at a concentration of about 0.6 mg/ml or less,
but
antigens and adjuvants may also be formulated from about 0.001 mg/ml to about
0.6
s mg/ml or from about 0.6 mg/ml to about 6 mg/ml. In addition, antimicrobial
agents
such as methylparaben and propylparaben may be included.
Combinations of Carbomer 940 and Pluronic F87 {e.g., 1.5% Carbomer 940,
0.5% Pluronic F87, 0.6 mg/ml LT, 0.3% methylparaben, 0.1 % propylparaben, 2.5%
lactose, in 1x PBS) may be used. Pluronics are another class of hydrogel that
contain repeating segments of ethylene oxide-propylene oxide-ethylene oxide.
The
amount of LT and antimicrobial agents in the formulation may be identical.
Other formulations may enhance delivery using penetration enhancers and
carbomers. For example, a gel may comprise Carbomer 940 with Pharmasolve
(e.g.,
1.5% Carbomer 940, 10% Pharmasolve, 0.6 mg/ml LT, 0.3% methylparaben, 0.1
propylparaben, 2.5% lactose, in 1x PBS) while the final gel may contain
Carbomer
940, glycerol, and isopropanol (e.g., 1.5% Carbomer 940, 10% glycerol, 10%
isopropanol, 0.6 mg/ml LT, 0.3% methylparaben, 0.1 % propylparaben, 2.5%
lactose,
in 1x PBS). The concentration of LT and antimicrobial agents may remain
identical to
the above formulations, or may be in other ranges specified.
~o
All references (e.g., articles, books, patents, and patent applications) cited
above are indicative of the level of skill in the art and are incorporated by
reference.
All modifications and substitutions that come within the meaning of the claims
and the range of their legal equivalents are to be embraced within their
scope. A
25 claim using the transition "comprising" allows the inclusion of other
elements to be
within the scope of the claim; the invention is also described by such claims
using
the transitional phrase "consisting essentially of (i.e., allowing the
inclusion of other
elements to be within the scope of the claim if they do not materially affect
operation
of the invention) and the transition "consisting" (i.e., allowing only the
elements listed
3o in the claim other than impurities or inconsequential activities which are
ordinarily
associated with the invention) instead of the "comprising" term. No particular
relationship between or among limitations of a claim is meant unless such
relationship is explicitly recited,in the claim (e.g., the arrangement of
components in
a product claim or order of steps in a method claim is not a limitation of the
claim
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52
unless explicitly stated to be so). Thus, all possible combinations and
permutations
of the individual elements disclosed herein are intended to be considered part
of the
invention.
From the foregoing, it would be apparent to a person of skill in this art that
the
invention can be embodied in other specific forms without departing from its
spirit or
essential characteristics. The described embodiments should be considered only
as
illustrative, not restrictive, because the scope of the legal protection
provided for the
invention will be indicated by the appended claims rather than by this
specification
