Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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NANOFIBER STRUCTURES ON ASPERITIES
FOR SEQUESTERING, CARRYING AND
TRANSFERRING SUBSTANCES
This application claims priority based on provisional application Serial No.
60/798,928, filed
May 9, 2006, provisional application Serial No. 60/848,506, filed September
29, 2006, provisional
application Serial No. 60/848,507, filed September 29, 2006, provisional
application Serial No.
60/848,504, filed September 29, 2006, provisional application Serial No.
60/848,505, filed
September 29, 2006, and provisional application Serial No. 60/848,213, filed
September 29, 2006,
the contents of which are incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
The present invention relates to structures that contain one or more fiber
and/or nanofiber
structures on asperities, including, but not limited to, pins,
microprojections, and microneedles. In
one embodiment, the present invention relates to structures that contain one
or more fiber and/or
nanofiber structures on asperities including pins, microprojections, and
microneedles where the
nanofiber and/or fiber structures are designed to sequester, carry and/or
encapsulate one or more
substances. In still another embodiment, the present invention relates to a
process for forming one or
more fibers, nanofibers or structures made therefrom on asperities, such as
pins, microprojections,
and microneedles where such fibers and/or structures are designed to
sequester, carry, and/or
encapsulate one or more substances and to deliver such substances, such as
therapeutic drugs and
preventive medicines, through the skin.
BACKGROUND OF THE INVENTION
There is an interest in methods to sequester, entrap, encapsulate and/or
deposit various
compounds or substances on the surfaces of and/or within various structures
(e.g., polymer, metal,
or ceramic structures). One such method that holds promise is the use of
fibers and/or nanofibers
that are designed to carry one or more compounds or substances, where such
fibers, nanofibers, or
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structures made therefrom are placed, deposited or formed on one or more
surfaces of a material
(e.g.. a polymer, metal, ceramic or other material).
Such fibers could be used as a vehicle for the topical delivery of drugs.
Topical delivery of
drugs is a useful method for achieving systemic or localized pharmacological
effects. An example is
disclosed in U.S. Pat. No. 3,964,482 (by Gerstel), in which an array of either
solid or hollow
microneedles is used to penetrate through the stratum comeum, into the
epidermal layer, but not to
the dermal layer; however, hollow microneedles create engineering issues and
require a reservoir or
other source of fluid. Solid needles do not offer sufficient loading of the
substance, let alone
sufficient preservation and stabilization of the substance..
Thus, there is a need in the art for products that can be designed to
incorporate various fibers
and/or nanofibers, where the fibers and/or nanofibers are designed to carry,
sequester and/or
encapsulate one or more compounds or substances, thereby providing for
efficient loading of the
substance, sufficient preservation and stabilization of the substance, and
desired release
characteristics.
SUMMARY OF THE INVENTION
In accordance with an aspect of the present invention, there is provided a
device which
includes a substrate comprising a surface and a plurality of asperities
extending from said surface,
and an electrospun material in contact with said substrate. The electrospun
material may be any of a
variety of forms, including, but not limited to, fibers, such as nanofibers
and microfibers, including
fiber mats, three-dimensional films, spheres, tubes, or any other geometrical
shape, and the like. In
one non-limiting embodiment, the electrospun material is in the form of
nanofibers. In a non-
limiting embodiment the electrospun material may be formed from a polymer,
including but not
limited to those described hereinbelow.
The electrospun material, in one embodiment, comprises at least one carrier
substance, such
as a polymer, capable of carrying a biologically active agent, and at least
one biologically active
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agent.
The present invention also is directed to administering at least one
biologically active agent,
such as a drug, or a prophylactic or therapeutic agent, to a human or non-
human animal by providing
a device including a substrate, and an electrospun material, which includes
the biologically active
agent, in contact with the substrate as hereinabove described. The human or
non-human animal is
contacted with the device such that the at least one biologically active agent
is released into the
human or non-human animal.
In accordance with yet another aspect of the present invention, there is
provided a device
which comprises an ionically cross-linked macromolecular assembly and at least
one biologically
active agent, such as those described herein, contained in said assembly. In a
non-limiting
embodiment, the ionically cross-linked macromolecular assembly comprises an
ionically cross-
linked water soluble polymer.
In another non-limiting embodiment, the device further comprises a substrate
which
comprises a surface and a plurality of asperities, such as microprojections
and microneedles,
extending from the surface. The macromolecular assembly containing the at
least one biologically
active agent is in contact with the substrate.
In a non-limiting embodiment, the ionically cross-linked macromolecular
assembly
containing the at least one biologically active agent may be in the form of an
electrospun material
which may be any of a variety of forms, including, but not limited to, fibers,
such as nanofibers and
microfibers, including fiber mats, three-dimensional films, spheres, tubes, or
any other geometrical
shape, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention now will be described with respect to the drawings, wherein:
Figure I is a low magnification optical image of grounded carbon nanofibers
coated with
polyethylene oxide nanofibers;
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Figure 2 shows a carbon fiber tip coated with nanofibers, and spanning
nanofibers near the
tip;
Figure 3 shows the tip of a carbon fiber coated with a ball of nanofibers at
the tip;
Figure 4 shows a drawing of an asperity which has an increased surface area
near the tip, and
has corners that protect the nanofibers from shearing forces;
Figure 5 shows a drawing of an embodiment of an asperity having barbs and
circumferential
grooves;
Figure 6 shows a drawing of another embodiment of an asperity having barbs;
Figure 7A shows one embodiment of a two-polymer device, wherein the
microneedle array
comprises a soluble first layer which dissolves upon administration (Figure
7B) thereby releasing a
relatively less soluble second layer into the skin;
Figure 8 shows one embodiment of a flexible microneedle array(s);
Figures 9A and 9B show one embodiment of a device comprising an elastomeric
element
which, upon pressure, changes form to hold the array(s) on the skin;
Figure 10 is a scanning electron microscope picture showing an overview of one
embodiment of a microneedle array with a particular density;
Figure 11 is a scanning electron microscope picture showing a close up, side
view of one
embodiment of a single microneedle (e.g. within an array) with a particular
shape and height;
Figure 12 is a scanning electron microscope picture showing a close up, top
view of one
embodiment of a single microneedle (e.g. within an array) with eight sides
(i.e. eight surfaces)
coming together at the top to form a point or tip;
Figure 13 shows a prospective microneedle array with a higher density;
Figure 14 shows an embodiment wherein a hydrogel is fabricated on the surface
of an
embodiment of the device of the present invention (e.g., a microneedle array)
using aqueous based
methods;
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Figure 15 shows an alternative embodiment wherein physiologically active
substances can
be dissolved or dispersed in a solution of a cross-linker (e.g., a solution
having multivalent or
polyvalent ions);
Figure 16 shows a C. botulinum type A toxin amino acid sequence;
Figure 17 shows another C. botulinum type A toxin amino acid sequence;
Figure 18 shows the amino acid sequence of wild-type anthrax protective
antigen;
Figure 19 shows the nucleotide sequence encoding wild-type anthrax protective
antigen;
Figure 20 shows the amino acid sequences of human 1- 34 PTH and bovine 1- 34
PTH.
(The present invention is not limited only to administering human hormones.
The hormone
homolog from other species (e.g., salmon, porcine, etc.) may have equal or
better results);
Figure 21 shows the amino acid sequences for GLP-1, Exendin-4, Exendin-4 (3 -
39), and
Exendin-4 (9 - 39);
Figure 22 shows the nucleotide sequence of the coding region for the C.
botulinum type A
neurotoxin;
Figure 23 shows C. botulinum type A toxin expression constructs; constructs
used to provide
C. botulinum or C. difficile sequences also are shown;
Figure 24 shows the nucleotide sequence of the C. Botulinum C fragment gene
sequences
contained within pAlterBot;
Figure 25 shows the C. botulinum type A toxin expression constructs;
constructs used to
provide C. botulinum sequences also are shown;
Figure 26 shows the nucleotide sequence present in the pETHisa vector which
encodes the
pHisBot fusion protein;
Figure 27 shows the amino acid sequence of the pHisBot protein; and
Figure 28 shows the expected results in a test of electrospun PA
functionality.
DETAILED DESCRIPTION OF THE INVENTION
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' As used herein the word "asperities" means the microscopic surface
elevations present on the
surface of a material due to surface roughness of a material, such as pins,
microprojections, and
microneedles. The asperities, microprojections, and microneedles preferably
are in the form of
piercing elements which are dimensioned to penetrate into or through a desired
body part such as a
tissue, or organ, or which may deliver a biological material transdermally,
intradermally,
intraepidermally, or transmucosally. In a non-limiting embodiment, the
asperity, microprojection,
or microneedle is dimensioned such that it penetrates through the stratum
corneum into the
underlying epidermis layer, and in some embodiments, the dermal layer of the
skin.
The term "transdermal" are used herein means the delivery of an agent into
and/or through at
least the top layer of the skin. The term "intradermal" means the delivery or
release within the skin.
The term "intra-epidermal" means the delivery or release specifically within
the epidermal layer of
the skin.
While the microneedle embodiment (described below) may be employed, other
systems and
apparatus that employ tiny skin piercing elements to enhance transdermal agent
delivery are also
contemplated, as disclosed in U.S. Pat. Nos. 5,879,326, 3,814,097, 5,250,023,
3,964,482, Reissue
U.S. Pat. No. 25,637, and PCT Publication Nos. WO 96/37155, WO 96/37256, WO
96/17648, WO
97/03718, WO 98/11937, WO 98/00193, WO 97/48440, WO 97/48441, WO 97/48442, WO
98/00193, WO 99/64580, WO 98/28037, WO 98/29298, and WO 98/29365; all
incorporated herein
by reference in their entireties.
It is not intended that the present invention be limited to a precise geometry
or topology of
the microneedles. In one embodiment, the microneedles are defined by a
plurality of surfaces
sloping upwards from a relatively broad base to a tip (e.g. a pyramidal
shape). In another
embodiment, the microneedles have a generally conical-shaped body (e.g. a
single curved surface).
Furthermore, within the scope of the present invention, the asperities, such
as
microprojections and microneedles, in one embodiment, may include one or more
indentations
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and/or barbs, which aid in retaining the nanofibers on the asperities.
It is not intended that the present invention be limited, by the precise
dimensions of the
microneedles. In one embodiment, the microneedles described herein have a
microneedle height to
width (measured at the base) ratio of between 1.2 and 2.0 (and in one
embodiment between 1.4 and
1.7), and the structure is predominantly solid, rather than hollow. These
factors contribute to the
force required to penetrate human skin being smaller than that required to
break the penetrating
elements. This applies to both single microneedles and various microneedle
arrays, for which
penetration forces are different depending on the number of penetrating
elements, their height and
spacing between adjacent microneedles.
In one embodiment, a prototypical microneedle has a diameter of between 200
and 500
microns (and in another embodiment, between 300 and 400 microns) at its broad
end (e.g., at the
base), and tapers to a sharp tip or chisel edges with a somewhat smaller
diameter at its other end.
The diameter of the tip may, for example, be in the range from about 50
microns to about 1 microns.
In one embodiment, the microprojections or microneedles have a length (or
height)
or less than 1000 microns, and in one embodiment less than 700 microns, but
more than 250
microns. In another embodiment, the microneedles have a height of between 550
and 650
microns (such as, for example, between 580 and 620 microns) with a height to
width ratio of
between 1.5 and 1.7. The microprojections may be formed in different shapes,
such as needles,
blades, pins, punches, and combinations thereof. In one embodiment, the
microneedles are
pyramidal in shape (e.g., having between 6 and 12 sides, and in one
embodiment, eight sides).
The term "fiber" includes not only structures that are cylindrical, but also
includes structures
which vary from a cylindrical shape, such as, for example, structures which
are spherical, acicular,
droplet shaped, or flattened or ribbon shaped. A "fiber-forming" material is
capable of being
fabricated into fibers. The fiber-forming materials that may be used in this
invention include
polymers, which can be in a liquid state, such as melted, in solution, or in
suspension.
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Water, organic solvents, or mixtures thereof.or heat can be used to convert
solid polymer into a
liquid "fiber-forming" material. In one embodiment, electrospinning creates
"pearls on a string"
or encapsulated balls on very thin fibers. This technique may be useful when
compounds do not
mix well, or when one needs to meet extended release requirements, or when one
wants to deliver
balls and the fibers are just a simple carrier.
Additionally, as used herein, nanofibers are fibers having an average diameter
in the range of
about 1 nanometer to about 25,000 nanometers (25 microns). In another
embodiment, the
nanofibers of the present invention are fibers having an average diameter in
the range of about 1
nanometer to about 10,000 nanometers, or about 1 nanometer to about 5,000
nanometers, or about 3
nanometers to about 3,000 nanometers, or about 7 nanometers to about 1,000
nanometers, or even
about 10 nanometers to about 500 nanometers, In another embodiment, the
nanofibers of the present
invention are fibers having an average diameter of less than 25,000
nanometers, or less than 10,000
nanometers, or even less than 5,000 nanometers. In still another embodiment,
the nanofibers of the
present invention are fibers having an average diameter of less than 3,000
nanometers, or less than
about 1,000 nanometers, or even less than about 500 nanometers.
In another embodiment, the nanofibers may have a diameter as small as 0.3
nanometers. In
yet another embodiment the nanometers have a diameter between 3 nanometers and
about 25
microns. In a further embodiment, the nanofibers have a diameter of from about
100 nanometers to
about 25 microns. In still another embodiment the nanofibers have a diameter
of from about 100
nanometers to about a micron. Such small diameters provide a high surface area
to mass ratio, such
as, for example, about 300m2/g. Within the scope of the present invention, a
fiber may be any
length. The term "fiber" also encompasses particles that are drop-shaped,
flat, or that may otherwise
form a cylindrical shape. Additionally, it should be noted that here, as well
as elsewhere in the text,
ranges may be combined.
The at least one fiber-forming material used in this invention may be selected
from any
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fiber-forming material which can be dissolved and is otherwise compatible with
the biological
material to be preserved. Fiber-forming materials which may be used in the
practice of the method
of the present invention include water soluble polymers, for example. In
particular, acceptable fiber-
forming materials, by way of example and not of limitation, include polyvinyl
pyrrolidone) (PVP),
polyethyl oxazoline (PEOZ), polyethylenlmine (PEI), polyethylene oxide (PEO)
and mixtures
thereof. Other polymers of interest include polyvinylalcohol, poly(ethylene
glycol),
polyoxymethylene, poly(hydroxyethyl methacrylate), carboxymethyl cellulose,
hydroxypropylcellulose, alginic acid, chitosan, poly(glutamic acid),
poly(isobutylacrylamide),
poly(butyl methacrylate), poly(ethyl methacrylate), poly(vinylidene fluoride)
poly(hydroxyvalerate),
poly(L-lactic acid), polycaprolactone, poly(lactide-co-glycolide),
poly(hydroxybutyrate),
poly(hydroxybutyrate-co-valerate), polydioxanone, polyorthoester,
polyanhydride, poly(glycolic
acid), poly(vinyl methyl ether), polyvinylidene chloride, polyacrylonitrile,
poly(trimethylene
carbonate), poly(iminocarbonate), and copolymers thereof. Other illustrative
polymers are set forth
in Table 1 below.
TABLE 1
A. Suitable Hydrophilic Polymers
Polyacrylamide
Poly(Acrylic Acid-Co-Hypophosphite)
Polyacrylic Acid, Sodium Salt
Poly(Alkyl(C 16-22)Acrylate)
Poly(Ethylene Glycol) or Poly(Propylene Glycol)
Poly(Vinyl Alcohol)
Polypyrrolidone or Polyvinylpyrrolidone
Polysaccharides Such As Chitosan, Alginate, Amylose
Water Soluble Fractions Of Proteins Such As Collagen, Gelatin
B. Suitable Biostable, Hydrophobic Polymers
Poly(Divinylbenzene-Co-Ethylstyrene)
Polyisobutylene
Polylimonene
Polymaleic Acid Polyoxyethylene Dioleate
Poly(Vinyl Acetate) or Poly(Vinyl Chloride)
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Polystyrene, Polyurethane, Polyurethane-Poly(Ethylene Oxide) Graft Copolymer
Poly(Ether Urethane) or Poly(Ether Urethane Urea)
Polyethylene
Polycarbonate
Poly(Ester Amide)
Polyacrylonitrile
Poly(Aryl Ether Keton)
Poly(Dimethyl Siloxane) And Other Polysiloxanes
Poly(Ethylene Terephthalate) And Other Polyesters
Poly(2-Hydroxyethylmethacrylate) or Polymethylmethacrylate
C. Suitable Biodegradable, Hydrophobic Polymers
Poly(Glycolic Acid), Poly(Lactic Acid) And Co-Polymers Thereof
Polyhydroxybutyrate (Phb)
Polyhydroxyvalerate (Phv), And Co-Polymers Thereof
Polycaprolactone
Polydioxanone And Other Synthetic Degradable Polyesters, Blends Thereof, And
Copolymers Thereof
Polyanhydrides
Poly(Amino Acids) Such As Poly(Benzyl Glutamate)
"Pseudo"-Poly(Amino Acids) Such As Tyrosine-Derived Polycarbonates And
Polyarylates And Copolymers Thereof With Poly(Ethylene Glycol)
Poly(Ortho Ester)
Polyphosphazenes Poly(Propylene Fumarate)
Although not specifically limited to any one production method, the fibers
and/or nanofibers
of the present invention are, in one embodiment, formed via an electrospinning
process. The
electrospinning of liquids and/or solutions capable of forming fibers, also
known within the fiber
forming industry as electrostatic spinning, is well known and has been
described in a number of
patents as well as in the general literature. The process of electrospinning
generally involves the
creation of an electrical field at the surface of a liquid. The resulting
electrical forces create ajet of
liquid that carries electrical charge. Thus, the liquid jets may be attracted
to other electrically
charged objects at a suitable electrical potential. As the jet of liquid
elongates and travels, it will
harden and dry. The hardening and drying of the elongated jet of liquid may be
caused by cooling
of the liquid, i.e., where the liquid is normally a solid at room temperature:
evaporation of a solvent,
e.g., by dehydration, (physically induced hardening), or by a curing mechanism
(chemically induced
hardening) or by a combination of such methods. The produced fibers are
collected on a suitably
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located, oppositely charged receiver and subsequently removed from it as
needed, or directly applied
to an oppositely charged or grounded generalized target area. In a non-
limiting embodiment, the
fibers are collected on a receiver such as, for example, a polystyrene or
polyester net or a foil slide.
Fibers produced by this process have been used in a wide variety of
applications, and are
known, from United States Patent Nos. 4,043,331: 4,878,908; and 6,753,454, all
of which are
incorporated herein by reference in their entireties. One of the major
advantages of electrospun
fibers is that very thin fibers can be produced having diameters, usually on
the order of about 50
nanometers to about 25,000 nanometers (25 microns), or even on the order of
about 50 nanometers
to about 5,000 nanometers (5 microns), or even on the order of about 50
nanometers to about 1,000
nanometers.
It will be appreciated that, because of the very small diameter of the fibers,
the fibers have a
high surface area per unit of mass. This high surface area to mass ratio
permits fiber-forming
material solutions to be transformed from solvated fiber-forming materials to
solid nanofibers in
fractions of a second. When biological materials are dissolved or suspended in
a fiber-forming
material solution which then is formed into fibers, the samples experience a
rapid loss of excess
solvent. This invention thereby also provides a fiber containing a mixture of
at least one fiber-
forming material and at least one preserved biological material and optionally
other materials (e.g.
carriers, fillers, etc.). The mixture may, in some embodiments, be
substantially homogeneous;
however, the present invention also contemplates non-homogeneous mixtures.
As one skilled in the art will recognize, the fibers may be spun using a wide
variety of
conditions such as potential difference, flow rate, and gap distance. These
parameters will vary with
conditions such as humidity or other environmental conditions, the size of the
biological material or
other additive, the solution viscosity, the collection surface, and the
polymer conductivity, among
others. The at least one fiber-forming material is in a liquid state when it
is electrospun with the
biological material to form a fiber containing a mixture of the at least one
fiber-forming material and
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the at least one biological material. In one embodiment, the present invention
contemplates mixtures
of the at least one fiber-forming material and at least one biological
material, including mixtures
where the biological material is soluble in the at least one fiber-forming
material in its liquid state
(homogeneous or single phase systems) and those mixtures in which the at least
one biological
material is insoluble in the at least one fiber-forming material in its liquid
state (heterogeneous or
multi-phase systems). When the biological material is insoluble in the at
least one fiber-forming
material in its liquid state, the biological material may take the form of a
suspension or emulsion in
the fiber-forming material. Whether the biological material is soluble or
insoluble in the fiber-
forming material, the biological material and the fiber-forming material may
be mixed by any
method which forms a substantially uniform mixture, including, for example,
mechanical shaking or
stirring, although other methods (such as sonication) may be used. As one
skilled in the art will
recognize, solubility of the biological material in the fiber-forming material
solution will depend on
the characteristics of the material itself, as well as factors such as, for
example, the requirements of
the material for a specific pH range, osmolarity, or the presence of co-
factors for the material.
By way of a non-limiting example, polyethylene oxide (PEO) (Mw 400,000), and
polyvinylpyrrolidone (PVP) (Mw 360,000) can be used as polymer substrates for
the electrospinning
of fibers. In case of PEO, about 2.5 mg/ml of bioactive agent(s) in
approximately 6% w/v polymer
solution can be used in electrospinning the sample. A cone cap is typically
used to do the spinning at
room temperature, using a voltage of 14 KV, a current of 80 nA, and positive
polarity. The gap
distance can be 28.5 cm, and the sample can be collected on polyester net or
other suitable substrate.
By way of a non-limiting example, PVP fibers can be electrospun from a
solution containing
about 2.5 mg/ml of bioactive agent(s) in 22% w/v polymer. A cone cap is again
typically used to do
the spinning at room temperature, using a voltage of 22 KV, a current of 360
nA, and positive
polarity. The gap distance can be 23 cm, and the sample collected on a
polyester net or other
suitable substrate.
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Although not limited solely hereto, suitable arrays of asperities can made by
the growth of
elongated cylindrical crystals by a vapor-liquid-solid process; growth of
polycyanoacrylate fibers
from small deposits of catalyst material; MEMs technology of the sort utilized
in the semiconductor
electronics industry; removing, by dissolution, fracture, or decomposition,
the matrix from a
composite that contains acicular particles; and in many other ways known in
the art.
In one non-limiting embodiment, the asperities are microneedles which are
anisotropically
etched MEMs microneedles in silicon.
In one embodiment, the solid microneedles are fabricated in a crystal silicon
material
suitable for use in the administration of the various preparations discussed
herein. Without limiting
the invention in any manner to any particular mechanism, it is believed that
the biologically active
materials or drug(s) is delivered by microporation of the stratum corneum, and
polymer-drug
deposition within the patient's skin and subsequent dissolution or erosion of
the polymer. The drug
becomes thereby bioavailable; it can dissolve and diffuse to the biological
target, or alternatively, it
can remain at the site of administration. Micropores are made into the stratum
comeum by means of
a microneedle array penetration, which optionally can be enhanced further by
applying energy in the
form of ultrasonic, heat and/or electric signals across or through the skin.
As is discussed above, it is possible to incorporate, encapsulate, entrap
and/or deposit one or
more compounds or substances on, in or around fibers and/or nanofibers. One
manner by which this
can be accomplished is by dissolving and/or dispersing the desired
substance(s) or compound(s) in a
polymer solution that is to be electrospun before it is electrospun. In one
embodiment, the material
inside the nanofiber is sequestered and preserved by the polymer matrix
provided by the nanofiber.
Pharmaceutically active or bioactive substances which may be included in the
electrospun
material are listed in the Physicians' Desk Reference, 57th Edition (2003),
and include allergens,
amebicides and trichomonacides, analeptic agents, analgesics, anorexics,
antacids, antihelmintics,
antialcohol preparations, antiarthritics, antiasthma agents, antibacterials
and antiseptics, antibiotics,
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antiviral antibiotics, anticancer preparations, anticholinergic drug
inhibitors, anticoagulants,
anticonvulsants, antidepressants, antidiabetic agents, antidiarrheals,
antidiuretics, antienuresis
agents, antifibrinolytic agents, antifibrotics (systemic), antiflatulents,
antifungal agents,
antigonadotropin, antihistamines, antihyperammonia agents, anti-inflammatory
agents,
antimalarials, antimetabolites, anti-migraine preparations, antinauseants,
antineoplastics, anti-
obesity preparations, antiparasitics, anti-parkinsonism drugs, antipruritics,
antipyretics,
antispasmodics and antichloinergics, antitoxoplasmosis agents, antitussives,
antivertigo agents,
antiviral agents, bone metabolism regulators, bowel evacuants, bronchial
dilators, calcium
preparations, cardiovascular preparations, central nervous system stimulants,
cerumenolytics,
chelating agents, choleretics, cholesterol reducers and anti-hyperlipemics,
colonic content acidifiers,
cough and cold preparations, decongestants, expectorants and combinations,
diuretics, emetics,
enzymes and digestants, fertility agents, fluorine preparations,
galactokinetic agents, geriatrics,
germicides, hematinics, hemorrhoidal preparations, histamine H. receptor
antagonists, hormones,
hydrocholeretics, hyperglycemic agents, hypnotics, immunosuppressives,
laxatives, mucolytics,
muscle relaxants, narcotic antagonists, narcotic detoxification agents,
ophthalmological osmotic
dehydrating agents, otic preparations, oxytocics, parashypatholytics,
parathyroid preparations,
pediculicides, premenstrual therapeutics, psychostimulants, quinidines,
radiopharmaceuticals,
respiratory stimulants, salt substitutes, scabicides, sclerosing agents,
sedatives, sympatholytics,
sympathomimetics, thrombolytics, thyroid preparations, toxins for therapeutic
vaccine use,
tranquilizers, tuberculosis preparations, uricosuric agents, urinary
acidifiers, urinary alkalinizing
agents, urinary tract analgesic, urological irrigants, uterine contractants,
vaginal therapeutics and
vitamins and each specific compound or composition listed under each of the
foregoing categories
in the Physicians' Desk Reference.
They include, but not limited to water-soluble molecules possessing
pharmacological
activity, such as a peptide, protein, enzyme, enzyme inhibitor, antigen,
cytostatic agent, anti-
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inflammatory agent, antibiotic, DNA-construct, RNA-construct, or growth
factor. Examples of
therapeutic proteins are interleukins, albumins, growth hormones,
aspariginase, superoxide
dismutase, monoclonal antibodies. Biological agents include also water-
insoluble drugs, such as
camptothecin and related topoisomerase I inhibitors, gemcitabine, taxanes and
paclitaxel derivatives.
Other compounds include, for example, peptides, including peptidoglycans, as
well as anti-tumor
agents, cardiovascular agents such as forskolin; anti-neoplastics such as
combretastatin, vinbiastine,
doxorubicin, maytansine; anti-infectives such as vancomycin, erythromycin;
anti-fungals such as
nystatin, amphotericin B, triazoles, papulocandins, pneumocandins,
echinocandins, polyoxins,
nikkomycins, pradimicins, benanomicins; anti-anxiety agents, gastrointestinal
agents, central
nervous system-activating agents, analgesics, fertility agents, anti-
inflammatory agents, steroidal
agents, anti-urecemic agents, cardiovascular agents, vasodilating agents,
vasoconstricting agents and
the like.
Other biologically active agents which may be included in the electrospun
material include
vaccine antigens. The vaccine antigens of the invention can be derived from a
cell, a bacteria or
virus particle or a portion thereof. The antigen can be a protein, peptide,
polysaccharide,
glycoprotein, glycolipid, or combination thereof which elicits an immunogenic
response in a human;
or in an animal, for example, a mammal, bird, or fish. The immunogenic
response can be humoral,
mucosal, or cell mediated. Examples are viral proteins, such as influenza
proteins, human
immunodeficiency virus (HIV) proteins, Herpes virus proteins, and hepatitus A
and B proteins.
Additional examples include antigens derived from rotavirus, measles, mumps,
rubella, and polio; or
from bacterial proteins and lipopolysaccharides such as Gram-negative
bacterial cell walls. Further
antigens may also be those derived from organisms such as Haemophilus
influenza, Clostridium
antigens, including but not limited to, Clostridium tetani, Corynebacterium
diphtheria, and
Nesisseria gonhorrhoae, as well as anthrax antigens.
In another embodiment, the bioactive agent in the electrospun formulation
comprises a
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cytokine. Cytokines are hormone-like substances secreted by a wide variety of
cells, including (but
not limited to) lymphocytes (e.g., T cells), macrophages, fibroblasts, and
endothelial cells. It is now
known that cytokines consist of a broad class of glycoproteins that have the
ability to regulate
intercellular communication (e.g., cell-cell interaction) in both normal and
pathologic situations.
Cytokines generally contain from approximately 60 to 200 amino acid residues,
with a relative
molecular weight of between 15 and 25 kd. At least 35 distinct cytokines have
been elucidated. It is
not intended that the present invention be limited by the particular cytokine.
Table 2 provides
illustrative examples.
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TABLE 2
ame bbr. y e Specific Name
nterferons FN I ha Leukocyte Interferon
eta Fibroblast Interferon
amma acro hage Activation Factor
nterleukins L-1 1 alpha ndogenous P ro en
I beta ymphocyte-Activating Factor
I ra L-1 Receptor Antagonist
L-2 -cell Growth Factor
L-3 ast Cell Growth Factor
L-4 -cell Growth Factor
L-5 osino hil Differentiation Factor
L-6 bridoma Growth Factor
L-7 m ho oietin
L-8 Granulocyte Chemotactic Protein
L-9 e akar oblast Growth Factor
L-10 Cytokine Synthesis Inhibitor
actor
L-11 Stromal Cell-Derived Cytokine
IL-12 4atural Killer Cell Stimulatory
actor
umor Necrosis Factors F alpha Cachectin
eta m hotoxin
Colony Stimulating CSF GM-CS Granulocyte-macrophage Colony-
actors Stimulating Factor
p-CS acrophage Growth Factor
G-CSF Granulocyte Colony-stimulating
actor
PO r hropoietin
17
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ransforming Growth GF eta 1 Cartilage-inducing Factor
Factor
eta 2 pstein-Barr Virus-inducing
actor
eta 3 issue-derived Growth Factor
Other Growth Factors IF eukemia Inhibitory Factor
W acrophage Migration-inhibiting
actor
CP onocyte Chemoattractant
rotein
EGF idermal Growth Factor
DGF latelet-derived Growth Factor
FGF lpha cidic Fibroblast Growth Factor
eta 3asic Fibroblast Growth Factor
LGF nsulin-like Growth Factor
GF erve Growth Factor
CGF .13-cell growth factor
Thus, the present invention provides a method of administering one or more
drugs to a
patient comprising the steps of penetrating at least an outer layer of the
skin of the patient with one
or more asperities, such as pins, microprojections, or microneedles extending
from a surface of a
substrate, wherein at least one of the one or more asperities, such as pins,
microprojections, or
microneedles (or portion thereof), is contacted or coated (uniformly or non-
uniformly) with an
electrospun polymer/drug(s) preparation, and releasing at least one of the one
or more drugs into the
patient.
The relative amounts of fiber-forming material and biological material that
may be
present in the fiber of the present invention may vary. In one embodiment, the
biological
material comprises between about 1 and about 50 percent (more typically 30
percent) by weight to
volume (w/v) of the mixture from which the fiber is electrospun. In another
example, the
biological material comprises 10 percent of the mixture or less. In still
another example, the
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biological material may be 25 percent, 5 percent, 0.5 percent of the mixture
by weight to volume. It
is envisioned that larger or smaller concentrations of biological material may
also be utilized.
It is advantageous to coat the asperities (including microprojections and
microneedles) with
nanofibers which contain useful substances inside or on the nanofiber.
Different substances can be
concentrated near the tips of the asperities. The high surface area per unit
volume of a fiber, in
particular a nanofiber, makes it possible to carry a high mass of particles
containing bioactive
material per unit volume on the outside surface of the fiber and/or nanofiber.
In one embodiment, it
is even more useful to coat the tips of the asperities with nanofibers
carrying one or more useful
substances, and to have nanofibers that sequester a second set of useful
substances span the regions
between the asperities.
If, in one embodiment, the surfaces with asperities are mechanically pressed
into contact
with a second surface, the nanofibers on top of the asperities will be
transferred onto, or impressed
into the second surface. If, in one embodiment, the second surface is a sheet
of material thinner than
the height of the asperities, the nanofibers may be pushed through the sheet
and available for
chemical reactions or other purposes on the other side of the sheet while the
nanofibers that span the
spaces between the asperities are pressed onto the first surface of the sheet.
In one, embodiment, the present invention permits a designer to specify the
relative amounts
and kinds of useful materials, in each of three regions: on the surface near
the tip of the asperity, a
ball on top of the asperity, (including microprojections and microneedles) and
in the space between
the asperities. Such designs can be manufactured by one or methods in
accordance with the present
invention.
In one embodiment, quantities of around 1 microgram of one or more bioactive
substances
per pin and/or asperities have been found to be useful. For a material with a
specific gravity near
one, a cube with edges that are 0.1 mm (100 microns) long, would weigh 1
microgram. For
nanofibers, the specific gravity is only about 0.2, so the volume required for
1 microgram is
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correspondingly larger The edge of a 1 microgram cube of nanofibers is about
170 microns long.
Coatings made of fibers and/or nanofibers for an array of asperities can be
designed to
produce a variety of useful arrangements of the nanofibers. Thus, in one
embodiment, the present
invention utilizes an attraction between electrically charged fibers and/or
nanofibers and a sharp
point. Further, because electrospun nanofibers are very long, the same
nanofiber may span the space
between adjacent tips and/or be attached to a large number of tips, resulting
in the creation of a thin,
non-woven sheet supported on the tips of the array of pins.
Other arrangements of the nanof bers, created by the methods of the present
invention
provide a number of advantages.
In one embodiment, the nanofibers are wrapped around the tip of each asperity,
such as
microprojections or microneedles, to form a relatively thick coating on the
tip, with relatively few
nanofibers spanning the spaces between the tips. This can be accomplished by
controlling the
electrospinning process so that the electrospinning jet is collected on the
tips held near the location
where the onset of the electrically driven bending instability occurs, and
further, the jet is nearly dry,
but is still soft and can readily bend. These conditions also favor mechanical
buckling as the jet is
caught and stopped on the tip. Via the present invention, the ability to
create such nanofiber
structures can be realized.
The combination of these effects, and the presence of the pin, causes the
nanofibers to
generate a cotton-swab-like ("Q-Tip"-like) collection of nanofibers near the
tip of the pin.
Conglutination of the nanofibers can also be controlled, so that the fibers
may range from
fibers that are not attached to each other and slide past each other at
crossing points, or fibers that
are mechanically attached to each other at crossing points. The material on
which the one or more
asperities are formed must be a good enough electrical conductor that charge
carried via or on the
arriving jet(s) is conducted away. A sufficient level of ionic conductivity
exists on the surface of
ordinary glass at a normal ambient relative humidity. If a material with an
excessively low
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conductivity is utilized, the effective conductivity of the asperities can be
increased sufficiently by
coating them with a ten to 1000 nanometer thick layer of carbon or other
conductor or conductive
material. This can be accomplished by any suitable technique including, but
not limited to, vapor
deposition, or by evaporation of carbon or metal onto the surface bearing the
one or more asperities.
It should be noted that cotton-swab-like shapes can be used as collectors for
electrospun
nanofibers where. in one embodiment, such collectors are placed on one or more
rotating mandrels
In another embodiment, the present invention does not require the use of a
rotating pin Instead, in
this embodiment. the arriving nanofiber rotates around the tip region of the
pin. This is made to
happen locally and separately at the tip of each asperity. When the arriving
fiber moves from one
asperity to an adjacent one, a nanofiber segment remains between the two
asperities, but the total
amount of material in the spanning nanofibers is much less than the total
amount accumulated near
the tips of all the asperities.
In another embodiment, the arriving nanofibers can be accumulated in a "ball"
attached to
the tip if the electrical conductivity of the collected fibers is kept at a
relatively high value as the
fiber accumulates. The conductivity of the solidifying nanofibers can be
adjusted by addition of
ions such as lithium or sodium, and, optionally. with a less volatile liquid
substance added to the
solvent used in the electrospinning process. The less volatile substance
supports ionic conduction of
the electrical charge on the arriving nanofiber to the asperity, which is
maintained thereby at an
attractive electrical potential. This condition allows the arriving nanofiber
(which can, in one
embodiment, be regarded as a jet solidified enough to retain its fiber-like
shape, but still quite soft,
sticky, and electrically conducting) to be collected on top of nanofibers that
have already been
collected. The less volatile solvent can be retained in the collected
nanofibers, or in another
embodiment this solvent can be evaporated in a time slightly longer than the
time required to collect
the desired mass of nanofibers.
In the absence of such ionic conduction through the collected fibers, charge
retained on the
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topmost layer will repel the arriving nanofiber, and cause it to move toward
uncoated pins. The
uncoated pins may be some distance away: so long lengths of spanning nanofiber
can be generated,
in form of a non-woven mat and utilized in some embodiments of the present
invention.
Three modes of collection of nanofiber are available to the device designer,
although the
present invention is not limited thereto. Two cause the nanofiber to be
collected on or in front of the
tip of the asperity, where the probability of its being pressed mechanically
through a thin layer of
material is high. The third mode is the a "non-woven mat" supported on the
tips of the asperities
Only a relatively small fraction of the material in the "non-woven mat' will
be carried through the
surface as the as the asperities are pressed against the surface. The
remaining matter is brought into
intimate contact with the outer surface of the thin layer, where it can be
used to protect the surface
or in other ways.
In one embodiment, the present invention relates to a structure where the tips
of one or more
asperities are coated with a cotton-swab-like arrangement of nanofibers
bearing one useful material.
Another set of useful materials is collected in balls attached to the tip of
each asperity. A third set of
useful materials is collected in nanofibers that span the spaces between
asperities.
In another embodiment, the present invention relates to multilayer structures
where material
is collected in the vicinity of the tip of the pin. in the 'cotton-swab-like"
part of the structure, the
material collected in "balls" in front of the tip of the asperity, and the
"spanning segments" that
extend from the tip of one asperity to the tip of an adjacent asperity.
An electrospinning jet that wrapped polyethylene oxide nanofibers around or on
the tip of
each asperity in an array, was demonstrated by collecting nanofibers on
closely spaced carbon fibers
of the sort used in reinforced composites, which are electrically conducting,
very stiff, and have
diameters of about 7 microns. The jet used was, in one embodiment. less than a
centimeter in length.
Potential differences between the electrospinning tip and the electrically
grounded carbon fibers
were in the range between about one and about two kilovolts, although the
present invention is not
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limited to just these differences. The diameters of the collected nanofibers
were about one micron.
The velocity of the jet as it arrived at the tips of the carbon fibers was
low, perhaps less than
about one meter per second, or perhaps even about one tenth of one meter per
second. The
specifications above were of a representative experiment and are not intended
to act as limitations
on the scope of the present invention.
The carbon nanofibers, separated by various distances, were mounted on
adhesive tape at
lateral distances ranging from about one millimeter to side by side contact.
The carbon fibers were
projected at different distances toward the jet. Cottonswab-like coatings were
formed on fibers
separated by less than one millimeter. Balls were grown on fibers that
projected furthest in the
direction from which the jet arrives. The balls and cotton-swab-like
structures sometimes formed at
the tips of two carbon fibers which were separated by less than a few tens of
microns A relatively
small number of spanning nanofibers extended between fibers that are about one
millimeter apart.
Figure 1 is a low magnification optical image of 7 micron diameter carbon
fibers supported
on transparent tape. The divisions in the scale shown in the image are one
millimeter apart. The tips
of the fibers were coated with polyethylene oxide nanofibers by the
electrospinning methods
described in this invention. The carbon nanofibers were grounded, and then
held about 1 cm below
the tip at which the electrospinning jet was created. The orifice from which
the jet issued was at the
end of a slender glass tube which had an internal diameter of about 60
microns. The potential
difference between the electrospinning tip and the carbon nanofibers was about
1.5 kV. The
position at which the electrically driven bending instability occurred is
noted, and the tips of the
carbon fibers were held. by hand and moving slightly, near this position. The
collection of the
nanofibers required about 10 or 20 seconds, and was terminated when white
spots are visible at the
ends of the carbon fibers. The nanofiber coating extended all the way around
the tip of each of the
carbon fibers.
Figure 2 shows a coated carbon fiber tip at higher magnification. The diameter
of the coated
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fiber was about 70 microns, so the radial thickness of the coating was about
30 microns. Several
spanning nanofibers can be seen near the tip. showing that the number of
spanning nanofibers was
smaller than the number of nanofibers on the tip. The end of the coated fiber
is rounded. The
diameter varied slightly at different places along the axis. The upper part of
the figure is out of
focus, and is seen, by focusing on this region, to be smaller in diameter than
the region near the tip.
Figure 3 shows the tip of a carbon fiber, of the same sort as in Figures 1 and
2, coated with a
ball of nanofibers at the tip. This carbon fiber is extended beyond its
neighbors, and is in a position
to collect more, less dry fibers, which is consistent with the mechanisms
described in this disclosure,
for the creation of balls on the tips by enhanced electrical conductivity of
the collected fibers.
If the jet were wet when collected, balls or droplets also form. The fluid jet
carries liquid into
the droplet until the charge accumulates to a high enough value to cause the
jet to move to a new
location, usually leaving a spanning fiber to mark its path. These droplets do
not dry as rapidly as
nanofibers, but probably still provide significant sequestration and
protection to bioactive substances
in the fluid. The distinctions between balls of nanofibers and fluid droplets
are noted but not
emphasized or critical to the present invention.
In one embodiment, the devices of the present invention are employed for the
injection of
bioactive substances through the skin. Bioactive substances, (for therapeutic,
diagnostic,
vaccination, immunization, behavior modification, health monitoring, and other
such purposes) need
to be injected through the skin. This can be accomplished by coating the
bioactive substance on a
tapered pin which pierces the skin and deposits the bioactive material inside,
either as a particle or
by dissolving it in blood or other body fluids.
In practice it is desirable to use arrays of many pins, or microprojections,
or microneedles to
achieve efficacious concentrations of the bioactive material inside the body.
In most instances
below, references to a pin or microprojection or microneedle are intended to
describe both a single
pin, microprojection, or microneedle, and an array of a large number of
similar pins,
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microprojections, or microneedles. A prototypical pin, microprojection, or
microneedle for this
purpose, in one embodiment, has a diameter of about 100 microns at its broad
end and tapers to a
sharp tip of chisel edges with somewhat smaller diameter at its other end. The
diameter of the tip
may for example, be in the range from about 50 microns to about 5 microns.
Such a pin,
microprojection, or microneedle can be inserted through the skin and withdrawn
with minimal
damage to the skin tissue.
It is advantageous to coat the tip with nanofibers which contain a useful
substance inside or
on the nanofiber. When the coated pin, microprojection, or microneedle is
inserted through the skin,
the coating material is distributed between the outer surface of the skin, the
surface of the hole
created in the skin, and the interior of the body.
In another embodiment, the present invention relates to nanofibers that are
carried on a pin,
microprojection, or microneedle that will and/or are designed to absorb
substances from inside the
body and retain a sample of these substances when the pin is withdrawn. The
retained substances
can then be analyzed for diagnosis, control, or other purposes. Glucose
concentration monitoring
provides an example.
Nanofibers adhere tightly to the tips and to each other during insertion. When
wet with
bodily fluids the fibers may become slippery, dissolve, degrade, release
bioactive substances by
leaching or chemical reaction or produce other useful effects. The material
used for the nanofiber
and the known possibilities for forming nanofibers from complex mixtures of
substances provide
many options, which are included in this invention.
The bioactive materials are to be injected through the skin, as described
herein. The spanning
nanofibers are applied in a thin layer. For example, the spanning fibers could
be a nitric oxide (or
chlorine dioxide) releasing structure of the sort described in other co-owned
University of Akron
patents. This sort of non-woven mat applied over the top of pins,
microprojections, or microneedles,
already coated with the bioactive substances for injection through the skin,
could be used both to
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keep the device sterile between manufacture and use, and to sterilize the skin
during and after use.
The non-injected material in the spanning nanofibers contributes to an
advantageous design that
utilizes both injected and non-injected components of the nanofibers.
Because the pins used to carry the nanofibers through the skin are much
thicker than the
carbon fibers, and in ordered arrays, it is clear from the results obtained
with the thin carbon fibers,
that microgram quantities of nanofibers can be placed at desired locations,
described above, on the
arrays of pins created by MEMs fabrication methods.
The cotton-swab like shape is a desirable arrangement of nanofibers, since the
majority of
the useful material is attached directly to the part of the pin that will be
inside the skin when the
device is used, Reactions between reagents in dry fibers that span the spaces
between pins can
release a material to disinfect the skin, or to adhere to the skin after the
pins are removed and
manage fluids escaping from the puncture holes, or for other purposes.
Because, in one embodiment, the nanofibers of the present invention are much
smaller than
the asperities needed to penetrate the skin, the shape of the tips of the pins
can be modified to carry
more nanofibers through the skin.
Figure 4 shows radial steps which both increase the surface area near the tip
and provide
corners that protect some of the nanofibers from the shearing forces that
would otherwise wipe the
nanofibers off the tip as it is forced through the skin.
Figure 5 provides even more protection of against wiping off of material to be
injected,
obtained by the creation of forward facing barbs and also illustrates the use
of circumferential
grooves to protect the fibers from wiping.
Figure 6 has backward facing barbs that protect some of the nanofibers from
wiping as the
point is inserted, and also tends to carry some of the nanofibers as the tip
is withdrawn. The
withdrawn material would then be available for analysis. Only the region close
to the tip of the pin
is shown in these three figures. The patterns shown are not limiting. These
patterns may continue in
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the same way or with variations along the entire surface of the pin.
In another embodiment, two drugs are employed in an electrospun formulation.
In some
embodiments, the first drug and the second drug are different, but selected
from antidepressant
compounds, analgesic compounds, anti-inflammatory steroidal compounds
(corticosteroids), non-
steroidal anti-inflammatory compounds (NSAIDs), antibiotic compounds, anti-
fungal compounds,
antiviral compounds, antiproliferative compounds, antiglaucoma compounds,
immunomodulatory
compounds, cell transport/mobility impeding agents, cytokines and
peptides/proteins skin-treating
compounds sunscreens skin protectants, leukotrienes such as LTB4,
antimetabolite compounds,
antipsoriatic compounds, keratolytic compounds, anxiolytic compounds, and
antipsychotic
compounds. In a preferred embodiment, the polymer/two drug electrospun
preparation is delivered
transdermally, intradermally, intra-epidermally, transmucosally, or
subcutaneously. Alternatively,
the electrospun material including the two drugs is delivered IP, IV, or IM.
The present invention further contemplates, in one embodiment, a drug delivery
system
comprising a) a substrate comprising asperities such as microprojections or
microneedles, and b) an
embodiment of the various electrospun preparations (mentioned above). The term
"substrate", as
used herein, includes materials out of which the asperities are made
including, but not limited to,
silicon. When microprojections or microneedles are used, they may be, in one
embodiment, solid.
In another embodiment, they may be hollow. It is not intended that the present
invention be limited
to the manner in which the microneedles are contacted with the electrospun
preparation. In one
embodiment, the microneedles are contacted only at the microneedle tips. In
one embodiment, the
preparation contacts the entire substrate (or substantially the entire
substrate) such that the
preparation contacts even the spaces between an array of microneedles. It is
not intended that the
present invention be limited to uniform coatings of the preparation. In one
embodiment, the
preparation is present on the substrate in a non-uniform manner. In one
embodiment, an electrospun
polymer/drug(s) preparation is sprayed on said microneedles. In one
embodiment, said
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microneedles are dipped into an electrospun preparation.
In one embodiment of the multidrug microneedle array, the drugs are separated
on the array
into different regions or zones in the manner of a mosaic. Conceptually, the
mosaic approach
provides a means of packaging, storing and delivering to the patient drug
combinations without
encountering conventional formulation issues. Incompatible drugs need not be
chemically
"compatibilized" if they are deposited and stored at different addresses on a
microneedle array or
other type of skin patch.
One particularly useful application of the mosaic design comprises a multidrug
microneedle
embodiment where a non-polar and a polar drug are to be co-administered. While
such drugs are not
easily mixed into a single vehicle, the mosaic design permits both drugs to be
on the same delivery
platform, albeit separated. Another particularly useful application of the
mosaic design comprises a
multidrug microneedle embodiment wherein it is desired to carry drugs that
cancel one another's
side effects or that have synergistic effects. Such uses would be particularly
appropriate where
traditional formulation happens to be problematic.
It is not intended that the present invention be limited by the nature of the
substrate
comprising said microneedles. In one embodiment, the microneedles are
formulated out of polymer.
In another embodiment, the microneedles are made with a mold. In a preferred
embodiment, the
microneedles are etched out of a silicon substrate. In a preferred embodiment,
said silicon
microneedles are solid and the electrospun polymer/drug(s) formulation is
deposited on said
microneedles.
It is not intended that the present invention be limited to a single polymer
or a single layer on
said microneedles. In one embodiment, a first layer comprises a first polymer
and a second layer a
second polymer, where said second polymer is less soluble (e.g. in water) than
said first polymer
(Figure 7A). When the microneedles are administered, the microenvironment of
the skin causes the
first layer to dissolve (or substantially dissolve) thereby releasing the
second (or top) layer from the
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microneedles (Figure 7B). The bioactive agent (e.g. drug, antigen or other
substance to be delivered)
can be in the first layer, second layer, or both. Preferably, it is in the
second layer. For example, in
one embodiment, the present invention contemplates a substance delivery device
comprising: a
substrate having a back surface and a front surface; a plurality of solid
microneedles extending
upwards from the front surface of the substrate; a first polymer layer in
contact with said
microneedles; and a second polymer layer in contact with said first polymer
layer, said second
polymer layer comprising at least one bioactive substance.
It is also not intended that the present invention be limited to inflexible
microneedle arrays.
Indeed, embodiments of flexible microneedle arrays are contemplated. In one
embodiment of a
flexible microneedle, the present invention contemplates separating
microneedles into individual
"islands" by cutting into (and even through) the substrate so as to define
such islands or regions
separated by channels or streets (which can be, in one embodiment, filled or
partially filled with
polymer or drug). In one embodiment, the present invention contemplates
mounting the substrate
onto an adhesive material (e.g. adhesive tape) and dicing or cutting through
the substrate to generate
flexible arrays (Figure 8). In this manner, the risk of breakage when pushing
against the back of the
silicon substrates, when applying the patch to the skin, is reduced.
Various features can be added to the microneedle arrays to assure proper
delivery. In one
embodiment, the present invention contemplates the use of a plastic or
otherwise elastomeric device
positioned above the array relative to the skin (or attached or incorporated
into the substrate or upper
layer) that snaps into place once pressure is applied against the patch to
push and keep the array of
microneedles in the skin while the patch is on (to make sure needles are
inside the skin and to avoid
the need for an applicator in the final product, which is fully disposable in
this embodiment). In one
embodiment, the elastomeric element takes a first form prior to administration
(Figure 9A) and then
takes a second form after application of pressure (Figure 9B). In other words,
the elastomeric
element (which can be arched, curved or generally U-shaped) undergoes a shape
change or
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WO 2008/024141 PCT/US2007/011228
deformation upon receiving the pressure from pushing the array into contact
with the skin (e.g. from
concave to convex).
Thus, the present invention contemplates formulations and devices, including
but not limited
to delivery devices, as well as methods of making formulations and devices. In
one embodiment, the
present invention contemplates a method of creating a substance delivery
device, comprising:
providing i) an electrospun polymer formulation comprising at least one
substance, and ii) a
substrate comprising a plurality of microprojections; and depositing at least
a portion of said
preparation onto at least a portion of said substrate, so as to create a
substance delivery device. The
present invention also contemplates, as a device, the treated substrate
prepared according to the
above-described method. In one embodiment, said electrospun formulation
comprises nanofibers.
It is not intended that the present invention be limited to a particular water-
soluble polymer.
In one embodiment, said water-soluble polymer is selected from the group
consisting of PVP,
hydroxyethylcellulose, carboxymethyl cellulose, alginic acid, chitosan, and
poly(glutamic acid).
It is not intended that the present invention be limited by the nature of the
substance. In one
embodiment, said substance is a therapeutic protein. In another embodiment,
said substance is a
vaccine antigen. In one embodiment, first and second substances are used
(e.g., a vaccine antigen as
a first substance, and adjuvant as the second substance).
The present invention, as mentioned above, also contemplates methods of
administering
substances (including but not limited to pharmaceuticals). In one embodiment,
the present invention
contemplates a method of administering a substance, comprising: providing a
subject and the
delivery device described above; and contacting said subject with said
delivery device under
conditions such at least a portion of said substance is released from said
device. The term "subject"
includes human and non-human animals. In the case of humans, the term includes
more than
patients. The term also includes healthy, asymptomatic recipients. In one
embodiment, said
contacting comprises piercing the subject's skin with said asperities such as
microprojections or
CA 02650197 2008-10-21
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microneedles.
The present invention also contemplates, in one embodiment, a substance
delivery device
comprising: a substrate having a back surface and a front surface; and a
plurality of solid
microneedles extending upwards from the front surface of the substrate, the
microneedles
comprising an electrospun polymer formulation, said formulation comprising at
least one substance.
In one embodiment, said formulation provides for a sustained release of
substance. In another
embodiment, said formulation provides for various release rates of said
substance.
When in an array, the density of the microprojections is, in one embodiment,
at least 10
microprojections/cm2, in another embodiment, at least 200
microprojections/cm2, and, in some
embodiments, at least 1000 microprojections/cm2. In one embodiment, each
microneedle is spaced
(when measured center to center with another microneedle) between 300 microns
and 2.7 mm apart.
In one embodiment, the spacing is approximately three times the height of the
microneedle, i.e. for a
microneedle that is 600 microns (plus or minus 200 microns) in height, the
spacing may be 1.8 mm,
while for a microneedle that is 900 microns in height, the spacing may be 2.7
mm, while for a
microneedle that is 300 microns in height, the spacing may be 900 microns.
The figures herein provide illustrative examples of microneedles and
corresponding arrays.
Figure 10 is a scanning electron microscope picture showing an overview of one
embodiment of a
microneedle array (each microneedle is approximately 620 microns, plus or
minus 20 microns, in
height) with a particular density (the distance between microneedles, when
measured center to
center, is approximately 1.8 mm, plus or minus 200 microns. Figure I1 is a
scanning electron
microscope close up, side view of one embodiment of a single microneedle (e.g.
within the array) of
approximately 620 microns (plus or minus 20 microns) in height showing the
pyramidal shape.
Figure 12 is a scanning electron microscope close up, top view of one
embodiment of a single
microneedle (e.g. within the array) with eight sides (i.e. eight surfaces)
coming together at the top to
form a point or tip, wherein the width is approximately 385 microns, plus or
minus 5 microns, and
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the height is approximately 620 microns, plus or minus 20 microns.
It is not intended that the present invention be limited to a particular
density. Figure 13
shows a prospective microneedle array with a higher density of microneedles.
In another non-limiting embodiment, the present invention comprises a
microfabricated
device for transdermal, intra-epidermal, intradermal or transmucosal drug
delivery comprising
ionically cross-linked macromolecular assemblies, which provides sufficient
mechanical strength,
sufficient capacity of loading various amounts of drug, stability, and release
characteristics. After
the device is brought in contact with a subject's skin, the substance is
released.
lonically cross-linked macromolecular assemblies are polymer networks that are
formed
through the establishment of reversible links between the macromolecular
chains, as described in "J.
Berger, M Reist, J.M. Mayer, 0. Felt, N.A. Peppas, R. Gurny, Structure and
interactions in
covalently and ionically crosslinked chitosan hydrogels for biomedical
applications, European
Journal of Pharmaceutics and Biopharmaceutics 57 (2004) 19-34. " They can
include
polyelectrolyte complexes; multilayer polyelectrolyte materials, complexes of
polyelectrolytes with
low molecular weight ions; assemblies of dendrimers; ionically cross-linked
hydrogels, which can
be in the form of film; micro-and nanospheres; micro- and nanocapsules; micro-
and nanofibers. The
properties of cross-linked hydrogels depend mainly on their cross-linking
density, namely the ratio
of moles of cross-linking units to the moles of polymer repeating units,
polymer characteristics, and
microstructure of the material. lonically cross-linked assemblies include
those in which gradient
exists in the density of the cross-linking agent.
lonically cross-linked macromolecular assemblies of the present invention
comprise a
substance to be delivered. The preferred substances of the present invention
are biologically active
agents such as those described herein, including but not limited to,
pharmaceutically active
substances, such as therapeutic drugs, which can include small molecule drugs,
proteins, peptides,
nucleic acids, hormones, or vaccines, which can include antigens and
adjuvants.
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The formulation containing the pharmaceutically active substance can be in the
form of an
ionically cross-linked three dimensional film, sphere, tube, fiber, such as a
nanofiber or microfiber,
or in any other geometrical shape, and is attached the surface of the device
by chemical or physical
means.
The polymers of this aspect of the present invention may be water-soluble
polymers. In one
embodiment they are polyelectrolytes - polymers containing ionic or ionizable
groups. In another
embodiment they are polymers capable of establishing ionic complexes, such as
sodium -
poly(ethylene oxide), or sodium -poly[di(methoxyethoxyethoxy)phosphazene
complex. In one
embodiment the polymers are biodegradable polymers. Typical examples of such
polymers are
poly(ethylene glycol), polyvinylpyrrolidone, polyvinylalcohol, poly(ethylene
oxide),
polyoxymethylene, poly(hydroxyethyl methacrylate), carboxymethylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose, alginic acid, chitosan,
poly(glutamic acid),
poly(isobutylacrylamide), poly(ethylenimine), and copolymers thereof. The
polymers can be linear,
branched, brush- or comb-like in their macromolecular architecture. Copolymers
can be random or
block copolymers, or biomolecules (such as fibrin, fibrinogen, cellulose,
starch, collagen and
hyaluronic acid). In yet another embodiment, the polymers can be hydrophobic,
but contain
hydrophilic groups or hydrophilic segments capable of forming ionic cross-
links or complexes.
Examples of hydrophobic polymers are poly(butyl methacrylate), poly(ethyl
methacrylate),
poly(vinylidene fluoride) poly(hydroxyvalerate), poly(L-lactic acid),
polycaprolactone, poly(lactide-
co-glycolide), poly(hydroxybutyrate), poly(hydroxybutyrate-co-valerate),
polydioxanone,
polyorthoester, polyanhydride, poly(glycolic acid), poly(vinyl methyl ether),
polyvinylidene
chloride, polyacrylonitrile, poly(trimethylene carbonate),
poly(iminocarbonate), and other
derivitized polyurethanes, and poly(organosiloxanes).
It is contemplated that the ionically cross-linked macromolecular
assembly/drug(s)
preparation can be deposited on a surface (in a uniform or non-uniform
manner). In one
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embodiment, the preparation is deposited (e.g. by dipping, coating, spin
coating, spraying, or by
suitable applicator) on an array of asperities such as those described herein,
such as microprojections
or microneedles for transdermal, intradermal, or transmucosal delivery of the
bioactive substance.
While the microneedle embodiment (described herein) is preferred, other
systems
and apparatus that employ tiny skin piercing elements to enhance transdermal
agent delivery are
also contemplated, as disclosed in U.S. Pat. Nos. 5,879,326, 3,814,097,
5,250,023, 3,964,482,
Reissue U.S. Pat. No. 25,637, and PCT Publication Nos. WO 96/37155, WO
96/37256, WO
96/17648, WO 97/03718, WO 98/11937, WO 98/00193, WO 97/48440, WO 97/48441, WO
97/48442, WO 98/00193, WO 99/64580, WO 98/28037, WO 98/29298, and WO 98/29365;
all
incorporated herein by reference in their entirety.
In one non-limiting embodiment, the macromolecular assembly can be prepared by
ionic
cross-linking or complexation of nanofibers. Polymeric materials can be
fabricated in fibers, nano-
and microfibers. The term "fiber" includes not only structures that are
cylindrical, but also includes
structures which vary from a cylindrical shape, such as for example,
structures which are flattened
or ribbon shaped. When fine fibers are randomly distributed they can form an
interlocking net (non-
woven fine fiber).
A"fiber-forming" material is capable of being fabricated into fibers. The
fiber-forming
materials used in this invention include polymers, which can be in a liquid
state, such as melted, in
solution, or in suspension. Water, organic solvents, or mixtures thereof or
heat can be used to
convert solid polymer into a liquid "fiber-forming" material. Electrostatic
solution spinning is one
method of making nanofibers and microfiber. Such electrospun material is then
treated with a
solution of an ion-complexing agent for cross-linking.
In one embodiment, the macromolecular assembly can be fabricated on the
surface of the
device using an aqueous based method (Figure 14). The advantages of such
method for making
ionically cross-linked hydrogels are that it avoids the use of organic
solvents, heat, and, complicated
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WO 2008/024141 PCT/US2007/011228
manufacturing equipment. In one embodiment, the method involves the following
steps. First, a
solution of containing polymer and pharmaceutically active substance can be
applied to a device
using methods, such as dipcoating, spin coating, electrospinning, spraying, or
with a brush or other
suitable applicator. The preferred solution is a solution in water or in
aqueous buffer; however
organic solutions can be used or added if beneficial. The surface of a device
can be dried after the
application if desired. Secondly, a solution containing multivalent or
polyvalent ions (cross-linker),
is applied to the surface using the previously described methods.
Multivalent ions can be selected from the group if inorganic ions, such as
calcium, zinc
bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, or cadmium, or
organic ions, such
as spermine, spermidine, or polylysine. In one embodiment, the salt of the
monovalent ion is a
calcium salt, such as calcium chloride, or CaC l2. The salt of the multivalent
ion may be present in
the solution at any concentration and pH, preferably from about 1% to about
25% and a pH from 4
to 9. The Ca2+ ions in the solution serve as a cross-linker, whereby the film
is stabilized when they
contact the solution containing the calcium salt.
The device can then be dried by freeze-drying, treating it with ethanol or
other water
miscible organic solvent, or under vacuum.
Alternatively, a physiologically active substance can be dissolved or
dispersed in a solution
of the cross-linker (multivalent or polyvalent ions) (Figure 15). In one
embodiment of such a
method, the physiologically active substance is introduced in the film at the
same stage as the film is
stabilized.
In one embodiment, the ionic cross-linking process can be applied to the total
volume of the
polymer film. In another embodiment the cross-linking process is limited to
portion of the film, such
as a portion localized on the elevated part of the device. In yet another
embodiment, the cross-
linking process is only applied to a portion of the film, which is localized
on the tip of the elevated
part of the device.
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It is realized that the nature of the cross-linking agent used in the process
can influence the stability of the film
and thus the disintegration time and the release kinetics upon the
administration. It is generally known that the
disintegration and release time increases as the stability of the ionic links
in hydrogel matrices
increases in the following way: two valent ionic cross-linker, such as calcium
ion, three valent ionic
cross-linker, such as aluminum ion, multivalent organic ions, such as spermine
or spermidine,
polyvalent organic ions, such as poly(L-lysine). It is also understood that an
increase in the cross-
linking density of the hydrogel matrix can also lead to the increase in the
disintegration of the
hydrogel formulation and release time of the pharmaceutically active
substance.
In one embodiment multiple ions are used to cross-link hydrogel matrix. In
another
embodiment the gradient of ions is achieved through the depth of the film. In
yet another
embodiment, the cross-linking density is also varied through the volume of the
film. This can be
achieved via varying the cross-linker and its concentration with time in the
cross-linking solution.
Alternatively, the polymer hydrogel is composed of interpenetrating networks
of ionically cross-
linked polyelectrolytes.
In a preferred embodiment, only one type of a cross-linker is used and no
additional cross-
linking agent is added in the course of the process.
In yet another embodiment, the type of cross-linker and the concentration of
the cross-linkers
varies in the various zones of the device. Thus, various modes of release
kinetics can be achieved
using the same device.
The present invention contemplates administering one or more of the various
embodiments
of the formulations (described herein) comprising a drug, such as a
prophylactic or therapeutic to
humans and animals. In one embodiment, the present invention contemplates a
method comprising:
a) providing i) a preparation comprising at least one drug in a ionically-
cross-linked macromolecular
assembly and ii) a subject, wherein said subject is a human or non-human
animal; and b) contacting
said subject with said preparation (e.g. under conditions such that at least a
portion of said drug is
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released into said subject). In one embodiment, the present invention
contemplates a method
comprising: a) providing i) a preparation comprising one or more drugs in a
macromolecular
assembly formulation and ii) a subject, wherein said subject is a human or non-
human animal; and
b) contacting said subject with said preparation (e.g. under conditions such
that at least a portion of
said drug is released into said subject).
In one embodiment, two drugs are employed in a macromolecular assembly
formulation. In
some embodiments, first drug and second drug are different, but selected from
antidepressant
compounds, analgesic compounds, anti-inflammatory steroidal compounds
(corticosteroids), non-
steroidal antiinflammatory compounds (NSAIDs), antibiotic compounds, anti-
fungal compounds,
antiviral compounds, antiproliferative compounds, antiglaucoma compounds,
immunomodulatory
compounds, cell transport/mobility impeding agents, cytokines and
peptides/proteins skin-treating
compounds sunscreens skin protectants, leukotrienes such as LTB4,
antimetabolite compounds,
antipsoriatic compounds, keratolyiic compounds, anxiolytic compounds, and
antipsychotic
compounds. In a preferred embodiment, the hydrogel/two drug preparation is
delivered
transdermally, intradermally, intra-epidermally, transmucosally,
subcutaneously, IP, IV or IM.
The present invention further contemplates, in one embodiment, a drug delivery
system
comprising a) a substrate comprising asperities such as those described
herein, such as
microprojections or microneedles, and an embodiment of the various ionically
cross-linked
preparations (mentioned above). When microneedles are used, they may be
hollow, but preferably
they are solid. It is not intended that the present invention be limited to
the manner in which the
microneedles are contacted with the macromolecular assembly preparation. In
one embodiment, the
microneedles are contacted only at the microneedle tips. In a preferred
embodiment, the preparation
contacts the entire substrate (or substantially the entire substrate) such
that the preparation contacts
even the spaces between an array of microneedles. It is not intended that the
present invention be
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limited to uniform coatings of the preparation. In one embodiment, the
preparation is present on the
substrate in a non-uniform manner. In one embodiment, a ionically cross-linked
macromolecular
assembly/drug(s) preparation is sprayed on said microneedles. In one
embodiment, said
microneedles are dipped into a preparation.
Thus, the present invention contemplates formulations and devices, including
but not limited
to delivery devices, as well as methods of making formulations and devices. In
one embodiment, the
present invention contemplates a method of creating a substance delivery
device, comprising:
providing i) an ionically cross-linked macromolecular assembly which includes
at least one
substance, such as a biologically active agent as described herein and ii) a
substrate comprising a
plurality of asperities such as those described herein, such as
microprojections and microneedles;
and depositing at least a portion of said preparation onto at least a portion
of said substrate, so as to
create a substance delivery device. The present invention also contemplates,
as a device, the treated
substrate prepared according to the above-described method. In one embodiment,
said ionically
cross-linked macro-molecular assembly comprises an ionically cross-linked
water-soluble polymer.
In a preferred embodiment, said ionically cross-linked macromolecular assembly
has a multilayer
structure. In another embodiment, said ionically cross-linked macromolecular
assembly comprises
ionically cross-linked nanofibers.
It is not intended that the present invention be limited to a particular water-
soluble polymer.
In one embodiment, said water-soluble polymer is selected from the group
consisting of
carboxymethyl cellulose, alginic acid, chitosan, poly(glutamic acid).
It is not intended that the present invention be limited by the nature of the
nanofibers. In one
embodiment, wherein said ionically cross-linked nanofibers comprise ionically
cross-linked chitosan
nanofibers.
It is not intended that the present invention be limited by the nature of the
substance. In one
embodiment, said substance is a therapeutic protein. In another embodiment,
said substance is
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vaccine antigen. In one embodiment, first and second substances are used (e.g.
a vaccine antigen as
a first substance, and adjuvant as the second substance).
The present invention, as mentioned above, also contemplates methods of
administering
substances (including but not limited to pharmaceuticals). In one embodiment,
the present invention
contemplates a method of administering a substance, comprising: providing: a
subject and the
delivery device described above; and contacting said subject with said
delivery device under
conditions such at least a portion of said substance is released from said
device. In one embodiment,
said contacting comprises piercing the subject's skin with said
microprojections.
The present invention also contemplates, in one embodiment, a substance
delivery device
comprising: a substrate having a back surface and a front surface; and a
plurality of solid
microneedles extending upwards from the front surface of the substrate, the
microneedles
comprising a ionically cross-linked macromolecular assembly formulation, said
formulation
comprising at least one substance. In one embodiment, said ionically cross-
linked macromolecular
assemblies provide for a sustained release of substance. In another
embodiment,said ionically cross-
linked macromolecular assemblies provide for various release rates of said
substance.
"Polyelectrolytes" are defined here as polymers that contain ionic (ionized or
ionizable)
groups, which groups impart to the polymer anionic, cationic, or amphiphilic
character. The ionic
groups can be in the form of a salt, or, alternatively, an acid or base that
is, or can be, at least
partially dissociated. Polyelectrolytes can also contain non-ionic side
groups. Polyelectrolyte can be
biodegradable (e.g. to prevent eventual deposition and accumulation of polymer
molecules in the
body) or non-biodegradable under the conditions of use. A preferred
polyelectrolyte is a polyanion
and contains ionic groups that include carboxylic acid, sulfonic acid,
hydroxyl, or phosphate
moieties.
Pharmaceutically active substances which may be included in the resulting
preparation
which includes the macromolecular assembly hereinabove described may be those
described herein.
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In one non-limiting embodiment, a hydrogel/drug(s) preparation is delivered
transdermally,
intra-epidermally, intradermally, or transmucosally using anisotropically
etched MEMS
microneedles in silicon. In one embodiment, the invention contemplates solid
asperities such as
those herein described, such as microprojections or microneedles which are
fabricated in a crystal
silicon material suitable for use in the administration of the various
preparations discussed above.
Without limiting the invention in any manner to any particular mechanism, it
is believed that the
drug(s) is delivered by microporation of the stratum corneum, and polymer-drug
deposition within
the patient's skin and subsequent dissolution or erosion of the polymer. The
drug becomes thereby
bioavailable; it can dissolve and diffuse to the biological target, or
alternatively, it can remain at the
site of administration. Micropores are made into the stratum corneum by means
of a microneedle
array penetration, which can optionally be further enhanced by applying energy
in the form of
ultrasonic, heat and/or electric signals across or through the skin.
In one embodiment, the present invention contemplates a method of
administering one or
more drugs to a patient comprising the steps of penetrating at least an outer
layer of the skin of the
subject with one or more microneedles extending from a front surface of a
substrate, at least one of
the one or more microneedles (or portion thereof) contacted or coated
(uniformly or non-uniformly)
by a macromolecular assembly/drug(s) preparation (discussed above), and
releasing at least one of
the one or more drugs into the subject.
In another, non-limiting embodiment, the at least one biologically active
agent is a toxin.
For example, several species of Clostridium produce toxins of significance to
human and
animal health. [C.L. Hatheway, Clin. Microbiol. Rev. 3:66-98 (1990).] The
effects of these toxins
range from diarrheal diseases that can cause destruction of the colon, to
paralytic effects that can
cause death (see Table 3). Particularly at risk for developing clostridial
diseases are neonates and
humans and animals in poor health (e.g., those suffering from diseases
associated with old age or
immunodeficiency diseases).
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Clostridium botulinum produces the most poisonous biological toxins known. The
lethal
human dose is a mere 10-9 mg/kg bodyweight for toxin in the bloodstream. THe
closely related
isoforms of botulinal toxin blocks nerve transmission to the muscles,
resulting in flaccid paralysis.
When the toxin reaches airway and respiratory muscles, the consequence is
respiratory failure that
can cause death. [S. Arnon, J. Infect. Dis.154:201-206 (1986).]
C. botulinum spores are carried by dust and are found on vegetables taken from
the soil, on
fresh fruits, and on agricultural products such as honey. Under conditions
favorable to the organism,
the spores germinate to vegetative cells which produce toxin. [S. Amon, Ann.
Rev. Med. 31:541
(1980).] Botulism disease may be grouped into four types, based on the method
of introduction of
the toxin isoform into the bloodstream. Food-borne botulism results from
ingesting improperly
preserved and inadequately heated food that contains botulinal toxin. [K.L.
MacDonald et al., Am. J.
Epidemiol. 124:794 (1986).] The death rate due to botulinal toxin is 12% and
can be higher in
particular risk groups. [C.O. Tacket et al., Am. J. Med. 76:794 (1984).] Wound-
induced botulism
results from C. botulinum penetrating traumatized tissue and producing toxin
that is absorbed into
the bloodstream.
TABLE 3
Clostridium Species of Medical and Veterinary Importance*
Species Disease
C. aminovalericum Bacteriuria (pregnant women)
C. argentinense Infected wounds; Bacteremia; Botulism; Infections of amniotic
fluid
C. baratii Infected war wounds; Peritonitis; Infectious processes of the eye,
ear
and prostate
C. beijerinckikii Infected wounds
C. bifermentans Infected wounds; Abscesses; Gas Gangrene; Bacteremia
C. botulinum Food poisoning; Botulism (wound, food, infant)
C. butyricum Urinary tract, lower respiratory tract, pleural cavity,
and abdominal infections;
Infected wounds; Abscesses; Bacteremia
C. cadaveris Abscesses; Infected wounds
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C. carnis Soft tissue infections; Bacteremia
C. chauvoei Blackleg
C. clostridioforme Abdominal, cervical, scrotal, pleural, and other
infections; Septicemia;
Peritonitis; Appendicitis
C. cochlearium Isolated from human disease processes, but role in disease
unknown.
C. difficile Antimicrobial-associated diarrhea; Pseudomembranous
enterocolitis;
Bacteremia;
Pyogenic infections
C. fallax Soft tissue infections
C. ghnoii Soft tissue infections
C. glycolicum Wound infections; Abscesses; Peritonitis
C. hastiforme Infected war wounds; Bacteremia; Abscesses
C. histolyticum Infected war wounds; Gas gangrene; Gingival plaque isolate
C. indolis Gastrointestinal tract infections
C. innocuum Gastrointestinal tract infections; Empyema
C. irregulare Penile lesions
C. leptum Isolated from human disease processes, but role in disease unknown
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TABLE 3
Clostridium Species of Medical and Veterinary Importance*
Species. Disease
C. limosum Bacteremia; Peritonitis; Pulmonary infections
C. malenominatum Various infectious processes
C. novyi Infected wounds; Gas gangrene; Blackleg, Big head (ovine);
Redwater disease (bovine)
C. oroticum Urinary tract infections; Rectal abscesses
C. paraputrificum Bacteremia; Peritonitis; Infected wounds; Appendicitis
C. perfringens Gas gangrene; Anaerobic cellulitis; Intra-abdominal abscesses;
Soft tissue infections; Food poisoning; Necrotizing pneumonia;
Empyema; Meningitis; Bacteremia; Uterine Infections; Enteritis
necrotans; Lamb dysentery Struck; Ovine Enterotoxemia
C. putrefaciens Bacteriuria (Pregnant women with bacteremia)
C. putrificum Abscesses; Infected wounds; Bacteremia
C. ramosum Infections of the abdominal cavity, genital tract, lung, and
biliary
tract; Bacteremia
C. sartagoforme Isolated from human disease processes, but role in disease
unknown.
C. septicum Gas gangrene; Bacteremia; Suppurative infections; Necrotizing
enterocolitis; Braxy
C. sordellii Gas gangrene; Wound infections; Penile lesions; Bacteremia;
Abscesses; Abdominal and vaginal infections
C. sphenoides Appendicitis; Bacteremia; Bone and soft tissue infections;
Intraperitoneal infections; Infected war wounds; Visceral gas
gangrene; Renal abscesses
C. sporogenes Gas gangrene; Bacteremia; Endocarditis; central nervous system
and europulmonary infections; Penile lesions; Infected war
wounds; Other pyogenic infections
C. subterminale Bacteremia; Empyema; Biliary tract, soft tissue and bone
infections
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C. symbiosum Liver abscesses; Bacteremia; Infections resulting due to
bowel flora
C. tertium Gas gangrene; Appendicitis; Brain abscesses; Intestinal tract and
soft tissue infections; infected war wounds; penodonitis;
Bacteremia
TABLE 3
Clostridium Species of Medical and Veterinary Importance*
Species isease
C. tetani etanus; Infected gums and teeth; Corneal ulcerations;
4astoid and middle ear infections; Intraperitoneal
infections; Tetanus neonatorum; Postpartum uterine
infections; Soft tissue infections, especially related to
rauma (including abrasions and lacerations); Infections
elated to use of contaminated needles
C. thermosaccharolyticum solated from human disease processes, but role in
disease
nknown.
* Compiled from P.G. Engelkirk et al. "Classification", Principles and
Practice of Clinical
Anaerobic Bacteriology, pp. 22-23, Star Publishing Co., Belmont, CA (1992); J.
Stephen and R.A.
Petrowski, "Toxins Which Traverse Membranes and Deregulate Cells," in
Bacterial Toxins, 2d ed.,
pp. 66-67, American Society for Microbiology (1986); R. Berkow and A.J.
Fletcher (eds.),
Bacterial Diseases," Merck Manual of Diagnosis and Therapy, 16th ed., pp. 116-
126, Merck
Research Laboratories, Rahway, N.J. (1992); and O.H. Sigmund and C.M. Fraser
(eds.), "Clostridial
Infections, "Merck Veterinary Manual, 5th ed., pp. 396-409, Merck & Co.,
Rahway, N.J. (1979).
[M.N. Swartz, "Anaerobic Spore-Forming Bacilli: The Clostridia," pp. 633-646,
in B.D.
Davis et al.,(eds.), Microbiology, 4th edition, J.B. Lippincott Co. (1990).]
Inhalation botulism has
been reported as the result of accidental exposure in the laboratory [E.
Holzer, Med. Klin. 41:1735
(1962)] and could arise if the toxin is used as an agent of biological warfare
[D.R. Franz et al., in
Botulinum and Tetanus Neurotoxins, B.R. DasGupta, ed., Plenum Press, New York
(1993), pp. 473-
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476]. Infectious infant botulism results from C. botulinum colonization of the
infant intestine with
production of toxin and its absorption into the bloodstream. It is likely that
the bacterium gains entry
when spores are ingested and subsequently germinate. [S. Arnon, J. Infect.
Dis. 154:201 (1986).]
Different strains of Clostridium botulinum each produce one of seven
structurally similar but
antigenically distinct toxin isoforms designated by the letters A-G. Serotype
A toxin has been
implicated in 26% of the cases of food botulism; types B, E and F have also
been implicated in a
smaller percentage of the food botulism cases [H. Sugiyama, Microbiol. Rev.
44:419 (1980)].
Wound botulism has been reportedly caused by only types A or B toxins [H.
Sugiyama, supra].
Nearly all cases of infant botulism have been caused by bacteria producing
either type A or type B
toxin. (Exceptionally, one New Mexico case was caused by Clostridium botulinum
producing type F
toxin and another by Clostridium botulinum producing a type B-type F hybrid.)
[S. Arnon,
Epidemiol. Rev. 3:45 (1981)] Type C toxin affects waterfowl, cattle, horses
and mink. Type D toxin
affects cattle, and type E toxin affects both humans and birds.
A trivalent antitoxin derived from horse plasma is commercially available from
Connaught
Industries Ltd. as a therapy for toxin types A, B, and E. However, the
antitoxin has several
disadvantages. First, extremely large dosages must be injected intravenously
and/or intramuscularly.
Second, multiple administrations of antitoxin can trigger serious side
effects, such as acute
anaphylaxis which can lead to death, and serum sickness. Finally, the efficacy
of the antitoxin is
uncertain and the treatment is costly.
[C.O. Tacket et al., Am. J. Med. 76:794 (1984).]
A heptavalent equine botulinal antitoxin which uses only the F(ab')2 portion
of the antibody
molecule has been tested by the United States Military. [M. Balady, USAMRDC
Newsletter, p. 6
(1991).] This was raised against impure toxoids in those large animals and is
not a high titer
preparation.
A pentavalent human antitoxin has been collected from immunized human subjects
for use
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as a treatment for infant botulism. The supply of this antitoxin is limited
and cannot be expected to
meet the needs of all individuals stricken with botulism disease. In addition,
collection of human
sera must involve screening out HIV and other potentially serious human
pathogens. [P.J. Schwarz
and S.S.on, Western J. Med. 156:197 (1992).]
Immunization of subjects with toxin preparations has been done in an attempt
to induce
immunity against botulinal toxins. A C. botulinum vaccine comprising
chemically inactivated (i.e.,
formaldehyde-treated) type A, B,C, D and E toxin is commercially available for
human usage.
However, this vaccine preparation has several disadvantages. First, the
efficacy of this vaccine is
variable (in particular, only 78% of recipients produce protective levels of
anti-type B antibodies
following administration of the primary series). Second, immunization is
painful (deep subcutaneous
inoculation is required for administration), with adverse reactions being
common (moderate to
severe local reactions occur in approximately 6% of recipients upon initial
injection; this number
rises to approximately 11% of individuals who receive booster injections)
[Informational Brochure
for the Pentavalent (ABCDE) Botulinum Toxoid, Centers for Disease Control].
Third, preparation of
the vaccine is dangerous as active toxin must be handled by laboratory
workers.
What are needed are safe and effective vaccine preparations for administration
to those at
risk of exposure to C. botulinum toxins.
The present invention contemplates, in a non-limiting embodiment, a
preparation comprising
at least one polymer and at least one Clostridium antigen or toxin (or
fragment thereof) such as those
selected from a species set forth in Table 3. The present invention
contemplates, in one embodiment,
such an antigen or toxin (or fragment thereof) in a preparation suitable for
transdermal, intradermal,
or transmuscosal delivery. In a preferred embodiment, the species is C.
botulinum. In one
embodiment, the antigen or toxin (or fragment thereof) is in a hydrogel
formulation. In one
embodiment, the antigen or toxin (or fragment thereof) is attached or linked
to a carrier. In one
embodiment, the antigen or toxin (or fragment thereof) is part of a fusion
protein. In one
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embodiment, the antigen or toxin (or fragment thereof) is in a micelle
formulation. In one
embodiment, the antigen or toxin (or fragment thereof) is in or on nanofibers,
nanotubes,
nanospheres, or nanocapsules. In one embodiment, the antigen or toxin (or
fragment thereof) is
complexed with one or more dendrimers. In one embodiment, the antigen or toxin
(or fragment
thereof) is in a liposome preparation (e.g. for liposome delivery). In a
preferred embodiment, the
antigen or toxin (or fragment thereof) is on the surface of a substrate, e.g.
on a skin patch,
microneedle array, or the like. Combinations of the above are specifically
contemplated, including
but not limited to, substrates comprising the Clostridium antigen(s) or
toxin(s) (or fragment(s)
thereof) in a polymer formulation, or in an assembly of nanofibers. In a
particularly preferred
embodiment, the toxin is functionally impaired (e.g. it will not cause
toxicity).
In one embodiment, the Clostridium antigen or toxin (or fragment thereof) is
in a hydrogel
formulation. It is not intended that the present invention be limited by the
nature of the hydrogel
since a variety of types can be used, including but not limited to ionically
cross-linked hydrogels or
hydrogel films. It is contemplated that the hydrogel/antigen(s) or
hydrogel/toxin(s) preparation can
be deposited on a surface (in a uniform or non-uniform manner). In one
embodiment, the
preparation is deposited (e.g. by dipping, coating, spin coating, spraying, or
by suitable applicator)
on an may of microprojections or microneedles for transdermal, intradermal or
transmucosal
delivery of the antigen(s). In one embodiment, the present invention
contemplates a method
comprising: a) providing i) a preparation comprising one or more Clostridium
antigens or toxins (or
fragments thereof) in a hydrogel formulation and ii) a subject, wherein said
subject is a human or
non-human animal; and b) contacting said subject with said preparation (e.g.
under conditions such
that said subject generates an immune response to said one or more antigens or
toxins).
When polymers are used, it is not intended that the present invention be
limited by the nature
of the polymer since a variety of types can be used (see Table 1). In one
embodiment, the polymer
comprises polyvinylpyrrolidone or hydroxy-propylcellulose. In one embodiment,
the polymer
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comprises hydroxyethylcel lulose.
It is not intended that the present invention be limited to the manner in
which the preparation
of polymer and antigen (or toxin) is prepared. However, in a preferred
embodiment, the preparation
is electrospun to create fibers or fiber mats. It is preferred that at least a
portion of the antigen (or
toxin) in the electrospun fibers or mats be capable of release or escape from
the polymer (e.g. upon
contact with an aqueous or semi-aqueous environment). In certain embodiments
(e.g. cosmetic), it is
desired that the toxin be functional. In other embodiments (e.g. vaccines), it
is not necessary that the
toxin be functional (e.g. mutants and chemically treated toxins can be used).
In a preferred embodiment, the toxin polypeptides comprise one or more
Clostridium
botulinum neurotoxins. The invention contemplates the use of polypeptides
derived from C.
botulinum toxin as immunogens for the production of vaccines and antitoxins,
as well as for
cosmetic purposes. The C. botulinum vaccines and antitoxins find use in humans
and other animals.
In one embodiment, the present invention contemplates a fusion protein
comprising a non-toxin
protein sequence and a portion of the Clostridium botulinum type A toxin. In
one embodiment, the
C. botulinum type A toxin sequences comprise a portion of the sequence shown
in Figure 16. In
another embodiment, the C. botulinum type A toxin sequences comprise a portion
of the sequence
shown in Figure 17. It is not intended that the present invention be limited
by the nature of the
fusion protein. For example, the fusion protein may comprise the Clostridium
botulinum type A
toxin sequence as shown in Figure 17 along with a poly-histidine tract. The
toxins are conveniently
made in host cells containing a recombinant expression vector, wherein the
vector encodes a fusion
protein comprising a non-toxin protein sequence and a portion of the
Clostridium botulinum type A
toxin sequence shown in Figure 16. In this embodiment, the host cell is
capable of expressing the
encoded Clostridium botulinum type A toxin protein as a soluble protein at a
level greater than or
equal to 0.25% to 10% of the total cellular protein and preferably at a level
greater than or equal to
0.75% of the total cellular protein. It is not intended that the present
invention be limited by the
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nature of the fusion protein expressed by the recombinant vector in the host
cell. For example, the
fusion protein may comprise the Clostridium botulinum type A toxin sequence as
set forth shown in
Figure 17, along with a poly-histidine tract.
Regardless of whether a wild-type, mutant, or toxin fusion proteins are used,
the present
invention contemplates administering one or more of the various embodiments of
the preparations
described above as a vaccine to humans and animals, or as a cosmetic. In one
embodiment, the
present invention contemplates a vaccine-related method comprising: a)
providing i) a preparation
comprising at least one polymer and one or more C. botulinum toxins and ii) a
subject, wherein said
subject is a human or non-human animal; and b) contacting said subject with
said preparation (e.g.
under conditions such that said subject generates an immune response to said
one or more antigens).
In a preferred embodiment, the toxin is electrospun with one or more polymers
such as those
described herein so as to create an electrospun preparation. As discussed
below, in a preferred
embodiment, the electrospun preparation is part of a vaccine delivery system
or a cosmetic.
The present invention further contemplates, in one embodiment, a vaccine
delivery system
comprising a) a substrate such as described herein comprising asperities such
as those described
herein, such as microprojections or microneedles, and an embodiment of the
various preparations
(mentioned above) comprising polymer and toxin. In a preferred embodiment, the
asperities, such
as microprojections or microneedles have dimensions such that they penetrate
into the stratum
corneum, the epidermal layer, and, in some embodiments, the dermal layer. In
one embodiment, the
microprojections or microneedles have a length (or height) or less than 1000
microns, more
preferably less than 500 microns, and still more preferably less than 250
microns. The
microprojections may be formed in different shapes, such as needles, blades,
pins, punches, and
combinations thereof. When in an array, the density of the microprojections is
at least 10
microprojections/cm2, more preferably, at least 200 microprojections/cm2, and,
in some
embodiments, at least 1000 microprojections/cm2. The microneedles may be
hollow or solid.
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It is not intended that the present invention be limited by the nature of the
substrate
comprising said microneedles. In one embodiment, the microneedles are
formulated out of polymer.
In one embodiment, the microneedles are formulated (at least in part) out of a
polymer/antigen(s) or
polymer/toxin(s) preparation. In another embodiment, the microneedles are made
with a mold. In a
preferred embodiment, the microneedles are etched out of a silicon substrate.
In a preferred
embodiment, said silicon microneedles are solid and an electrospun
polymer/antigen(s) preparation
is deposited on said microneedles.
Thus, the present invention contemplates novel conjugates, formulations,
delivery devices,
methods of conjugation, methods of formulation, and methods of delivery. In
one embodiment, the
present invention contemplates a method of creating a formulation: providing
i) at least one fiber-
forming material, and ii) a preparation comprising at least one Clostridium
toxin; mixing said fiber-
forming material with said preparation so as to create a mixture; and
electrospinning said mixture to
create an electrospun formulation. In one embodiment, the method further
comprises: depositing at
least a portion of said electrospun material onto at least a portion of a
substrate to create a treated
substrate (e.g. microneedles, including but not limited to etched solid
microneedles). The present
invention also contemplates, as a device, the treated substrate prepared
according to the above-
described method. In one embodiment, said fiber-forming material comprises
hydroxypropylcellulose, while in another embodiment said fiber-forming
material comprises PVP.
In one embodiment, said preparation comprises first and second Clostridium
toxins. In one
embodiment, said first toxin comprises at least a portion of a C. difficile
toxin and said second toxin
comprises at least a portion of C. botulinum toxin. In one embodiment, said
first and second toxin
are part of a fusion protein.
In one embodiment, the present invention contemplates a method of creating a
toxin delivery
device: providing i) at least one fiber-forming material, ii) a preparation
comprising at least one
Clostridium toxin, and iii) a substrate comprising a plurality
microprojections; mixing said fiber-
CA 02650197 2008-10-21
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forming material with said preparation so as to create a mixture;
electrospinning said mixture to
create an electrospun material; and depositing at least a portion of said
electrospun material onto at
least a portion of said substrate, so as to create a toxin delivery device. In
one embodiment, said
Clostridium toxin is a C. botulinum toxin. The present invention also
contemplates, as a device, the
treated substrate prepared according to the above-described method. In one
embodiment, said fiber-
forming material comprises hydroxypropylcellulose, while in another embodiment
said fiber-
forming material comprises PVP.
The present invention, as mentioned above, also contemplates methods of
administering
antigens and vaccines. In one embodiment, the present invention contemplates a
method of
administering antigen, comprising: providing: a subject and the toxin delivery
device described
above; and contacting said subject with said antigen delivery device under
conditions such at least a
portion of said antigen is released from said device. In one embodiment, said
contacting of step b)
comprises piercing the subject's skin with said microprojections.
The present invention also contemplates, in one embodiment, a toxin delivery
device
comprising: a substrate having a back surface and a front surface; a plurality
of solid microneedles
extending upwards from the front surface of the substrate, the microneedles
comprising a
formulation, said formulation comprising at least one polymer and at least one
Clostridium toxin
(e.g. a C. botulinum toxin).
As used herein, the term "fusion protein" refers to a chimeric protein
containing the protein
of interest (i.e., C. botulinum toxin A or B and fragments thereof) joined to
an exogenous protein
fragment (the fusion partner which consists of a non-toxin protein). The
fusion partner may enhance
solubility of the C. botulinum protein as expressed in a host cell, may
provide an affinity tag to
allow purification of the recombinant fusion protein from the host cell or
culture supernatant, or
both. If desired, the fusion protein may be removed from the protein of
interest (i.e., toxin protein or
fragments thereof) prior to immunization by a variety of enzymatic or chemical
means known to the
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art.
As used herein, the term "poly-histidine tract" when used in reference to a
fusion protein
refers to the presence of two to ten histidine residues at either the amino-
or carboxy-terminus of a
protein of interest. A poly-histidine tract of six to ten residues is
preferred. The poly-histidine tract is
also defined functionally as being a number of consecutive histidine residues
added to the protein of
interest which allows the affinity purification of the resulting fusion
protein on a nickel-chelate
column.
The botulinal neurotoxin is synthesized as a single polypeptide chain which is
processed into
a heavy (H) and a light (L) chain; these two chains are held together via
disulfide bonds in the active
toxin [B.R. DasGupta and H. Sugiyama, Biochem. Biophys. Res. Commun. 48:108
(1972);
reviewed in B.R. DasGupta, J. Physiol. 84:220 (1990) and H. Sugiyama,
Microbiol. Rev. 44:419
(1980)]. Antisera raised against purified preparations of isolated H and L
chains have been shown to
protect mice against the lethal effects of the toxin; however, the
effectiveness of the two antisera
differ with the anti-H sera being more potent (H. Sugiyama, supra).
While the different botulinal toxins show structural similarity to one
another, the different
serotypes are reported to be immunologically distinct (i.e., sera raised
against one toxin type does
not cross-react to a significant degree with other types). Thus, the
generation of multivalent vaccines
may require the use of more than one type of toxin. Purification methods have
been reported for
native toxin types A, B, C, D, E, and F [reviewed in G. Sakaguchi, Pharmac.
Ther. 19:165 (1983)].
As the different botulinal toxins are structurally related, the invention
contemplates
polymer/toxin formulations with any of the botulinal toxins (e.g., types A-30
F) as soluble
recombinant fusion proteins, and in particular electrospun formulations. In
one preferred
embodiment, toxins A and B of C. difficile are contemplated as immunogens
(either separately or
together). In one embodiment, recombinant C. botulinum toxin proteins are used
as antigens in
mono- and multivalent vaccine preparations. For example, soluble,
substantially endotoxin-free
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recombinant C. botulinum type A toxin proteins may be used alone or in
conjunction with either
recombinant or native toxins or toxoids from C. botulinum, C. difficile and C.
tetani as antigens for
the preparation of these mono- and multivalent vaccines. It is contemplated
that, due to the structural
similarity of C. botulinum and C. tetani toxin proteins, a vaccine comprising
C. difficile and
botulinum toxin proteins (native or recombinant or a mixture thereof) be used
to stimulate an
immune response against C. botulinum, C. tetani and C. difficile.
The adverse consequences of exposure to botulinal toxin would be avoided by
immunization
of subjects at risk of exposure to the toxin with nontoxic preparations which
confer immunity such
as chemically or genetically detoxified toxin. In general, chemical
detoxification of bacterial toxins
using agents such as formaldehyde, glutaraldehyde or hydrogen peroxide can be
used for the
generation of vaccines or antitoxins. A delicate balance must be struck
between too much and too
little chemical modification. If the treatment is insufficient, the vaccine
may retain residual toxicity.
If the treatment is too excessive, the vaccine may lose potency due to
destruction of native
immunogenic determinants.
Vaccines which confer immunity against one or more of the toxin types A, B, E
and F would
be useful as a means of protecting humans from the deleterious effects of
those C. botulinum toxins
known to affect man. Vaccines which confer immunity against one or more of the
toxin types C, D
and E would be useful for veterinary applications.
The C fragment of the C. botulinum type A neurotoxin heavy chain has been
evaluated as a
vaccine candidate. The C. botulinum type A neurotoxin gene has been cloned and
sequenced [D.E.
Thompson et al., Eur. J. Biochem. 189:73 (1990)]. The C fragment of the type A
toxin was
expressed as either a fusion protein comprising the botulinal C fragment fused
with the maltose
binding protein (MBP) or as a native protein [H.F. LaPenotiere et al., supra].
The plasmid construct
encoding the native protein was reported to be unstable, while the fusion
protein was expressed in
inclusion bodies as insoluble protein. Immunization of mice with crudely
purified MBP fusion
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protein resulted in protection against IP challenge with 3 LD50 doses of toxin
[LaPenotiere et al.,
supra]. However, this recombinant C. botulinum type A toxin C fragment/MBP
fusion protein is not
a suitable immunogen for the production of vaccines as it is expressed as an
insoluble protein in E.
coli. Furthermore, this recombinant C. botulinum type A toxin C fragment/MBP
fusion protein was
not shown to be substantially free of endotoxin contamination. Experience with
recombinant C.
botulinum type A toxin C fragment/MBP fusion proteins shows that the presence
of the MBP on the
fusion protein greatly complicates the removal of endotoxin from preparations
of the recombinant
fusion protein.
Inclusion body protein must be solubilized prior to purification and/or
administration to a
host. The harsh treatment of inclusion body protein needed to accomplish this
solubilization may
reduce the immunogenicity of the purified protein. Ideally, recombinant
proteins to be used as
vaccines are expressed as soluble proteins at high levels (i.e., greater than
or equal to about 0.75% of
total cellular protein) in E. coli or other host cells. This facilitates the
production and isolation of
sufficient quantities of the immunogen in a highly purified form (i.e.,
substantially free of endotoxin
or other pyrogen contamination). The ability to express recombinant toxin
proteins as soluble
proteins in E. coli is advantageous due to the low cost of growth compared to
insect or mammalian
tissue culture cells.
In one embodiment, the vaccine comprises the C fragment of the C. botulinum
type A toxin
and a poly-histidine tract (also called a histidine tag). In a particularly
preferred embodiment, a
fusion protein comprising the histidine tagged C fragment (expressed using the
pET series of
expression vectors (Novagen)) is employed in one of the polymer/toxin
formulations of the present
invention, and in particular an electrospun formulation. The pET expression
system utilizes a vector
containing the T7 promoter which encodes the fusion protein and a host cell
which can be induced
to express the T7 DNA polymerase (i.e., a DE3 host strain). The production of
C fragment fusion
proteins containing a histidine tract is not limited to the use of a
particular expression vector and
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host strain. Several commercially available expression vectors and host
strains can be used to
express the C fragment protein sequences as a fusion protein containing a
histidine tract (For
example, the pQE series (pQE-8, 12, 16, 17, 18, 30, 31, 32, 40, 41, 42, 50,
51, 52, 60 and 70) of
expression vectors (Qiagen) which are used with the host strains M15[pREP4]
(Qiagen) and
SG13009[pREP4] (Qiagen) can be used to express fusion proteins containing six
histidine residues
at the amino-terminus of the fusion protein).
When recombinant clostridial toxin proteins produced in gram-negative bacteria
(e.g., E.
coli) are used as vaccines, they are purified to remove endotoxin prior to
administration to a host
animal. In order to vaccinate a host, an immunogenicallyeffective amount of
purified substantially
endotoxin-free recombinant clostridial toxin protein is administered in any of
a number of
physiologically acceptable carriers known to the art. When administered for
the purpose of
vaccination, the purified substantially endotoxin-free recombinant clostridial
toxin protein may be
used alone or in conjunction with known adjutants, including potassium alum,
aluminum phosphate,
aluminum hydroxide, Gerbu adjuvant (GmDP; C.C. Biotech Corp.), RMI adjuvant
(MPL; RJBI
Immunochemical research, Inc.), QS21 (Cambridge Biotech). The alum and
aluminum-based
adjutants are particularly preferred when vaccines are to be administered to
humans.
A variety of routes of immunization may be used, such as nasal, oral,
intramuscular, intra-
peritoneal, etc. However, it is preferred that immunization be through the
skin (e.g. subcutaneous, or
more preferably transdermally, intradermally or transmucosally using
asperities such as
microprojections or microneedles, as discussed herein).
In one embodiment, the present invention contemplates using Clostridium
antigen(s) and/or
toxin(s) in polymer formulations for vaccines and cosmetics. In a preferred
embodiment, such
antigens and toxins are electrospun with one or more polymers or hydrogels,
such as those discussed
herein.
In a preferred embodiment, the preparation comprising Clostridium antigen(s)
or toxin(s) is
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capable, after electrospinning, of acting as an antigen by eliciting an immune
response in a human or
animal. It is envisioned that in such an embodiment, a medically acceptable
fiber-forming material
may be used to preserve the antigen for later rehydration and use as a
vaccine. In general,
rehydration of the fiber of the present invention may be accomplished by
mixing the fiber with a
solvent for the fiber-forming material. When the fiber is used to preserve an
antigen for use in a
vaccine, the solvent will optimally be a medically acceptable compound.
It is not intended that the present invention be limited to the method by
which the
electrospun antigen(s) or toxin(s) is administered. The resulting vaccine may
be injected or an
ingested vaccine. However, in a preferred embodiment, the vaccine is
administered at or in the skin.
In a particularly preferred embodiment, the electrospun polymer/ antigen(s)
material is delivered on
microneedles or on a patch such that antigen(s) is released into the skin.
In one non-limiting embodiment, the toxin may be contained in an ionically
cross-linked
macromolecular assembly, such as those hereinabove described, including but
not limited to
hydrogels, such as those hereinabove described.
In one embodiment, the present invention contemplates a method of
administering one or
more antigens such as the toxins hereinabove described, to a patient
comprising the steps of
penetrating at least an outer layer of the skin of the patient with one or
more microneedles extending
from a front surface of a substrate, at least one of the one or more
microneedles (or portion thereof)
contacted or coated (uniformly or non-uniformly) by an antigen preparation
(discussed above), and
releasing at least one of the one or more antigens into the patient.
One use of microneedle vehicle for delivery of the polymer/toxin(s)
preparations described
above involves cosmetic applications, and in particular, facial cosmetic
applications. The
administration of an appropriate dose of, for example, botulinum toxin, to
attenuate tone of muscles
about the eyes and forehead can in many cases remove wrinkles characteric of
aging in skin
overlying the muscle while inducing only mild, often acceptable muscle
weakness. Such
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chemodenervating agents also may be used to induce cosmetic improvement in
hemifacial paralysis
by intentionally inducing partial paralysis in the contralateral side of the
face thereby to improve
bilateral facial symmetry.
Administration of low doses of the agents into muscles activating the jaw can
retard tooth
wear caused by involuntary or unconscious clenching of the teeth.
Examples of direct effects are the treatment of unwanted involuntary
pathologic muscle
stimulation, i.e., spasm, rigidity, or hyperstimulation, by direct
administration throughout, or in the
area of innervation of, the affected muscle or muscles. Thus, diseases
involving muscle spasticity in
general can be treated, typically without regard for its cause. The drug may
be used to alleviate
overstimulation, rigidity, or spasticity in muscle or muscle groups caused by
stroke, cerebral palsey,
multiple sclerosis, unilateral or bilateral parkinsonism, and other diseases
characterized by
spasmodic or continuous muscle hyperstimulation.
Accordingly, it is an object of the invention to provide a novel method of
standardizing
chemodenervating neurotoxin-derived pharmaceuticals such as botulinumderived
pharmaceuticals
by using microneedle administration. Another object is to standardize
botulinum toxin preparations
with respect to their zone of denervation when administered in vivo. Another
object is to provide
novel (lower) dosage forms of such agents. Yet another object is to provide
novel therapies for
muscle spasticity andlor hyperactivation heretofore untreatable or treatable
only imperfectly with
systemic drugs or surgery.
While many such neurotoxins are known, the currently most promising reagents
of this type
are the family of toxins derived from Clostridium botulinum, and the most
preferred is
pharmaceutical grade botulinum toxin Type A available commercially from
Allergan
Pharmaceuticals, Inc. under the tradename OCULINUM. The therapetic effects are
achieved at
dosage levels in the range between a few I.U. to 500 to 1000 I.U., preferably
no more than 500 I.U.,
and most preferably no more than 300 I.U., and still most preferably between
50 I.U. and 200 I.U.,
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administered on a microneedle array about a muscle or muscle group. The
dimensions of the
microneedle array control the zone of destruction of a subset of the
neuromuscular junctions
innervating the muscle, leaving others in a functional state.
Facial expression lines such as the transverse forehead lines or the
nasolabial fold are created
by attachments of projections of facial muscles into the dermis. Contraction
of facial muscles
generally is well known to produce the various characteristic forms of facial
expressions such as
smiling, grimacing, etc. In addition, exaggeration of facial lines also is
associated with the aging
process. The general principle of the application of the toxin is to limit the
tonic contractile state of
facial muscles so as to reduce muscle tone and to improve or change the
quality and characteristics
of facial expression.
The transverse forehead lines may be reduced in intensity by administering a
quantity of
toxin with a diffusion field of approximately 5 to 10 mms at the superior
border of the forehead and
at a point approximately 15 mms superior to the brow. This can be done
symmetrically on both sides
of the forehead. The glabellar lines (the frowning lines in the mid position
of the forehead) may be
targeted by treated the glabellar muscles with a toxin quantity producing a
field of denervation of 5
to 10 mms The toxin is administered 15 mms. above the brow line in the mid
position of the
forehead. The nasal labial fold lines can be diminished in their intensity by
treating the zygomatic
major and minor muscles which emanate from the zygomatic arch and extend
diagonally to the
position of the nasal labial fold.
Thus, in one embodiment, the present invention contemplates a method of
cosmetically
decreasing facial wrinkling in a subject, the method comprising: providing i)
a subject, and ii) a
microneedle array comprising a chemodenervating pharmaceutical in a polymer
formulation; and
placing said microneedle array on the skin of said subject under conditions
whereby said wrinkling
is reduced. In a preferred embodiment said denervating pharmaceutical
comprises a neurotoxin
derived from Clostridium botulinum. In a particularly preferred embodiment,
said neurotoxin is in
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the form of a fusion protein. In one embodiment, said chemodenervating agent
is Acetyl
hexapeptide-3 (trade name Argireline). Argireline is manufactured by a Spanish
company Lipotec
and is a hexapeptide (a chain of 6 amino acids) attached to the acetic acid
residue. It is believed to
work by inhibiting the release of neurotransmitters. When applied to the skin,
Argireline relaxes
facial tension leading to the reduction in facial lines and wrinkles with
regular use.
In a further embodiment, the present invention contemplates a method of
decreasing
sweating (e.g. excessive sweating of the underarms, palms, feet, etc.) of a
subject, the method
comprising: providing i) a subject, and ii) a microneedle array comprising a
chemodenervating
pharmaceutical in a polymer formulation; and placing said microneedle array on
the skin of said
subject under conditions whereby said sweating is reduced. In a preferred
embodiment said
denervating pharmaceutical comprises a neurotoxin derived from Clostridium
botulinum. In a
particularly preferred embodiment, said neurotoxin is in the form of a fusion
protein.
Alternatively, the present invention contemplates, in one embodiment, that
alpha-
Bungarotoxin molecules can be used in the manner of botulinum toxin A. In one
embodiment,
native alpha-Bungarotoxin is employed. As used herein, "native"
alphabungarotoxin molecules are
those found in the venom of Bungarus multicinctus. The complete amino acid
sequence of native
alpha-bungarotoxin molecules and the DNA sequences that encode them have been
published (see,
e.g., GenBank accession numbers X91990, AF056400-AF056417, AJ131356, Y17057,
Y17058,
Y 17693, Y 17694). In one embodiment, the native sequence is
IVCHTTATSPISAVTCPPGENLCYRKMWCD-
AFCSSRGKV VELGCAATCPSKKPYEEVTCCSTDKCNPHPKQRPG.
In a preferred embodiment, modified alpha-bungarotoxin molecules that have
altered binding
specificities are employed. In particular, modification of residues 38 (Lys)
and 42 (Leu) to Pro and
Gln, respectively, altered the binding specificity of alphabungarotoxin from
alpha7-containing
nicotinic acetylcholine receptors to alpha3beta.2-containing nicotinic
acetylcholine receptors.
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Additional modifications to these amino acid residues and other amino acid
residues are possible.
In another non-limiting embodiment, the at least one biologically active agent
is an anthrax
antigen. Anthrax is an acute infectious disease caused by the bacterium B.
anthracis. It occurs in
wild and domestic lower vertebrates (cattle, sheep, goats, etc.), but can also
occur in humans (e.g. do
to exposure to infected animals or because of bioterrorism). The virulence of
B. anthracis is
dependent on Anthrax Toxin and the capsule, a polymer of gamma-D-glutamic
acid. Anthrax Toxin
(AT) consists of two enzymatic moieties, edema factor (EF) and lethal factor
(LF), and a single
receptor-binding moiety, protective antigen (PA).
The capsule is a poor immunogen. Wild-type PA (the amino acid sequence of
which is
shown in Figure 18) is also a poor immunogen. Indeed, the currently licensed
human anthrax
vaccine (AVA, BioPort Corporation, Lansing Mich.) requires six vaccinations
over eighteen months
followed by yearly boosters to induce and maintain protective anti-PA titers
(Pittman et al., Vaccine
20:1412-20, 2002; Pittman et al., Vaccine 20:972-78, 2001). In some vaccines,
this regimen is
associated with undesirable side effects (Pittman et al., Vaccine 20:972-78,
2001). Thus, there is a
need for an effective and safe vaccine that would require fewer doses to
confer immunity to anthrax.
The present invention, in a non-limiting embodiment, an anthrax antigen (or
fragment
thereof) in a preparation suitable for transdermal, intra-epidermal,
intradermal or intramucosal
delivery. In another embodiment, antigen from Y. pestis or F. tularensis is
contemplated in a
preparation suitable for transdermal delivery. In a preferred embodiment, the
present invention
contemplates mutant anthrax protective antigen (e.g. the mutant antigens set
forth in Table 4 below,
or fragment thereof) in a preparation suitable for transdermal delivery.
Table 4. amino acid residues at which Cys substitutions inhibited PA acrivity
by at least 100-
fold.
Residues
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Domain
1 1210, K225, T240, K245
2 S337, G342, W346, T357, I364, P379, T380, S382, T390, T393, K397,
N399, Y411, I419, A420, N422, F427, S248, D451, D453, V455, N458
3 E515
4 1656, N657, I665, D683, L687
In one embodiment, the mutant protective antigen (or fragment thereof) is
attached or linked
to a carrier. In one embodiment, the mutant protective antigen (or fragment
thereof) is part of a
fusion protein. In one embodiment, the mutant protective antigen (or fragment
thereof) is in a
micelle formulation. In one embodiment, the mutant protective antigen (or
fragment thereof) is in or
on nanofibers, nanotubes, nanospheres, or nanocapsules. In one embodiment, the
mutant protective
antigen (or fragment thereof) is complexed with one or more dendrimers. In one
embodiment, the
mutant protective antigen (or fragment thereof) is in a liposome preparation
(e.g. for liposome
delivery). In a preferred embodiment, the mutant protective antigen (or
fragment thereof) is on the
surface of a substrate, e.g. on a skin patch, such as an array, of asperities
such as those described
herein, such as microprojections or microneedles or the like. Combinations of
the above are
contemplated specifically, including but not limited to substrates comprising
the mutant protective
antigen (or fragment thereof) in a polymer formulation, or in an assembly of
nanofibers. In a
particularly preferred embodiment, the mutant is functionally impaired (e.g.
it will not form pores
or channels, or will otherwise not cause toxicity).
The present invention contemplates, in one embodiment, a preparation suitable
for
transdermal, intradermal, intra-epidermal, transmucosal, subcutaneous,
intravenous, or
intramuscular delivery comprising a first anthrax antigen (or fragment
thereof) in a mixture with a
second anthrax antigen (or fragment thereof). The present invention
contemplates, in one
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embodiment, a preparation suitable for transdermal, transmucosal,
subcutaneous, intravenous, or
intramuscular delivery comprising a first anthrax antigen (or fragment
thereof) conjugated to a
second anthrax antigen (or fragment thereof). These first and second antigens
(or fragments thereof)
can be prepared simply as a mixture or, alternatively, they can be linked
together in a conjugate. In
one embodiment, the first and second antigens (or fragments thereof) are in a
micelle formulation. In
one embodiment, the first and second antigens (or fragments thereof) are in or
on nanofibers,
nanotubes, nanospheres, or nanocapsules. In one embodiment, the first and
second antigens (or
fragments thereof) are complexed with one or more dendrimers. In one
embodiment, the first and
second antigens (or fragments thereof) are in a liposome preparation (e.g. for
liposome delivery). In
a preferred embodiment, the first and second antigens (or fragments thereof)
are on the surface of a
substrate, e.g. on a skin patch, microneedle array, or the like. Combinations
of the above are
specifically contemplated, including but not limited to substrates comprising
the first and second
antigens (or fragments thereof) in a polymer formulation, or in an assembly of
nanofibers. While
edema factor and/or lethal factor (or fragments thereof) can be used as first
and second antigens, in a
preferred embodiment, said first and second antigens comprises (respectively)
poly-glutamic acid
and protective antigen (or fragment thereof). The present invention
contemplates, in one
embodiment, a preparation suitable for transdermal delivery comprising wild-
type protective antigen
or mutant protective antigen (e.g. the mutant antigens set forth in Table 4)
conjugated to poly-
glutamic acid. In a preferred embodiment, the present invention contemplates
an array of
microprojections or microneedles comprising anthrax antigen(s) selected from
the group consisting
of wild-type protective antigen (or fragment thereof), mutant protective
antigen (or fragment
thereof), a conjugate of wild-type protective antigen (or fragment thereof)
and poly-glutamic acid,
and a conjugate of mutant protective antigen (or fragment thereof) and poly-
glutamic acid. In a
particularly preferred embodiment, the mutant is functionally impaired (e.g.
it will not form pores or
channels, or will otherwise not cause toxicity). In a preferred embodiment,
the microprojections or
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microneedles have dimensions such that they penetrate into the stratum comeum,
the epidermal
layer, and, in some embodiments, the dermal layer. In one embodiment, the
microprojections or
microneedles have a length (or height) or less than 1000 microns, more
preferably less than 500
microns, and still more preferably less than 250 microns. The microprojections
may be formed in
different shapes, such as needles, blades, pins, punches, and combinations
thereof. When in an array,
the density of the microprojections is at least microprojections/cm2, more
preferably, at least 200
microprojections/cm2, and, in some embodiments, at least 1000
microprojections/cmz.
The present invention further contemplates, in one embodiment, a preparation
comprising at
least one polymer and at least one anthrax antigen (or fragment thereof). In
another embodiment,
antigen from Y. pestis or F. tularensis is contemplated in such a polymer
preparation. It is not
intended that the present invention be limited by the nature of the polymer
since a variety of types
can be used (see Table 1). In one embodiment, cross-linked polymers are
employed. However, in a
preferred embodiment, the polymer comprises polyvinylpyrrolidone or
hydroxypropylcellulose. It is
not intended that the present invention be limited by the nature of the
antigen(s) in the polymer
preparation. While anthrax edema factor and/or lethal factor (or fragments
thereof) can be use, in
one embodiment, the antigen(s) is/are selected from the group consisting of
wild-type anthrax
protective antigen (or fragment thereof), mutant protective antigen (or
fragment thereof), a
conjugate of wild-type protective antigen (or fragment thereof) and poly-
glutamic acid, and a
conjugate of mutant protective antigen (or fragment thereof) and poly-glutamic
acid. In a
particularly preferred embodiment, the mutant is functionally impaired (e.g.
it will not form pores
or channels, or will otherwise not cause toxicity). It is contemplated that
the polymer/antigen(s)
preparation can be administered by a variety of routes (e.g. IP, IM, IV,
inhalation, transdermal,
intradermal, intra-epidermal, transmucosal, subcutaneous, etc.)
It is not intended that the present invention be limited to the manner in
which the preparation
of polymer and antigen(s) is prepared. However, in one embodiment, the
preparation is electrospun
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to create fibers or fiber mats. It is preferred that at least a portion of the
antigen in the electrospun
fibers or mats be capable of release or escape from the polymer (e.g. upon
contact with an aqueous
or semi-aqueous environment).
While edema factor and/or lethal factor can be used as antigens, in one
embodiment, said
antigen in said polymer preparation comprises poly-glutamic acid and
protective antigen (or
fragment thereof). These antigens can be prepared simply as a mixture.
However, in a preferred
embodiment, the antigen comprises a conjugate of poly-glutamic acid and
protective antigen (or
fragment thereof). In one embodiment, the conjugate is prepared by covalently
linking poly-
glutamic acid to protective antigen (or fragment thereof). In one embodiment,
the protective
antigen (or fragment thereof) is wild-type. In a preferred embodiment, the
protective antigen (or
fragment thereof) is a mutant. In a particularly preferred embodiment, the
mutant is functionally
impaired (e.g. it will not form pores or channels, or will otherwise not cause
toxicity).
It is not intended that the present invention be limited to a particular
mutant. A variety of
mutants have been described that are functionally impaired. For example, U.S.
Patent No.
7,037,503 (hereby incorporated by reference) to Collier et al. describes a
group of dominant
negative mutants having point mutations at positions 397 and/or 425 of the PA
protein (see Figure
18 for the wild-type sequence of the PA protein). In addition, Mourez et al.
(PNAS 100:13803
2003) describe 568 mutants, thirty-three of which demonstrate reduced ability
to mediate toxicity
(see Table 4). The present invention contemplates various embodiments wherein
one or more of
such mutants are employed (whether conjugated or unconjugated).
It is not intended that the present invention be limited to the nature of the
conjugates (when
conjugates are used). U.S. Patent Application 20060134143 to Schneerson et al.
(hereby
incorporated by reference) describes immunogenic conjugates of poly-gamma-
glutamic acid (PGA)
made using a variety of different PGA types (including but not limited to PGA
comprising D-
glutamic acid and PGA comprising L-glutamic acid) extracted from culture
supernatants of B.
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anthracis. In one example, recombinant wild-type PA was derivatized with
adipic acid dihydrazide
and reacted. On the other hand, Aulinger et al. (Infection & Immunity 73:3408
2005) describes
conjugating PA (or a PA mutant) to purified PGA via a carbodiimide reaction.
Poly-L-glutamic acid
and poly-D-glutamic acid sodium salt are commercially available from Sigma-
Aldrich (St. Louis).
Protective antigen and fragments thereof (i.e. the 63kDa and 20kDa fragments)
are commercially
available from List Biological Laboratories, Inc. When a fragment is used, it
is preferred that the
63kDa fragment is used (or a mutant thereof).
The present invention further contemplates, in one embodiment, a preparation
comprising at
least one anthrax antigen (or fragment thereof) in a hydrogel formulation. In
another embodiment,
antigen from Y. pestis or F. tularensis is contemplated in such a preparation.
It is not intended that
the present invention be limited by the nature of the hydrogel since a variety
of types can be used,
including but not limited to ionically cross-linked hydrogels or hydrogel
films. It is not intended that
the present invention be limited by the nature of the antigen(s) in the
hydrogel preparation. While
edema factor and/or lethal factor (or fragments thereof) can be use, in one
embodiment, the
antigen(s) is/are selected from the group consisting of wild-type protective
antigen (or fragment
thereof), mutant protective antigen (or fragment thereof), a conjugate of wild-
type protective
antigen (or fragment thereof) and poly-glutamic acid, and a conjugate of
mutant protective antigen
(or fragment thereof) and poly-glutamic acid. In a particularly preferred
embodiment, the mutant is
functionally impaired (e.g. it will not form pores or channels, or will
otherwise not cause toxicity). It
is contemplated that the hydrogel/antigen(s) preparation can be deposited on a
surface (in a uniform
or non-uniform manner). In one embodiment, the preparation is deposited (e.g.
by dipping, coating,
spin coating, spraying, or by suitable applicator) on an array of
microprojections or microneedles for
transdermal or transmucosal delivery of the antigen(s).
Regardless of whether a mutant or wild-type is used, the present invention
contemplates
administering one or more of the various embodiments of the preparations
described above as a
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vaccine to humans and animals. In one embodiment, the present invention
contemplates a method
comprising: a) providing i) a preparation comprising at least one polymer and
one or more anthrax
antigens (or fragments thereof) and ii) a subject, wherein said subject is a
human or non-human
animal; and b) contacting said subject with said preparation (e.g. under
conditions such that said
subject generates an immune response to said one or more antigens). In one
embodiment, the present
invention contemplates a method comprising: a) providing i) a preparation
comprising one or more
anthrax antigens (or fragments thereof) in a hydrogel formulation and ii) a
subject, wherein said
subject is a human or non-human animal; and b) contacting said subject with
said preparation (e.g.
under conditions such that said subject generates an immune response to said
one or more antigens).
In a preferred embodiment, a conjugate of two anthrax antigens is employed (as
discussed above). In
a preferred embodiment, the conjugate is electrospun with one or more polymers
so as to create an
electrospun preparation. As discussed below, in a preferred embodiment, the
electrospun preparation
is part of a vaccine delivery system. In a preferred embodiment, the
electrospun preparation is
delivered transdermally, intradermally, intra-epidermally, transmucosally,
subcutaneously, IP, IV or
IM.
The present invention further contemplates, in one embodiment, a vaccine
delivery system
comprising a) a substrate comprising microprojections or microneedles,and an
embodiment of the
various preparations (mentioned above) comprising either i) antigen(s) alone
(or fragments thereof),
ii) antigen(s) (or fragments thereof) and carrier alone, iii) polymer (or
hydrogel) and antigen(s) (or
fragments thereof), iv) conjugates of poly-glutamic acid and wild-type or
mutant protective antigen
(or fragment thereof), or v) iv) polymer (or hydrogel) and conjugates of poly-
glutamic acid and
wild-type or mutant protective antigen (or fragment thereof). When
microneedles are used, they may
be solid or hollow. It is not intended that the present invention be limited
to the manner in which
the microneedles are contacted with the preparation. In one embodiment, the
microneedles are
contacted only at the microneedle tips. In a preferred embodiment, the
preparation contacts the
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entire substrate (or substantially the entire substrate) such that the
preparation contacts even the
spaces between an may of microneedles. It is not intended that the present
invention be limited to
uniform coatings of the preparation. In one embodiment, the preparation is
present on the substrate
in a non-uniform manner.
In one embodiment, a polymer/antigen(s) preparation is sprayed on said
microneedles. In
one embodiment, said microneedles are dipped into a preparation comprising at
least one polymer
and at least one anthrax antigen (or fragment thereof). In a preferred
embodiment, an electrospun
polymer/antigen(s) preparation is deposited on said microneedles. In another
embodiment, antigen
from Y. pestis or F. tularensis is contemplated in such an electrospun
polymer/antigen(s) preparation
and this preparation is deposited on said microneedles.
In one embodiment, the present invention contemplates using at least two
different anthrax
protective antigens (or fragments thereof). For example, in order to generate
effective immunity
against more virulent strains of Bacillus anthracis, such as Vollum 1 B, the
Ames strain, two or more
different sources of protective antigens (or fragments thereof) are
contemplated in one embodiment,
and mutants in another embodiment. Protective antigen from variants of such
strains are also
contemplated.
Thus, the present invention contemplates novel conjugates, formulations,
delivery devices,
methods of conjugation, methods of formulation, and methods of delivery. In
one embodiment, the
present invention contemplates a method of creating a formulation: providing
i) at least one fiber-
forming material, and ii) a preparation comprising at least one anthrax
antigen; mixing said fiber-
forming material with said preparation so as to create a mixture; and
electrospinning said mixture to
create electrospun formulation. In one embodiment, the method further
comprises: depositing at
least a portion of said electrospun material onto at least a portion of a
substrate to create a treated
substrate (e.g. a substrate comprising microneedles). The present invention
also contemplates, as a
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device, the treated substrate prepared according to the above-described
method. In one embodiment,
said fiber-forming material comprises hydroxypropylcellulose, while in another
embodiment said
fiber-forming material comprises PVP. In one embodiment, said preparation
comprises first and
second anthrax antigens (e.g. said first anthrax antigen comprises poly-gamma-
glutamic acid and
said second anthrax antigen comprises protective antigen). In one embodiment,
said protective
antigen is wild-type, while in another embodiment, said protective antigen is
mutant. In a preferred
embodiment, said first and second antigens are linked to form a conjugate.
In one embodiment, the present invention contemplates a method of creating an
antigen
delivery device, comprising: providing i) at least one fiber-forming material,
ii) a preparation
comprising at least two anthrax antigens, and iii) a substrate comprising a
plurality of
microprojections; mixing said fiber-forming material with said preparation so
as to create a mixture;
electrospinning said mixture to create electrospun material; and depositing at
least a portion of said
electrospun material onto at least a portion of said substrate, so as to
create an antigen delivery
device. In one embodiment, said two anthrax antigens comprises poly-gamma-
glutamic acid and
protective antigen (e.g. linked to form a conjugate). In one embodiment, said
protective antigen is
wild-type, while in another embodiment, said protective antigen is mutant. In
one embodiment, said
fiber-forming material comprises hydroxypropylcellulose, while in another
embodiment said fiber-
forming material comprises PVP. The present invention also contemplates, as a
device, the antigen
delivery device prepared according to the above-described method.
The present invention also contemplates, in one embodiment, a drug delivery
device
comprising: a substrate having a back surface and a front surface; a plurality
of solid microneedles
extending upwards from the front surface of the substrate, the microneedles
comprising a
formulation, said formulation comprising at least one polymer and at least one
anthrax antigen (e.g.
a two antigen conjugate as described above).
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Anthrax toxin is a member of the binary bacterial toxins, a subset of
intracellularly acting
toxins in which the enzymatic and receptor-binding moieties are secreted by
the bacteria as discrete
monomeric proteins. While an understanding of the mechanism of action of these
toxins is not
needed for the successful practice of the various embodiments of the present
invention, it is believed
these proteins assemble at the surface of receptor-bearing eukaryotic cells to
form toxic hetero-
oligomeric complexes. The complexes are internalized and delivered to an
acidic compartment,
where the receptor-binding moiety inserts into the membrane and mediates
translocation of the
enzymatic moiety to the cytosol. Within the cytosol, the enzymatic moiety
modifies a molecular
target, disrupting cell physiology and causing cytopathic effects.
Anthrax toxin consists of two enzymatic moieties, edema factor (EF; 89 kDa)
and lethal
factor (LF; 90 kDa), and a single receptor-binding moiety, protective antigen
(PA; 83 kDa), named
for its ability to elicit protective immunity. The amino acid sequence for
wild-type PA is shown in
Figure 18. The nucleic acid sequence encoding wild-type PA is shown in Figure
19. EF is a Ca 2+
and calmodulin-dependent adenylate cyclase, and LF is a ZnZ+ -dependent
protease that cleaves
mitogen-activated protein kinase kinases.
The crystallographic structure of intact PA has been solved and shows that the
protein is
organized into four domains. Domain 1, the N-terminal domain, contains the
furin cleavage site and
therefore encompasses PA20. That portion of domain 1 within PA63, termed
domain F, contains two
calcium atoms and participates in EF/LF binding and oligomerization. Domain 2
contains a flexible
loop (2132-2133) that is believed to form the transmembrane region of the
pore. While not limiting
the invention in any manner by reference to proposed mechanisms, it is
believed (based on ion
conductance experiments on Cys-substitution mutants) that the 2132-2133 loops
from the seven
subunits of the prepore combine to form a transmembrane 14-strand 13 -barrel.
Domain 3 is
believed to be involved in oligomerization, and domain 4, the C-terminal
domain, contains the
receptor-binding site. In addition to monomeric PA, the structure of a soluble
form of the PA63
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heptamer has been solved; it is believed to represent the conformation of the
heptamer before pore
formation and has been termed the prepore.
PA mutants have been described that block the intoxication process. There has
been some
interest in mutations that make the protein dominantly negative (DN), thereby
converting it into a
potent antitoxin. Without limiting the invention in any manner by reference to
proposed
mechanisms, it is believed that DN-PA co-oligomerizes with wild-type PA63 and
inhibits its ability
to form pores and mediate translocation. Point mutations at D425 and F427, a
deletion or
substitution of the 2132-2133 loop, or combinations of these mutations, have
been shown to confer
varying degrees of DN activity on PA. Interestingly, the conjugation of such
a=DN mutant to PGA
has been reported to generate a much improved immune response (as compared to
wild-type PA
alone or even wild-type PA conjugated to PGA). Aulinger et al. (Infection &
Immunity 73:3408
2005). In one embodiment, the present invention contemplates transdermal
delivery of such
conjugates, wherein said mutant comprises a substitution at K397, D425 or
both. In a preferred
embodiment, said mutant is selected from the group consisting of K397A, K397D,
K397C, and
K397Q. In another embodiment, said mutant is selected from the group
consisting of D425A,
D425N, D425E, and D425K. In another embodiment, said mutant is defined by a
substitution at
position F427 (including but not limited to F427A, F427D, and F427K). In one
embodiment, the
present invention contemplates transdermal delivery of such conjugates,
wherein said mutant
comprises a substitution at K397, D425 and F427. In one embodiment, the
present invention
contemplates transdermal delivery of such conjugates, wherein said mutant
comprises a deletion or
substitution of the 213z-2 133 loop.
The analysis of large arrays of PA mutants has recently become feasible,
following
identification of ways to obtain small-scale preparations of PA mutants and
screen them rapidly and
reliably for defects in PA functions. To search PA for additional DN sites,
researchers at the
University of Oklahoma developed a protocol to mutate each of the 568 amino
acids of PA63 to Cys
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and characterize the resulting mutants. They restricted their study to PA63
because PA20 does not
appear to play a role in intoxication besides preventing toxin assembly in
solution. Cys-
replacements were chosen because this amino acid is absent from PA and because
the thiol-
containing side chain is amenable to specific derivatization, facilitating
structure-function studies.
These researchers first screened for mutations that a caused large (>100-fold)
reduction in PA's
ability to mediate toxicity (these mutations are listed in Table 4). Such
mutations were identified in
all four domains, but a majority were in domain 2. Mutations conferring the DN
phenotype were
found exclusively within domain 2, in the 2136 strand, the 2137 strand, or the
21310-21311 loop.
Because DN mutants prevent the conformational transition of PA63 from the
prepore to the pore
state, they concluded that these structural elements play major roles in this
change.
As noted above, in another embodiment, antigen from Y. pestis or F. tularensis
is
contemplated in a preparation suitable for transdermal delivery. It is not
intended that the present
invention be limited by the particular antigens from these organisms. However,
in one embodiment,
an Fl capsule antigen from Y. pestis is employed in a polymer formulation
(e.g. electrospun
formulation) deposited on a microneedle array. In another embodiment, the LcrV
protein (or
fragment thereof) from Y. pestis is similarly employed. In a particular
embodiment, these two
antigens are used together (as a mixture, conjugate or fusion protein). In
still another embodiment,
one or more virulence-associated factors are employed, including but not
limited to catalase-
peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and Fl
capsule antigen (Can)
from Y. pestis. In one embodiment, a specific sequence of the outer membrane
protein encoded by
the fopA gene of F. tularensis is employed.
In one embodiment, the present invention contemplates using new and previously
identified
PA mutants (or fragments thereof) in vaccines. In a preferred embodiment, such
mutants are
conjugated to PGA and electrospun with one or more polymers.
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In a preferred embodiment, the preparation comprising anthrax antigen(s) is
capable, after
electrospinning, of acting as an antigen by eliciting an immune response in a
human or animal. It is
envisioned that in such an embodiment, a medically acceptable fiber-forming
material may be used
to preserve the antigen for later rehydration and use as a vaccine. In
general, rehydration of the fiber
of the present invention may be accomplished by mixing the fiber with a
solvent for the fiber-
forming material. When the fiber is used to preserve an antigen for use in a
vaccine, the solvent will
optimally be a medically acceptable compound.
It is not intended that the present invention be limited to the method by
which the
electrospun anthrax antigen(s) is administered. The resulting vaccine may be
injected or an ingested
vaccine. However, in a preferred embodiment, the vaccine is administered at or
in the skin. In a
particularly preferred embodiment, the electrospun polymer/anthrax antigen(s)
material is delivered
on microneedles or on a patch such that antigen(s) is released into the skin.
In one embodiment, the anthrax antiget(s) may be contained in an ionically
cross-linked
macromolecular assembly such as those hereinabove described, including a
hydrogel, as described
hereinabove.
In one embodiment, the present invention contemplates a method of
administering one or
more anthrax antigens to a patient comprising the steps of penetrating at
least an outer layer of the
skin of the patient with one or more microneedles extending from a front
surface of a substrate, at
least one of the one or more microneedles (or portion thereof) contacted or
coated (uniformly or
non-uniformly) by an anthrax antigen preparation (discussed above), and
releasing at least one of the
one or more anthrax antigens into the patient.
In another non-limiting embodiment, the at least one biologically active agent
is a hormone.
Osteoporosis is a bone disorder characterized by progressive bone loss that
predisposes an
individual to an increased risk of fracture, typically in the hip, spine and
wrist. The progressive bone
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loss, which typically begins between the ages of 40 and 50, is mainly
asymptomatic until a bone
fracture occurs, leading to a high degree of patient morbidity and mortality.
Eighty percent of those
affected by osteoporosis are women and, based on recent studies, during the
six years following the
onset of menopause, women lose one third of their bone mass.
Parathyroid hormone (PTH) is a hormone secreted by the parathyroid gland that
regulates the
metabolism of calcium and phosphate in the body. PTH has stirred great
interest in the treatment of
osteoporosis for its ability to promote bone formation and, hence,
dramatically reduced incidence of
fractures. Large-scale clinical trials have shown that PTH effectively and
safely reduces the
percentage of vertebral and non-vertebral fractures in women with
osteoporosis. PTH-based agents
have also stirred interest in the treatment of bone fractures (in both men and
women) by virtue of
their ability to accelerate bone healing.
To this end, various stabilized formulations of PTH-based agents have been
developed that
can be reconstituted for subcutaneous injection, which, as discussed below, is
the conventional
means of delivery. Illustrative are the formulations disclosed in U.S. Pat.
No. 5,563,122 ("Stabilized
Parathyroid Hormone Composition") and U.S. patent application Pub. No.
2002/0107200
("Stabilized Teriparatide Solutions"), which are incorporated by reference
herein in their entirety.
A currently approved injectable PTH-based agent is FORTEO (an rDNA derived
teriparatide
injection), which contains recombinant human parathyroid hormone (1-34),
(rhPTH (1-34)).
FORTEOTM is typically prescribed for women with a history of osteoporotic
fracture, who have
multiple risk factors for fracture, or who have failed or are intolerant of
previous osteoporosis
therapy, based on a physician's assessment. In postmenopausal women with
Osteoporosis,
FORTEOTM has been found to increase bone mineral density (BMD) and reduce the
risk of vertebral
and non-vertebral fractures.
FORTEOTM has also been found to increase bone mass in men with primary or
hypogonadal
osteoporosis who are at high risk for fracture. These include men with a
history of osteoporotic
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fracture, or who have multiple risk factors for fracture, or who have failed
or are intolerant to
previous osteoporosis therapy. In men with primary or hypogonadal
osteoporosis, FORTEOTM has
similarly been found to increase BMD.
Despite the efficacy of PTH in treating disorders such as osteoporosis, there
are several
drawbacks and disadvantages associated with the disclosed prior art methods of
delivering PTH,
particularly, via subcutaneous injection. A major drawback is that
subcutaneous injection is a
difficult and uncomfortable procedure, which often results in poor patient
compliance.
What is needed is a more comfortable hormone delivery approach with good
patient
compliance.
The present invention contemplates, in one embodiment, a hormone, including
but not
limited to PTH (or a fragment thereof, such as the 1-34 fragment), in a
preparation suitable for
transdermal, intradermal, intra-epidermal or intramucosal delivery. In one
embodiment, the hormone
(or fragment thereof) is attached or linked to a carrier. In one embodiment,
the hormone (or
fragment thereof) is part of a fusion protein. In one embodiment, the hormone
(or fragment thereof)
is in a micelle formulation. In one embodiment, the hormone (or fragment
thereof) is in or on
nanofibers, nanotubes, nanospheres, or nanocapsules. In one embodiment, the
hormone (or fragment
thereof) is complexed with one or more dendrimers. In one embodiment, the
hormone (or fragment
thereof) is in a liposome preparation (e.g. for liposome delivery). In a
preferred embodiment, the
hormone (or fragment thereof) is on the surface of a substrate, e.g. on a skin
patch, an array, of
asperities such as those herein described, such as microprojections or
microneedles, or the like.
Combinations of the above are contemplated specifically, including but not
limited to substrates
comprising the hormone (or fragment thereof) in a polymer formulation, or in
an assembly of
nanofibers. In a particularly preferred embodiment, the hormone is functional
and not functionally
impaired.
The present invention contemplates, in one embodiment, a preparation suitable
for
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transdermal, intradermal, intra-epidermal, transmucosal, subcutaneous,
intravenous, or
intramuscular delivery comprising a first hormone (or fragment thereof) in a
mixture with a second
hormone (or fragment thereof). These first and second hormones (or fragments
thereof) can be
prepared simply as a mixture or, alternatively, they can be linked together in
a conjugate. In one
embodiment, the first and second hormones (or fragments thereof) are in a
micelle formulation. In
one embodiment, the first and second hormones (or fragments thereof) are in or
on nanofibers,
nanotubes, nanospheres, or nanocapsules. In one embodiment, the first and
second hormones (or
fragments thereof) are complexed with one or more dendrimers. In one
embodiment, the first and
second hormones (or fragments thereof) are in a liposome preparation (e.g. for
liposome delivery).
In a preferred embodiment, the first and second hormones (or fragments
thereof) are on the surface
of a substrate, e.g. on a skin patch, microneedle array, or the like.
Combinations of the above are
specifically contemplated, including but not limited to substrates comprising
the first and second
hormones (or fragments thereof) in a polymer formulation, or in an assembly of
nanofibers. In a
preferred embodiment, said first hormone is PTH (or the 1-34 fragment thereof)
and the second
hormone is calcitonin (e.g. human or salmon). In a preferred embodiment, the
hormone(s)
preparation is placed on microprojections or microneedles having dimensions
such that they
penetrate into the stratum corneum, the epidermal layer, and, in some
embodiments, the dermal
layer. In one embodiment, the microprojections or microneedles have a length
(or height) or less
than 1000 microns, more preferably less than 500 microns, and still more
preferably less than 250
microns. The microprojections may be formed in different shapes, such as
needles, blades, pins,
punches, and combinations thereof. When in an array, the density of the
microprojections is at least
microprojections/cm2, more preferably, at least 200 microprojections/cmz, and,
in some
embodiments, at least 1000 microprojections/cm2.
The present invention further contemplates, in one embodiment, a preparation
comprising at
least one polymer and at least one hormone (or fragment thereof). It is not
intended that the present
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invention be limited by the nature of the polymer since a variety of types can
be used (see Table 1).
In one embodiment, cross-linked polymers are employed. However, in a preferred
embodiment, the
polymer comprises polyvinylpyrrolidone or hydroxypropylcellulose. In another
embodiment, said
polymer is hydroxyethylcellulose. It is not intended that the present
invention be limited by the
nature of the hormone(s) in the polymer preparation. However, in a
particularly preferred
embodiment, a functional PTH (or the 1-34 PTH fragment) is contemplated for
use in the polymer
formulation. Human and bovine sequences of 1-34 PTH are shown in Figure 20. It
is contemplated
that the polymer/hormone(s) preparation can be administered by a variety of
routes (e.g. IP, IM, IV,
inhalation, transdermal, intradermal, 'infra-epidermal, transmucosal,
subcutaneous, etc.).
It is not intended that the present invention be limited to the manner in
which the preparation
of polymer and hormone(s) is prepared. However, in a preferred embodiment, the
preparation is
electrospun to create fibers or fiber mats. It is preferred that at least a
portion of the hormone in the
electrospun fibers or mats be capable of release or escape from the polymer
(e.g. upon contact with
an aqueous or semi-aqueous environment). In one embodiment, the electrospun
polymer/hormone(s)
fiber mat is placed directly on the skin. In another embodiment, the
polymer/hormone(s) fiber mat is
part of a delivery vehicle (e.g. skin patch, microneedle array, etc.).
The present invention further contemplates, in one embodiment, a preparation
comprising at
least one hormone (or fragment thereof) in a hydrogel formulation. It is not
intended that the present
invention be limited by the nature of the hydrogel since a variety of types
can be used, including but
not limited to ionically cross-linked hydrogels or hydrogel films. It is not
intended that the present
invention be limited by the nature of the hormone(s) in the hydrogel
preparation. In a particularly
preferred embodiment, the hormone comprises a functional PTH (or 1-34 fragment
thereof). It is
contemplated that the hydrogel/hormone(s) preparation can be deposited on a
surface (in a uniform
or non-uniform manner). In one embodiment, the preparation is deposited (e.g.
by dipping, coating,
spin coating, spraying, or by suitable applicator) on an array of asperities
such as microprojections
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or microneedles as described herein for transdermal, intradermal, intra-
epidermal, or transmucosal
delivery of the hormone(s).
Other systems and apparatus that employ tiny skin piercing elements to enhance
transdermal
agent delivery are disclosed in U.S. Pat. Nos. 5,879,326, 3,814,097,
5,250,023, 3,964,482, Reissue
U.S. Pat. No. 25,637, and PCT Publication Nos. WO 96/37155, WO 96/37256, WO
96/17648, WO
97/03718, WO 98/11937, WO 98/00193, WO 97/48440, WO 97/48441, WO 97/48442, WO
98/00193, WO 99/64580, WO 98/28037, WO 98/29298, and WO 98/29365; all
incorporated herein
by reference in their entirety.
The present invention contemplates administering one or more of the various
embodiments
of the preparations described above as a prophylactic or therapeutic to humans
and animals. In one
embodiment, the present invention contemplates a method comprising: a)
providing i) a preparation
comprising at least one polymer and one or more hormones (or fragments
thereof) and ii) a subject,
wherein said subject is a human or non-human animal; and b) contacting said
subject with said
preparation (e.g. under conditions such that at least a portion of said
hormone is released into said
subject). In one embodiment, the present invention contemplates a method
comprising: a) providing
i) a preparation comprising one or more hormones (or fragments thereof) in a
hydrogel formulation
and ii) a subject, wherein said subject is a human or non-human animal; and b)
contacting said
subject with said preparation (e.g. under conditions such that at least a
portion of said hormone is
released into said subject). In a preferred embodiment, two hormones are
employed (as discussed
above). In a preferred embodiment, the hormones are electrospun with one or
more polymers so as
to create an electrospun preparation. As discussed below, in a preferred
embodiment, the
electrospun preparation is part of a hormone delivery system. In a preferred
embodiment, the
electrospun preparation is delivered transdermally, intradermally, intra-
epidermal, transmucosally,
subcutaneously, IP, IV or IM.
The present invention further contemplates, in one embodiment, a hormone
delivery system
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comprising a) a substrate comprising microprojections or microneedles, and an
embodiment of the
various preparations (mentioned above) comprising either i) hormone(s) alone
(or fragments
thereof), ii) hormone(s) (or fragments thereof) and carrier alone, or iii)
polymer (or hydrogel) and
hormone(s) (or fragments thereof). When microneedles are used, they may be
solid or hollow. It is
not intended that the present invention be limited to the manner in which the
microneedles are
contacted with the preparation. In one embodiment, the microneedles are
contacted only at the
microneedle tips. In a preferred embodiment, the preparation contacts the
entire substrate (or
substantially the entire substrate) such that the preparation contacts even
the spaces between an
array of microneedles. It is not intended that the present invention be
limited to uniform coatings of
the preparation. In one embodiment, the preparation is present on the
substrate in a non-uniform
manner. In one embodiment, a polymer/hormone(s) preparation is sprayed on said
microneedles. In
one embodiment, said microneedles are dipped into a preparation comprising at
least one polymer
and at least one hormone (or fragment thereof). In a preferred embodiment, an
electrospun
polymer/hormone(s) preparation is deposited on said microneedles.
It is not intended that the present invention be limited by the nature of the
substrate
comprising said microneedles. In one embodiment, the microneedles are
formulated out of polymer.
In one embodiment, the microneedles are formulated (at least in part) out of a
polymer/hormone(s)
preparation. In another embodiment, the microneedles are made with a mold. In
a preferred
embodiment, the microneedles are etched out of a silicon substrate. In a
preferred embodiment, said
silicon microneedles are solid and an electrospun polymer/hormone(s)
preparation is deposited on
said microneedles.
Thus, the present invention contemplates novel formulations, delivery devices,
methods of
formulation, and methods of delivery. In one embodiment, the present invention
contemplates a
method of creating a formulation: providing i) at least one fiber-forming
material, and ii) a
preparation comprising at least one hormone (or fragment thereof); mixing said
fiber-forming
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material with said preparation so as to create a mixture; and electrospinning
said mixture to create
electrospun formulation. In one embodiment, the method further comprises:
depositing at least a
portion of said electrospun material onto at least a portion of a substrate
(e.g. a substrate comprising
microneedles, including but not limited to etched solid microneedles) to
create a treated substrate..
The present invention also contemplates, as a device, the treated substrate
prepared according to the
above-described method. In one embodiment, said fiber-forming material
comprises hydroxypropyl-
cellulose, while in another embodiment said fiber-forming material comprises
PVP. In one
embodiment, said preparation comprises first and second hormones. In a
preferred embodiment, said
first hormone comprises PTH (1-34).
In one embodiment, the present invention contemplates a method of creating a
hormone
delivery device, comprising: providing i) at least one fiber-forming material,
ii) a preparation
comprising providing i) at least one fiber-forming material, ii) a preparation
comprising at least one
hormone (or fragment thereof), and iii) a substrate comprising a plurality
microprojections; mixing
said fiber-forming material with said preparation so as to create a mixture;
electrospinning said
mixture to create electrospun material; and depositing at least a portion of
said electrospun material
onto at least a portion of said substrate, so as to create a hormone delivery
device. In a preferred
embodiment, said hormone comprises PTH (1-34). In one embodiment, said fiber-
forming material
comprises hydroxypropyl-cellulose, while in another embodiment said fiber-
forming material
comprises PVP. The present invention also contemplates, as a device, the
hormone delivery device
prepared according to the above-described method.
The present invention, as mentioned above, also contemplates methods of
administering
hormones. On one embodiment, the method of administering hormone comprises:
providing: a
subject and the hormone delivery device described above; and contacting said
subject with said
delivery device under conditions such at least a portion of said hormone is
released from said
device. In one embodiment, said contacting of step b) comprises piercing the
subject's skin with said
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microprojections.
The present invention also contemplates, in one embodiment, a hormone delivery
device
comprising: a substrate having a back surface and a front surface; a plurality
of solid microneedles
extending upwards from the front surface of the substrate, the microneedles
comprising a
formulation, said formulation comprising at least one polymer and at least one
hormone (or
fragment thereof). In a preferred embodiment, said hormone comprises PTH (1-
34).
Importantly, the present invention contemplates utilizing, in some
embodiments, peptide
hormones that are protease resistant. In one embodiment, such protease-
resistant peptides are
peptides comprising protecting groups. In some embodiments, the peptide
hormone (such as 1-34
PTH) has the amino terminus blocked by standard methods to prevent digestion
by exopeptidases,
for example by acetylation. In another embodiment, the carboxyl terminus is
blocked by standard
methods to prevent digestion by exopeptidases, for example, by amidation. In
some embodiments,
both the amino terminus and the carboxyl terminus are blocked, e.g. the amino
terminus is blocked
with an acetal group, and said peptide's carboxyl terminus is blocked with an
acetate group. In
another embodiment, endoprotease-resistance is achieved using peptides which
comprise at least
one D-amino acid.
The present invention contemplates a variety of hormones and hormone
combinations in the
above-indicated formulations and delivery vehicles. Vertebrate hormones fall
into three chemical
classes: Amine derived hormones (examples are catecholamines and thyroxine, as
well as melatonin
and serotonin); Peptide hormones (examples are TRH and vasopression, as well
as leuteinizing
hormone, follicle-stimulating hormone, EPO, angiotensin, gastrin, growth
hormone and thyroid-
stimulating hormone; preferred embodiments of peptide hormones include
glucagon, glucagon-like
peptide GLP-1, and Exendin-4); Lipid and phospholipids (examples are steroid
hormones such as
testosterone and cortisol, sterol hormones such as calcitriol, and eicosanoids
such as prostaglandins).
The most commonly-prescribed hormones are estrogens and progestagens,
thyroxine (such as
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levothyrroxin) and steroids. Of course, insulin is used by diabetics.
A "pharmacologic dose" of a hormone is a medical usage referring to an amount
of a
hormone far greater than naturally occurs in a healthy body. The effects of
pharmacologic doses of
hormones may be different from responses to naturally-occurring amounts and
may be
therapeutically useful. An example is the ability of pharmacologic doses of
glucocorticoid to
suppress inflammation.
In a preferred embodiment, the present invention contemplates formulations of
a PTH-based
agent, such as PTH-based agent is selected from the group consisting of hPTH(1-
34), hPTH salts
and analogs, teriparatide and related peptides. Throughout this application,
the terms "PTH-based
agent" and "PTH(1-34)" include, without limitation, recombinant hPTH(I-34),
synthetic hpTH(1-
34), PTH(1-34), teriparatide, hPTH(I-34) salts, simple derivatives of hPTH(1-
34), such as hPTH(1-
34) amide, and closely related molecules, such as hPTH(1-33) or hPTH(1-31)
amide, or any other
closely related osteogenic peptide. Synthetic hPTH(1-34) is the most preferred
PTH agent (however,
1-34 PTH from other species is also contemplated).
Examples of pharmaceutically acceptable hPTH salts include, without
limitation, acetate,
propionate, butyrate, pentanoate, hexanoate, heptanoate, levulinate, chloride,
bromide, citrate,
succinate, maleate, glycolate, gluconate, glucuronate, 3-hydroxyisobutyrate,
tricarballylicate,
malonate, adipate, citraconate, glutarate, itaconate, mesaconate, citramalate,
dimethylolpropinate,
tiglicate, glycerate, methacrylate, isocrotonate,l3-hydroxibutyrate,
crotonate, angelate, hydracrylate,
ascorbate, aspartate, glutamate, 2-hydroxyisobutyrate, lactate, malate,
pyruvate, fumarate, tartarate,
nitrate, phosphate, benzene, sulfonate, methane sulfonate, sulfate and
sulfonate.
Preferably, the PTH-based agent is present in the coating formulation at a
concentration in
the range of approximately 1-30 wt. %.
More preferably, the amount of PTH-based agent contained in the solid
biocompatible
coating (i.e., microprojection member or product) is in the range of
approximately l g-1000 g,
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even more preferably, in the range of approximately 10-100 g.
In a further embodiment of the invention, the coating formulation includes at
least one
polymeric material or polymer that has amphiphilic properties, which can
comprise, without
limitation, cellulose derivatives, such as hydroxyethylcellulose (HEC),
hydroxypropylmethylcellulose (HPMC), hydroxypropycellulose (HPC),
methylcellulose (MC),
hydroxyethylmethylcellulose (HEMC), or ethylhydroxy-ethylcellulose (EHEC), as
well as
pluronics.
In one embodiment of the invention, the concentration of the polymer
presenting amphiphilic
properties in the coating formulation is in the range of approximately 0.01-20
wt. %, and in one
embodiment in the range of approximately 0.03-10 wt.% of the coating
formulation.
In another embodiment, the coating formulation includes a hydrophilic polymer
selected
from the following group: hydroxyethyl starch, carboxymethyl cellulose and
salts of, dextran,
poly(vinyl alcohol), poly(ethylene oxide), poly(2-hydroxyethylmethacrylate),
poly(n-vinyl
pyrolidone), polyethylene glycol and mixtures thereof, and like polymers.
In one embodiment, the concentration of the hydrophilic polymer in the coating
formulation
is in the range of approximately 1-30 wt. %, and in another embodiment, in the
range of
approximately 1-20 wt. % of the coating formulation.
In one embodiment, the present invention contemplates utilizing an electrospun
formulation
(e.g. alone or on a delivery vehicle described herein) comprising at least one
substance to improve
the look and/or feel of skin, wherein said substance is selected from the
group consisting of vitamins
(e.g. A, C, D and/or E), polysaccharides, botanicals, proteins/peptides (e.g.
collagen, keratin, elastin,
and peptide fragments thereof, including but not limited to pentapeptides such
as KTTKS), fatty
acids (omega-3, omega-6, etc.), and enzymes/coenzymes. In one embodiment,
bioactive factors
from whey (e.g. lactalbumin, lactoferrin, lactose, etc.) are contemplated for
such formulations.
With respect to botanicals, both plant extracts and purified bioactive agents
from plants (e.g.
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in electrospun polymer formulations) are contemplated in some embodiments. It
is not intended that
the present invention be limited to a particular plant or group of plants.
Illustrative plants (from
which extracts can be made or agents can be purified) include but are not
limited to aloe vera, St.
John's Wort (hypericum crean), Noni (Morinda citrifolia), coffee plant, white
and green tea
(camellia sinensis), avocado (persea Americana), jojoba (simmondsia
chinensis), almond (prunus
dulcis), olive oils (olea europea), Sea Kulp, Yacon (smallanthus
sonchifolius), soy (glycine max),
comfrey plant (symphytum uplandicum), gotu cola (centella asiatica), and
African Baobab Tree
(adansonia digitata). In one embodiment, the present invention contemplates
extracts or purified
agents useful for treating cellulite, wherein said plants are selected from
the group consisting of
meadowsweet (spiraea ulmaria), coffee plant (caffeine), ginkgo (ginkgo
biloba), birch (betula),
common ivy (hedera helix), and lemon (citrus lemon). In one embodiment, the
present invention
contemplates extracts or purified agents useful for treating acne, wherein
said plants are selected
from the group consisting of saw palmetto (sereonnoa repens), green tea
(camellia sinensis), soy
(glycyne soja), burdock (arctium lappa), tea tree (melaleuca alternifolia),
wild pansy (viola tricolor),
kiwi (actinidia chinensis), kokum (garcinia indica), and licorice (glycyrrhiza
glabra). In one
embodiment, the present invention contemplates extracts or purified agents
useful as skin whiteners,
wherein said plant is selected from the group consisting of bearbeny
(arctostaphylos uva-ursi), aloe
vera, lemon citrus, white mulberry (moms alba), Chinese sage (salvia
miltrionhiza), watercress
(nasturtium officinale), and amla (embelica officinalis). In one embodiment,
the present invention
contemplates extracts or purified agents useful for specific skin conditions,
wherein said plant is
selected from the group consisting of yarrow flowers (achillea millefolium),
oats (avena sativa), pot
marigold (calendula officinalis), chesnut bark (castaneva sativa), hawthorn
fruits (crataegus
monogina), cucumber seeds (cucumis sativus), semitake mushrooms (cordyceps
sabolifera),
wormwood (artemesia carvifolia), oriental horsetail (equisetum arvense), and
tamanu (calophyllum
inophyllum).
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In one embodiment, the present invention contemplates utilizing a delivery
vehicle described
herein comprising at least one hormone (e.g. electrospun hormone) to treat (or
prevent) a skin
condition. In one embodiment, the hormone (e.g., estradiol, or a vitamin) is
prescribed to treat a
condition, including but not limited to a skin condition that is the result of
photodamage or
photoaging. In one embodiment, the present invention contemplates treating a
skin condition
associated with inflammation with such a delivery device described herein
comprising one or more
retinoids (the vitamin A family). In yet another embodiment, the present
invention contemplates
utilizing a delivery vehicle described herein (e.g. microneedle array)
comprising Vitamin D (and/or
analogs) for the treatment of psoriasis. In one embodiment of the present
invention, the delivery
device comprising Vitamin E is utilized for the prevention and treatment of
skin scars and/or
keloids. In another embodiment, the present invention contemplates utilizing
one of the delivery
devices described herein comprising selenium in order to a) protect
keratinocytes against damage
from ultraviolet radiation, b) promote the healing of wounds and burns, and c)
reduce the chance of
skin cancer (i.e. an anti-oncogenic treatment). In one embodiment, the present
invention
contemplates such a delivery device comprising combinations of the above-named
vitamins and
minerals (e.g. vitamin D in combination with selenium) in a formulation (e.g.
an electrospun
formulation or hydrogel formulation).
In one embodiment, the present invention contemplates utilizing a delivery
vehicle described
herein (e.g. microneedle array) for delivery of Vitamin C (e.g. in an
electrospun polymer
formulation) in subjects (e.g. subjects who cannot take Vitamin C orally)
through the skin. In
another embodiment, the present invention contemplates the delivery of Vitamin
C to the skin
without utilizing a microneedle may (e.g. in an electrospun polymer
formulation or on a skin patch).
In still another embodiment, Vitamin C is administered using both of the above
approaches are
utilized so as to achieve both local and systemic administration.
In one embodiment, the present invention contemplates utilizing a delivery
vehicle described
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herein comprising at least one hormone (e.g. electrospun hormone) to treat (or
prevent) type II
diabetes. In one embodiment, the hormone is GLP-1. In another embodiment, the
hormone is
Exendin-4 or fragments thereof such as Exendin-4 (3-39) and Exendin-4 (9-39).
As noted above, in
some embodiments, protease resistance versions of such peptide hormones are
contemplated,
including but not limited to N-acetal GLP-1 (commercially available from
Phoenix Pharmaceuticals,
Inc.). The amino acid sequences for such peptide hormones are shown in Figure
21.
In one embodiment, the present invention contemplates electrospun hormones,
including but
not limited to PTH(1-34). In a preferred embodiment, such hormones are
electrospun with one or
more polymers such as those described herein.
In a preferred embodiment, the preparation comprising hormone(s) is capable,
after
electrospinning, of releasing some functional hormone into the human or
animal. It is envisioned
that in such an embodiment, a medically acceptable fiber-forming material may
be used to preserve
the hormone(s) for later rehydration and use. In general, rehydration of the
fiber of the present
invention may be accomplished by mixing the fiber with a solvent for the fiber-
forming material.
When the fiber is used to preserve a hormone, the solvent will optimally be a
medically acceptable
compound.
It is not intended that the present invention be limited to the method by
which the
electrospun hormone(s) is administered. However, in a preferred embodiment,
the hormone(s) is
administered at or in the skin. In a particularly preferred embodiment, the
electrospun
polymer/hormone(s) material is delivered on microneedles or on a patch such
that hormone(s) is
released into the skin.
In a non-limiting embodiment, the hormones(s) is (are) contained in an
ionically cross-linked
macromolecular assembly such as those hereinabove described, including but not
limited to a
hydrogel, such as those hereinabove described.
lonically cross-linked macromolecular assemblies of the present invention
comprise a
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substance to be delivered such as one or more hormones. The formulation
containing the
pharmaceutically active substance can be in the form of an ionically cross-
linked three dimensional
film, sphere, tube, fiber, or in any other, geometrical shape and is attached
the surface of the device
by chemical or physical means.
It is contemplated that the ionically cross-linked macromolecular
assembly/drug(s)
preparation can be deposited on a surface (in a uniform or non-uniform
manner). In one
embodiment, the preparation is deposited (e.g. by dipping, coating, spin
coating, spraying, or by
suitable applicator) on an array of microprojections or microneedles for
transdermal, intradermal or
transmucosal delivery of the hormone(s).
In one embodiment, the present invention contemplates a method of
administering one or
more hormones to a patient comprising the steps of penetrating at least an
outer layer of the skin of
the patient with one or more asperities such as those hereinabove described,
such as
microprojections or microneedles extending from a front surface of a
substrate, at least one of the
one or more microneedles (or portion thereof) contacted or coated (uniformly
or non-uniformly) by
an hormone preparation (discussed above), and releasing at least one of the
one or more hormones
into the patient.
Other applications for spatially controlled deposition of electrospun fibers:
Appropriate
nanofibers could be applied to the edge of a blade, such as a razor blade, or
on or between multiple
blades or to the piercing edges of a syringe needle. The fibers could aid in
the lubrication, prevent
corrosion, or minimize bleeding for example.
The needle could be coated with the fibers. These fibers may be adjuvant
polymers, such as
PVP. Upon injection of the vaccine, the adjuvant During injection, the coating
provides lubrication
during the insertion. The same or another kind of fiber can also serve as an
adjuvant.
Multiple layers of fibers or composites that have varied chemistries, like the
NO delivering
patches, with Vitamin C spun out on one pin and nitrite spun out on another,
or fibers containing
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each are spun onto the same needle The fibers deliver the NO when moisture
from the skin, or from
a puncture of the skin activates the reagents. For some applications the
reagents may be placed on
the separate pins which are inserted at the same time.
The spinning methods utilized in the present invention can also be used to
coat only the
surface of a stent, without forming a web between the struts of a stent. Only
the metal is coated;
thus there is not made a stent graft but a fibrous coating that allows fluids
to flow through the open
interstices of the stent during insertion, and fluids can have direct access
to the interior surface of the
blood vessel through these interstices after the stent is inserted
The steerable (directable) nature of the jets used in this invention can
improve application of
fibers to skin. Fine details of the wound can be coated with less "bridging"
than when a typical
bending and coiling jet is used. Before when it was attempted to spin out
fibers in attempts to
control bleeding wounds, such as, for example, when fibers were spun onto a
liver wound on a dog,
the fibers tented over the wound surface. Now the fibers may be delivered
directly to a specific
surface, such as a grounded surface on a wound, where one would want only to
deliver a polymer
containing a small quantity of growth factor to enhance healing of the wound.
This may be useful in
plastic surgery, where only a small incision is treated in this way. A small
wire may be laid on top
of the incision, and function as the target or ground, that provides even
greater control of the place at
which the fiber is attached to the wound.
Controlled jets of the sort described in this invention can be used in
laparoscopic surgery to
cover, protect. medicate, mechanically stabilize or treat tissues to which a
surgeon has only very
limited access.
In another embodiment, the asperities coated with nanofibers in accordance
with the present
invention may be employed as ion and fluid transport membranes for utilization
of hierarchical
structures of multiwall nanotubes on carbon nanofibers.
The hierarchical structure described includes, in its simplest concept, carbon
nanofibers with
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radial branches which may or may not have metal particles at the end of each
radial branch. Each
radial branch is connected electrically through a path that is a part of the
carbon hierarchical
structure, to an external electrical circuit. The methods described in this
invention can be used to
coat the end of each branch and metal particle with a porous network of
polymer nanofibers. If these
pores are filled with an ionized network this network can function as a
selective ion transport
membrane, which is an essential component of a fuel cell. Thus, in one
embodiment, the present
invention makes it possible to make radical new designs for fuel cells,
batteries, and bioelectric
contacts. It could also lead to new designs for electrochemical sensors which
depend on the motion
of ions in the vicinity of an electrode, which is a large class of sensors.
The ability to deposit one kind of nanofiber at the tips of the carbon
nanotube branches and
other kinds of nanofibers to span the interstices between the tips of the
branches makes it feasible to
consider new ways of creating essential paths for the flow of gases and
liquids through the
hierarchical structures of carbon and other materials, including the
electrically conducting variations
of titanium oxide known as Magneli phases, and other such conducting or
semiconducting materials,
some of which are listed in the following reference by J. R. Smith, et al.
The use of electrospun nanofibers to form durable ion transport membranes is
also an
enabling technology for the manufacture of light emitting diodes and
photoelectric diodes that
depend on the motion of ions in the vicinity of asperities on a surface.
("Electrodes Based on
Magnelipahse Titanium Oxides", J. R. Smith, F. C. Walsh, and R L Clarke,
Journal of Applied
Electrochemistry, Vol. 28 (1988), pages 1021-1033. Reviews in Applied
ElectrochemisU, Number
50).
EXAMPLES
The invention now will be described with respect to the following examples,
however, the
scope of the present invention is not intended to be limited thereby.
EXAMPLE 1
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Construction And Expression Of C. botulinum C Fragment Fusion Proteins
The C. botulinum type A neurotoxin gene has been cloned and sequenced
[Thompson, et al.,
Eur. J. Biochem. 189:73 (1990)]. The nucleotide sequence of the toxin gene is
available from the
EMBL/GenBank sequence data banks under the accession number X52066; the
nucleotide sequence
of the coding region is set forth in Figure 22. The amino acid sequence of the
C. botulinum type A
neurotoxin is shown in Figure 16. The type A neurotoxin gene is synthesized as
a single
polypeptide chain which is processed to form a dimer composed of a light and a
heavy chain linked
via disulfide bonds. The 50 kD carboxy-terminal portion of the heavy chain is
referred to as the C
fragment or the Hc domain.
A number of attempts by others to express polypeptides comprising the C
fragment of C.
botulinum type A toxin as a native polypeptide (e.g., not as a fusion protein)
in E. coli have been
unsuccessful [H.F. LaPenotiere, et al. in Botulinum and Tetanus Neurotoxins,
DasGupta, Ed.,
Plenum Press, New York (1993), pp. 463-466]. Expression of the C fragment as a
fusion with the E.
coli MBP was reported to result in the production of insoluble protein (H.F.
LaPenotiere, et al.,
supra).
In order to produce soluble recombinant C fragment proteins in E. coli, Dr.
Williams has
shown that it is desirable to construct fusion proteins comprising a synthetic
C fragment gene
derived from the C. botulinum type A toxin and either a portion of the C.
difficile toxin protein or
the MBP. See Williams et al., U.S. Patent 5,919,665 (hereby incorporated by
reference).
a) Construction Of Plasmids Encoding C Fragment Fusion Proteins
Fusion proteins comprising a fusion between the MBP, portions of the C.
difficile toxin A repeat domain (shown to be expressed as a soluble fusion
protein) and the C
fragment of the C. botulinum type A toxin have been described. See Williams et
al., U.S. Patent
5,919,665 (hereby incorporated by reference). A fusion protein comprising the
C fragment of the C.
botulinum type A toxin and the MBP was also constructed by Dr. Williams.
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Figure 23 provides a schematic representation of the botulinal fusion proteins
along with the
donor constructs containing the C. difficile toxin A sequences or C. botulinum
C fragment
sequences which were used to generate the botulinal fusion proteins. In Figure
23, the solid boxes
represent C. difficile toxin A gene sequences, the open boxes represent C.
botulinum C fragment
sequences and the solid black ovals represent the E. coil MBP. When the name
for a restriction
enzyme appears inside parenthesis, this indicates that the restriction site
was destroyed during
construction. An asterisk appearing with the name for a restriction enzyme
indicates that this
restriction site was recreated at the cloning junction.
In Figure 23, a restriction map of the pMA1870-2680 and pPA1100-2680
constructs which
contain sequences derived from the C. difficile toxin A repeat domain are
shown; these constructs
can be used as the source of C. difficile toxin A gene sequences for the
construction of plasmids
encoding fusions between the C. botulinum C fragment gene and the C. difficile
toxin A gene. Dr.
Williams demonstrated that the pMA 1870-2680 expression construct expresses
high levels of
soluble, intact fusion protein (20 mg/liter culture) which can be affinity
purified on an amylose
column. See Williams et al., U.S. Patent 5,919,665 (hereby incorporated by
reference). The
pAlterBot construct was used by Dr. Williams as the source of C. botulinum C
fragment gene
sequences for the botulinal fusion proteins (obtained from J. Middlebrook and
R. Lemley at the U.S.
Department of Defense). pAlterBot contains a synthetic C. botulinum C fragment
inserted in to the
pALTER-1 vector (Promega). This synthetic C fragment gene encodes the same
amino acids as does
the naturally occurring C fragment gene. The naturally occurring C fragment
sequences, like most
clostridial genes, are extremely A/T rich (Thompson et al., supra). This high
A/T content creates
expression difficulties in E. coli and yeast due to altered codon usage
frequency and fortuitous
polyadenylation sites, respectively. In order to improve the expression of C
fragment proteins in E.
coli, a synthetic version of the gene was created by Dr. Williams in which the
non-preferred codons
were replaced with preferred codons. See Williams et al., U.S. Patent
5,919,665 (hereby
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incorporated by reference).
The nucleotide sequence of the C. botulinum C fragment gene sequences
contained within
pAlterBot is shown in Figure 24. The first six nucleotides (ATGGCT) encode
methionine and
alanine residues, respectively. These two amino acids result from the
insertion of the C. botulinum
C fragment sequences into the pALTER vector and provide the initiator
methionine residue. The
amino acid sequence of the C. botulinum C fragment encoded by the sequences
contained within
pAlterBot is shown in Figure 17. The first two amino acids (Met Ala) are
encoded by vector-
derived sequences. From the third amino acid residue onward (Arg), the amino
acid sequence is
identical to that found in the C. botulinum type A toxin gene.
. The pMA1870-2680, pPA1100-2680 and pAlterBot constructs were used by Dr.
Williams as
progenitor plasmids to make expression constructs in which fragments of the C.
difficile toxin A
repeat domain were expressed as genetic fusions with the C. botulinum C
fragment gene using the
pMAL-c expression vector (New England BioLabs). See Williams et al., U.S.
Patent 5,919,665
(hereby incorporated by reference). The pMAL-c expression vector generates
fusion proteins which
contain the MBP at the amino-terminal end of the protein. A construct, pMBot,
in which the C.
botulinum C fragment gene was expressed as a fusion with only the MBP was
constructed by Dr.
Williams (Figure 23). Fusion protein expression can be induced from E. coli
strains harboring the
above plasmids, and induced protein was affinity purified on an amylose resin
column.
i) Construction Of pBlueBot
In order to facilitate the cloning of the C. botulinum C fragment gene
sequences into a
number of desired constructs, the botulinal gene sequences can be removed from
pAlterBot and
were inserted into the pBluescript plasmid (Stratagene) to generate pBlueBot
(Figure 23). pBlueBot
was constructed by Dr. Williams as follows. Bacteria containing the pAlterBot
plasmid were grown
in medium containing tetracycline and plasmid DNA was isolated using the
QlAprep-spin Plasmid
Kit (Qiagen). One microgram of pAlterBot DNA was digested with Ncol and the
resulting 3'
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recessed sticky end was made blunt using the Klenow fragment of DNA polymerase
I (here after the
Klenow fragment). The pAlterBot DNA was then digested with Hindlll to release
the botulinal gene
sequences (the Bot insert) as a blunt (filled Nco1 site)-Hind111 fragment.
pBluescript vector DNA
was prepared by digesting 200 ng of pBluescript DNA with SmaI and.HindIIl. The
digestion
products from both plasmids were resolved on an agarose gel. The appropriate
fragments were
removed from the gel, mixed and purified utilizing the Prep-a-Gene kit
(BioRad). The eluted DNA
was then ligated using T4 DNA ligase and used to transform competent DH5a
cells (Gibco-BRL).
Host cells were made competent for transformation using the calcium chloride
protocol of
Sambrook et al., supra at 1.82-1.83. Recombinant clones were isolated and
confirmed by restriction
digestion using standard recombinant molecular biology techniques (Sambrook et
al, supra). The
resultant clone, pBlueBot, contains several useful unique restriction sites
flanking the Bot insert
(i.e., the C. botulinum C fragment sequences derived from pAlterBot) as shown
in Figure 23. See
Williams et al., U.S. Patent 5,919,665 (hereby incorporated by reference).
ii) Construction Of C. difficile/C. botulinum/MBP Fusion Proteins Constructs
encoding fusions between the C. difficile toxin A gene and the C. botulinum C
fragment gene and
the MBP were made by Dr. Williams utilizing the same recombinant DNA
methodology outlined
above; these fusion proteins contained varying amounts of the C. difficile
toxin A repeat domain.
See Williams et al., U.S. Patent 5,919,665 (hereby incorporated by reference).
The pMABot clone contains a 2.4 kb insert derived from the C. difficile toxin
A gene fused
to the Bot insert (i.e, the C. botulinum C fragment sequences derived from
pAlterBot). pMABot
(Figure 23) was constructed by mixing gel-purified DNA from NotUHindII1
digested pBlueBot (the
1.2 kb Bot fragment), SpeI/NotI digested pPA1100-2680 (the 2.4 kb C. difficile
toxin A repeat
fragment) and XbaUHindlll digested pMAL-c vector. Recombinant clones were
isolated by Dr.
Williams, confirmed by restriction digestion and purified using the QlAprep-
spin Plasmid Kit
(Qiagen). This clone expresses the toxin A repeats and the botulinal C
fragment protein sequences
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as an in-frame fusion with the MBP.
The pMCABot construct contains a 1.0 kb insert derived from the C. difficile
toxin A gene
fused to the Bot insert (i.e, the C. botulinum C fragment sequences derived
from pAlterBot).
pMCABot was constructed by digesting the pMABot clone with EcoRl to remove the
5' end of the
C. difficile toxin A repeat (see Figure 23, the pMAL-c vector contains a EcoRI
site 5' to the C.
difficile insert in the pMABot clone). The restriction sites were filled and
religated together after gel
purification. The resultant clone (pMCABot, Figure 23) generated an in-frame
fusion between the
MBP and the remaining 3' portion of the C. difficile toxin A repeat domain
fused to the Bot gene.
See Williams et al., U.S. Patent 5,919,665 (hereby incorporated by reference).
The pMNABot clone contains the I kb SpeUEcoRI (filled) fragment from the C.
difficile
toxin A repeat domain (derived from clone pPA 1100-2680) and the 1.2 kb C.
botulinum C fragment
gene as a Ncol (filled)/HindI1l fragment (derived from pAlterBot). These two
fragments were
inserted by Dr. Williams into the pMAL-c vector digested with XbaUHindI1l. The
two insert
fragments were generated by digestion of the appropriate plasmid with EcoRI
(pPA 1100-2680) or
Ncol (pAlterBot) followed by treatment with the Klenow fragment. After
treatment with the
Klenow fragment, the plasmids were digested with the second enzyme (either
Spel or HindIII). All
three fragments were gel purified, mixed and Prep-a-Gene purified prior to
ligation. Following
ligation and transformation, putative recombinants were analyzed by
restriction analysis; the EcoRl
site was found to be regenerated at the fusion junction, as was predicted for
a fusion between the
filled EcoRI and Ncol sites. See Williams et al., U.S. Patent 5,919,665
(hereby incorporated by
reference).
A construct encoding a fusion protein between the botulinal C fragment gene
and the MBP
gene was constructed (i.e., this fusion lacks any C. difficile toxin A gene
sequences) and termed
pMBot. The pMBot construct was made by Dr. Williams by removal of the C.
difficile toxin A
sequences from the pMABot construct and fusing the C fragment gene sequences
to the MBP. This
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was accomplished by digestion of pMABot DNA with Stul (located in the pMALc
polylinker 5' to
the Xbal site) and Xbal (located 3' to the Notl site at the toxA-Bot fusion
junction), filling in the
Xbal site using the Kienow fragment, gel purifying the desired restriction
fragment, and ligating the
blunt ends to circularize the plasmid. Following ligation and transformation,
putative recombinants
were analyzed by restriction mapping of the Bot insert (i.e., the C. botulinum
C fragment
sequences). See Williams et al., U.S. Patent 5,919,665 (hereby incorporated by
reference).
b) Expression Of C. botulinum C Fragment Fusion Proteins In E. coli
Large scale (I liter) cultures of the pMAL-c vector, and each recombinant
construct described above in (a) can be grown, induced, and soluble protein
fractions isolated as
described in Williams et al., U.S. Patent 5,919,665 (hereby incorporated by
reference), who reported
yields in excess of 20 mg fusion protein per liter culture.
EXAMPLE 2
Production Of Soluble C. botulinum C Fragment
Protein Substantially Free Of Endotoxin Contamination
In order to determine if the solubility of the botulinal C fragment was
enhanced by
expressing the C fragment protein as a native protein, an N-terminal His-
tagged protein or as a
fusion with glutathione-S-transferase (GST), alternative expression plasmids
have been constructed.
See Williams et al., U.S. Patent 5,919,665 (hereby incorporated by reference).
Figure 25 provides a
schematic representation of the vectors described below.
In Figure 25, the following abbreviations are used. pP refers to the pET23
vector. pHIS
refers to the pETHisa vector. pBlue refers to the pBluescript vector. pM
refers to the pMAL-c vector
and pG refers to the pGEX3T vector. The solid black lines represent C.
botulinum C fragment gene
sequences; the solid black ovals represent the MBP; the hatched ovals
represent GST; "HHHHH"
represents the poly-histidine tag. In Figure 25, when the name for a
restriction enzyme appears
inside parenthesis, this indicates that the restriction site was destroyed
during construction. An
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asterisk appearing with the name for a restriction enzyme indicates that this
restriction site was
recreated at a cloning junction.
i) Construction Of pPBot
In order to express the C. botulinum C fragment as a native (i.e., non-fused)
protein, the
pPBot plasmid (shown schematically in Figure 25) was constructed by Dr.
Williams as follows. The
C fragment sequences present in pAlterBot were removed by digestion of
pAlterBot with NcoI and
Hindlll. The NcoUHindll l C fragment insert was ligated to pETHisa vector
which was digested
with Ncol and HindIII. This ligation creates an expression construct in which
the Ncol-encoded
methionine of the botulinal C fragment is the initiator codon and directs
expression of the native
botulinal C fragment. The ligation products were used to transform competent
BL21(DE3)pLysS
cells (Novagen). Recombinant clones were identified by restriction mapping.
See Williams et al.,
U.S. Patent 5,919,665 (hereby incorporated by reference).
ii) Construction Of pHisBot
In order to express the C. botulinum C fragment containing a poly-histidine
tag at the amino-
terminus of the recombinant protein, the pHisBot plasmid (shown schematically
in Figure 8) was
constructed by Dr. Williams as follows. The NcoUHindlll botulinal C fragment
insert from
pAlterbot was ligated into the pETHisa vector which was digested with Nhel and
Hindill. The Ncol
(on the C fragment insert) and Nhel (on the pETHisa vector) sites were filled
in using the Klenow
fragment prior to ligation; these sites were then blunt end ligated (the Ndel
site was regenerated at
the clone junction as predicted). The ligation products were used to transform
competent
BL21(DE3)pLysS cells and recombinant clones were identified by restriction
mapping. See
Williams et at., U.S. Patent 5,919,665 (hereby incorporated by reference).
The resulting pHisBot 'clone expresses the botulinal C fragment protein with a
histidine-
tagged N-terminal extension having the following sequence:
MetGlyHisHisHisHisHisHisHisHisHisHisSerSerGlyHislleGluGlyArgHisMetAla; the
amino acids
CA 02650197 2008-10-21
WO 2008/024141 PCT/US2007/011228
encoded by the botulinal C fragment gene are underlined and the vector encoded
amino acids are
presented in plain type. The nucleotide sequence present in the pETHisa vector
which encodes the
pHisBot fusion protein is set forth in Figure 26. The amino acid sequence of
the pHisBot protein is
set forth in Figure 27.
iii) Construction Of pGBot
The botulinal C fragment protein was expressed as a fusion with the
glutathione-S-
transferase protein by constructing the pGBot plasmid (shown schematically in
Figure 24). This
expression construct was created by Dr. Williams by cloning the NotUSall C
fragment insert present
in pBlueBot into the pGEX3T vector which was digested with Smal and Xhol. The
Notl site
(present on the botulinal fragment) was made blunt prior to ligation using the
Klenow fragment. The
ligation products were used to transform competent BL21 cells. See Williams et
al., U.S. Patent
5,919,665 (hereby incorporated by reference).
iv) solubility and purification
The pGBot protein was almost entirely insoluble under the utilized conditions,
while the
pHisBot protein was soluble. See Williams et al., U.S. Patent 5,919,665
(hereby incorporated by
reference). For improved affinity purification of the soluble pHisBot protein
(in terms of both yield
and purity), an alternative poly-histidine binding affinity resin (Ni-NTA
resin; Qiagen) is desirable.
The Ni-NTA resin has a superior binding affinity (Kd= I x 10-13 at pH 8.0;
Qiagen user manual)
relative to the His-bind resin.
Based upon the foregoing disclosure, it should now be apparent that
Clostridium toxins, and
in particular, C. botulinum toxins, together with polymer (e.g. in an
electrospun formulation) can be
used as vaccines. A particularly desirable administration approach is
transdermal, via
microprojections (e.g. an array of microneedles). A variety of polymers, as
well as delivery vehicles,
can be used without departing from the spirit of the invention. In one
embodiment, the electrospun
polymer/toxin(s) fiber mat is placed directly on the skin. In another
embodiment, the
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polymer/toxin(s) fiber mat is part of a delivery vehicle (e.g. skin patch,
microneedle array, etc.)
EXAMPLE 3
Mutagenesis. To generate a complete library of Cys mutants of PA63 one can use
the
QuikChange (Stratagene) method to generate point mutations, in conjunction
with automated
systems currently used for high-throughput DNA sequencing. The pag gene
encoding PA is AT rich
(34% GC) and does not present significant technical hurdles. Automated systems
can be used to
synthesize the mutagenic oligonucleotide pairs, and the quality of mutagenic
oligonucleotides can be
confirmed by automated capillary electrophoresis. In this manner, a full plate
can be processed in a
day, and the oligonucleotides needed for complete mutagenesis of PA63 can be
synthesized and
checked in 12 days. After manually performing the PCR mutagenesis in 96-well
plates and
transforming the resulting mutated plasmids into E. coli, automation can again
be used for preparing
DNA from the colonies and sequencing of the region of interest. Clones bearing
the desired
mutation can be used for functional screens.
As noted above, researchers at the University of Oklahoma used this protocol
to generate
568 mutants, corresponding to the replacement of every amino acid of PA63 with
Cys. Thirty three
mutants (6% of the total number of mutants tested) were both strongly
defective and well expressed.
As shown in Table 3, the 33 mutations were distributed among the four domains
of PA, with a
majority in domain 2.
Some Inactive Mutants Are Dominant. The 33 inactive mutants were tested by the
researchers at the University of Oklahoma for ability to inhibit the toxicity
of a mixture of PA and
LFNDTA toward CHO cells at ratios of mutated to wild-type PA up to 8:1. Those
that showed
measurable inhibitory activity under these conditions were defined, for the
purpose of this study, as
"dominant negative" (DN). Nine preparations of mutant PA were inhibitory:
1364, T380, S382,
T393, K397, N399, Y41 1, N422, and F427. The present invention contemplates
using such mutant
protective antigens both alone and in conjugates (as described above). Like
the DN mutants
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characterized earlier, the purified DN Cys-mutants showed defects specifically
in pore formation
and translocation. PA bearing T393C, T380C, or S382C showed strong DN
activity, only slightly
weaker than that of the K397D + D425K double mutant characterized earlier.
N399C and N422C
showed moderate DN activity, and 1364C and Y411 C showed weak inhibitory
activity.
One of the strongest of the five DN mutants, T393C, was tested by researchers
at the
University of Oklahoma for ability to protect Fisher 344 rats from a lethal
challenge with a mixture
of PA and LF. Like the other DN mutants tested, T393C prevented symptoms of
intoxication until
the time of death, whereas rats challenged with native toxin alone became
moribund 75 rain after
challenge (data not shown). In the end, 33 well-expressed mutants with defects
in toxicity were
described by the Oklahoma group.
In contrast to the Cys substitutions, Ala substitutions created by the
Oklahoma group at
selected sites gave different results: Y411A showed strong DN activity; T380A
and N399A showed
weak DN activity; and N422 and S382A showed no DN activity. At position 393,
Cys or Lys gave
strong DN activity; Asp or Ala gave moderate DN activity; and Ser gave no DN
activity. The
present invention contemplates employing these Ala substituted mutant
protective antigens alone or
as conjugates (as described above).
Conjugating wild-type and mutant PA to PGA. Conjugates are readily synthesized
as
follows. To I mg of PA in 0.5 ml phosphate-buffered saline (PBS; pH 7.0), 0.5
mg of degraded
PGA can be added. After PGA has dissolved, 5 mg of I-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride should be added, and the
solution mixed on a
rotary shaker at 25C for 4 hours. One can monitor the conjugation process by
SDS-PAGE.
Moreover, the conjugates can be purified on a PDIO column (Amersham
Biosciences), verified by
SDS-PAGE, and stored at -20C until use.
Alternatively, gamma-PGA polypeptides can be synthesized (with various
lengths, e.g. 5,
10, 15 or 20 residues) and can be bound to carrier proteins at either the C-or
N-termini (for further
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CA 02650197 2008-10-21
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reactions). The various types of these polypeptides are described in U.S.
Patent Application
20060134143 to Schneerson et al. (hereby incorporated by reference) including
but not limited to
PGA comprising D-glutamic acid and PGA comprising L-glutamic acid:
Type I: NBrAcGly3_yDPGAn- COOH(Br-G 1 y3_7DPGA,,-C)
NBrAc-GIy3--yLPGAõ-COOH(Br-GIy3--yLPGAn-C)
Type II: NAc-L-Cys-G 1 y3-yDPGAn,-COOH(Cys-Gly3-yDPGA,,-C)
NAc-L-Cys-G 1 y3-7LPGA,,,-COOH(Cys-GIy3-yLPGA,,-C)
Type III: NAc-yDPGA,,-G I y3-L-Cys-CONH2(N- yDPGAõ-G 1 y3-Cys)
NAc-yLPGAn-Gly3-L-Cys-CONH2(N-yLPGA,,-G I y3-Cys)
Type IV: CHO-G 1 y3- yDPGAõ-COOH
Type V: NAc-yDPGAr,-GIy3-CO-AH
NAc-yDPGAn-CO-AH
Type VI: NAc--yDPGAõ-Cys-CONHZ
In one example, recombinant wild-type PA is derivatized with adipic acid
dihydrazide and
reacted with one or more of these polypeptides.
Using Y. pestis antigens. As noted above, in another embodiment, antigen from
Y. pestis is
contemplated in a preparation suitable for transdermal delivery. It is not
intended that the present
invention be limited by the particular antigens from these organisms. However,
in one embodiment,
an Fl capsule antigen from Y. pestis is employed in a polymer formulation
(e.g. electrospun
formulation) deposited on a microneedle array. In another embodiment, a
protein (or fragment) from
the injectisome from Y. pestis is employed. In a preferred embodiment, the
LcrV protein (or
fragment thereof) from the Y. pestis injectisome is similarly employed. With
regard to the latter,
Table 5 shows the amino acid sequence for LcrV of Y. pestis, as well as from
some other sources.
TABLE 5
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CA 02650197 2008-10-21
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Y pews IOM LdV' MIFtAYEptaPqF1YIEDL~(VFtVD~L7 GE9GõSvi L'ELV~1
VKGQQiIbISIlCYDPR~BVt
Y. 8YV&O=WM I.CN MIFAY6QIPQIPIEDLEKVFCVFMR'6FK;S
YpMtMtbLCrV i9FtA IEDLEttVAt+~ T+Gi11~S81'YF2SUM
P. eatwnt7sA
Y. POWs i0M I.GYV AHRV27'DDIP.I.LIQCILATIPLP!*DATLAOGHYDHOIaGTKAVDM'L68..
SPHYOWARA
Y.OVWDMWW ICN A~KVtTDO1xLIlacI1AYPLFBII91IL~t~6i.ItEtvftEE6SS..S~l7T~IAA
Y (JB9UdfbLCYv A10RVITDOI6LL?.IQIAYYLPrLU%ILKfG}lYD20N(X4iiIlLT9..SP2MP1PLi1A
P aw*waw pav
Y Q@64`9IOM li7V
EMVDIP`SLZIDRIPDDItd1;ViV1SMdiDGDAdiBXLRB8d1iBL4A8LtfIYSVIpAEIHQ(N
Y endOVOON,ia9laY
ERaP1~HPSLSADEtIDADxIdCYIVf~S~tiAGlbajLSlcLtiB8I11RLTABI;iEIYSVI(YU3ItazN
Y. pswurtoPbltrV iM PSL~DRIL~7 A9teLMASLiAS1WCiYSVICRBIM
P S&Wkrom Pc.v Elmuft
~I~I~~~~I~~LT~s+-I~-~
Y, pEbtslOM I CN iSS862IIdIffiX<CSIMkNOdLYC.YTD. E3IPPU49MYICITEFQWQTTI WD...
:... ....
YoarawmKm LAfv L488G7LNIIVRSIIVII4DI0II,YOYTD.&3IFY 4ABYi{I1~PQ{PQPF
Y Psetftb LaY IS IFTISDRSIETl3M47LYGYTD.=FKaB4BYR1 I
P. emofncw abrv I jM4[h&,YC~.~~ . . . . . . .
V. peaNafOM (.nN
cSomi4SlmoG+i*Bl1KTITGAi.CiLiQtfiYS1RlMM*IIMLSNI'ATY'CSDKSRP11lmLV80K
YaaBmG1pwc9 L.orv Ivs 8 RI ~ SY~L~lA2~CSD[i3RPLrDLVSax
YpmaabdbLcrV rvsn~ s _ r~rrCSDtcsRela+nivs
P. esrtlDTse R:N sI
Y. p6SVS FdM LOV TTWDITSR@WAI8l1I2RFICjLyDSt?s3tyLpD'r6GR
Y. fJ-VBIl7CIOW608 L.GYY TTQS'JDITSREN9AIF.AU@tFTQiiYDSVKiXX[ LaD7M
Y pssumbPh LcrV
P. aert,gJnxa PCtV 3
T D QiSUD3v I~
Because of the similarity between three of the organisms (but not P.
aeruginosa), the present
invention contemplates, in one embodiment, using the LcrV protein (or fragment
thereof) of any of
these three organisms in order to generate protection against Y. pestis. If a
fragment is used,
typically the fragment will be at least twenty (20) amino acids and selected
in a region of high
conservation (see Table 5).
Where Fl capsule antigen and LcrV protein are made as a fusion protein, the
fusion protein
may be made by selecting appropriate coding sequences from GenBank accession
No. AF07461 1.
Using F. tularensis antigens. As noted above, in another embodiment, antigen
from F. tularensis is
contemplated in a preparation suitable for transdermal delivery. It is not
intended that the present
invention be limited by the particular antigens from these organisms. However,
in one embodiment,
a specific sequence of the outer membrane protein (or fragment thereof)
encoded by the fopA gene
of F. tularensis is employed.
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Alternatively, a protein (or fragment thereof) encoded by the tul4 gene is
employed. The
PCR primers that will conveniently amplify these genes are given below:
*tu14 primers (forward, ATTACAATGGCAGGCTCCAGA; reverse,
GCCCAAGTTTTATCGTTCTTCTCA) will specifically amplify an 89-bp fragment of the
tul4 gene (GenBank accession no. M32059) encoding a 17-kDa lipoprotein.
* fopA primers (forward, AACAATGGCACCTAGTAATATTTCTGG; reverse,
CCACCAAAGAACCATGTTAAACC) will amplify an 86 bp fragment of the fopA gene
(GenBank accession no. AF097542) which encodes a 43-kDa outer membrane
protein.
The proteins can be expressed in vitro or in vivo and then formulated (e.g. in
an electrospun
formulation) for application to the microneedle array.
Electrospinning Anthrax Protective Antigen. In this example, the protocol for
electrospinning anthrax antigen is described. Pure native PA (List Biological
Laboratories) will be
reconstituted in I ml of distilled water containing 0.1 % Bovine Serum Albumin
(BSA) at a
concentration of 1 mg/ml. 400 Al of the solution will be further diluted with
equal volume of
aqueous 0.1% BSA to prepare the PA solution which will be mixed appropriately
with PVP as
described below. The "unspun" 500 g/ml PA solution will be used as the
control for the evaluation
of retention of functionality (although this is not required for a vaccine).
Two electrospinning
solutions will be prepared utilizing the PA solution. The first will consist
of 400 l of PA solution,
and 1600 Al of 0.075 mM Polyvinylpyrrolidone (PVP) dissolved in absolute
ethanol, and the second
will have distilled water as the solvent. Both solutions will be utilized in
preparation of electrospun
mats, and tested in the assay of functionality to confirm that protein
activity has been retained upon
introduction of polymer solution.
Because an approximate concentration of 100 g/ml of PA is required for the
functionality
test, and the volume needed for the cell-based assay is 1 ml, 100 mg of the
material is deposited onto
the collector. It is estimated that the mixture of PA and PVP in ethanol will
be electrospun at 20
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uUmin and 16 kV, while the completely aqueous mixture will be electrospun at 5-
10 uUmin and 20
kV. From previous experiments it can be estimated that, regardless of which
solution is electrospun,
only 50% to"75% of the total material will be deposited on the collector. 1500
l of each solution is
electrospun onto aluminum foil, coated with Teflon to ease the removal of the
electrospun mat from
the foil. The entire material deposited on the sheet will be peeled off and
dissolved in 1 ml of
MatTek tissue culture medium to provide samples for the test of functionality.
This stock solution
will be further diluted with culture medium to a final estimated concentration
of 10 g/ml; this
concentration is 50-100 times the required amount for the test of
functionality.
The cells used in this test are MonoMac 6 cells (DSMZ, Braunschweig, Germany))
maintained in RPMI 1640 (Hyclone) medium supplemented with 10% fetal bovine
serum (FBS),
2mM L-glutamine, 1% non-essential amino acids, 1 mM oxaloacetate, 0.45 mM
pyruvate, 0.2 U/ml
insulin (OPI media supplement, Sigma, St.Louis, MO), and 1% (v/v) penicillin
streptomycin
solution (final concentrations 100 IU penicilin G and 100 gg streptomycin per
ml, Sigma). Cells are
washed with Hank's Balanced Salt Solution (HBSS; Hyclone) at 37 C once prior
to commencing
experiments, which are all carried in serum free media without antibiotics.
The functionality experiments are based on examination of Lethal Factor (LF)
proteolytic
activity in the cytosol of viable cells. We have shown in previous experiments
that LF added to the
surrounding medium cannot enter MonoMac 6 cells unless functionally active PA
is present to
facilitate its transport from the medium into the cytosol. MonoMac 6 cells are
washed with warm
DPBS and are resuspended in Macrophage Serum Free Medium (Gibco) for plating
at a density of 1
x 106/ml/well in 24 well microplates. After addition of each of the PA
solutions to be tested (two
different dissolved electrospun preparations and an aliquot of the "unspun"
solution) and a 100
ng/mi solution of LF (List Biolabs) to separate wells of cells, the cells are
incubated for different
periods of time, and at each designated time point a 200 Rl aliquot of cell
suspension is removed.
Cells are pelleted by centrifugation and are then lysed in a buffer containing
0.1% Nonidet P-40
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(NP40), 150 mM NaCI, 40 mM Tris @H 7.2), 10% 5 glycerol, 5 mM NaF, 1 mM Na
pyrophosphate, 1mM Na o-vanadate, 10 mM ophenanthroline, and 100 ng/ml
phenylmethyl sulfonyl
fluoride (PMSF) at 70 C for 10 min.
The lysates are separated by SDS-polyacrylamide gel electrophoresis and the
presence of
intact or cleaved MEK-2 is quantitated by immunoblotting. Aliquots of 7.5-10
ug total cell lysate
protein per well are loaded onto 10% SDS gels (Novex) and electrophoresed
followed by transfer to
nitrocellulose membranes in an electrotransfer apparatus (Novex) for 1 h at 30
V. The membranes
are blocked with 5% low-fat milk in This-buffered saline buffer, pH 7.6,
containing 0.1% Tween 20
for 1 h at room temperature and then probed with anti-MEK-2 rabbit polyclonal
antibody (Santa
Cruz) at a dilution of 1:2000. Goat anti-rabbit IgG is used at a dilution of
1:2000 as the secondary
antibody.
Blots are washed after probing and processed with the Enhanced
Chemiluminescence
detection kit according to the manufacturer's directions.
The predicted results for a successful delivery of LF are illustrated in
Figure 28.
As can be seen in Figure 28, it is expected that after 4 hours of incubation
of MonoMac 6
cells with LF alone or with PA alone, there is no degradation of the MAP
Kinase MEK-2 detected in
an immunoblot, or "Western blot," of the cell lysates, whereas when LF and PA
are both present,
MEK-2 is destroyed within the cells in 4 hours. If the electrospun PA behaves
in a~manner similar to
unspun PA (as is to be expected), one can anticipate seeing MEK-2 cleavage as
shown in Figure 28.
Delivering Electrospun PA To A Tissue Model. In this example, the protocol for
delivering
electrospun PA through MatTek EpiDerm 200 epidermal equivalent tissue, using
600 um
microspike-bearing silicon chips and collecting the surrounding medium from
the base of the
MatTek units for challenge with LF and MonoMac 6 cells, is described.
Testing Antigenicity of Electrospun PA. In this example, the protocol for
testing for
preservation of antigenicity of the electrospun PA using a commercially
available polyclonal goat
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anti-PA antibody from List Biological Laboratories is described. This antibody
recognizes PA even
after it has been subjected to SDS-polyacrylamide gel electrophoresis,
indicating that, at least in
goats, the PA can be prepared as an immunogen to generate a robust immune
response. Aliquots of
the dissolved samples of electrospun PA generated from the two solvent systems
will be separated
by SDS polyacrylamide gel electrophoresis along with aliquots of the "unspun"
PA control and
transferred to nitrocellulose membranes as described above. The membranes will
be probed with an
appropriate dilution of the goat anti-PA antibody, while a rabbit anti-goat
polyclonal antibody will
be used as the secondary antibody. The membranes will then be examined by
enhanced
chemiluminescence as described above. This test is less stringent than the
functionality assay (and
all that is needed for a vaccine) because even PA which has been denatured by
the electrospinning
protocol (which is not expected) will retain its capacity to be recognized by
the goat antibody.
However, we can still quantitate the delivery of immunoreactive PA after
electrospinning and even
after delivery across an epidermal equivalent model by using a
semiquantitative immunoblotting
procedure known as a "slot blot." The slot-blot apparatus allows direct
application of samples
collected after electrospinning, or after delivery through a skin equivalent,
onto a nitrocelulose
membrane. The apparatus can accommodate 48 samples at a time, including a
series of sequential
dilutions of the "unspun" PA control, the PA concentrations of which are all
precisely known. After
all the samples are applied, the membrane is removed and incubated with goat
anti-PA primary
antibody and rabbit-antigoat secondary antibody, and examined by enhanced
chemiluminescence as
described above.
Based upon the foregoing disclosure, it should now be apparent that anthrax
antigens, and
in particular, certain conjugates of two anthrax antigens (one of which being
a mutant), taken alone
or together with polymer (e.g. in an electrospun formulation) can be used as
vaccines. A particularly
desirable administration approach is transdermal, via microprojections (e.g.
an array of
microneedles). A variety of polymers and mutants, as well as delivery
vehicles, can be used without
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departing from the spirit of the invention. In one embodiment, the electrospun
polymer/antigen(s)
fiber mat is placed directly on the skin. In another embodiment, the
polymer/antigen(s) fiber mat is
part of a delivery vehicle (e.g. skin patch, microneedle array, etc.)
EXAMPLE 4
Parathyroid Hormone (PTH 1-34) Polyvinylpyrrolidone (PVP) molecular weight of -
1,300,000 from Sigma-Aldrich was dissolved at a concentration of 0.075mM in
0.4% Human Serum
Albumin (HSA) in water and magnetically stirred for 3 hours. A 550 pg/ml
peptide solution
supplied by Immunotopics as part of its PTH 1-34 Elisa kit was used as the
hormone source. The
electrospinning PTH 1-34 solution was prepared by mixing the PVP and PTH
solutions at an 80% /
20% (v/v) PVP/PTH ratio. The electrospinning method was utilized to deposit a
nanocomposite of
the solution onto the substrates. A total of 1.0 ml was electrospun onto the
target. The coating was
created at the flow rate of 80min, voltage of 20 kV, and a distance between
the collector and needle
of 10 cm. The coated devices were attached to microcentrifuge tubes with crazy
glue to add
stability, and ease the handling process. The transdermal delivery experiments
were carried out
using EpiDerm Full Thickness (EFT-300) tissue models from MatTek Corporation.
The samples
were placed on top of the tissue model units with sufficient pressure to
ensure that the spikes had
entered the tissue, thereby beginning the time release study. At each time
point the tissue model its
with the devices in place were moved into pre-warmed media, and supernatant
was collected. After
the 24 hour harvest, the devices were removed, the tissue was cut out of the
holders and
homogenized into 500 l of tissue culture media in a microfuge tube using a
small motor-driven
pestle. The ground tissue was centrifuged, and analysis of the supernatant
medium was performed.
To test tissue viability after the 24 hour time point, mitochondrial
dehydrogenase activity was
assayed using Alamar Blue reduction. Due to this assay's interaction with the
HRP assay only two
tissue model units from each set were examined for viability.
Based upon the foregoing disclosure, it should now be apparent that hormones,
and in
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particular, PTH(1-34), together with polymer (e.g. in an electrospun
formulation) can be used
prophylatically (to prevent) or therapeutically (to treat) bone loss. A
particularly desirable
administration approach is transdermal, via microprojections (e.g. an array of
microneedles). A
variety of polymers and hormones (or hormone combinations), as well as
delivery vehicles, can be
used without departing from the spirit of the invention. For example, iIn one
embodiment, the
electrospun polymer/hormone(s) fiber mat is placed directly on the skin. In
another embodiment, the
polymer/hormone(s) fiber mat is part of a delivery vehicle (e.g. skin patch,
microneedle array, etc.).
The disclosures of all patents and publications (including published patent
applications) are
hereby incorporated by reference to the same extent as if each patent and
publication were
individually and specifically incorporated by reference.
Although the invention has been described in detail with particular reference
to certain
embodiments detailed herein, other embodiments can achieve the same results.
Variations and
modifications of the present invention will be obvious to those skilled in the
art and the present
invention is intended to cover in the appended claims all such modifications
and equivalents.
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