CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No.
62/151,922, filed April 23, 2015, U.S. Provisional Application No. 62/194,067, filed
July 17, 2015, and U.S. Provisional Application No. 62/303,224, filed March 3, 2016,
each of which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
This invention relates to treatments for allergies, e.g., peanut allergies, and to
useful compositions comprising Glucopyranosyl Lipid A (GLA) and allergens, such
as peanut allergens, for allergy treatments.
BACKGROUND
Peanut allergy is characterized by an aberrant T-helper-type 2 (Th-2) immune
response towards the normally innocuous antigens present in peanuts. The
prevalence of peanut allergy in North American school-aged children is
approximately 1% and is often a lifelong condition. It is the most common foodrelated
cause of fatal allergic reactions in Western countries.
SUMMARY
The present invention is based, at least in part, on the determination that
administering a TLR4 agonist along with allergen, such as peanut allergen(s),
sublingually can modulate pathogenic, allergen-specific immune responses.
Specifically, glucopyranosyl lipid adjuvant (GLA) can be administered along with
allergens, e.g., peanut proteins, in order to treat allergy in a patient, such as peanut
allergy. The disclosed pharmaceutical compositions that include one or both of GLA
and peanut proteins are sublingual immunotherapies (SLIT) intended for treatment of
allergy, e.g., peanut allergy.
Accordingly, in one aspect, the present specification provides a
pharmaceutical composition comprising a plurality of glucopyranosyl lipid adjuvant
(GLA) particles (e.g., GLA liposomes, micelles, aggregates, or mixtures thereof), 1,2-
dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and a therapeutically effective
amount of allergen(s), e.g., peanut proteins, in a carrier (e.g., an aqueous carrier, i.e., a
carrier in which water acts as a solvent), wherein the molar ratio of GLA to DPPC is
in a range of about 1:1 to about 1:3 and at least some of the allergen(s), e.g., peanut
proteins, are at least partially (e.g., completely) disposed within and/or on at least one
of the lipid particles (e.g., liposomes) and/or free in the carrier (e.g., aqueous carrier).
In another aspect, the present specification provides a pharmaceutical
composition comprising glucopyranosyl lipid adjuvant (GLA) and allergen(s), e.g.,
peanut proteins, wherein the pharmaceutical composition is in a solid or semi-solid
dosage form.
In embodiments of the pharmaceutical compositions described herein, where
an aqueous carrier is used, the aqueous carrier can include water. A composition
comprising GLA can be in a form comprising a plurality of lipid particles, such as
liposomes, micelles, and/or aggregates. The average lipid particle size in the plurality
(e.g., the mean hydrodynamic diameter (Z-Average diameter) of the lipid particle) can
be about 10 nm to about 2000 nm, e.g., about 16 nm to about 1800 nm, about 50 nm
to about 1000 nm, about 80 nm to about 500 nm, e.g., about 90 nm, 100 nm, about
200 nm, about 300 nm or about 400 nm. A composition comprising GLA can be in a
colloidal dispersion. The particle size can be measured using a Malvern Zetasizer,
which provides the mean hydrodynamic diameter (Z-Average diameter) and the
polydispersity index (PDI) of the particles. In some embodiments of any
pharmaceutical compositions described herein, allergens(s) (e.g., peanut proteins
and/or other allergen) may be included and completely enclosed within lipid particles
or partially enclosed within lipid particles, or both, and/or disposed on lipid particle
surface(s). In other embodiments, some allergens (e.g., peanut protein(s)) may be
enclosed and/or partially enclosed by the lipid particles while others are free in the
pharmaceutical composition and not enclosed and/or partially enclosed within lipid
particles. In still other embodiments, substantially all allergens, e.g., peanut
protein(s), are free in the pharmaceutical composition and not enclosed or partially
enclosed within lipid particles.
The concentration of GLA in pharmaceutical compositions described herein
can be, e.g., from about 0.2 mg/mL to about 5 mg/mL, e.g., about 2 mg/mL, 10 mg/mL,
20 ug/mL, 50 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL,
400 mg/mL, 500 mg/mL, 0.8 mg/mL, 1 mg/mL or about 1.6 mg/mL. For example, the
concentration of GLA in the composition can be about 0.01 mg/mL to about 5
mg/mL, e.g., about 0.02 mg/mL to 0.2 mg/mL, or 0.16 mg/mL. Pharmaceutical
compositions can include 0.001% to 0.1% DPPC, e.g., 0.01% to 0.05% DPPC, e.g.,
0.02% to 0.03% DPPC. For example, a composition can contain about 0.010%
DPPC, 0.015% DPPC, 0.020% DPPC, 0.025% DPPC, 0.030% DPPC, 0.035% DPPC,
0.040% DPPC, 0.045% DPPC, or about 0.050% DPPC. In some instances, the molar
ratio of GLA to DPPC can be about 1:2. In one embodiment, a GLA composition
includes a preservative, such as glycerol. For example, a GLA composition for use in
a SLIT formulation can include 0.025% DPPC, 60% glycerol and water.
The concentration of peanut proteins in a pharmaceutical composition for
sublingual administration can be about 2 mg/mL to about 25,600 mg/mL, e.g., about
2,000 mg/mL to about 7,000 mg/mL, e.g., about 5,000 mg/mL. In some embodiments,
the concentration of peanut proteins in a pharmaceutical composition for sublingual
administration is about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL,
about 7 mg/mL, about 8 mg/mL, about 10 mg/mL, about 20 mg/mL, about 23 mg/mL,
about 24 mg/mL, about 25 mg/mL, about 26 mg/mL, about 50 mg/mL, about 100
mg/mL, about 200 mg/mL, about 500 mg/mL, about 1 mg/mL, about 5 mg/mL, about
10 mg/mL, or about 20 mg/mL. In some embodiments, the concentration of peanut
proteins in a pharmaceutical composition for sublingual administration is about 6.4
mg/mL or about 12.8 mg/mL. For example, the concentration of peanut proteins in a
pharmaceutical composition described herein can be about 5 mg/mL to about 25,600
mg/mL or about 1,000 mg/mL to about 7,000 mg/mL.
Other allergens can be included in a pharmaceutical composition or method of
making a pharmaceutical composition described herein, as an alternative, or in
addition, to peanut allergens. For example, in some instances, a food allergen other
than peanut allergen(s) can be included. Examples of such food allergens include
milk allergen (e.g., whole milk or extract thereof, casein (e.g., alpha Sl-casein),
and/or beta-lactoglobulin), seafood allergen (e.g., allergens from vertebrates (e.g.,
salmon, cod, mackerel, sardines, herring, anchovies, tuna, trout, haddock, eel, and/or
rays) and/or invertebrates (e.g., crustaceans (e.g., shrimp, crab, crayfish, and/or
lobster allergen) and/or mollusks (e.g., clams, mussels, oysters, octopus, squid and/or
scallops allergen)), egg allergen, mustard allergen, sesame allergen, soy allergen,
wheat allergen (e.g., gluten), fruit allergen (such as Bet v 1 or homologues thereof,
lipid transfer protein, and/or profilin, and/or allergens from strawberry, apple,
avocado, blueberry, date, kiwi fruit, peach, raspberry, fig, grape, plum, cherry,
grapefruit, and/or prune), vegetable allergen (e.g., alfalfa, cauliflower, cucumber,
mushroom, radish, broad bean, eggplant, spinach, zucchini, broccoli, and/or pepper
allergen) or tree nut allergen (e.g., walnut, almond, cashew, pistachio, and/or pecan
allergen), to treat allergies to each of those foods. Alternatively or in addition, house
dust mite (HDM) allergen, an aeroallergen (e.g., pollen allergen) and/or grass allergen
can be included, e.g., for the treatment of an allergy, such as a seasonal allergy.
Further, compositions that include such allergens can be used in the methods for
treating allergy (i.e., an allergy to the allergen included in the pharmaceutical
composition) described herein.
In some embodiments, the pharmaceutical composition can be formulated to
deliver a dose of about 0 .1 mg to about 80 mg GLA, e.g., 0.5 mg to 40 mg, e.g., 0.8 mg
to 20 mg GLA. In some embodiments, the pharmaceutical sublingual formulation can
include about 0.5 mg, about 1 mg, about 2 mg, about 5 mg, about 10 mg, about 20 mg,
about 40 mg or about 50 mg GLA.
In some embodiments, a pharmaceutical composition described herein is
formulated to deliver a dose of about 50 ng to about 30 grams or more peanut
proteins, e.g. 100 ng to about 20 grams or more peanut proteins, about 300 ng to about
15 grams of more peanut proteins, about 500 ng to about 10 grams or more peanut
proteins; 800 ng to about 9 grams or more peanut proteins; about 1000 ng to about
8.5 grams or more peanut protein; about 2 mg to about 8.3 grams or more peanut
protein; e.g., about 5 , 10 , 20 m , 40 m , 80 m , 100 m , 160 m , 250 m , 320 m ,
500 mg, 640 mg, 1000 mg, 1280 mg, 2000 mg, 2560 mg, 3000 mg , 4000 mg , 5000 mg ,
5120 mg, 6000 mg , 7000 mg , 10 mg, 20 mg, 40 mg, 50 mg, 100 mg, 500 mg, 1000,
mg, 1500 mg, 2000 mg, or about 5000 mg, or more, peanut protein. The composition
can be in a range from pH 4 to pH 8.5, e.g., about pH 4.5, pH 5.5, pH 7.5, or about pH
8.5. In some embodiments, the composition is at about pH 8.0, pH 8.1, pH 8.2 or
about pH 8.3. The pharmaceutical composition can be formulated to deliver a dose of
about 100 m to about 8 grams or more peanut protein; e.g., about 500 mg to about 7.5
grams or more peanut protein.
Peanut proteins in the pharmaceutical compositions described herein can
comprise or consist of one or more of peanut allergen components Ara hi, Ara h2,
Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara h i 1, Ara hl2,
Ara hl3, Ara hl4, Ara hl5, Ara hl6, and Ara hl7, or any combination thereof. In
some embodiments, the peanut allergens included in the pharmaceutical compositions
consist of peanut allergen components Ara hi, Ara h2, Ara h3, and Ara h6. In other
embodiments, the peanut allergens included in the pharmaceutical compositions
consist of peanut allergen components Ara hi, Ara h2, and Ara h6. In still other
embodiments, the peanut allergens included in the pharmaceutical compositions
consist of peanut allergen components Ara h2 and Ara h6.
Pharmaceutical compositions described herein can be in the form of, e.g., a
liquid, a semi-solid, a tablet (e.g., a fast disintegrating tablet (FDT), a gel capsule, a
thin film, a sublingual drop, or a sublingual spray. Pharmaceutical compositions
described herein may comprise a bioadhesive component. Pharmaceutical
compositions can be in the form of a tablet or a multi-particulate granule or coated
microsphere, e.g., packaged in a sachet form. FDTs described herein can include a
superdisintegrant, e.g., cross-linked cellulose, cross-linked polyvinylpyrrolidone,
cross-linked starch, or cross-linked alginic acid, or a mixture thereof. Compositions
described herein can include a gelling agent, a binder, a glidant, an antiadherant, a
flavoring agent, a sweetening agent, and/or a colorant, or any combination thereof.
Thin films described herein can include, e.g., plasticized hydrocolloid. In certain thin
films described herein, the GLA or allergen(s) (e.g., peanut proteins), or both, can be
disposed within the thin film. In other thin films, the GLA or allergen(s) (e.g., peanut
proteins), or both, are disposed on the surface of the thin film. In still other thin films,
the GLA or allergen(s) (e.g., peanut proteins), or both, are disposed both within and
on the surface of the thin film.
In another aspect, the specification provides a method of treating an allergy in
a patient, comprising administering sublingually to a patient having an allergy a
plurality of glucopyranosyl lipid adjuvant (GLA) particles and allergens (e.g., peanut
proteins), in amounts effective to treat the allergy in the patient. The method can
further comprise administering sublingually to the patient 1,2-dipalmitoyl-sn-glycero-
3-phosphocholine (DPPC). In some embodiments, the plurality of GLA particles are
formulated as a first pharmaceutical composition and the allergen(s) (e.g., peanut
proteins) are formulated as a second, separate pharmaceutical composition. The first
and second pharmaceutical compositions can be, e.g., administered simultaneously, or
the first formulation can be administered to the patient prior to the second
formulation, or the second formulation can be administered to the patient prior to the
first formulation. In other embodiments, the GLA and the allergen(s) (e.g., peanut
proteins) are formulated in a single pharmaceutical composition comprising a
plurality of GLA particles and allergen(s) (e.g., peanut proteins) in an aqueous carrier.
The pharmaceutical composition can further comprise l,2-dipalmitoyl-sn-glycero-3-
phosphocholine (DPPC), and in some instances the molar ratio of GLA to DPPC in
the pharmaceutical composition can be in a range of about 1:1 to about 1:3.
In another aspect, the specification provides a method of treating an allergy in
a patient, comprising administering sublingually to a patient having an allergy a
pharmaceutical composition comprising glucopyranosyl lipid adjuvant (GLA) and
allergen(s) (e.g., peanut proteins), wherein the pharmaceutical composition is in a
solid or semi-solid dosage form. The pharmaceutical composition can be, e.g., a thin
film or a tablet or capsule, as described herein.
In the methods of treating an allergy described herein, where peanut allergens
are included in the treatment, the allergy can be a peanut allergy, an allergy to birch
tree pollens, an allergy to peach and peach related fruits, or a combination thereof.
The methods of treating an allergy described herein can further include
performing a basophil activation test on the patient prior to administering the
treatment. Patients treated using the presently described methods can be, e.g., a
human, e.g., an adult human (18 years or older) or juvenile human (17 years or
younger). The patient can be an adolescent (age 12 years to 17 years), or a child ( 11
years of age or younger). The patient can be a child 4 to 11 years old.
In one embodiment, treatment of an allergic patient with a sublingual
formulation described herein can prevent future allergic reactions of the patient upon
exposure to peanut proteins. For example, in peanut allergy treatments, treatment of a
patient can prevent or reduce the severity of an allergic reaction of a patient following
exposure (such as by ingestion) of the patient to up to 20 grams or more of peanut
proteins. For example, treatment of a patient with the adjuvanted sublingual
formulation described herein can prevent or reduce the severity of an allergic reaction
of a patient following exposure of the patient to 100 mg to 10 grams or more of
peanut proteins, e.g, about 200 mg, 300 mg, 500 mg, 800 mg, 1000 mg, 2000 mg,
3000 mg, 4000 mg, or about 5000 mg of peanut proteins. Typically, treatment of a
patient with the sublingual formulation results in the prevention of an anaphylactic
reaction following accidental exposure to peanut proteins.
In still another aspect, the specification provides a method of making a
pharmaceutical composition, the method comprising: co-dissolving glucopyranosyl
lipid adjuvant (GLA) and l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at a
1:2 molar ratio in chloroform to thereby form a GLA/DPPC mixture; adding
allergen(s) (e.g., peanut proteins) to the GLA/DPPC mixture, to thereby form a
GLA/DPPC/allergen(s) (e.g., peanut proteins) mixture; removing the chloroform from
the GLA/DPPC/allergen(s) (e.g., peanut proteins) mixture; adding water to the
GLA/DPPC/allergen(s) (e.g., peanut proteins) mixture; and agitating the
GLA/DPPC/allergen(s) (e.g., peanut proteins) mixture, to thereby form a
pharmaceutical composition.
In still another aspect, the specification provides a method of making a
pharmaceutical composition, the method comprising co-dissolving glucopyranosyl
lipid adjuvant (GLA) and l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at a
1:2 molar ratio in chloroform to thereby form a GLA/DPPC mixture; removing the
chloroform from the GLA/DPPC mixture; adding water to the GLA/DPPC mixture;
agitating the GLA/DPPC mixture; and adding allergen(s) (e.g., peanut proteins) to the
GLA/DPPC mixture, to thereby form a pharmaceutical composition.
In still another aspect, the specification provides a method of making a
pharmaceutical composition, comprising mixing glucopyranosyl lipid adjuvant (GLA)
and l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at a 1:2 molar ratio in
water, e.g., water that is heated, such as to a temperature of about 40°C to 80°C, e.g.,
to 50°C to 70°C, to thereby form a GLA/DPPC mixture. The GLA/DPPC mixture is
then agitated at the elevated temperature (e.g., at about 70°C); and then allergen(s)
(e.g., peanut proteins) are added to the GLA/DPPC mixture, to thereby form a
pharmaceutical composition.
In yet another aspect, the specification provides a method of making a
pharmaceutical composition, comprising mixing glucopyranosyl lipid adjuvant (GLA)
and a surfactant; adding water to the GLA/surfactant mixture; agitating the
GLA/surfactant mixture; and adding allergen(s) (e.g., peanut proteins) to the
GLA/surfactant mixture, to thereby form a pharmaceutical composition. The
surfactant can be, e.g., Sodium Lauryl Sulfate, polysorbate-80, poloxamer 407 or
poloxamer 188, or a combination of lecithin and taurocholate, or a combination
thereof. In one example, the surfactant can be polysorbate-80, and the mixture can
further include glycerol from 40% w/v to 80% w/v, e.g., glycerol at about 50% w/v,
about 60% w/v, or about 70% w/v. In some instances, in the methods of making a
pharmaceutical composition described herein, agitating can include sonication,
microfluidization, high pressure homogenization, or mechanical agitation, such as
with an Ultra-Turrax system, or any combination thereof. The methods can include
compression molding the pharmaceutical composition to form a tablet. The methods
can include lyophilizing or spray drying the pharmaceutical composition. The
methods can include the steps of: forming a thin polymer film by a method selected
from the group consisting of solvent casting, semisolid casting, hot melt extrusion,
solid dispersion extrusion, and rolling; and disposing the pharmaceutical composition
within thin film and/or on at least one surface of the thin film.
In any of the pharmaceutical compositions and methods of making the
pharmaceutical compositions described herein, the peanut proteins can include one or
more of the peanut allergen components Ara hi, Ara h2, Ara h3, Ara h4, Ara h5, Ara
h6, Arah7, Ara h8, Arah9, ArahlO, Ara hi 1, Arahl2, Arahl3, Arahl4, Arahl5,
Ara hi 6, and Ara hi 7, or any combination thereof. In the pharmaceutical
compositions and methods of making the pharmaceutical compositions described
herein, the peanut proteins can consist of one or more of peanut allergen component
Ara hi, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara
hi 1, Arahl2, Arahl3, Arahl4, Arahl5, Arahl6, and Arahl7, or any combination
thereof. For example, the peanut proteins can consist of peanut allergen components
Ara hi, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara
hi 1, Arahl2, Arahl3, Arahl4, Arahl5, Arahl6, and Arahl7. For example, the
peanut proteins can consist of peanut allergen components Ara hi, Ara h2, Ara h3,
and Ara h6. As another example, the peanut proteins can consist of peanut allergen
components Ara hi, Ara h2, and Ara h6. As still another example, the peanut
proteins can consist of peanut allergen components Ara h2 and Ara h6.
The methods of making the pharmaceutical compositions can further include
prior to adding the peanut proteins to the GLA/DPPC mixture, performing a basophil
activation test on the peanut proteins to measure the potency of the peanut proteins.
Also provided herein is the use of GLA and allergen(s), e.g., peanut
allergen(s), together or separately in a sublingual formulation(s) for the treatment of
allergy (e.g., peanut allergy) in a patient. Also provided herein is the use of at least
GLA and/or allergen(s), e.g., peanut allergens(s), in the manufacture of a sublingual
medicament for treatment or prevention of allergy, e.g., peanut allergy. The
medicament can be used in a method for treating allergy in a patient. The medicament
can be in any form as described herein, e.g., liquid, semisolid, or solid, compositions.
The terms "effective amount" and "effective to treat," as used herein, refer to
an amount or concentration of a composition described herein utilized for a period of
time (including acute or chronic administration and periodic or continuous
administration) that is effective within the context of its administration for causing an
intended effect or physiological outcome. Effective amounts of a composition
described herein for use in the present invention include, for example, amounts that
prevent or reduce the intensity of an allergic reaction to ingestion or exposure to
peanuts or products that include peanut-based ingredients, reduce the risk of such
allergic reactions, reduce one or more symptoms of such allergic reactions, and/or
improve the outcome of other peanut allergy treatments. An effective amount may be
determined by one skilled in the art.
The terms "treat" and "treatment" are used herein to describe delaying the
onset of, inhibiting, or alleviating the detrimental effects of a condition, e.g., peanut
allergy or symptoms of allergic reaction to peanuts or products that include peanutbased
ingredients.
The term "patient" is used throughout the specification to describe an animal,
human or non-human, to whom treatment according to the methods of the present
invention is provided. Veterinary and non-veterinary applications are contemplated
by the present invention. Human patients can be adult humans or juvenile humans
(e.g., humans below the age of 18 years old). In addition to humans, patients include
but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and
primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee,
gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits),
lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other
domestic, farm, and zoo animals.
Unless otherwise defined, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which this invention belongs. Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of the present invention,
suitable methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present specification, including
definitions, will control. The materials, methods, and examples are illustrative only
and not intended to be limiting.
DESCRIPTION OF DRAWINGS
FIG. 1A is a bar graph illustrating the effect of GLA and peanut proteins
treatments (at varying concentration) on anaphylaxis score in mice following
challenge with crude peanut extract (CPE). ††= p< 0.001 compared to vehicle +
control (CT) group; * p<0.05 compared to vehicle + CPE/CT group.
FIG. IB is a bar graph illustrating the effect of GLA and peanut proteins
treatments (at varying concentration) on anaphylaxis score in mice following
challenge with crude peanut extract. The CPE-alone group is omitted from this
presentation. ††= p< 0.001 compared to vehicle + control (CT) group; * p<0.05
compared to vehicle + CPE/CT group.
FIG. 2A is a bar graph illustrating the effect of GLA and peanut proteins
treatment (0.5 mg) on core body temperature drop in mice following challenge with
crude peanut extract. †= p< 0.05 compared to vehicle + control (CT) group.
FIG. 2B is a bar graph illustrating the effect of GLA and peanut proteins
treatment (0.5 mg) on core body temperature drop in mice following challenge with
crude peanut extract. The CPE-alone group is omitted from this presentation.
†= p< 0.05 compared to vehicle + control (CT) group.
FIG. 3 is a diagram of a typical peanut extraction process.
FIG. 4A is a histogram illustrating the effect of sublingual administration of
GLA alone on antigen-specific T cell proliferation in draining cervical nodes.
FIG. 4B is a histogram illustrating the effect of sublingual administration of
GLA alone on antigen-specific T cell proliferation in the spleen.
FIG. 4C is a histogram illustrating the effect of sublingual administration of
Ova peptide alone on antigen-specific T cell proliferation in draining cervical lymph
nodes.
FIG. 4D is a histogram illustrating the effect of sublingual administration of
Ova peptide alone on antigen-specifc T cell proliferation in the spleen.
FIG. 4E is a histogram illustrating the effect of sublingual co-administration of
GLA and Ova peptide on antigen-specific T cell proliferation in draining cervical
lymph nodes.
FIG. 4F is a histogram illustrating the effect of sublingual co-administration of
GLA and Ova peptide on antigen-specific T cell proliferation in the spleen.
FIG. 5 is a bar graph illustrating that sublingual administration of GLA with
antigen enhances antigen-specific T cell proliferation in the draining lymph nodes in
mice.
DETAILED DESCRIPTION
The present specification is based, at least in part, on formulations comprising
allergens such as peanut proteins and the TLR-4 agonist GLA, which are particularly
suitable for sublingual administration, e.g., for the treatment of peanut allergy.
Accordingly, provided herein are compositions and methods for treating an allergy in
a patient, e.g., a peanut allergy. In general, treatment can be accomplished by
administering to a patient, via the sublingual route, GLA in combination with at least
one type of allergen. The GLA and allergen(s), e.g., peanut allergen, can be
administered to patients either in separate sublingual formulations or in a single
sublingual formulation comprising both GLA and allergen, e.g., peanut allergen. The
disclosed pharmaceutical formulations containing one or both of GLA and allergen(s),
such as peanut allergen, are sublingual immunotherapies (SLIT) intended, e.g., for
treatment of peanut allergy.
Glucopyranosyl Lipid Adjuvant (GLA)
The presently described compositions comprise the adjuvant GLA, which is an
art-known and commercially available compound. Typically, the GLA can be of
formula (I):
GLA Structure (A)
or a pharmaceutically acceptable salt thereof, where: Rl, R3, R5 and R6 are CI 1-C20
alkyl; and R2 and R4 are C12-C20 alkyl; in a more specific embodiment, the GLA
has the formula (I) set forth above wherein Rl, R3, R5 and R6 are CI 1-14 alkyl; and
R2 and R4 are C12-15 alkyl. In a further more specific embodiment, the GLA has the
formula (I) set forth above wherein Rl, R3, R5 and R6 are CI 1 alkyl, or undecyl; and
R2 and R4 are C13 alkyl, or tridecyl. In yet a further specific embodiment, the GLA
has the formula (I) set forth above wherein Rl, R3, R5 and R6 are undecyl and R2
and R4 are tridecyl. GLA is known in the art and can be prepared by skilled
practitioners and/or obtained from a variety of commercial sources.
For example, particularly useful in the present invention is GLA of formula
(II), which can be obtained, e.g., from Avanti Polar Lipids, Inc.:
GLA Structure (B)
A pharmaceutically acceptable salt of GLA suitable for use in a sublingual
formulation featured in the invention can be an aluminum salt or ammonium salt, or a
benzathine salt. Other suitable salts include calcium, ethylene diamine, lysine,
magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.
Anomeric variants of GLA can also be used in the sublingual formulations featured in
the invention.
A GLA suitable for use in a sublingual formulation featured in the invention
can be the free acid form of GLA with no counter ion salt (see Structure (A) above).
As used herein, "alkyl" means a straight chain or branched, noncyclic or
cyclic, unsaturated or saturated aliphatic hydrocarbon containing from 1 to 20 carbon
atoms, and in certain preferred embodiments containing from 11 to 20 carbon atoms.
Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl,
n-pentyl, n-hexyl, and the like, including undecyl, dodecyl, tridecyl, tetradecyl,
pentadecyl, hexadecyl, heptadecyl, octadecyl, etc.; while saturated branched alkyls
include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and
cyclohexenyl, and the like. Cyclic alkyls are also referred to herein as "homocycles"
or "homocyclic rings." Unsaturated alkyIs contain at least one double or triple bond
between adjacent carbon atoms (referred to as an "alkenyl" or "alkynyl",
respectively). Representative straight chain and branched alkenyls include ethylenyl,
propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-lbutenyl,
2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; while
representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-
butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-l-butynyl, and the like. For
example, "CI 8-13 alkyl" and "C6-11 alkyl" mean an alkyl as defined above,
containing from 8-13 or 6-11 carbon atoms, respectively.
As used herein, "acid functional group" means a functional group capable of
donating a proton in aqueous media (i.e. a Bronsted-Lowry acid). After donating a
proton, the acid functional group becomes a negatively charged species (i.e. the
conjugate base of the acid functional group). Examples of acid functional groups
include, but are not limited to:-OP(=0)(OH) 2 (phosphate), -OS(=0)(OH) 2 (sulfate), -
OS(OH)2 (sulfite), -OC(OH)2 (carboxylate), -OC(=0)CH(NH 2)CH2C(=0)OH
(aspartate),
-OC(=0)CH 2CH2C(=0)OH (succinate), and -OC(=0)CH 2OP(=0)(OH) 2
(carboxymethylphosphate).
As used herein, "hydrocarbyl" refers to a chemical moiety formed entirely
from hydrogen and carbon, where the arrangement of the carbon atoms may be
straight chain or branched, noncyclic or cyclic, and the bonding between adjacent
carbon atoms maybe entirely single bonds, that is, to provide a saturated hydrocarbyl,
or there may be double or triple bonds present between any two adjacent carbon
atoms, i.e., to provide an unsaturated hydrocarbyl, and the number of carbon atoms in
the hydrocarbyl group is between 3 and 24 carbon atoms. The hydrocarbyl may be an
alkyl, where representative straight chain alkyls include methyl, ethyl, n-propyl, nbutyl,
n-pentyl, n-hexyl, and the like, including undecyl, dodecyl, tridecyl, tetradecyl,
pentadecyl, hexadecyl, heptadecyl, octadecyl, etc.; while branched alkyls include
isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative
saturated cyclic hydrocarbyls include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, and the like; while unsaturated cyclic hydrocarbyls include cyclopentenyl
and cyclohexenyl, and the like. Unsaturated hydrocarbyls contain at least one double
or triple bond between adjacent carbon atoms (referred to as an "alkenyl" or
"alkynyl," respectively, if the hydrocarbyl is non-cyclic, and cycloalkeny and
cycloalkynyl, respectively, if the hydrocarbyl is at least partially cyclic).
Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-
butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-l-butenyl, 2-
methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; while representative straight
chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-
pentynyl, 2-pentynyl, 3-methyl- 1-butynyl, and the like.
Compounds of formula I and/or II may be obtained by synthetic methods
known in the art, for example, the synthetic methodology disclosed in PCT
International Publication No. WO 2009/035528. Skilled practitioners will also
appreciate that GLA may be obtained commercially, e.g., from Avanti Polar Lipids,
Inc.
Allergens
As used herein, an "allergen" is any antigenic substance capable of producing
an allergen-specific allergic reaction in a patient. An allergen can be, e.g., a protein,
glycoprotein, carbohydrate, lipid, glycolipid, or other organic compound.
Some exemplary allergens useful in the present compositions and methods include(a)
a full length antigen, (2) an immunogenic fragment of an antigen, (3) an immunogenic
variant of a full length antigen or immunogenic fragment, (4) a chimeric fusion that
includes portions from different polypeptides/antigens, and (5) a conjugate
comprising two or more of such examples.
Peanut proteins contain multiple allergens, which are particularly useful in the
compositions and methods described herein. Compositions described herein can
include at least one type of peanut allergen. A peanut allergen used in a composition
described herein can be, e.g., (a) a full length antigen, (2) an immunogenic fragment
of the antigen, (3) an immunogenic variant of the full length antigen or immunogenic
fragment, (4) a chimeric fusion that includes portions from different peanut
polypeptides, and/or (5) a conjugate comprising two or more of such peanut allergens.
In some instances, the compositions include a single type of isolated peanut
allergen, a mixture of isolated peanut allergens, or whole peanut extract, which is a
mixture of peanut proteins that includes peanut allergens. As used herein, the term
"isolated" means that a material is removed from its original environment (e.g., the
natural environment if it is naturally occurring). With respect to individual types of
peanut allergens, at least 17 of such allergens have been identified. Among them are
Ara h 1, Ara hi, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO,
Ara hi 1, Arahl2, Arahl3, Arahl4, Arahl5, Arahl6, and Arahl7. Genbank
Accession Numbers for the cDNA sequences of exemplary allergens include
L34402.1 (Ara hi), AY007229.1 (Arah2.0101), AY158467.1 (Arah2.0201),
AF093541.1 (Arah3.0101), AF086821.1 (Ara h3.0201), AF059616 (Arah5),
AF092846.1 (Arah6), AF091737.1 (Arah7), EU046325.1 (Arah7.0201),
AY328088.1 (Arah8.0101), EF436550.1 (Ara h8.0201), EU159429.1 (Ara h9.0101),
and EU161278.1 (Arah9.0201), AY722694.2 (ArahlO.0101), AY722695.1 (Ara
hlO.0201), DQ097716.1 (Ara hi 1), EY396089.1 (Arahl2), EY396019.1 (Arahl3),
AF325917.1 (Arahl4.0101), AF325918 (Ara hl4.0102), DQ368496 (Ara hi 4 .0 103),
and AY722696 (Ara hl5), respectively (see, e.g., Leon et al, The peanut allergy
epidemic: allergen molecular characterisation and prospects for specific therapy.
Expert Rev. Mol. Med. Vol. 9, Issue 1, January 2007; see also Arkwright et al., IgE
Sensitization to the Nonspecific Lipid-Transfer Protein Ara h 9 and Peanut-
Associated Bronchospasm, BioMed Research International, vol. 2013, Article ID
746507). Allergens Ara hi, 2, 3, 6, 8, and 9 are important markers of peanut
sensitization and can be predictive of an allergic response. Ara hi, 2, and 3 are seed
storage proteins. Ara h2 is a more important predictor of clinical peanut allergy than
Ara hi and 3, and is often associated with severe reactions. Ara h6 elicits antibodies
that cross-react with Ara h2 and sensitization to Ara h6 and Ara h2 often occur
together. Ara h8 is a pathogenesis-related (PR)- 10 protein, and sensitization is often
associated with mild, localized symptoms. Ara h8 cross-reacts with certain pollens
(e.g., Birch and Birch-related tree pollen). Ara h9 is a lipid transfer protein, and
sensitization can result in systemic reactions, including anaphylaxis. Patients with
sensitivity to Ara h9 are often also sensitive to Ara hi -3. Ara h9 often cross-reacts
with pitted fruits, such as peaches. Compositions described herein can comprise, for
example, at least one type of peanut allergen selected from the group consisting of:
Ara hi, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara
i 1, Ara h!2, Ara h!3, Ara h!4, Ara h!5, Ara h!6 and Ara h 17, or an allergic
reaction-inducing portion of Ara hi, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7,
Arah8, Arah9, ArahlO, Arahll, Arahl2, Ara h i 3, Ara hi 4, Arahl5, Ara h 6, and
Ara h 7. It is contemplated, for example, that an extract of the whole peanut may be
used, that a single peanut allergen or allergic reaction-inducing portion thereof may be
used, and that any of the isolated peanut allergens and/or allergic reaction-inducing
portions thereof can be used in any combination. For example, instead of whole
peanut extract, the compositions may include any combination of at least 2, e.g., at
least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17, of Arahl, Ara h2, Ara h3, Ara
h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara h 11, Ara hl2, Ara h 3, Ara
hl4, Ara hl5, Arahlb and Ara h 17, or allergic reaction-inducing portion(s) of Arahl,
Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara hi 1,
Arahl2, Arahl3, Ara h 4, Ara h 5, Ara h 6 or Arahl?. For example, a
composition described herein may include Ara hi, Ara h2, and Ara h6, or allergic
reaction-inducing portion(s) thereof. As another example, the composition may
include Ara h2, and Ara h6, or allergic reaction-inducing portion(s) thereof. As still
another example, the composition may include Ara hi, Ara h2, Ara h3, and Ara h6, or
allergic reaction-inducing portion(s) thereof. In yet another example, the composition
may include Ara h i 2 and Ara h i 3, or allergic reaction-inducing portion(s) thereof. It
is contemplated that combinations of any full-length allergen(s) and any allergic
reaction-inducing portions of allergens may be used together.
In some instances, compositions described herein can include a mixture of
full-length Ara h proteins, a mixture of allergic reaction-inducing portions of Ara
proteins (i.e., immunogenic fragments), or a combination of both full-length and
allergic reaction-inducing portions of Ara h proteins. For example, an immunogenic
fragment can include at least 5, e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, 60, 70, 80, 90, or at least 100, or more,
contiguous amino acids of the protein. Immunogenic fragments can be small, e.g.
about 50 amino acids or less, or between about 6-10, 10-15, 15-20, 20-30, 30-40, 40-
50, 50-60, 60-70, 70-80, 80-90, 90-100, or more contiguous amino acids. The
immunogenic fragments may comprise a sufficient number of contiguous amino acids
that form a linear epitope and/or may comprise a sufficient number of contiguous
amino acids that permit the fragment to fold in the same (or sufficiently similar) threedimensional
conformation as the full-length polypeptide from which the fragment is
derived to present a non-linear epitope or epitopes (also referred to in the art as
conformational epitopes). Identifying an immunogenic region and/or epitope of an
antigen can be readily determined by a person skilled in the art and/or by computer
analysis and computer modeling, using methods and techniques that are routinely
practiced by persons skilled in the art.
In some instances, compositions described herein can include a peanut extract,
such as an extract made from roasted peanuts, or an extract made from raw
(unroasted) peanuts. A pharmaceutically acceptable peanut extract is suitable for use
in humans, and is referred to as a drug substance or a drug product (see, e.g., FIG. 3).
A pharmaceutically acceptable peanut extract in the form of a drug substance can be
further formulated for administration to humans in the form of a drug product. A
peanut extract that is not processed to the stage that is acceptable for administration to
humans is referred to herein as a "crude peanut extract." Examples of a crude peanut
extract include a peanut flour extract, peanut oil, and crude peanut powder extract, or
liquid extract. A drug substance, which can also be a "new active substance", can be
a lyophilized peanut powder extract, a liquid peanut extract concentrate, or a diluted
liquid peanut extract. A drug substance can also be a purified flour or oil extract.
A pharmaceutically acceptable peanut extract, which is a drug substance or a
drug product, can be characterized in various ways, such as by measuring one or more
of Ara h protein content, total protein content, lipid content, polysaccharide content
metal content, aflatoxin content, microbial content, water content (particularly if the
extract is lyophilized) and the like. For example, the extract can be characterized
according to the amount of one or more of aflatoxin products Bl, B2, Gl and G2,
such as by HPTLC (high performance thin layer chromatography). Microbial content
can include, for example, total aerobic microbial count (TAMC), total yeasts and
molds count (TYMC) and measurement of specific microorganisms. In some
embodiments, a pharmaceutically acceptable peanut extract can be characterized
according to the amount of certain impurities, such as pesticides, solvents or other
organic or inorganic materials contained in the extract.
In some embodiments, the extract is characterized by how much of a particular
Ara h protein, or a combination of Ara h proteins, is contained in the extract. For
example, a pharmaceutically acceptable peanut extract, or a crude protein extract
(e.g., a crude protein extract with a known protein content) can be characterized by
the percentage of 2 or more Ara h proteins in the extract, e.g., at least 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, or 17, of Ara hi, Arah2, Arah3, Arah4, Arah5, Arah6,
Ara h7, Ara h8, Ara li9, Ara hlO, Ara h i 1, Ara hl2, Ara hl3, Ara hl4, Ara hl5, Ara
6, and Ara h protein. A peanut extract, such as a pharmaceutically acceptable
extract can be characterized by the ratio of 2 or more Ara h proteins in the extract.
For example, the Ara h2/Ara h6 ratio can range from about 3.0 to 1.0 (e.g., about 3.0,
about 2.5, about 2.2, about 2.0, about 1.9, about 1.7, about 1.5, about 1.4, about 1.3,
about 1.2, about 1.1, or about 1.0).
The total protein content of a pharmaceutically acceptable extract can be from
about 12% to about 6% (e.g., about 11.5%, about 11%, about 10.5%, about 10%,
about 9.5%, about 9%, about 8.5%, about 8%, about 7.5% or about 7%).
In one embodiment, the extract is characterized by the percent content of Ara
hi, Ara h2, and Ara h6 content and total protein content. In another embodiment, the
extract is characterized by the percent content of Ara hi, Ara h2, and Ara h6 content,
the total protein content, and the lipid content. In yet another embodiment, the extract
is characterized by at least (i) the percent content of Ara hi, Ara h2, and Ara h6
content, (ii) the total protein content, (iii) the lipid content, and (iv) the polysaccharide
content.
The components of a peanut extract, such as a pharmaceutically acceptable
peanut extract, can be assayed by methods known in the art. For example, the
proteins present in the extract and the total protein content can be assayed by
electrophoresis, such as by Bradford assay, SDS-PAGE and Western Blot, or
Coomassie Stain; by ELISA assay, or by chromatographic methods, such as HPLC
(high performance liquid chromatograph), or HPLC-UV or mass spectrometry
methods, such as LC-MS (Liquid Chromatography-Mass Spectrometry). Such assays
can further be used to identify the protein profile (e.g., ratio of Ara h proteins, such as
Ara hi : Ara h2: Ara h6) of a peanut extract. The Ara h content of a peanut extract
can be measured, for example, by ELISA, using specific antibodies (e.g., antibodies
that specifically bind Ara hi, Ara h2 or Ara h6), or by LC-MS. Lipid content can be
measured, for example, by HPTLC or HPLC-ESLD (Evaporating light scattering
detector). Metal content can be measure, for example, by ICP-MS (inductively
coupled plasma mass spectrometry).
A peanut extract, such as a pharmaceutically acceptable peanut extract, can be
assessed qualitatively, such as by its color or clarity in solution.
In some embodiments a pharmaceutically acceptable peanut extract can be
characterized functionally, such as by its ability to bind IgE and IgG in a biological
sample (e.g., a serum sample) of a peanut allergic patient. In other embodiments, a
pharmaceutically acceptable peanut extract can be characterized by a biological
activity of the extract, such as by the ability of the extract to activate one or both of
basophils or PBMCs (Peripheral Blood Mononuclear Cells) in a biological sample
(e.g., a blood or serum sample) of a peanut allergic patient. A pharmaceutically
acceptable peanut extract can be can be evaluated by measuring the potency of the
extract, such as by using a basophil activation test as described in the examples below.
A pharmaceutically acceptable peanut extract can be characterized and
established as an in house reference product (IHRP) to verify batch-to-batch
consistencies. A pharmaceutically acceptable peanut extract can have a protein
composition determined to be the same as an IHRP. A pharmaceutically acceptable
peanut extract can have a protein content of 80% to 120% of the reference product,
and individual allergens (e.g., certain Ara h proteins, such as Ara hi, Ara h2 and Ara
h6 proteins) that are 50% to 200% of the amount identified in a reference product. In
some instances, a lyophilized pharmaceutically acceptable peanut extract will have for
example, not more than 5% water content. In some instances, a pharmaceutically
acceptable peanut extract will meet the acceptance criteria set forth in the European
Pharmacopoeia Monograph on Allergen Products (European Pharmacopoeia 7.0
01/2010:1063).
Allergens may be immunogenic variants of a one or more naturally occurring
polypeptide antigens (e.g., an Arah protein) that retain at least 90% amino acid
identity over at least 10 contiguous amino acids of an antigen, or at least 85% amino
acid identity over at least 15 contiguous amino acids of an antigen. Other examples
include at least or about 70%, e.g., at least or about 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or at least or about 99% identity over at least
50 contiguous amino acids of the antigen, or at least or about 70%, e.g., at least 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least or about
99% identity over at least 100 contiguous amino acids of the antigen. These
polypeptide immunogenic variants retain the ability to cross-react with
immunoglobulins that are specific for the native antigen.
Variants can include at least one amino acid substitution, insertion, or deletion
in an amino acid sequence. Conservative substitutions of amino acids are well known
and may occur naturally in the polypeptide or may be introduced when the
polypeptide is recombinantly produced. Amino acid substitutions, deletions, and
additions may be introduced into a polypeptide using well-known and routinely
practiced mutagenesis methods. Alternatively, random mutagenesis techniques, such
as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis,
and oligonucleotide-directed mutagenesis may be used to prepare variants.
A variety of known criteria indicate whether an amino acid that is substituted
at a particular position in a peptide or polypeptide is conservative (or similar). For
example, a similar amino acid or a conservative amino acid substitution is one in
which an amino acid residue is replaced with an amino acid residue having a similar
side chain. Similar amino acids may be included in the following categories: amino
acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic
side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,
histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with betabranched
side chains (e.g., threonine, valine, isoleucine), and amino acids with
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, which is
considered more difficult to classify, shares properties with amino acids that have
aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In certain
circumstances, substitution of glutamine for glutamic acid or asparagine for aspartic
acid may be considered a similar substitution in that glutamine and asparagine are
amide derivatives of glutamic acid and aspartic acid, respectively.
A variety of methods are known in the art for recombinant production of
polypeptide allergens. Methods that may be used for isolating and purifying a
recombinant polypeptide, by way of example, may include obtaining supernatants
from suitable host/vector systems that secrete the recombinant allergen or antigen into
culture media and then concentrating the media using a commercially available filter.
Following concentration, the concentrate may be applied to a single suitable
purification matrix or to a series of suitable matrices, such as an affinity matrix or an
ion exchange resin. One or more reverse phase HPLC steps may be employed to
further purify a recombinant polypeptide. A variety of alternative purification
methods are known in the art.
A composition comprising the allergen/antigen can in some instances be in the
form of a composition comprising a recombinant expression vector that expresses the
allergen/antigen. Thus, all references herein to a composition comprising an allergen
or antigen apply equally to a composition comprising a viral vector carrying a
nucleotide that encodes the allergen(s) or antigen(s).
Alternatively or in addition, a composition can include an allergen that is a
chimeric fusion that includes multiple portions from different allergens, e.g., a fusion
protein that includes a first full-length Ara h protein fused to a second full-length Ara
h protein, a fragment of a first Ara h protein fused to a fragment of a second Ara h
protein, or a first full-length Ara h protein fused to a fragment of a second Ara h
protein. Methods of making fusion proteins are well known to those of ordinary skill
in the art, and skilled practitioners will appreciate that many variations are possible,
including fusion proteins that include whole or portions of at least 2, e.g., at least 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or at least 17, allergens, e.g., Ara h proteins.
Other allergens can be included in a pharmaceutical composition or method of
making a pharmaceutical composition described herein, as an alternative, or in
addition, to peanut allergens. For example, in some instances, a food allergen other
than peanut allergen(s) can be included. Examples of such food allergens include
milk allergen (e.g., whole milk or extract thereof, casein (e.g., alpha Sl-casein),
and/or beta-lactoglobulin), seafood allergen (e.g., allergens from vertebrates (e.g.,
salmon, cod, mackerel, sardines, herring, anchovies, tuna, trout, haddock, eel, and/or
rays) and/or invertebrates (e.g., crustaceans (e.g., shrimp, crab, crayfish, and/or
lobster allergen) and/or mollusks (e.g., clams, mussels, oysters, octopus, squid and/or
scallops allergen)), egg allergen, mustard allergen, sesame allergen, soy allergen,
wheat allergen (e.g., gluten), fruit allergen (such as Bet v 1 or homologues thereof,
lipid transfer protein, and/or profilin, and/or allergens from strawberry, apple,
avocado, blueberry, date, kiwi fruit, peach, raspberry, fig, grape, plum, cherry,
grapefruit, and/or prune), vegetable allergen (e.g., alfalfa, cauliflower, cucumber,
mushroom, radish, broad bean, eggplant, spinach, zucchini, broccoli, and/or pepper
allergen) or tree nut allergen (e.g., walnut, almond, cashew, pistachio, and/or pecan
allergen), to treat allergies to each of those foods. Alternatively or in addition, house
dust mite (HDM) allergen, an aeroallergen (e.g., pollen allergen) and/or grass allergen
can be included, e.g., for the treatment of an allergy, such as a seasonal allergy.
Further, compositions that include such allergens alternative or in addition to peanut
allergens can be used in the methods for treating allergy (i.e., an allergy to the
allergen included in the pharmaceutical composition) described herein.
Preparation of Compositions for Sublingual Administration
Compositions that include GLA and separately allergens (e.g., separate GLA
and peanut protein compositions), or a combination of both GLA and allergens (e.g., a
combination of GLA and peanut proteins in a single composition), in a sublingual
administration dosage form(s), can be prepared by a variety of methods. For example,
a composition useful in the present invention is a composition comprising peanut
allergens. Methods of extracting peanut proteins and producing allergen preparations
are known to those of skill in the art. For example, a method of making a crude
peanut extract is provided in U.S. Patent No. 6,486,311. In some methods, peanuts are
provided (e.g., from a commercial source), optionally roasted using methods known
in the peanut-processing arts, and defatted with hexane (after roasting, if applicable)
for 13 to 6 minutes at 163° C to 177° C. The powdered crude peanut can then be
extracted in 1 mol/L NaCl, 20 mmol/L sodium phosphate (pH 7.0) and 8 mol/L urea
for 4 hours at 4° C. The extract can be clarified by centrifugation at 20,000 g for 60
minutes at 4° C. Another exemplary' method is described in US. Patent Publication
No. 2014/0363470, that includes grinding peanuts to peanut powder; incubating the
peanuts in acetone for 30 minutes using 5 grams peanut powder per 50 mL acetone to
provide defatted peanut powder, drying the defatted peanut powder, suspending the
powder in a buffer with a pH between 7 and 9, and isolating the resulting supernatant
to provide a whole peanut extract. Alternatively or in addition, peanut extract, peanut
flour (comprising, e.g., grinded and defatted peanuts) and/or individual allergens can
be obtained from a private, academic, government, or commercial source (e.g., Greer
(North Carolina, US); Golden Peanut (Texas, US); Amanda Nut Processing Plant
(Germany); INDOOR biotechnologies (Virginia, US)).
A composition described herein comprising peanut proteins (whether alone or
in combination with GLA) can include peanut proteins in the range of about 0.3
mg/100ml to about 26,000 m ΐ , e.g., 0.5 m IOOpiI to 25,600 mg/ml, e.g.,
5 mg/100ml to about 10,000 mg/ml, e.g., about 3 ΐ to about 5,000 mg/ml. For
example, compositions may include peanut proteins in the range of about 100 m ΐ
to about 25,600 mg/ml, or about 2000 mg/ml to about 10,000 mg/ml. A composition
described herein can include peanut proteins of at least or about 1 mg/ml, e.g., at least
or about 3 mg/ml, 10 mg/ml, 20 mg/ml, 24 mg/ml, 25 mg/ml, 26 mg/ml, 50 mg/ml, 100
m p , 200 mg/ml, 400 mg/ml, 500 mg/ml, 1000 mg/ml, 2000 mg/ml, 3000 mg/ml, 4000
mg/ml, 5000 mg/ml, 6000 mg/ml, 6400 mg/ml, 6500 mg/ml, 6600 mg/ml, 7000 mg/ml,
8000 mg/ml, 9000 mg/ml, 10,000 mg/ml, 15,000 mg/ml, 20,000 mg/ml, 25,000 mg/ml,
or 26,000 mg/ml.
A composition comprising peanut proteins can include a salt, such as sodium
chloride, or sodium citrate, in an amount of 0.05% to 1%, e.g., 0.1% to 0.5%, e.g.,
0.2% to 0.3%, e.g., 0.25%. A composition comprising peanut proteins can contain a
preservative, such as glycerol, in an amount of 30% glycerol to 80% glycerol, e.g.,
40% glycerol to 60% glycerol, e.g., 45% glycerol, 50% glycerol or 55% glycerol. A
composition comprising peanut proteins can be at neutral or basic pH, e.g., at pH 7 to
pH 9, e.g., pH 7.5, pH 8.0, pH 8.2, pH 8.5.
In one embodiment, a composition comprising peanut proteins for sublingual
administration (with or without GLA) comprises 0.25% NaCl, 0.27% NaHC0 , 60%
glycerol, and water. In one embodiment, the composition has a pH of about 8.2.
A composition comprising GLA can be prepared in any number of art-known
ways. In one exemplary method, GLA can be synthesized or obtained from
commercial suppliers (e.g., Avista Pharma Solutions (Durham, NC), Avanti Polar
Lipids (Alabaster, AL), Immune Design Corporation (Seattle, WA)). The GLA can
be combined with a lipid, such as l,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC; commercially available, e.g., from Avanti Polar Lipids, Inc.), in a molar ratio
of about 1:2 (GLA:DPPC), and optionally dissolved in chloroform by vortexing or by
other art-known mixing means. The chloroform can then be evaporated (e.g., under
vacuum) and the resulting thin film solid mixture can be rehydrated and diluted, e.g.,
in water. The mixture can then be agitated (e.g., by one or more of mechanical
agitation, such as by Ultra-Turrax® (IKA®, Wilmington NC), by BecoMix (A.
Berents GmbH & Co. KG, Stuhr, Germany), or by sonication, microfluidization or
high pressure homogenization, or any combination thereof, such as to generate
nanoparticles) and filtered. Sonication of the formulation during the methods yields a
formulation that includes GLA lipid particles, such as liposomes, micelles, and/or
aggregates, or combinations thereof. In some instances, the final, average lipid
particle size is less than 100 nm. In some instances, the final, average size of the lipid
particles in the formulations produced by methods described herein is about 10 nm to
about 2000 nm, e.g., about 16 nm to about 1800 nm, about 50 nm to about 1000 nm,
about 80 to about 500 nm, about 85 nm to about 300 nm, about 95 nm to about 200
nm, about 90 nm to about 100 nm. In some instance, the final, average lipid particle
size is less than 500 nm.
A composition comprising GLA can contain DPPC at a concentration of, e.g.,
0.001% to 0.1% DPPC, e.g., 0.01% to 0.05% DPPC, e.g., 0.02% to 0.03% DPPC,
e.g., 0.025% DPPC. A composition comprising GLA can contain a preservative, such
as glycerol, in an amount of 30% glycerol to 80% glycerol, e.g., 40% glycerol to 60%
glycerol, e.g., 45% glycerol, 50% glycerol or 55% glycerol. A composition
comprising peanut proteins can be at neutral or basic pH, e.g., at pH 7 to pH 9, e.g.,
pH 7.5, pH 8.0, pH 8.2, pH 8.5.
In one embodiment, a composition comprising GLA (with or without peanut
allergen) comprises 0.025% w/v DPPC, 60% w/v glycerol and water. The
formulation can be comprised in water. In some embodiments, the formulation can be
filtered.
In some instances, it may be beneficial to not use chloroform in the
preparation methods. Accordingly, another exemplary method omits using
chloroform in the procedure. For example, GLA and DPPC can be combined in a
molar ratio of 1:2. Warm purified water (e.g., at 50°C to 75 °C) is added and the
mixture is agitated, such as by mechanical agitation or sonication and filtered. For
example, in one embodiment, GLA and 0.025% DPPC are combined. Warm purified
water (e.g., at 70°C) is added, and the mixture is mechanically agitated using Ultra-
Turrax. In some embodiments, the mixture is further filtered.
Importantly, skilled practitioners will appreciate that lipids or surfactants
alternative or in addition to DPPC can be used. Exemplary alternative lipids or
surfactants include, e.g., Sodium Lauryl Sulfate; Polyoxy ethylene (20) sorbitan
monooleate (also known as polysorbate-20 and Tween-80); poloxamer (also known
under the trade names Synperonics, Pluronics, and Kolliphor), e.g., poloxamer 407 or
polxamer 188; camphor; Tween-20; or mixtures thereof. In one instance, the
preparation will utilize polysorbate (e.g., polysorbate-80) with 50% or 60% glycerol,
which may be useful to maintain a preservative free formulation. In one example, the
sublingual formulation will include one or both of lecithin and taurocholate.
In one embodiment, the sublingual formulation is free of preservatives.
Of particular usefulness in the presently described methods is Tween-80 (e.g.,
at 0.5%-10% of the formulation) which, when used with GLA, may reduce (or
eliminate) the need to sonicate the GLA solution to form a nanosuspension, which is
particularly useful, e.g., during scaled-up processes.
The concentration of GLA in a composition produced by methods described
herein can be in a range of about 0.002 mg/mL to about 8.0 mg/ml, about 0.005
mg/mL to about 7.0 mg/ml, about 0.01 mg/mL to about 6.0 mg/ml, about 0.02 mg/mL
to about 5.5 mg/ml, e.g., about 0.05 mg/ml to about 5.0 mg/ml, about 0.1 mg/ml to
about 4.0 mg/ml, about 0.2 mg/ml to about 3.5 mg/ml, about 0.3 mg/ml to about 3
mg/ml, about 0.4 mg/ml to about 2.5 mg/ml, about 0.5 to about 2.0 mg/ml, or about
0.6 to about 2 mg/ml. For example, the GLA concentration can be about 0.02 mg/mL,
about 0.1 mg/mL, about 0.8 mg/ml or about 1.6 mg/ml. Further, in the methods of
preparing the GLA described herein, the molar ratio of GLA to DPPC can be in a
range of from about 1:1 to about 1:3, e.g., about 1:2. In some instances, in addition to
GLA, at least one other type of lipid and/or adjuvant (e.g., at least 2, at least 3, or at
least 4 types of lipid and/or at least 2, at least 3, or at least 4 types of adjuvant) can be
included in the compositions described herein. Alternatively or in addition, in some
instances, in addition to DPPC, at least one other type of surfactant (e.g., at least 2, at
least 3, or at least 4 types of surfactants) can be included in the compositions
described herein.
A composition comprising both GLA and peanut proteins can be prepared by
mixing an aqueous solution of the peanut proteins described above (e.g., whole peanut
extract or individual allergens described herein) with GLA and the surfactant (e.g.,
DPPC). Alternatively, GLA may be co-precipitated with the antigen. In one
example, the method includes co-dissolving glucopyranosyl lipid adjuvant (GLA) and
l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at a 1:2 molar ratio in a
solvent, such as chloroform, to form a GLA/DPPC mixture and then adding peanut
allergen, such as in the form of peanut proteins, to the mixture. The solvent, e.g.,
chloroform, is then removed from the GLA/DPPC/ peanut proteins mixture, e.g.,
under vacuum. Water is then added to the GLA/DPPC/ peanut proteins mixture to
rehydrate the mixture and then the mixture is agitated, e.g., via sonication and/or
microfluidization. Another exemplary method includes co-dissolving glucopyranosyl
lipid adjuvant (GLA) and l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at a
1:2 molar ratio in chloroform to form a GLA/DPPC mixture. The chloroform is then
removed from the GLA/DPPC mixture, e.g., under vacuum. Water is then added to
the GLA/DPPC mixture and then the mixture is agitated (e.g., using mechanical
agitation, such as by Ultra-Turrax, or sonication and/or microfluidization). Peanut
protein extract is then added to the formulation in amounts appropriate to achieve the
desired concentration of proteins.
In another exemplary method, GLA and DPPC are co-dissolved at a 1:2 molar
ratio in chloroform to form a GLA/DPPC mixture. The chloroform is then removed
from the GLA/DPPC mixture, e.g., under vacuum. The mixture is then spray-dried to
form a solid particle of GLA/DPPC. The peanut proteins are then layered on the
surface of the GLA/DPPC particle by a fluid bed coating process. The final
composition is a dry powder containing GLA and peanut proteins.
Alternatively, skilled practitioners will appreciate that separate compositions
of GLA and peanut proteins can be created as described herein and mixed together
shortly before administration to a patient.
The compositions may conveniently be formulated with a pharmaceutically
acceptable diluent, carrier or excipient suitable for administration. Details of
pharmaceutical excipients may be found in "Handbook of Pharmaceutical
Excipients", 7th Ed. (2012), The Pharmaceutical Press, London, Editors: Rowe et al.
Suitable physiologically acceptable carriers and diluents include, e.g., sterile water or
5% dextrose water solution. Sterile saline and/or phosphate buffered saline at
physiological pH may be used. Preservatives, stabilizers, dyes and/or flavoring agents
may be provided in the pharmaceutical compositions described herein. For example,
sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as
preservatives. Antioxidants and/or suspending agents may be included.
Sublingual administration of the presently described compositions is
particularly useful in the present invention. A gel or other viscous formulation may be
useful due to increased antigen contact with the sublingual surface. Skilled
practitioners will appreciate that a formulation need not physically or chemically bind
to the mucosal tissue.
Formulations suitable for sublingual administration may include aqueous and
non-aqueous sterile solutions. Such formulations may include anti-oxidants, buffers,
bacteriostatic compounds and solutes that render the formulation isotonic with the
bodily fluid, e.g., the mucus, of the individual. The aqueous and non-aqueous sterile
suspensions may include suspending agents or thickening agents.
Specifically, compositions described herein, including compositions
comprising GLA alone or antigen alone, and compositions comprising both GLA and
antigen, can be incorporated into forms for sublingual delivery, such as gels, capsules,
gel capsules, lozenges, tablets, sprays, drops, strips, or films, or disposed in devices
such patches (e.g., for extended release delivery) or pumps or sprayers. The
compositions can be administered by a metered dosing pump, for example. The
compositions can alternatively be incorporated into multi-particulate granules or
microspheres and optionally packaged in a container, such as such as sachet.
By way of example, a spray or drop formulation can be prepared for delivering
peanut proteins and GLA, either in separate formulations or as a combined
formulation. A spray or drop formulation useful in the present invention may include
about 0.5% NaCl, 0.54% Na-bicarbonate, and 50% glycerol. Alternatively, a spray or
drop formulation useful in the present invention may include about 0.5% NaCl, 0.54%
Na-bicarbonate, and 60% glycerol. The formulation can have a pH of about 4.0 to
about 8.4 (e.g., pH 5.0, pH 5.5, pH 6.0, pH 6.8, pH 7.0, pH 7.5 or pH 8.0). One
exemplary spray or drop formulation includes a flavoring, such as a citric flavoring
(e.g., orange or cherry), and has pH 4 to 4.5.
In another example, the compositions may be in the form of salves, pastes,
gels, solutions, powders and the like. Gels may be formulated using carbopol, also
known as carbomer, a carboxyvinyl polymer, or a cellulose-based thickening agent
such as hydroxy ethyl cellulose, hydroxypropyl cellulose or hydroxypropyl
methylcellulose, carboxymethylcellulose calcium, carboxymethylcellulose sodium,
ethyl cellulose, methylcellulose. Gels may also be conveniently formulated using:
acacia, alginic acid, bentonite, carrageenan, cetostearyl alcohol, gelatin, guar gum,
magnesium aluminium silicate, maltodextrin, polyvinyl alcohol, propylene carbonate,
propylene glycol alginate, colloidal silicon dioxide, sodium alginate, tragacanth,
and/or xanthan gum. Particularly useful are carbopol and the cellulose-based agents.
A pharmaceutical composition formulated as a sublingual tablet is particularly
useful in the methods described herein. Sublingual tablets are placed under the
tongue and are usually small and flat, compressed lightly to yield a soft tablet. The
tablet should dissolve easily allowing the composition to be absorbed quickly.
Various techniques can be used to formulate rapidly disintegrating or dissolving
tablets (see, e.g., Fu et al., Crit Rev Ther Drug Carrier Syst. 2004; 21: 433-476).
Direct compression is one of these techniques, which can involve incorporation of a
superdisintegrant into the formulation or the use of highly water-soluble excipients to
achieve fast tablet disintegration. Direct compression does not require the use of
water or heat during the formulation procedure and is an ideal method for moisture
and heat-labile medications. Superdisintegrants include, for example, cross-linked
cellulose, cross-linked polyvinylpyrrolidone, cross-linked starch, and cross-linked
alginic acid. A sublingual tablet can include, for example, one or more of a binding
agent, such as gelatin, a microcrystalline cellulose, sucrose with dextrins, polyvidone,
tragacanth, acacia, starch, or methylcellulose; a matrix supporting/disintegrating agent
such as mannitol, L-alanine, cornstarch, alginic acid, cellulose or a cellulose
derivative, povidone or sodium crosscarmelose; a lubricant, such as stearic acid,
stearate (e.g., magnesium stearate), sodium stearyl fumarate, or talc; a buffer; a
surface active agent; an anti-adhering agent selected from colloidal silicon dioxide,
calcium sulfate, calcium chloride, talc, com starch; polar and non-polar dissolving
agents selected from water, ethyl alcohol, acetone, isopropyl myristate,
polyoxypropylenes, propylene glycol, polyethylene glycol, glycerol, 70% sorbitol,
polyethylene glycols, mineral oil, petrolatum, lanoline, vegetable waxes, animal
waxes, vegetable oils such as olive oil, cottonseed oil, corn oil, or a mixture thereof;
and a sweetener (e.g., dextrose, sucralose, stevia, aspartame, acesulfame potassium,
fructose, glucose, mannitol, sorbitol, sugar, and sucrose) or other flavoring agent (e.g.,
chocolate, menthol, vanillin, cinnamon, sorbitol, citric acid, cherry flavor, orange
flavor, pineapple flavor, peach flavor, grape flavor, or berry flavor, such as strawberry
or blueberry flavor). In one embodiment, the flavoring is a citric flavor such as
SyrSpend cherry, SyrSpend grape, or Ora-Sweet cherry. The flavoring agent can be
present in a ratio from 0.0001% to 5.0%.
Exemplary tablets include fast disintegrating sublingual tablets (FDT) (which
can be lyophilisates), bioadhesive sublingual tablets, compressed tablets (ODT- orally
disintegrating tablets), and lipid matrix sublingual tablets. Exemplary methods of
making such tablets are described, e.g., in Nibha et al, International Journal of
Research in Pharmaceutical and Biomedical Sciences, p . 913-923, vol. 3 (2) Apr. -
June 2012. A sublingual tablet can include, for example, peanut extract (from roasted
or raw peanuts), GLA, com starch, and lactose monohydrate magnesium stearate. A
sublingual tablet can include, as another example, peanut extract (from roasted or raw
peanuts), GLA, mannitol, microcrystalline cellulose, a sodium
carboxymethylcellulose, such as croscarmellose sodium, colloidal anhydrous silica,
magnesium stearate and lactose monohydrate. Still another example of a sublingual
tablet is one that includes peanut extract (from roasted or raw peanuts), GLA, gelatin,
mannitol, and sodium hydroxide. In some instances, sublingual tablet formulations
can contain one of, but not both of, GLA or peanut extract.
Also useful are thin films or strips. A thin film or strip dissolves when in
contact with liquid, e.g., the saliva of a patient, and is placed under the tongue for an
appropriate amount of time to allow the film or strip to dissolve. Fast-dissolving
films can be made of plasticized hydrocolloids. Films are typically stable to moisture,
are flexible, and may be formulated to resist sticking to packaging materials and
fingers. Exemplary methods of making thin films or strips for sublingual
administration include solvent casting, semisolid casting, hot melt extrusion, solid
dispersion extrusion, and/or rolling (see, e.g., Nibha et al, International Journal of
Research in Pharmaceutical and Biomedical Sciences, p . 913-923, vol. 3 (2) Apr. -
June 2012). Skilled practitioners will appreciate that thin films or strips of the present
invention can take a number of different configurations. For example, the GLA or
peanut proteins (e.g., peanut extract made from roasted or raw peanuts), or both, can
be disposed within the thin film. Alternatively, the GLA or peanut proteins (e.g.,
peanut extract made from roasted or raw peanuts), or both, can be disposed on the
surface of the thin film. Alternatively, GLA or peanut proteins (e.g., peanut extract
made from roasted or raw peanuts), or both, can be disposed both within and on the
surface of the thin film.
A thin film or strip for sublingual administration typically contains one or
more of, e.g. a polymer, such as pullulan or microcrystalline cellulose with
maltodextrin; a plasticizer, such as glycerol, propylene glycol, a low molecular weight
polyethylene glycol, a phthalate derivative, such as dimethyl, diethyl, or dibutyl
phthalate, a citrate derivative with as tributyl, triethyl, acetyl citrate, triacetin or castor
oil; a sweetener; a coloring agent, and thickening and stabilizing agents.
Of particular usefulness are compounds that can be added to the formulations
to enhance sticking or retention of the formulation under the tongue of a patient, i.e., a
mucoadhesive agent. Exemplary mucoadhesive agents include chitosan, hyaluronate,
alginate, gelatin, collagen, poly(acrylic acid), poly(methacrylic acid), poly(L-lysine),
poly(ethyleneimine), poly(ethylene oxide), poly(2-hydroxyethyl methacrylate), and
derivatives or copolymers thereof, and any combination thereof. Skilled practitioners
will appreciate that a mucoadhesive can be added to any of the formulations or dosage
forms described herein.
There are various commercial sources for manufacturing solid dosage forms,
such as Catalent®, which uses Zydis® technology; Colorcon®, which produces
Suglets® Sugar Spheres; Umang Pharmatech, which produces Sprayspheres™
microcrystalline cellulose (MCC) pellets.
The formulations may be presented in unit-dose or multi-dose containers, for
example, sealed ampoules and vials and may be stored in a freeze-dried condition
requiring only the addition of the sterile liquid carrier immediately prior to use.
Treatments and Administration of Compositions
While not necessarily required for carrying out treatments described herein,
patients may be diagnosed with peanut allergy and/or monitored according to standard
clinical criteria prior to and/or during treatment. Standard clinical criteria may
include, for example, a history of atype-1 hypersensitivity reaction that is temporally
related to peanut ingestion (e.g. hives, swelling, wheezing, abdominal pain, vomiting,
breathlessness). The presence of peanut-specific IgE by positive skin prick test
(wheal diameter >3 mm) or ImmunoCap serum IgE > 0.35 kU/1, may also be
indicative of peanut allergy. A patient's blood can be assayed, e.g., using the basophil
activation test described in detail in the Examples section, below.
The compositions are administered in a manner compatible with the dosage
formulation, and in such amount as will be prophylactically and/or therapeutically
effective. The quantity to be administered depends on the patient to be treated, the
capacity of the patient's immune system to synthesize antibodies, and the degree of
protection desired. In some instances a patient can be treated with a series of
administrations which will include a rising peanut proteins dose regime. The precise
amounts of active ingredients administered may depend on the judgment of the
practitioner and may be peculiar to each individual patient. The composition may be
given in a single dose schedule, or in a multiple dose schedule.
In one exemplary dosage regimen, a composition comprising GLA, such as a
solid or liquid composition comprising GLA, is administered first, followed by
administration of a composition (e.g., a solid or liquid composition) comprising
allergen, e.g., peanut allergen(s). In another exemplary dosage regimen, both the
GLA and peanut allergen(s) are administered at approximately the same time. In one
regimen, a GLA composition that includes GLA (e.g., a liquid formulation at 1
mg/50 ml or 10 mg/50ml, or in a range between these two values, inclusive) and DPPC
(or GLA, DPPC and glycerol, or GLA, DPPC, and Tween 80) is provided. An
amount of the GLA composition is administered sublingually, i.e., under the tongue of
the patient (e.g., about 50m1to about 500m1of a liquid formulation). Following and/or
during administration of the GLA composition to the patient, an amount of a peanut
proteins composition (e.g., about 50 mΐ to about 200 mΐ of a liquid formulation having
protein concentration of about 6 mg/ml to about 25.6 mg/ml, inclusive, e.g., a protein
concentration of 6 mg/ml or 25.6 mg/ml) is administered sublingually, under the
tongue of the patient. Here, the peanut protein composition can include, e.g., 50%
glycerol or 60% glycerol or 70% glycerol, and, optionally, 0.4% phenol as a
preservative, 0.2% to 0.5% NaCl, 0.20% to 0.45% sodium bicarbonate, and 50%
glycerol, at a pH of between 6.8 to 8.4. In one embodiment, the peanut protein
composition includes, e.g., 60% glycerol, 0.25% NaCl, 0.27% sodium bicarbonate,
60% glycerol, and water at a pH of about 8.2.
In another exemplary regimen, a composition that includes GLA (e.g., a solid
formulation) and a compound to aid the solubilization of GLA, such as DPPC or a
polysorbate, such as PS80 or PS20, is provided. The GLA composition can include
one or more of DPPC, a polysorbate, glycerol, or Tween (e.g., Tween 80). An
amount of the GLA composition is administered sublingually, i.e., under the tongue of
the patient. A solid formulation, in the form of a capsule or tablet, for example, can
contain a therapeutically effective amount of GLA, such as 0.5 mg, 1 mg, 1.5 mg, 2 mg,
3 mg, 5 mg, 7 mg, 10 mg, 12 mg, 15 mg, 20 mg or more of GLA, for sublingual
administration.
In some instances, a solid formulation comprising GLA and separate solid
formulation comprising peanut protein are administered to the patient at about the
same time, e.g., by placing the solid formulations together under the tongue or on
opposite sides under the tongue of the patient. In other instances, a single solid
formulation comprising both GLA and peanut protein is placed under the tongue of
the patient. In other instances, a liquid formulation comprising GLA and a separate
liquid formulation comprising peanut protein are administered to the patient at about
the same time, e.g., by placing the liquid formulations together under the tongue or on
opposite sides under the tongue of the patient. In still other instances, a single liquid
formulation comprising both GLA and peanut protein is placed under the tongue of
the patient. In yet other instances, one of each type of composition, i.e., one solid
(comprising GLA or peanut protein) and one liquid (comprising GLA or peanut
protein, i.e., whichever component is not comprised within the solid composition),
can be administered to the patient.
The frequency with which a patient is dosed with the compositions can vary
depending upon the needs of the patient and the level of protection required. For
example, in some instances, only a single dose on a single day of each composition
(i.e., the GLA composition and the peanut proteins composition) is administered to a
patient. In other instances, the patient can be dosed more than once on a single day,
such as twice, thrice, or more times in a single day. In still other instances, the patient
can be dosed once a day for multiple days (e.g., for at least 2, e.g., at least 3, 4, 5, 6, or
7 days), or for several weeks (e.g., at least 2, e.g., at least 3, 4, 5, 6, 7, 8, or more than
8, weeks) or for several months (e.g., at least 2, e.g., at least 3, 4, 5, 6, or more than 6,
months), or more than once each day (e.g., at least twice, or at least thrice), for
multiple days (e.g., for at least 2, e.g., at least 3, 4, 5, 6, or 7 days), or for several
weeks (e.g., at least 2, e.g., at least 3, 4, 5, 6, 7, 8, or more than 8, weeks) or for
several months (e.g., at least 2, e.g., at least 3, 4, 5, 6, or more than 6, months, up to
multiple years). Skilled practitioners will appreciate that the methods can be adapted
for delivery of a combined GLA/peanut protein formulation to patients. Such a
formulation may take the form of a liquid combined GLA/peanut protein formulation
or a solid, dissolvable tablet combined GLA/peanut protein formulation.
Subjects suitable for administration of the GLA and peanut SLIT formulations
featured herein include children 1 year old and older, e.g., children 4 years old and
older. Patients suitable for administration of the GLA and peanut SLIT formulations
featured herein include children 2 to 12 years of age, e.g., 4 to 10 years of age,
adolescents 13 to 20 years of age, and adults 2 1 years of age and older.
The efficacy of the peanut protein/ GLA combination sublingual therapy
described herein can be measured by examining expression of certain markers in the
patient, e.g., markers of the immune system, such as cytokines and interleukins. For
example, the patient can be assessed for the effect of the formulation on expression of
a Thl cytokine, such as IL- I b, IL-6, or IFN-g , IFN-b or a Th2 cytokine such as IL-4,
IL-5, IL-10, or IL-13. In some embodiments, the patient is assessed for an effect on
expression of IL-10, IL-7, IL-8, IL-2, IL-12, IL-17, GM-CSF, CRP (C-Reactive
Protein), fibrinogen, RSAD2, IFIT1B, TLR4, TNFa, TNFy, CXCL2, CXCL10,
CCL4, CCL7, CD154, a Type I interferon and/or TGFp in a biological sample, such
as a saliva or blood or serum sample, or a sample (e.g., a swab or a biopsy) from the
sublingual mucosa. For example, administration of a formulation comprising GLA
and antigen can prevent upregulation of CD154 or IL-13 expression following
exposure to antigen, and can maintain or increase IL-10 expression levels, or prevent
a decrease in IL-10 expression. The effect on biomarker expression can be assayed
in peripheral blood mononuclear cells (PBMCs) isolated from the patient. Expression
can be monitored by assaying for changes in mRNA or protein levels. The patient can
also be monitored for upregulation of dendritic cells (DCs) or macrophage cell surface
expression of one of more of CD40, CD80, CD83, CD86, and MHC II. For example,
the patient can be monitored for upregulation of macrophage expression of CD80 and
CD86. The patient can also be monitored for the expression of one or more of
salivary peanut allergen-specific IgA, peanut allergen-specific IgE, peanut allergenspecific
IgG, or allergen-specific Ig4. In one embodiment, the patient is assessed for
the effect of the formulation on IL-6 levels in a biological sample, such as in a sample
from the sublingual mucosa of the patient. Typically, administration of a GLA/peanut
allergen formulation will cause IL-6 levels to rise at a dose-dependent manner at the
site of sublingual administration.
In some embodiments, a basophil activation test will be used to measure the
effect of the GLA/peanut allergen sublingual therapy. For example, the basophil
activation test can be used to sample biomarker expression in a blood sample from the
patient. Exemplary biomarkers include IgE, CD203c, HLS-DR, CD123, CD63, and
Lin. Typically CD203c, CD63 and basophil degranulation (e.g., histamine and beta
hexosaminidase release) will be decreased, in a blood sample from a patient treated
with the GLA/peanut allergen sample, as compared to levels prior to treatment. Also,
peanut allergen-induced memory T cell activation will be decreased, which will be
evident by a decrease in allergen-induced proliferation, and IL-2 and IL-13
expression, and an increase in IFNy and IL-10 expression. The patient can also be
monitored for an expected increase in T regulatory cell activity, which is typically
evidenced by an increase in FoxP3 positive T cells, IL-10 expression, and increased
epigenetic changes at the FoxP3 locus of regulatory T cells. The patient can also be
monitored for an increase in peanut allergen-specific IgGs, including IgGl, IgG2a,
IgG2c, and IgG4.
Further, combination treatments are contemplated by the present invention.
For example, a skilled practitioner will appreciate that the treatment methods
described herein can be administered to a patient in conjunction with other, art-known
treatments for allergy e.g., peanut allergy. Such combination treatments may include
known treatments for the allergy itself, and/or one or more symptoms of allergy in the
patient.
Further, since certain peanut allergens cross-react in the body with other, nonpeanut
allergens, skilled practitioners will appreciate that the presently described
compositions may be useful for treating other types of allergies. For example, the
compositions and methods described herein that include peanut allergen(s) may be
useful for treating allergies to birch tree pollens. Alternatively or in addition, the
compositions and methods may be useful for treating allergies to pitted fruits, such as
peaches and peach-related fruits and products.
The invention will be described with reference to the following examples that
are intended to be illustrative only and not limiting.
EXAMPLES
Example 1. GLA and Peanut Allergen Co-Administered Sublingually Provides a
Protective Effect Against Peanut Allergy
The present Example describes in vivo mouse studies demonstrating that a
combination of GLA and peanut allergen(s), when administered sublingually, confers
protection against subsequent challenge with peanut allergens. All experiments were
performed in mouse model C3H/HeOuJ. Briefly, mice were sensitized with 1mg
crude peanut extract (CPE), and/or 10 mg cholera toxin (CT) on days 0, 1, 2, 7, 14,
and 21. Mice were then treated with CPE (0.5 mg, 5.0 mg, or 50 mg), GLA (aqueous
formulation; GLA-AF), and methyl cellulose (MC) (1.875%) sublingually on days 28,
35, 42, and 49. Finally, the mice were challenged with 500 mg CPE intraperitoneally
(IP) on day 56. The experiment was terminated on day 57. The experimental
protocol is summarized in the below Table 1.
Table 1. Mouse allergen challenge experiment
Results are provided in FIGs. 1A, IB, 2A, and 2B. In each figure, the x-axes
provide a description of the treatments administered to a given group of mice. All
mice were challenged with 500 mg CPE intraperitoneally, which is indicated below
the x-axes. In FIGs. 1A and IB, the y-axes provide the anaphylaxis score observed in
the mice on a scale of 0 to 5 for each treatment. In FIGs. 2A and 2B, the y-axes
provide the change in core body temperature observed in the mice following
challenge. A severe reaction is manifested as an increase in anaphylaxis score (FIGs.
1A and IB) or a drop in body temperature (FIGs. 2A and 2B). The greater the
decrease in core temperature, or increase in anaphylaxis, then the more severe the
reaction. A protective affect is noted when, following IP challenge, a less severe drop
in body temperature or a lower anaphylaxis score is observed.
Methods: For sublingual administration, a needleless syringe was used to
apply 5 mΐ of compound sublingually under the tongue of anesthetized mice. The
mice were monitored for 10 minutes to allow time for the dose to be absorbed, before
the mice were returned to their cages.
Saline was 0.9% NaCl. CPE was formulated in DPBS (Dulbecco's Phosphate
Buffered Saline), and diluted in saline. GLA-AF was formulated by mixing GLA and
DPPC in a 1:2 molar ratio in water. Immediately before dosing, methylcellulose was
added to the final concentration to create a gelatinous mixture. The mixture was
vortexed vigorously and then the mixture was sonicated for 10 minutes.
For co-formulated CPE and GLA-AF, CPE was diluted to 0.4 mg/ml in saline.
A 1:2 molar ratio mixture GLA-AF:DPPC in water was added to the 0.4 mg/mL CPE
mixture to yield a 0.8 mg/mL GLA/ 0.2 mg/mL CPE solution. Methylcellulose was
then added to the 0.8 mg/mL GLA/ 0.2 mg/mL CPE solution to make a final solution
of 0.4 mg/mL GLA/ 0.1 mg/mL CPE/ 1.875% methylcellulose solution. This mixture
was vortexed vigorously and then sonicated for 10 minutes. Mice were administered
5 of this final solution sublingually, which delivered a dose of 2.0 mg GLA-AF +
0.5 mg CPE.
Example 2 . Sublingual co-administration of GLA and antigen induces antigenspecific
T cell proliferation in the draining cervical lymph nodes
Ova-specific OT-II TcR transgenic T cells were labeled with cell-tracker violet
dye and adoptively transferred into syngeneic wild type mice. Mice then received
sublingual administration of GLA (0.2 mg) alone (FIGs. 4A and 4B), O v a 2 -
peptide (10 mg) alone (FIGs. 4C and 4D), or GLA co-administered with Ova peptide
(FIGs. 4E and 4F). Four days later, the draining cervical lymph nodes (FIGs. 4A, 4C,
and 4E) and spleen (FIGs.4B, 4D, and 4F) were removed and OT-II T cell
proliferation (via the decrease in cell-tracker dye fluorescence) was measured by flow
cytometry. As shown in Figures 4A-4F, sublingual administration of GLA with Ova
antigen, but not GLA or antigen alone, induces antigen-specific T cell proliferation in
the draining lymph nodes but not the spleen.
Methods. Sublingual administration was performed as described above in
Example 1.
Formulations of Ova peptide and/or GLA included methylcellulose as
described in Example 1. Mixtures of methylcellulose + Ova peptide, and
methylcellulose + Ova peptide + GLA were prepared the day before dosing.
Example 3. Antigen-specific T cell proliferation in the draining cervical lymph nodes
after sublingual co-administration of GLA and Ova323-329 peptide
Ova-specific OT-II TcR transgenic T cells were labeled with cell-tracker violet
dye and adoptively transferred into syngeneic wild type mice. Mice then received
sublingual administration of GLA (0.2 mg, 0.02 mg or 0.002 mg) co-administered with
O v a 2 - peptide or peptide without GLA (0 mg). Four days later, cervical lymph
nodes were removed and OT-II T cell proliferation (via the decrease in cell-tracker
dye fluorescence) was measured by flow cytometry. As shown in FIG. 5, sublingual
administration of GLA with antigen enhances antigen-specific T cell proliferation in
the draining lymph nodes and in a dose-dependent manner.
Methods. Sublingual administration was performed as described above in
Example 1.
Formulations of Ova peptide and/or GLA included methylcellulose as described in
Example 1. Mixtures of methylcellulose + Ova peptide, and methylcellulose + Ova
peptide + GLA were prepared the day before dosing.
Example 4 . IL-6 Expression is Increased at the site of Sublingual Administration
mRNA gene expression analysis of sublingual mucosal brushings from
Non-Human Primates (NHPs) treated with 3 different doses of GLA revealed a
dose-dependent expression of IL-6 six hours after sublingual administration.
GLA was formulated in DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine)
as GLA:DPPC 1:2 (M:M) in Millipore (sterile) water. Animals were sedated with
ketamine (5 mg/kg to 10 mg/kg)/dexmedetomidine (0.01 mg/kg to 0.01 6 mg/kg), IM
(intramuscularly), while in their cage. Once sedated, they were brought into the
laboratory and placed onto a circulating water heating pad (Gaymar). Sublingual
(right side) and left cheek pouch mucosal brushings were taken 15 minutes prior to
dosing using Copan FLOQSwabs™ and placed into RLT buffer (QIAmp Minikit
Plus, Qiagen), vortexed and immediately frozen on dry ice. Sublingual dosing of
GLA (100 mΐ dosed via syringe) was performed under the right side of the animals
tongue and allowed to absorb into the mucosa for three minutes. Further sublingual
and cheek brushings were obtained at 6 hr, 24 hr and 48 hr post dosing.
RNA processing was performed from the cheek and sublingual swabs using a
QIAmp Minikit Plus (Qiagen) which includes a gDNA (genomic DNA) eliminator
column. Gene expression was quantified using the Comparative CT Method, which
calculates the relative gene expression quantitation between a target gene and an
endogenous control. All target genes were quantitated relative to the expression of
the housekeeping gene ARL1. Using the relative expression, the fold of baseline
expression was calculated as [(Relative gene expression at time point of interest) /
(Baseline relative gene expression)].
Expression was localized to the sublingual mucosa as IL-6 was not detected in
brushings from the cheek or in the blood at this time point. IL-6 expression was not
detected 24 hours after sublingual administration of GLA. Together the data suggest
that the current formulation of GLA acts very locally within the oral cavity, does not
last longer than 1 day and has a potential window of activity between 0 .1 and 5mg .
The results also suggest that IL-6 expression may be a biomarker for GLA activity.
Example 5. Basophil Activation Test
The present specification provides a basophil activation test. There are at least
two uses for the test. The first relates to its use in the clinic, e.g., a one tube assay for
measuring biomarkers in the sera of peanut allergic patients. The second relates to its
use as an assay to measure the potency (allergenicity) of a peanut extract, and which
can be used to standardize the potency of different batches of peanut extract that will
be used in generating compositions described herein.
Generally, an exemplary test can be performed as follows. Whole blood
sample from peanut allergic patients is obtained, and basophils are primed by diluting
the blood 1:1 in a BAT buffer (Basophil Activation Test Buffer), which contains 4
ng/mL rhIL-3 and 5000 IU/mL heparin (to a final concentration of 2 ng/mL rhIL-3),
and incubating the whole blood cells at 37°C for 10 minutes. The activated basophils
express IgE antibody. In an antigen stimulation step, 600 mΐ of the primed whole
blood is added to 600 mΐ of a 2x antigens preparation (e.g., peanut antigen
preparation) in BAT buffer, and the mixture is incubated for 30 minutes in a 37°C
water bath. The addition of the antigen stimulates (and degranulates) the cells. The
basophils are then surface stained, lysed and fixed. The basophils are then analyzed
by fluorescence-activated cell sorting (FACS), and visualization can be accomplished
by, e.g., CD63: lysosomal associated glycoprotein. The following antibody panel
represents an exemplary basophil antibody cocktail that can be used for monitoring
basophil activation:
i . FITC IgE
ii. PE CD203c
iii. PE-Cy7 CD123
iv. APC Lin*
v. V450 CD63
Lin* = Human Hematopoietic Lineage cocktail which contains CD2, CD3,
CD14, CD16, CD19, CD56, CD235a
Up-regulation of basophil proteins, or an increased rate of protein upregulation,
or overexpression of one or more of these markers over a longer period of
time may be indicative of a stronger allergic response, or a more potent (more
allergenic) antigen preparation.
When the sensitized basophils are exposed to antigen (e.g., peanut allergen),
IgE levels are expected to decrease over time, as the basophil becomes desensitized.
Basophils are typically identified by specific markers such as CCR3+/CD3 ,
CD123+/HLA-DR , IgE+/CD203c +, CD63+. Both CD63 and CD203c are commonly
used markers for in vitro basophil activation measurement. In some experiments, the
expression level of CD203c peaked earlier than CD63 in activated basophils, and thus
the timing of detection may be important. Further, IL3 priming enhances CD63
expression. The activation of CD203c is transient and more rapid than the expression
of CD63, so assays that use CD203c require careful consideration of the timing for
detection. Accordingly, a CD203c-based basophil activation test may best be
performed within 4 h after taking the blood samples. The robust assay based on
IgE-dependent upregulation of a cell surface antigen on basophils was the CD63-test,
and CD63 may be more specific for allergic (IgE-dependent) reactions and may be
less susceptible to non-specific upregulation by cytokines or other factors. One way
to measure basophil activation would be to employ both CD63 and CD203c.
In the first use of the test, i.e., in the clinic, the whole blood sample comes
from a patient in the clinic, and the antigen stimulation step involves adding a 2x
antigen preparation made from peanut extract or a known cocktail of peanut
peptides. Then a basophil antibody cocktail is added to look for biomarker
expression. An exemplary antibody cocktail includes antibodies that bind one of
more of the following biomarkers: IgE, CD203c, HLS-DR, CD123, CD63, and Lin.
A patient with a more severe peanut allergy will produce a blood sample that causes a
result in the basophil activation test where the activated basophils express higher
levels of the basophil markers, including one or more of IgE, CD203c, HLS-DR,
CD123, CD63, and Lin; express higher levels of one or more of the markers for a
longer period of time; or express levels of one or more of the markers that peak
sooner.
In the second use of the test, e.g., to measure potency of a peanut extract, sera
from peanut allergic donors can be spiked with a known quantity of a known antiantigen
antibody (e.g., anti-Ara h2 antibodies) to provide a standard, then the "antigen
stimulation" step will entail adding the synthesized peanut extract to be tested. Then
the "basophil antibody cocktail" will be added to look for biomarker expression
(which should match an expected standard). Exemplary biomarkers include IgE,
CD203c, HLS-DR, CD123, CD63, Lin. A more potent (i.e., more allergenic) peanut
extract will produce a result in the basophil activation test where the activated
basophils express higher levels of basophil activation markers, including one or more
of IgE, CD203c, HLS-DR, CD123, CD63, and Lin; express higher levels of one or
more of the markers for a longer period of time; or express levels of one or more of
the markers that peak sooner.

WHATIS CLAIMED IS:
1. A pharmaceutical composition, comprising a plurality of glucopyranosyl lipid
adjuvant (GLA) particles, l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
and a therapeutically effective amount of peanut proteins, in an aqueous carrier,
wherein the molar ratio of GLA to DPPC is in a range of about 1:1 to about 1:3
and at least some of the peanut proteins are disposed in at least one lipid particle
or free in the aqueous carrier, or both.
2 . A pharmaceutical composition, comprising glucopyranosyl lipid adjuvant (GLA)
and peanut proteins, wherein the pharmaceutical composition is in a solid or semi
solid dosage form.
3 . The pharmaceutical composition of claim 1, wherein the lipid particle is a
liposome or a micelle.
4 . The pharmaceutical composition of claim 1, wherein the aqueous carrier
comprises water.
5 . The pharmaceutical composition of claim 1, wherein the GLA particles in the
plurality have a Z-Average diameter of about 16 nm to about 1800 nm.
6 . The pharmaceutical composition of claim 1, wherein the GLA particles in the
plurality have a Z-average diameter of about 80 nm to about 500 nm.
7 . The pharmaceutical composition of claim 1, wherein the concentration of GLA in
the composition is from about 0.01 mg/mL to about 5 mg/mL.
8 . The pharmaceutical composition of claim 1, wherein the concentration of GLA in
the composition is about 0.02 mg/mL to 0.2 mg/mL.
9 . The pharmaceutical composition of claim 1, wherein the concentration of GLA in
the composition is about 0.16 mg/mL.
10. The pharmaceutical composition of claim 1, wherein the molar ratio of GLA to
DPPC in the composition is about 1:2.
11. The pharmaceutical composition of claim 1, wherein the concentration of peanut
proteins in the composition is about 5 g/mL to about 25,600 g/mL.
12. The pharmaceutical composition of claim 1, wherein the concentration of peanut
proteins in the composition is about 1,000 g/mL to about 7,000 g/mL.
13. The pharmaceutical composition of claim 1, wherein the concentration of peanut
proteins in the composition is about 5,000 mg/mL.
14. The pharmaceutical composition of claim 1 or 2, wherein the peanut proteins
comprise one or more of peanut allergen components Ara hi, Ara h2, Ara h3, Ara
h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara hi 1, Ara hl2, Ara hl3,
Ara hl4, Ara hl5, Ara hl6, and Ara hl7.
15. The pharmaceutical composition of claim 1 or 2, wherein the peanut proteins
consist of one or more of peanut allergen components Ara hi, Ara h2, Ara h3, Ara
h4, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara hi 1, Ara hl2, Ara hl3,
Ara hl4, Ara hl5, Ara hl6, and Ara hl7.
16. The pharmaceutical composition of claim 1 or 2, wherein the peanut proteins
comprise peanut allergen components Ara hi, Ara h2, Ara h3, and Ara h6.
17. The pharmaceutical composition of claim 1 or 2, wherein the peanut proteins
comprise peanut allergen components Ara hi, Ara h2, and Ara h6.
18. The pharmaceutical composition of claim 1 or 2, wherein the peanut proteins
comprise peanut allergen components Ara h2 and Ara h6.
19. The pharmaceutical composition of claim 1, wherein the pharmaceutical
composition is a liquid.
20. The pharmaceutical composition of claim 2, wherein the pharmaceutical
composition is a semi-solid.
21. The pharmaceutical composition of claim 2, wherein the pharmaceutical
composition is in the form of a tablet.
22. The pharmaceutical composition of claim 2, wherein the pharmaceutical
composition is in the form of a gel capsule.
23. The pharmaceutical composition of claim 1, wherein the pharmaceutical
composition is formulated as a sublingual drop or spray.
24. The pharmaceutical composition of claim 2, wherein the pharmaceutical
composition comprises a bioadhesive component.
25. The pharmaceutical composition of claim 2, wherein the pharmaceutical
composition is a multi-particulate granule or coated microsphere packaged in a
sachet form.
26. The pharmaceutical composition of claim 2, wherein the pharmaceutical
composition is a fast disintegrating tablet (FDT).
27. The pharmaceutical composition of claim 26, wherein the FDT comprises a
superdisintegrant.
28. The pharmaceutical composition of claim 27, wherein the superdisintegrant is
selected from the group consisting of: cross-linked cellulose, cross-linked
polyvinylpyrrolidone, cross-linked starch, and cross-linked alginic acid.
29. The pharmaceutical composition of claim 2, further comprising a gelling agent.
30. The pharmaceutical composition of claim 2, further comprising a binder.
31. The pharmaceutical composition of claim 2, further comprising a glidant.
32. The pharmaceutical composition of claim 2, further comprising an anti-adherant.
33. The pharmaceutical composition of claim 1 or 2, further comprising a flavoring
agent, a sweetening agent, or a colorant, or any combination thereof.
34. The pharmaceutical composition of claim 2, wherein the composition is a thin
film.
35. The pharmaceutical composition of claim 34, wherein the composition comprises
plasticized hydrocolloid.
36. The pharmaceutical composition of claim 34, wherein the GLA or peanut
proteins, or both, are disposed within the thin film.
37. The pharmaceutical composition of claim 34, wherein the GLA or peanut
proteins, or both, are disposed on the surface of the thin film.
38. The pharmaceutical composition of claim 34, wherein the GLA or peanut
proteins, or both, are disposed both within and on the surface of the thin film.
39. A method of treating an allergy in a patient, comprising administering
sublingually to a patient having an allergy a plurality of glucopyranosyl lipid
adjuvant (GLA) particles and peanut proteins, in amounts effective to treat the
allergy in the patient.
40. The method of claim 39, further comprising administering sublingually to the
patient l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC).
41. The method of claim 39, wherein the plurality of GLA particles is formulated as a
first pharmaceutical composition and the peanut proteins are formulated as a
second, separate pharmaceutical composition.
42. The method of claim 41, wherein the first and second pharmaceutical
compositions are administered simultaneously.
43. The method of claim 41, wherein the first formulation is administered to the
patient prior to the second formulation.
44. The method of claim 41, wherein the second formulation is administered to the
patient prior to the first formulation.
45. The method of claim 39, wherein the GLA and the peanut proteins are formulated
in a single pharmaceutical composition comprising a plurality of GLA particles
and peanut proteins in an aqueous carrier.
46. The method of claim 45, wherein the pharmaceutical composition further
comprises l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC).
47. The method of claim 46, wherein the molar ratio of GLA to DPPC in the
pharmaceutical composition is in a range of about 1:1 to about 1:3.
48. The method of claim 39, wherein the allergy is a peanut allergy.
49. The method of claim 39, wherein the allergy is allergy to birch tree pollens.
50. The method of claim 39, wherein the allergy is allergy to peach and peach related
fruits.
51. A method of treating an allergy in a patient, comprising administering
sublingually to a patient having an allergy a pharmaceutical composition
comprising glucopyranosyl lipid adjuvant (GLA) and peanut proteins, wherein the
pharmaceutical composition is in a solid or semi-solid dosage form.
52. The method of claim 51, wherein the pharmaceutical composition is a thin film.
53. The method of claim 51, wherein the pharmaceutical composition is a tablet or
capsule.
54. The method of claim 51, wherein the allergy is a peanut allergy.
55. The method of claim 51, wherein the allergy is allergy to birch tree pollens.
56. The method of claim 51, wherein the allergy is allergy to peach and peach related
fruits.
57. The method of claim 39 or 51, wherein the method comprises performing a
basophil activation test on the patient prior to administering the pharmaceutical
composition to the patient.
58. The method of claim 39 or 51, wherein the patient is an adult human.
59. The method of claim 39 or 51, wherein the patient is a juvenile human.
60. The method of claim 39 or 5 1 wherein the patient is 4 years old or older.
61. A method of making a pharmaceutical composition, the method comprising:
a . co-dissolving glucopyranosyl lipid adjuvant (GLA) and 1,2-dipalmitoylsn-
glycero-3-phosphocholine (DPPC) at a 1:2 molar ratio in chloroform to
thereby form a GLA/DPPC mixture;
b. adding peanut allergen to the GLA/DPPC mixture, to thereby form a
GLA/DPPC/ peanut proteins mixture;
c . removing the chloroform from the GLA/DPPC/ peanut proteins mixture;
d . adding water to the GLA/DPPC/ peanut proteins mixture; and
e . agitating the GLA/DPPC/ peanut proteins mixture, to thereby form a
pharmaceutical composition.
62. A method of making a pharmaceutical composition, the method comprising:
a . co-dissolving glucopyranosyl lipid adjuvant (GLA) and 1,2-dipalmitoylsn-
glycero-3-phosphocholine (DPPC) at a 1:2 molar ratio in chloroform to
thereby form a GLA/DPPC mixture;
b. removing the chloroform from the GLA/DPPC mixture;
c . adding water to the GLA/DPPC mixture;
d . agitating the GLA/DPPC mixture; and
e . adding peanut proteins to the GLA/DPPC mixture, to thereby form a
pharmaceutical composition.
63. A method of making a pharmaceutical composition, the method comprising:
a . mixing glucopyranosyl lipid adjuvant (GLA) and 1,2-dipalmitoyl-snglycero-
3-phosphocholine (DPPC) at a 1:2 molar ratio in water to thereby
form a GLA/DPPC mixture;
b. agitating the GLA/DPPC mixture at 70°C; and
c . adding peanut proteins to the GLA/DPPC mixture, to thereby form a
GLA/DPPC/peanut proteins mixture;
to thereby form a pharmaceutical composition.
64. A method of making a pharmaceutical composition, the method comprising:
a . mixing glucopyranosyl lipid adjuvant (GLA) and a surfactant;
b. adding water to the GLA/surfactant mixture;
c . agitating the GLA/surfactant mixture; and
d . adding peanut proteins to the GLA/surfactant mixture, to thereby form a
pharmaceutical composition.
65. The method of claim 63, wherein the water at step (a) is at 50°C to 70°C.
66. The method of any one of claims 6 1 to 64, wherein agitating comprises
sonication.
67. The method of any one of claims 6 1 to 64, wherein agitating comprises
microfluidization or high pressure homogenization, or both.
68. The method of any one of claims 6 1 to 64, wherein the method further comprises
compression molding the pharmaceutical composition to form a tablet.
69. The method of any one of claims 6 1 to 64, wherein the method further comprises
lyophilizing or spray drying the pharmaceutical composition.
70. The method of any one of claims 6 1 to 64, wherein the method further comprises:
forming a thin polymer film by a method selected from the group
consisting of solvent casting, semisolid casting, hot melt extrusion, solid
dispersion extrusion, and rolling; and
disposing the pharmaceutical composition within thin film and/or on at
least one surface of the thin film.
71. The method of any one of claims 6 1 to 64, wherein the peanut proteins comprise
one or more of peanut allergen components Ara hi, Ara h2, Ara h3, Ara h4, Ara
h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara hlO, Ara hi 1, Ara hl2, Ara hl3, Ara hl5,
Ara hl6, and Ara hl7.
72. The method of any one of claims 6 1 to 64, wherein the peanut proteins consist of
peanut allergen components Ara hi, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, Ara
h7, Ara h8, Ara h9, Ara hlO, Ara hi 1, Ara hl2 or Ara hl3, Ara hl5, Ara hl6, and
Ara hl7.
73. The method of any one of claims 6 1 to 64, wherein the peanut proteins consist of
peanut allergen components Ara hi, Ara h2, Ara h3, and Ara h6.
74. The method of any one of claims 6 1 to 64, wherein the peanut proteins consist of
peanut allergen components Ara hi, Ara h2, and Ara h6.
75. The method of any one of claims 6 1 to 64, wherein the peanut proteins consist of
peanut allergen components Ara h2 and Ara h6.
76. The method of any one of claims 6 1 to 64, further comprising, prior to adding the
peanut proteins to the GLA/DPPC mixture, performing a basophil activation test
on peanut allergens in the peanut proteins to measure the potency of the peanut
allergens.
77. The method of claim 64, wherein the surfactant is Sodium Lauryl Sulfate,
polysorbate-80, poloxamer 407 or poloxamer 188, or a combination of lecithin
and taurocholate.
78. The method of claim 64, wherein the surfactant is polysorbate-80, and the mixture
further includes 50% glycerol or 60% glycerol.