U.S. patent application number 15/989741 was filed with the patent office on 2019-02-07 for mixed allergen compositions and methods for using the same.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Kari C. Nadeau.
Application Number | 20190038741 15/989741 |
Document ID | / |
Family ID | 56689334 |
Filed Date | 2019-02-07 |
United States Patent
Application |
20190038741 |
Kind Code |
A1 |
Nadeau; Kari C. |
February 7, 2019 |
MIXED ALLERGEN COMPOSITIONS AND METHODS FOR USING THE SAME
Abstract
Mixed allergen compositions of two or more different allergens
are provided. In some instances, the mixed allergen compositions
include: a nut allergen; an animal allergen; and at least one of: a
non-nut plant allergen; a biotic agent; and a vitamin. Also
provided are methods of administering the mixed allergen
compositions to a subject. The mixed allergen compositions find use
in a variety of applications, including health maintenance, immune
balance, gut balance, immune support, health improvement and
therapeutic applications.
Inventors: |
Nadeau; Kari C.; (Los Altos
Hills, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
56689334 |
Appl. No.: |
15/989741 |
Filed: |
May 25, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15048609 |
Feb 19, 2016 |
10064936 |
|
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15989741 |
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62119014 |
Feb 20, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/35 20130101;
A61K 47/46 20130101; A61P 1/00 20180101; A61P 37/08 20180101; A61K
47/10 20130101; A61K 2039/542 20130101; A61K 35/747 20130101; A61K
2035/115 20130101; A61K 2039/575 20130101; A61K 2039/70 20130101;
A61P 43/00 20180101; A61K 2039/57 20130101; A61K 45/06 20130101;
A61K 2039/54 20130101; A61K 35/60 20130101; A23V 2002/00 20130101;
A61K 47/44 20130101; A61K 2039/572 20130101; A23L 33/10 20160801;
A61K 2039/55 20130101; A61P 37/02 20180101; A61P 29/00 20180101;
A61K 31/593 20130101; A61K 31/593 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 39/35 20060101
A61K039/35 |
Claims
1. A mixed allergen composition comprising: peanut complete
allergen; and at least one animal allergen selected from the group
consisting of shrimp complete allergen, cod complete allergen and
salmon complete allergen, wherein the peanut complete allergen and
at least one animal allergen are at about equal parts by protein
mass.
2. The mixed allergen composition of claim 1, wherein the animal
allergen is selected from the group consisting of shrimp complete
allergen and cod complete allergen, and the shrimp complete
allergen, cod complete allergen and the peanut complete allergen
are at about equal parts by protein mass.
3. The mixed allergen composition of claim 1, further comprising an
allergen selected from the group consisting of an almond allergen,
a walnut allergen, a cashew allergen, a hazelnut allergen, a pecan
allergen and a pistachio allergen.
4. The mixed allergen composition of claim 1, further comprising a
wheat allergen.
5. The mixed allergen composition of claim 1, further comprising an
egg allergen.
6. The mixed allergen composition of claim 1, further comprising a
crab allergen.
7. The mixed allergen composition of claim 1, further comprising an
allergen selected from the group consisting of a sesame allergen
and an oat allergen.
8. The mixed allergen composition of claim 1, further comprising a
soy allergen.
9. The mixed allergen composition of claim 1, further comprising a
milk allergen.
10. (canceled)
11. The mixed allergen composition of claim 1, further comprising a
physiologically acceptable delivery vehicle.
12. A food product comprising the unit formulation of a mixed
allergen composition of claim 21.
13. A method of treating a peanut allergy in a human subject in
need thereof, the method comprising administering the unit
formulation composition of claim 19 to the subject.
14.-18. (canceled)
19. A unit formulation composition for oral administration
comprising: 0.5 mg to 5 g of a mixed allergen composition, by
protein mass, wherein the mixed allergen composition comprises:
peanut complete allergen; a nut allergen selected from the group
consisting of walnut complete allergen, cashew complete allergen,
hazelnut complete allergen and combinations thereof; a soy
allergen; and an animal allergen selected from the group consisting
of shrimp complete allergen, cod complete allergen, salmon complete
allergen and combinations thereof; about 5 percent to about 95
percent, based on the total unit formulation composition, of a
pharmaceutically acceptable carrier.
20. The unit formulation composition for oral administration of
claim 19, wherein the mixed allergen composition comprises, by
total mass, about 15 mg to about 5000 mg of each allergen.
21. A unit formulation of a mixed allergen composition comprising:
about 15 mg to about 5000 mg cashew; about 15 mg to about 5000 mg
walnut; about 15 mg to about 5000 mg peanut; about 15 mg to about
5000 mg hazelnut; about 15 mg to about 5000 mg soy; about 15 mg to
about 5000 mg white and/or pink fish; and about 15 mg to about 5000
mg weight hen's egg.
22. A unit formulation composition for oral administration
comprising the mixed allergen composition of claim 21 and about 5
percent to about 95 percent, based on the weight of the total unit
formulation composition, of a pharmaceutically acceptable
carrier.
23. A unit formulation of a mixed allergen composition comprising:
about 1 mg to about 15000 mg cashew; about 1 mg to about 15000 mg
walnut; about 1 mg to about 15000 mg peanut; about 1 mg to about
15000 mg hazelnut; about 1 mg to about 15000 mg soy; about 1 mg to
about 15000 mg white and/or pink fish; and about 1 mg to about
15000 mg weight hen's egg; and about 5 to about 95 percent, based
on the weight of the total unit formulation composition, of a
pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE
[0001] Pursuant to 35 U.S.C. .sctn. 119 (e), this application
claims priority to the filing date of the U.S. Provisional Patent
Application Ser. No. 62/119,014, filed Feb. 20, 2015: the
disclosure of which is herein incorporated by reference.
INTRODUCTION
[0002] Allergy is a disorder of the immune system and is
characterized by the occurrence of allergic reactions to normally
harmless environmental substances. Allergies are caused by
allergens, which may be present in a wide variety of sources,
including but not limited to pollens or other plant components,
dust, molds or fungi, foods, additives, latex, transfusion
reactions, animal or bird danders, insect venoms, radiocontrast
medium, medications or chemicals. Common allergic reactions include
eczema, hives, hay fever, asthma, and reactions to venoms. Mild
allergies like hay fever are highly prevalent in the human
population and cause symptoms such as allergic conjunctivitis,
itchiness, and runny nose. In some people, severe allergies to
environmental or dietary allergens or to medication may result in
life-threatening anaphylactic reactions and potentially death, if
left untreated. Allergic reactions can occur in three distinct
patterns: a) an early phase reaction or acute response, b) late
phase reactions and c) potentially chronic allergic inflammation.
The early phase of the allergic reaction typically occurs within
minutes, or even seconds, following a first allergen exposure,
where this early phase is also commonly referred to as the
immediate allergic reaction. In the early stages of allergy, a
hypersensitivity reaction against an allergen, encountered for the
first time, causes a response in Th2 cells, which are a subset of T
cells that produce the cytokine interleukin-4 (IL-4). The Th2 cells
interact with B cells (lymphocytes that produce antibodies against
antigens) and, coupled with the effects of IL-4, stimulate the B
cells to begin production and secretion of Immunoglobulin E
(IgE).
[0003] IgE plays an important role in allergies and allergic
reactions. Upon introduction of an allergen, B cells of the
respective subject produce large amounts of IgE. The IgE elicits an
immune response by binding onto receptors found on basophils and
mast cells. When activated, these cells release chemical mediators
such as histamine and cytokines that cause the characteristic
symptoms of allergy.
[0004] A food allergy is an adverse immune response to a food
allergen, e.g., a food protein. Common food allergens are found in
shellfish, peanuts, tree nuts, fish, milk, eggs, soy and fresh
fruits such as strawberries, mango, banana, and apple.
Immunoglobulin-E (IgE)-mediated food allergies are classified as
type-I immediate hypersensitivity reactions. These allergic
reactions have an acute onset (from seconds to one hour) and the
accompanying symptoms may include angioedema (soft tissue swelling
of the eyelids, face, lips, tongue, larynx and trachea); hives;
itching of the mouth, throat, eyes, skin; gastrointestinal symptoms
such as nausea, vomiting, diarrhea, stomach cramps, or abdominal
pain; rhinorrhea or nasal congestion; wheezing, shortness of
breath, or difficulty swallowing; and even anaphylaxis, a severe,
whole-body allergic reaction that can result in death. Gupta, et al
(PEDIATRICS Vol. 128 No. 1 Jul. 1, 2011 pp. e9-e17) demonstrated
that 1 out of 12 children under the age of 21 years of age have a
doctor's diagnosis of food allergies. This epidemic has been
reported to be doubling every 10 years for certain nuts (CDC 2009).
Moreover, there are still deaths that occur every year due fatal
food allergies. Importantly, over $24 billion is spent per year on
health care/care costs for food allergic reactions (Gupta, et al.
JAMA PEDIATRICS November 2013, Vol. 167, No. 11). This cost is
largely due to about 90,000 visits to the ER per year in the U.S.
due to food induced anaphylactic symptoms.
SUMMARY
[0005] Mixed allergen compositions of two or more different
allergens are provided. In some instances, the mixed allergen
compositions include: a nut allergen; an animal allergen; and at
least one of: a non-nut plant allergen; a biotic agent; and a
vitamin. Also provided are methods of administering the mixed
allergen compositions to a subject. The mixed allergen compositions
find use in a variety of applications, including health
maintenance, immune balance, gut balance, immune support, health
improvement and therapeutic applications.
BRIEF DESCRIPTION OF THE FIGURES
[0006] FIG. 1: Th2 cell proliferation in ex vivo blood samples from
subjects fed different food allergen mixtures (units are cells/mL).
(It is noted that with respect to each of FIGS. 1 to 6, for each
group of bars on the graphs the order of test compositions from
left to right corresponds to the order of test compositions listed
in the column at the right of the figure from top to bottom. As
such, in FIG. 1, 4-6 months group, the bars from left to right are
the data for each of the compositions tested from top to bottom in
the column at the right hand side of the figure that lists the test
compositions)
[0007] FIG. 2: Specific IgG4 levels in plasma from subjects fed
different food allergen mixtures (units are mg IgG4/L).
[0008] FIG. 3. Specific IgE levels in plasma from subjects fed
different food allergen mixtures (units are mg IgE/L).
[0009] FIG. 4: Th2 cell proliferation in ex vivo blood samples from
subjects fed different food allergen mixtures (units are
cells/mL).
[0010] FIG. 5: Specific IgG4 levels in plasma from subjects fed
different food allergen mixtures (units are mg IgG4/L).
[0011] FIG. 6: Specific IgE levels in plasma from subjects fed the
food allergen mixtures (units are mg IgE/L).
[0012] FIG. 7: T cell proliferation in response to gluten, insulin,
tetanus and bacterial flagellin in ex vivo blood samples from
subjects fed different food allergen mixtures.
DETAILED DESCRIPTION
[0013] Mixed allergen compositions of two or more different
allergens are provided. In some instances, the mixed allergen
compositions include: a nut allergen; an animal allergen; and at
least one of: a non-nut plant allergen; a biotic agent; and a
vitamin. Also provided are methods of administering the mixed
allergen compositions to a subject. The mixed allergen compositions
find use in a variety of applications, including health
maintenance, immune balance, gut balance, immune support, health
improvement and therapeutic applications.
[0014] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to a
particular method or composition described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
invention will be limited only by the appended claims.
[0015] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0016] Unless defined otherwise, 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
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supersedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0017] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0018] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the peptide" includes reference to one or more
peptides and equivalents thereof, e.g., polypeptides, known to
those skilled in the art, and so forth.
[0019] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Mixed Allergen Compositions
[0020] As summarized above, aspects of the invention include mixed
allergen compositions. By mixed allergen composition is meant a
composition that includes two or more different allergens, where
any two given allergens are different if they are distinct from
each other, e.g., they are compounds described by different
chemical formula or compositions described by different components
(e.g., constituent compounds) and/or amounts thereof. The number of
different allergens in a composition may vary, as desired. In
certain embodiments, the mixed allergen compositions include 2 more
different allergens, such as 3 or more different allergens, 4 or
more different allergens, 5 or more different allergens, 6 or more
different allergens, 7 or more different allergens, 8 or more
different allergens, 9 or more different allergens, 10 or more
different allergens, 15 or more different allergens, 20 or more
different allergens, 25 or more different allergens, 30 or more
different allergens, 40 or more different allergens, 50 or more
different allergens, 75 or more different allergens, 100 or more
different allergens, where in some instances the number of
different allergens in a given composition is 100 or less, such as
75 or less, including 50 or less, e.g., 25 or less, 15 or less,
including 10 or less.
[0021] Allergens present in the composition may vary, where in some
instances the allergen present in the composition is one that
induces an allergy in a susceptible subject. Allergens include any
antigen, or active derivative thereof, that elicits a specific IgE
response. Antigens include any substance that can stimulate the
production of antibodies and can combine specifically with them.
Antigenic determinants or epitopes are antigenic sites on a
molecule. Allergens may have little or no intrinsic toxicity by
themselves, but cause a pathological condition due to their ability
to elicit an IgE-associated immune response, and, upon subsequent
exposure, due to their ability to elicit IgE- and/or T
cell-dependent hypersensitivity reactions. As such, an "allergen"
includes any substance which is capable of stimulating a typical
hypersensitivity reaction in atopic subjects. Allergens that may be
present in a given mixed allergen composition include any substance
found in a variety of different sources, such as but not limited
to: foods, drugs, perfume, plants, the environment or biological
systems (e.g., prokaryotic or eukaryotic cells or viruses), as well
as chemical allergens.
[0022] Allergens of interest include nut allergens. Nut allergens
are allergens that include one or more compounds found in nuts,
e.g., dry fruits that include an edible kernel or meat enclosed in
a woody or leathery shell. Nut allergens of interest include, but
are not limited to: peanut allergens, e.g., rAra h 1, rAra h 2,
rAra h 3; rAra h 8 PR-10, rAra h 9 LTP, peanut complete allergen
(the phrase "complete allergen" as used herein refers to all
possible antigenic components of a given food protein), etc.;
brazil nut allergens, e.g., rBer e 1, brazil nut complete allergen,
etc., hazelnut or filbert allergens, e.g., rCor a 1 PR-10, rCor a 8
LTP, hazel nut complete allergen, nCor a 9, rCor a 14, etc.; walnut
allergens, e.g., rJug r 1, rJug r 3 LTP, walnut complete allergen,
etc.; cashew allergens; pistachio allergens, e.g., pistachio
component allergens, pistachio complete allergen, etc.; pecan
allergens, e.g., pecan component allergens, pecan complete
allergen, etc.; tree nut component package allergens.sub.; such as
one or more allergens from cashew nut, walnut, hazelnut, brazil
nut; etc.
[0023] Allergens of interest include animal allergens. Animal
allergens are allergens that include one or more compounds found in
animals, including both vertebrates and invertebrates. Vertebrate
animal allergens that may be present in mixed allergen compositions
include avian allergens, such as egg allergens, e.g., nGal d 1
Ovomucoid, n Gal d 2 Ovalbumin, nGal d 3 Conalbumin, egg white
complete allergen, etc.; mammalian allergens, such as milk
allergens, e.g., nBos d 4 alpha-lactalbumin, nBos d 5
beta-lactoglobulin, nBos d 8 Casein, nBos d Lactoferrin, milk
complete allergen, etc., fish allergens, e.g., e,g., rCyp c 1, rGad
c 1, cod complete allergen, white fish allergens, pink fish
allergens, etc. Invertebrate animal allergens that may be present
in mixed allergen compositions include: crustacean allergens, such
as shrimp allergens, e.g., rPen a 1 tropomyosin, shrimp complete
allergen, etc.; insect allergens, e.g., bee sting venom allergen,
wasp sting venom allergen, mosquito bite allergen, etc.; and the
like.
[0024] Allergens of interest include non-nut plant allergens, i.e.,
plant allergens that are not nut allergens. Plant allergens are
allergens that include one or more compounds found in plants. Plant
allergens of interest include: wheat allergens, e.g., rTri a 19
Omega-5 Gliadin, wheat complete allergen, gliadin wheat, rTri a 14
LTP, etc.; kiwi allergens, e.g., rAct d 8 PR-10, kiwi complete
allergen, etc.; celery allergens, e.g., rApi g 1.01 PR-10, rPhl p
12, celery complete allergen, COD MUXF3 from Bromelain, etc.; soy
allergens, e.g., rGly m 4 PR-10, soy complete allergen, nGly m 5
Beta-conglycinin, nGly m 6 Glycinin, etc.; stone fruit allergens,
e.g., f419, f420, f421, f95, f242, o214 rPru p 1 PR-10, rPru p 3
LTP, stone fruit primary complete allergen, CCD MUXF3 from
Bromelain, etc.; oat allergens, e.g., oat component allergens, oat
complete allergen, etc.; sesame allergens, e.g., sesame seed
component allergens, sesame see complete allergen, etc.
[0025] Additional types of allergens that may be present in mixed
allergen compositions include, but are not limited to: non-food
animal allergens, e.g., cats or dog fur and dander, cockroach
calyx, dust mite excretion, etc.; drug allergens, e.g., e.g.,
penicillin, sulfonamides, salicylates, local anesthetics; mold
spore allergens, latex allergens; metal allergens; plant pollen
allergens, e.g., e.g. grass-ryegrass, timothy-grass, weeds-ragweed,
plantago, nettle, Artemisia, vulgaris, chenopodium album, sorrel,
trees-birch alder, hazel, hornbeam, aesculus, willow, poplar,
platanus, tilia, olea; etc.
[0026] The amount of a given allergen in a mixed allergen
composition may vary, as desired. In some instances, the amount of
a given allergen ranges from 5 to 15,000 mg, such as 10 to 10,000
mg, including 15 to 5,000 mg. The weight percentage of a given
allergen in a mixed allergen composition may vary, ranging in some
instances from 15 to 99.9 wt.%, such as 25 to 65 wt. %. A mixed
allergen composition may be a unit dosage composition, by which is
meant that it is present in a composition that is configured to be
administered to a subject as a single dose, which single dose may
or may not be part of a dosing schedule made up of two or more unit
dosages that are administered to a subject over a given a period of
time. While the mass of a given unit dosage may vary, in some
instances unit dosages have a mass ranging from 300 mg to 20 grams,
such as 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800mg, 900 mg, 1000
mg (1 g), 1.5 g, 2 g, 3 g, 4 g, 5 g, 10 g, 15 g, 20 g,and anywhere
in between. In certain embodiments, any two of the mixed allergens
are present in equal parts, e.g., in a 1:1 ratio, such that each
allergen is present in the composition in equal weight. In such
embodiments where the mixed allergen composition includes three
different allergens, the three different allergens are present in a
1:1:1 ratio. For example, a mixed allergen composition may include
the following allergens in equal parts (e.g., 1:1:1 etc. ratio):
walnut, hazelnut, shrimp, salmon, hen's egg, cow's milk, peanut,
cashew, almond, and wheat (e.g., about 30 mg of protein each; about
90 mg of each protein; or about 300 mg of each protein).
[0027] In some embodiments, the mixed allergen compositions include
a nut allergen and an animal allergen; and at least one of; a
non-nut plant allergen; a biotic agent; and a vitamin. As such, in
some embodiments, the mixed allergen compositions include a nut
allergen, an animal allergen and a non-nut plant allergen. In such
embodiments, the mixed allergen compositions may further include a
biotic agent or vitamin or both a biotic agent and a vitamin. In
some embodiments, the mixed allergen compositions include a nut
allergen an animal allergen, and further include a biotic agent. In
such embodiments, the mixed allergen compositions may further
include a vitamin. In some embodiments, the mixed allergen
compositions include a nut allergen an animal allergen, and further
include a vitamin. In such embodiments, the mixed allergen
compositions may further include a biotic agent.
[0028] Biotic agents may vary, and include both probiotics and
probiotics. A probiotic is generally a live eukaryotic or a
prokaryotic organism which has a beneficial property when given to
a subject. In one aspect, the probiotic complements the existing
microflora in the subject. Hence, the probiotic agent is a live
microorganism which can confer a health benefit to a host subject.
The probiotic agent may be a culture of microorganisms or provided
in a dietary supplement or may be freeze dried and reconstituted
prior to use. A prebiotic is an agent that facilitates or confers
growth, maintenance and/or beneficial properties of or on the
subject's microflora. A prebiotic may include an oligosaccharide
and soluble or insoluble fiber material. Examples of probiotic
agents include, but are not limited to, species of Lactobacillus
spp., Escherichia spp., Bacillus spp., Bifidobacterium spp.,
Saccharomyces spp. and Streptococcus spp. Specific probiotic agents
that may be present in the mixed allergen compositions include;
Lactobacillus spp., such as Lactobacillus acidophilus,
Lactobacillus casei, Lactobacillus easel Shirota, Lactobacillus
casei immunitass, Lactobacillus johnsonii, Lactococcus lactic,
Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus
rhamnosus (lactobacillus GG), Lactobacillus salivarius and
Lactobacillus helvetirus. The probiotic microorganisms may be
naturally occurring, attenuated or genetically modified to
introduce new or to alter existing traits. In one embodiment, the
probiotic has been genetically modified to introduce an allergen
gene or part or fragment or portion thereof which is expressed to
produce recombinant microorganisms which release or expose the
subjects immune system to the allergen or an antigenic fragment
thereof. Hence, the probiotic and allergen may be combined into a
single component of the mixed allergen composition. When present,
the amount of the biotic agent in the mixed allergen composition
may vary. In some instances, the biotic agent is present in an
amount ranging from 1.5 to 99.9 wt. %, such as 10 to 25 wt. %.
[0029] The mixed allergen compositions may include one or more
vitamins, as desired. Vitamins that may be present in the
compositions include, but are not limited to: vitamin A, e.g., in
an amount ranging from 1 to 35,000 IU; vitamin C, e.g., in an
amount ranging from 1 and about 1,000 mg; vitamin D, e.g., in an
amount ranging from 1 and 4,000 IU; vitamin E, e.g., in an amount
ranging from 1 to 450 IU; vitamin K, e.g., in an amount ranging
from 1 to 250 mcg; vitamin B-1 (thiamin), e.g., in amount ranging
from 1 to 15 mg; vitamin B-2 (riboflavin), e.g., in an amount
ranging from 1 to 17 mg; vitamin B-3 (niacin), e.g., in an amount
ranging from 1 to 200 mg; vitamin B-5 (pantothenic acid), e.g., in
an amount ranging from 1 to 100 mg; vitamin B-6 (pyridoxine), e.g.,
in an amount ranging from 1 to 30 mg; vitamin B-9 (folic acid),
e.g., in an amount ranging from 1 to 4,000 mcg; vitamin B-12
(cobalamin), e.g., in an amount ranging from 1 to 250 mcg; vitamin
H (biotin), e.g., in an amount ranging from 1 to 1,000 mcg of
vitamin H (biotin); etc.; and combinations thereof.
[0030] The mixed allergen compositions of the invention may be
present in different configurations. In some instances the mixed
allergen composition is present in a solid configuration, e.g., as
a powder. When present as a powder, the dimensions of the particles
making up the powder may vary, ranging in some instances from 0.1
to 1000 microns, such as 1 to 500 microns.
[0031] Also provided are physiological acceptable compositions that
include the mixed allergen compositions and a physiologically
acceptable delivery vehicle. The mixed allergen compositions can be
incorporated into a variety of formulations for administration to a
subject. More particularly, the mixed allergen compositions can be
formulated into physiological acceptable compositions by
combination with appropriate, physiologically acceptable carriers
or diluents, and may be formulated into preparations in solid,
semi-solid, liquid or gaseous forms, such as tablets, capsules,
powders, granules, ointments, solutions, suppositories, injections,
inhalants and aerosols and topical compositions. The formulations
may be designed for administration via a number of different
routes, including oral, buccal, sublingual, rectal, parenteral,
intraperitoneal, intradermal, transdermal, intracheal, etc.,
administration.
[0032] The physiological compositions may be in a form suitable for
oral use, for example, as foods, tablets, troches, lozenges,
aqueous or oily suspensions, dispersible powders or granules,
emulsions, hard or soft capsules, or syrups or elixirs, gums, etc.
Compositions intended for oral use may be prepared according to any
convenient protocol for the manufacture of pharmaceutical
compositions and such compositions may contain one or more agents
selected from the group consisting of sweetening agents, flavoring
agents, coloring agents and preserving agents in order to provide
palatable preparations.
[0033] Food formulations of interest include the mixed allergen
composition in combination with a food delivery vehicle. By food
delivery vehicle is meant a delivery vehicle that is a nourishing
substance that is eaten, drunk, or otherwise taken into the body to
sustain life, provide energy, promote growth, etc. Examples of food
delivery vehicles of interest include, but are not limited to: baby
formula, baby food, chips, cookies, breads, spreads, creams,
yogurts, liquid drinks, chocolate containing products, candies, ice
creams, cereals, coffees, etc.
[0034] Also of interest as oral formulations are food supplements.
Where the oral formulation is provided as a food supplement, the
food supplement may further include one or more of a sweetener, a
stabilizer, a flavoring or a colorant, etc. An oral formulation
according to the present disclosure may be provided in the form of
sugar-coated tablets or lozenges, pills, gelatin capsules, or
syrups. Oral formulations may be provided as a bulk sample, e.g., a
container having multiple doses in powder form that can be measured
out by a subject, or in unit dose form, e.g., a pill, pouch, single
use container, and the like.
[0035] Tablets may contain the active ingredient in admixture with
non-toxic physiologically acceptable excipients which are suitable
for the manufacture of tablets. These excipients may be for
example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example, magnesium stearate,
stearic acid or talc. The tablets may be uncoated or they may be
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monostearate or glyceryl distearate may be
employed. They may also be coated by the technique described in the
U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic
therapeutic tablets for control release.
[0036] Formulations for oral use may also be presented as hard
gelatin capsules wherein the mixed allergen component is mixed with
an inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the mixed
allergen component is mixed with water or an oil medium, for
example peanut oil, liquid paraffin, or olive oil.
[0037] Aqueous suspensions contain the mixed allergen component in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients may include suspending agents, for
example sodium carboxymethylcellulose, methylcellulose,
hydroxy-propylmethycellulose, sodium alginate,
polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethylene-oxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl,
p-hydroxybenzoate, one or more coloring agents, one or more
flavoring agents, and one or more sweetening agents, such as
sucrose, saccharin or aspartame.
[0038] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0039] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0040] The physiologically acceptable compositions of the invention
may also be in the form of oil-in-water emulsions. The oily phase
may be a vegetable oil, for example olive oil or arachis oil, or a
mineral oil, for example liquid paraffin or mixtures of these.
Suitable emulsifying agents may be naturally-occurring
phosphatides, for example soy bean, lecithin, and esters or partial
esters derived from fatty acids and hexitol anhydrides, for example
sorbitan monooleate, and condensation products of the said partial
esters with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0041] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, preservative and
flavoring and coloring agents. The pharmaceutical compositions may
be in the form of a sterile injectable aqueous or oleagenous
suspension. This suspension may be formulated according to the
known art using those suitable dispersing or wetting agents and
suspending agents which have been mentioned above. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parenterally-acceptable diluent or
solvent, for example as a solution in 1,3-butane dial. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid find use in the
preparation of injectables.
[0042] The mixed allergen components can be formulated into
preparations for injection by dissolving, suspending or emulsifying
them in an aqueous or nonaqueous solvent, such as vegetable or
other similar oils, synthetic aliphatic acid glycerides, esters of
higher aliphatic acids or propylene glycol; and if desired, with
conventional additives such as solubilizers, isotonic agents,
suspending agents, emulsifying agents, stabilizers and
preservatives.
[0043] The mixed allergen components can be utilized in aerosol
formulation to be administered via inhalation. The compounds of the
present invention can be formulated into pressurized acceptable
propellants such as dichlorodifluoromethane, propane, nitrogen and
the like.
[0044] Furthermore, the mixed allergen compositions can be made
into suppositories by mixing with a variety of bases such as
emulsifying bases or water-soluble bases. The compounds of the
present invention can be administered rectally via a suppository.
The suppository can include vehicles such as cocoa butter,
carbowaxes and polyethylene glycols, which melt at body
temperature, yet are solidified at room temperature.
[0045] Also of interest are topical compositions, e.g., where the
mixed allergen composition is combined with a topical delivery
vehicle component. The topical delivery vehicle component of the
delivery compositions of the invention may vary, as desired, where
the particular ingredients of a given delivery vehicle component
will depend, at least in part, on the nature of the particular
composition. Delivery compositions of interest include liquid
formulations, such as lotions (liquids containing insoluble
material in the form of a suspension or emulsion, intended for
external application, including spray lotions) and aqueous
solutions, semi-solid formulations, such as gels (colloids in which
the disperse phase has combined with the dispersion medium to
produce a semisolid material, such as a jelly), creams (soft solids
or thick liquids) and ointments (soft, unctuous preparations), and
solid formulations, such as topical patches. As such, delivery
vehicle components of interest include, but are not limited to:
emulsions of the oil-in-water (O/W) and the water in-oil (W/O)
type, milk preparations, lotions, creams, ointments, gels, serum,
powders, masks, packs, sprays, aerosols or sticks.
[0046] The amount of mixed allergen composition that may be
combined with the carrier materials to produce a single dosage form
will vary depending upon the host treated and the particular mode
of administration. For example, a formulation intended for the oral
administration of humans may contain from 0.5 mg to 5 g of mixed
allergen composition compounded with an appropriate and convenient
amount of carrier material which may vary from about 5 to about 95
percent of the total composition. It will be understood, however,
that the specific dose level for any particular patient will depend
upon a variety of factors including the age, body weight, general
health, sex, diet, time of administration, route of administration,
rate of excretion, drug combination and the severity of the
particular disease undergoing therapy. As such, unit dosage forms
for oral or rectal administration such as syrups, elixirs, and
suspensions may be provided wherein each dosage unit, for example,
teaspoonful, tablespoonful, tablet or suppository, contains a
predetermined amount of the composition containing one or more
inhibitors. Similarly, unit dosage forms for injection or
intravenous administration may include the inhibitor(s) in a
composition as a solution in sterile water, normal saline or
another pharmaceutically acceptable carrier. As described above
"unit dosage forms," include physically discrete units suitable as
unitary dosages for human and animal subjects, each unit containing
a predetermined quantity of mixed allergen composition calculated
in an amount sufficient to produce the desired effect in
association with a pharmaceutically acceptable diluent, carrier or
vehicle.
Methods
[0047] Aspects of the invention also include methods of
administering a mixed allergen composition, e.g., as described
above, to a subject. The administration route employed in a given
method may vary, e.g., depending on the nature of the mixed
allergen composition. As reviewed above, physiologically acceptable
compositions that include a mixed allergen composition may be
formulated for delivery to a subject using a variety of different
administration routes, such as but not limited to: oral, buccal,
sublingual, rectal, parenteral, intraperitoneal, intradermal,
transdermal, intracheal, etc., administration. As such, aspects of
the methods may include orally, buccaly, sublingually, rectally,
parenterally, intraperitonealy, intradermally, transdermally,
intracheally, etc., administering a mixed allergen composition or
physiologically acceptable composition that includes the same,
e.g., as described above, to a subject.
[0048] The methods described herein may be employed with a variety
of different types of subjects, i.e., animals, where the animals
are typically "mammals" or "mammalian," where these terms are used
broadly to describe organisms which are within the class mammalia,
including the orders carnivore (e.g., dogs and cats), rodentia
(e.g., mice, guinea pigs, and rats), lagomorpha (e.g., rabbits) and
primates (e.g., humans, chimpanzees, and monkeys). In embodiments
of the invention, the subjects are humans. The subject may be
infant, juveniles or adults, where in some instances the subject
may be pregnant adults, e.g., as described in greater detail below.
As such, in certain embodiments, the subject is an infant younger
than 1 year of age, whereas in other embodiments the subject is
older, e.g., 1 year old or more, 5 years old or more, etc., and
including adults.
[0049] The dosing schedule may vary as desired, and may depend on a
number of different factors, e.g., purpose of the administration,
age of the subject, condition of the subject, nature of the
physiologically acceptable composition, etc. In certain
embodiments, a mixed allergen composition or physiologically
acceptable composition that includes the same is administered to a
subject on an hourly basis, on an every few hours basis (e.g.,
every 2, 3, 4, 6 hours), on a daily basis, on a weekly basis, on a
bi-weekly basis, on a monthly basis, one a bimonthly basis, on a
quarter annual basis, on a semi-annual basis, on an annual basis,
etc., for a treatment period of time, which treatment period of
time may also vary, where in some instances the treatment period of
time is 1 day or longer, 1 week or longer, 2 weeks or longer, 1
month or longer, 3 months or longer, 6 months or longer, 1 year or
longer, 2 years or longer, 3 years or longer, 5 years or longer, 10
years or longer, etc., up to the life of the subject.
[0050] In some embodiments, the methods are methods of increasing
immune health of the subject. As the methods of these embodiments
are methods of increasing the immune health of a subject,
embodiments of such methods result in the subject being better able
to have a healthy immune response to a given challenge. Immune
health or immune balance may be characterized as as a state in the
body where there is not an imbalance of inflammation. For example,
when IgE is decreased and/or IgG4 increased in plasma and/or Th2
decreased (e.g., as described in greater detail below in the
Experimental section and accompanying figures). The magnitude of
immune health enhancement may vary, where in some instances the
magnitude is 2-fold or greater, e.g., 5-fold or greater, including
10-fold or greater, e.g., as compared to a suitable control.
[0051] In some embodiments, the methods are methods of increasing
gut health of the subject. As the methods of these embodiments are
methods of increasing the gut health of a subject, embodiments of
such methods result in the subject having an enhanced maintenance
of healthy microbiota or improving the resilience of microbiota,
for instance, by reducing the numbers or colonization of pathogenic
bacteria or viruses and by maintaining and improving the intestinal
integrity and barrier function. Gut health or gut balance may be
characterized as a state in the body where there is not an
imbalance of inflammation. For example, when T cell proliferation
is decreased (e.g., as described in greater detail below in the
Experimental section and accompanying figures). The magnitude of
gut health enhancement may vary, where in some instances the
magnitude is 2-fold or greater, e.g., 5-fold or greater, including
10-fold or greater, e.g., as compared to a suitable control.
[0052] In some embodiments, the methods are methods of enhancing
wellness or maintaining immune balance of the subject. As the
methods of these embodiments are methods of enhancing wellness of a
subject, embodiments of such methods result in the subject having a
quality or state of being healthy in body and mind. Wellness may be
characterized as a state of the body where there is not an
imbalance of immune problems (for example, less IgE, more IgG4,
less cell inflammation (e.g., as described in greater detail below
in the Experimental section and accompanying figures). The
magnitude of wellness enhancement may vary, where in some instances
the magnitude is 2-fold or greater, e.g., 5-fold or greater,
including 10-fold or greater, e.g., as compared to a suitable
control.
[0053] In some embodiments, the methods are methods of at least
decreasing the potential of the subject for developing an immune
mediated condition, such as an immune-mediated inflammatory disease
condition. By at least decreasing the potential of the subject for
developing an immune mediated condition is meant that the
probability of the subject for developing the immune mediated
condition is reduced, such that the risk of the subject for
developing the immune mediated condition is reduced. For example,
to determine risk reduction, if 100 different individuals were
administered the composition, 20% or more of the individuals would
show a decrease in their immune markers, e.g., IgE, as compared to
the control group. The magnitude of the decrease in potential may
vary, where in some instances the magnitude is 2-fold or greater,
e.g., 5-fold or greater, including 10-fold or greater, e.g., as
compared to a suitable control. In some instances, the methods are
methods of preventing the subject from developing an immune
mediated condition. As such, the methods of the invention include
administering a composition of the invention to a subject that is
not known to have or does not have an immune mediated condition.
While the subject may not have or may not be known to have the
immune mediated condition, the subject may be one that is suspected
to be or known to be at risk of developing the immune mediated
condition,
[0054] Aspects of the invention further include methods of treating
a subject for an immune mediated condition. By treating or
treatment is meant at least an amelioration of one or more symptoms
associated with the disease condition, e.g., immune mediated
condition, afflicting the subject, where amelioration is used in a
broad sense to refer to at least a reduction in the magnitude of a
parameter, e.g., symptom, associated with the pathological
condition being treated, etc. As such, treatment also includes
situations where the pathological condition, or at least symptoms
associated therewith, are completely inhibited, e.g., prevented
from happening, or stopped, e.g., terminated, such that the subject
no longer suffers from the pathological condition, or at least the
symptoms that characterize the pathological condition.
[0055] Treatment may also manifest in the form of a modulation of a
surrogate marker of the disease condition. For example, where the
target condition is an allergy, e.g., as described below, Th2 cell
proliferation may be reduced, e.g., as determined using the assay
described in the Experimental Section, below. The magnitude of Th2
cell proliferation reduction may vary, and in certain instances may
range from 1.2.times. to 10.times., such as 2.times. to 4.times..
Where the target condition is an allergy, e.g., as described below,
specific IgG4 levels may be increased, e.g., as determined using
the assay described in the Experimental Section, below. The
magnitude of IgG4 level increase may vary, and in certain instances
may range from 1.2.times. to 100.times., such as 2.times. to
6.times.. Where the target condition is an allergy, e.g., as
described below, specific IgE levels may be reduced, e.g., as
determined using the assay described in the Experimental Section,
below. The magnitude of IgE level reduction may vary, and in
certain instances may range from 1.1.times. to 7.times., such as
2.times. to 6.times..
[0056] As summarized above, immune mediated conditions that are the
targets of methods of the invention include immune-mediated
inflammatory conditions, where such conditions include, but are not
limited to conditions characterized by common inflammatory pathways
leading to inflammation, and which may result from, or be triggered
by, a dysregulation of the normal immune response. Examples of
immune-mediated inflammatory conditions include, but are not
limited to: allergy, autoimmune diseases, ankylosing spondylitis,
psoriasis, psoriatic arthritis, Behcet's disease, arthritis,
inflammatory bowel disease (IBD), cardiovascular disease,
neuromuscular disease, and infectious disease, etc.
[0057] In some instances, the target immune mediated condition is
an allergy. The target allergy may vary widely, where in some
instances the target allergy is food allergy, drug allergy,
environmental allergy, animal allergy, and insect and/or bee
allergy. As such, aspects of the invention include methods of
reducing the risk of a subject for developing an allergy. In
certain embodiments, the methods result in the subject having a
reduced risk of developing a food allergy to a food allergen that
is not present in the mixed allergen composition that is
administered to the subject. For example, the administered
composition may provide to the subject allergen protection for a
nut protein in the formulation as well as a nut protein not found
in the formulation. In certain embodiments, the reduced risk for a
first food allergen is reduced to a greater extent using the mixed
formulation than would be achieved using a formulation with only a
single allergen. For example, feeding a mixed allergen formulation
as described herein can result in lower risk of allergy development
for an allergen (e.g., with respect to Th2/IgE/IgG4 analyses, as
shown below) than if the formulation only included that single
allergen. In some cases, this phenomenon is referred to as
"synergy" (e.g., cashew synergizes with pistachio, walnut with
pecan, shrimp with lobster (and other crustacean), and vice-versa).
Where the method is a method of treating the subject for the
allergy, as reviewed above the method may result in at least an
amelioration of one or more symptoms associated with the allergy,
e.g., as described above in the introduction section. Allergy
symptoms that may be ameliorated, but are not limited to: eczema,
asthma, atopic dermatitis, bronchospasm, cough, rhinorrhea,
angioedema, gastric hypermotility, urticaria (hives), pruritis,
fatigue, bradycardia, and/or hypotension. The magnitude of the
symptom reduction may vary, where in some instances the magnitude
is 2-fold or greater, e.g., 5-fold or greater, including 10-fold or
greater, e.g., as compared to a suitable control. In some
instances, treatment of an allergy results the subject being cured
of the allergy, such that the subject no longer suffers from the
allergy. In some embodiments of allergy treatment methods, the
methods include administering to a subject a mixed allergen
composition that includes a nut allergen; an animal allergen; and
at least one of: a non-nut plant allergen a biotic agent; and a
vitamin, such as described above.
[0058] In some instances, the methods are methods of treating a
subject for an eosinophilic disorder. Eosinophilic disorders are
disorders characterized by the occurrence of eosinophils in
above-normal amounts in various parts of the body. Eosinophilic
disorders of interest include, but are not limited to: eosinophilic
esophagitis (esophagus); eosinophilic gastritis (stomach);
eosinophilic enteritis (small intestine); eosinophilic colitis
(large intestine); hypereosinophilic syndrome (blood and any
organ); and the like. In some instances the methods are methods of
reducing the risk of a subject for developing an eosinophilic
disorder. The magnitude of the risk reduction may vary, where in
some instances the magnitude is 2-fold or greater, e.g., 5-fold or
greater, including 10-fold or greater, e.g., as compared to a
suitable control.
[0059] In some instances the methods are methods of treating a
subject for inflammation, where the inflammation may be a symptom
of a variety of different disease conditions. Disease conditions in
which the inflammation thereof may be treated according to
embodiments of the invention include, but are not limited celiac
disease, multiple sclerosis, inflammatory bowel disease,
eosinophilic diseases, allergy, food allergy, etc.
[0060] Aspects of the invention further include methods of at least
decreasing the potential of a fetus or suckling infant for
developing an immune mediated condition, such as an immune-mediated
inflammatory disease condition, e.g., allergy, such as described
above. By at least decreasing the potential of the fetus or
suckling infant for developing an immune mediated condition is
meant that the probability of the fetus or suckling infant for
developing the immune mediated condition is reduced, such that the
risk of the fetus or suckling infant for developing the immune
mediated condition is reduced. The magnitude of the decrease in
potential may vary, where in some instances the magnitude is 2-fold
or greater, e.g., 5-fold or greater, including 10-fold or greater,
e.g., as compared to a suitable control. In some instances, the
methods are methods of preventing the fetus or suckling infant from
developing an immune mediated condition. As such, the methods of
the invention include administering a composition of the invention
to a mother of a fetus or suckling infant that is not known to have
or does not have an immune mediated condition. While the fetus or
suckling infant may not have or may not be known to have the immune
mediated condition, the fetus or suckling infant may be one that is
suspected to be or known to be at risk of developing the immune
mediated condition.
[0061] In such embodiments, the mixed allergen composition or
physiologically acceptable composition that includes the same is
administered to the mother of the fetus or suckling infant. For
example, the mixed allergen composition or physiologically
acceptable composition that includes the same may be administered
to a mother pregnant with the fetus. In such instances, the mixed
allergen composition or physiologically acceptable composition that
includes the same may be administered to the mother using any
convenient dosing schedule, e.g., as described above, starting at
any convenient time during the pregnancy, e.g., at the start of the
second trimester, at the start of the third trimester, etc. Where
the method is a method of reducing risk in a suckling infant, the
mixed allergen composition or physiologically acceptable
composition that includes the same may be administered to a mother
that is breast feeding the infant. In such instances, the mixed
allergen composition or physiologically acceptable composition that
includes the same may be administered to the breast feeding mother
using any convenient dosing schedule, e.g., as described above,
starting at any convenient time during the breast feeding, e.g., at
the start of lactation, 1 week after lactation commencement,
etc.
[0062] The following examples are provided by way of illustration
and not by way of limitation.
Experimental
I. Mixed Allergen Assay
A. Materials and Methods
1. Allergen Treatment
[0063] For each allergen or allergen mix listed below (the allergen
groups), five subjects from each of five different age groups were
fed a total of 300 mg of the allergen or allergen mix daily for one
year. The only exceptions are for Formula (i.e., dose) 1 at
3.times. and Formula (i.e., dose) 1 at 10.times., in which the
subjects were fed 900 mg and 3,000 mg of the formula on a daily
basis, respectively. Allergen mixes were formulated at a 1:1 ratio.
Thus, for an allergen mix with 2 allergens, the mix would include
150 mg of each. Five subjects were included in each age group that
were not fed an allergen (Not Treated, or NT group listed last).
The five age groups were: 4 to 6 months, 7 to 12 months, 1 to 3
years, 3 to 5 years, and 5 years or older. The subjects were not
selected based on observed or suspected food allergy (or other
allergy) profile, and thus the cohort of subjects tested included
those who may have, or have the propensity to develop, a food
allergy as well as those who do not. The total number of subjects
was 450 (5 subjects in 5 age groups for 18 different treatments,
including NT).
Allergen or Allergen Mix
[0064] 1. Cashew: Finely ground cashew from "Wellbee's", Spring
Valley, N.Y. 10977. [0065] 2. Cashew and pistachio: Finely ground
cashew from "Wellbee's", Spring Valley, N.Y. 10977. Pistachio flour
from "nuts.com", Cranford, N.J. 07016. [0066] 3. Walnut: Roasted
walnut from "Holmquist Hazelnut Orchards", Lynden Wash. 98264.
[0067] 4. Walnut and pecan: Roasted walnut from "Holmquist Hazelnut
Orchards", Lynden Wash. 98264. Ground pecan from "King Arthur
Flour", Norwich, Vt., 05055. [0068] 5. Formula 1: equal parts
walnut, hazelnut, shrimp, salmon, hen's egg, cow's milk, peanut,
cashew, almond, and wheat (30 mg of protein each) [0069] 6. Formula
1 at 3.times.x: equal parts walnut, hazelnut, shrimp, salmon, hen's
egg, cow's milk, peanut, cashew, almond, and wheat (90 mg of each
protein) [0070] 7. Formula 1 at 10.times.: equal parts walnut,
hazelnut, shrimp, salmon, hen's egg, cow's milk, peanut, cashew,
almond, and wheat (300 mg of each protein) [0071] 8. Non-treated
control (NT) [0072] 9. White fish: Pacific cod from "Seattle
Seafoods", Bellevue, Wash. 98008. [0073] 10. White fish and pink
fish: Pacific cod and sockeye salmon from "Seattle Seafoods",
Bellevue, Wash. 98008. [0074] 11. Shrimp: White shrimp from
"Seattle Seafoods", Bellevue, Wash. 98008. [0075] 12. Shrimp and
crab: White shrimp from "Seattle Seafoods", Bellevue, Wash. 98008.
[0076] 13. Peanut: Defatted peanuts from "Byrd Mill Company",
Ashland, Va. 23005. [0077] 14. Peanut and soy: Defatted peanuts
from "Byrd Mill Company", Ashland, Va. 23005. Stone ground soy from
"Bob's Red Mill", Milwaukie, Oreg. 97222. [0078] 15. Hazelnut:
Natural hazelnuts from "Holmquist Hazelnut Orchards", Lynden Wash.
98264. [0079] 16. Almond: Blanched almond flour from "Honeyville"
Rancho Cucamonga, Calif. 91730. [0080] 17. Milk: Organic non-fat
dry ilk powder from "Now Foods" Bloomingdale, Ill. 60108. [0081]
18. Egg: Powdered egg whites with sodium lauryl sulphate as an
anti-caking agent from "Honeyville Food Products", Honeyville, Utah
84314.
2. Stimulation and Enumeration of Th2 Cell Subsets
[0082] PBMCs from subjects were labeled with CFSE and cultured with
the same food allergen or food allergen mix that was fed to them at
100 .mu.g/mL (Byrd Mill) or anti-CD3/CD28 (to test for nonspecific
proliferation capacity) for 7 days to identify T cell subsets. At
day 7, cells were washed and stained for surface CD4, CD25, CD127,
CD45RO, CD45RA, CD40L, and CD69 and intracellular Foxp3 and IL-10
along with Live/Dead staining (Invitrogen) (see T-cell flow
cytometry method below). Th2 cells were defined as the cells that
proliferated in response to food allergen (CFSEIo) and were
CD411L-4, 1L-13 cells. Antigen-induced T cells were also identified
by isolating CD40L and CD69 double-positive cells after antigen
stimulation.
3. T-Cell Flow Cytometry
[0083] Cells were fixed with Lyse/Fix PhosFlow buffer (BD
Biosciences). For intracellular staining, fixed cells were
permeabilized with Perm Buffer III (BD Biosciences) at 48.degree.
C. for 30 minutes, followed by staining at 48.degree. C. for 20
minutes. Flow cytometry was performed with an LSRII flow cytometer
(BD Biosciences). Viable cells were identified with a Live/Dead
probe (Invitrogen). Phenotypes of T cells were detected with
antibodies against surface CD3 (UCHT1), CD4 (SK3), CD25 (4E3),
CD127 (SB199), CD45RO (UCHL1), CD45RA (HI100), CD62L (DREG-56),
CCR4 (1G1), and CCR8 from BD Biosciences; CCR7, CD69, and CD40L and
intracellular IL-10 (JES3-19F1), IL-4 (MP4-2502), and IL-13
(JES10-52A2) from BD Biosciences; Helios (22F6) from BioLegend;
anti-CD49b from BioLegend; anti-LAGS from R&D Systems
(Minneapolis, Minn); and Foxp3 (150D) from BioLegend and stained
per the manufacturer-recommended protocol.
4. Measurement of Antibody Titers
[0084] Total and allergen-specific blood IgE and IgG4 levels were
measured in all subjects in the Clinical Laboratories at Stanford
Hospital and Clinics using a standard ImmunoCAP assay (Phadia,
Uppsala, Sweden).
B. Results
1. Th2 Cell Proliferation
[0085] PBMCs from each subject in allergen groups 1 to 14 were
stimulated ex vivo with the allergen or allergen mix fed to the
subject for 7 days and Th2 cell proliferation was analyzed (as
described in the Methods section above). It is noted that for
Formula 1 3.times. and Formula 1 10.times. groups, the PBMCs were
stimulated with 3.times. and 10.times. the amount of allergen ex
vivo, respectively (i.e., 300 .mu.g/mL and 1000 .mu.g/mL,
respectively).
[0086] As shown in FIG. 1, subjects in allergen groups 1 to 4 and 9
to 14 showed a somewhat reduced Th2 proliferation in response to
allergen stimulation as compared to the non-treated control (Group
8; Baseline-NT). However, Th2 cell proliferation in PBMCs from
subjects in allergen groups 5 to 7 (fed increasing amounts of
Formula 1), who were fed formulations having 10 separate allergens,
as described above had significantly reduced Th2 cell proliferation
as compared to both the control group (NT) as well as all other
antigen groups (1 to 4 and 9 to 14). This phenomenon was seen for
all subjects in all allergen groups and at all ages tested.
2. Immunoglobulin Analyses
[0087] In addition to the Th2 proliferation assay above, plasma
from subjects in allergen groups 1 to 18 were analyzed for the
presence of IgG4 and IgE antibodies (using standard methods, as
described above).sub.; the former being an indicator of a
non-allergenic or non-inflammatory (or anti-allergenic or
non-inflammatory) state and the latter an indicator of
pro-allergenic or pro-inflammatory a sate.
[0088] As shown in FIG. 2, subjects in allergenic groups 1 to 4 and
9 to 18 showed moderately increased levels of IgG4 as compared to
the baseline group (Group 8; NT). However, allergen groups 5 to 7
(fed increasing amounts of Formula 1) showed significantly
increased levels of IgG4 as compared to both the baseline group as
well as all other antigen groups (1 to 4 and 8 to 18). This result
is consistent with the results for Th2 cell proliferation as
described above. Specifically, increased levels of IgG4 in the
plasma and reduced Th2 cell proliferation are indicators of a
reduced or non-allergenic or non-inflammatory state in a
subject.
[0089] As shown in FIG. 3, subjects in allergenic groups 1 to 4 and
9 to 17 showed significantly higher levels of IgE in the plasma as
compared to allergen groups 5 to 7 (fed increasing amounts of
Formula 1). Indeed, IgE levels in the allergen groups 5 to 7 were
below the level of detection of the assay. This result is
consistent with the results for Th2 cell proliferation and the IgG4
levels as described above. Specifically, low levels of IgE and
increased levels of IgG4 in the plasma coupled with reduced Th2
cell proliferation are indicators of a reduced or non-allergenic or
non-inflammatory state in a subject.
[0090] It is clear from the results above that continual feeding a
complex mixture of food allergens to subjects (e.g., in the form of
Formula 1 described above) at a very young age can prophylactically
protect a subject from developing an allergenic or inflammatory
immune profile to a wide variety of antigens. In other words, this
process can induce in a subject a non-allergenic or non
-inflammatory (or anti-allergenic or anti-inflammatory) state. Of
particular interest in the results shown herein is the indication
that feeding antigens in a complex mixture provides broad spectrum
protection against developing allergies/inflammation that is
superior to single or even double allergen formulas that that
target a single allergen (as in allergen groups 1, 3, 11, 13 and 15
to 18 above) or multiple related allergens (e.g., as in allergen
groups 2, 4, 10, 12, and 14 above).
[0091] The above shows that feeding subjects complex antigen
mixtures will not only protect against the development of allergies
and/or inflammation to the allergens/antigens in the complex
mixture, but also to allergens or antigens that are not present in
the complex mixture. In essence, feeding complex food antigen
mixtures creates a general anti-allergenic or anti-inflammatory
state in a subject that broadly prevents the development of
allergies or inflammatory state, even to allergens/antigens not yet
fed to the subject.
II. Mixed Allergen+Probiotics and/or Vitamin Assay
A. Mixed Allergen Compositions
[0092] In FIGS. 4-7, compositions of a 1:1 blend were used: [0093]
1 part dietary supplement mixture: 1 part probiotics (300 mg each
to be specific). Probiotic in this instance was Lactobacillus from
Culturelle brand; or [0094] 1 part dietary supplement mixture: 1
part vitamins (300 mg each to be specific) Vitamins in this
instance were Poly Vi Sol brand (for children); or [0095] 1 part
dietary supplement mixture: 1 part vitamins: 1 part probiotics (300
mg each to be specific)
[0096] Mixtures at pH 2 were incubated in vinegar for 40 min at
room temperature before using.
[0097] Mixtures that were baked were heated for 40 min at
350.degree. F.
[0098] Control received no dietary supplement.
B. Th2 Cell Proliferation
[0099] As shown in FIG. 4, PBMCs from each subject in allergen
groups 1 to 14 were stimulated ex vivo with the allergen or
allergen mix or with probiotics or with vitamins or in certain
conditions with differing temperatures or pH, for 7 days and Th2
cell proliferation was analyzed (as described in the Methods
section above).
[0100] As shown in FIG. 4, subject samples showed similar Th2
proliferation decreases with the mixture or under different
conditions with the mixture (i.e. baked food, skin cream, juice).
This phenomenon was seen for all subjects in all allergen groups
and at all ages tested.
C. Immunoglobulin Analyses
[0101] In addition to the Th2 proliferation assay above, plasma
from subjects in allergen groups were analyzed for the presence of
IgG4 and IgE antibodies (using standard methods, as described
above), the former being an indicator of a non-allergenic or
non-inflammatory (or anti-allergenic or anti-inflammatory) state
and the latter an indicator of pro-allergenic or pro-inflammatory
state.
[0102] As shown in FIGS. 5 and 6, subjects showed moderately
increased levels of IgG4 as compared to control group and
significantly higher levels of IgE in the plasma. As shown in FIG.
6, no IgE levels were observed in response to the different tested
mixed allergen compositions. There were similar effects seen with
the mixture or under different conditions with the mixture.
D. T Cell Proliferation
[0103] T cell proliferation was performed (please see methods
section above) and data presented in FIG. 7. The data show
anti-inflammatory properties to gluten (i.e., celiac agent), and
insulin (i.e., diabetes agent) and bacterial flagellin
(inflammatory bowel disease agent). This demonstrates that the
mixture could also decrease inflammatory states involved in
diseases like celiac, diabetes and inflammatory bowel diseases.
[0104] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
[0105] Accordingly, the preceding merely illustrates the principles
of the invention. It will be appreciated that those skilled in the
art will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
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