Note: Descriptions are shown in the official language in which they were submitted.
CA 02086101 2001-07-30
IMMUNE SUPPRESSIVE PRODUCT FROM MILK
Field of the Invention
This invention is directed to the discovery of polypeptide fractions in milk
which
contain peptides derived from allergens administered to a milk-producing
animal. These
fractions are useful in suppressing allergic responses in humans and animals.
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~1CK6ROUhtD OF THE I~11ENTIOP!
The allergic reaction in man and animals has been
extensively studied and the basic immune mechanisms involved i
are well known. The generic name for molecules that cause an ',
allergic reaction is allergen. There are numerous species of
allergens. Common examples include plant pollens, bee venom,
house dust, animal dander, and a wide array of food proteins. '
Many allergens are protein or polypeptide in nature, as
proteins and polypeptides are generally more antigenic than
carbohydrates or fats. The allergic reaction occurs when
tissue-sensitizing immunoglobulin of the IgE type reacts with
foreign allergen. The IgE antibody is bound to mast cells
and/or basophils, and these specialized Cells release
chemica'1 mediators of the allergic reaction when stimulated
to do so by divalent antigens bridging the antibody
molecules. Histamine, platelet activating factor,
arachidonic acid metabolites, and serotonin are among the
best known mediators of allergic reactions in man.
The symptoms of the allergic reaction vary, depending on
the location within the body where the IgE reacts with the
antigen. If the reaction occurs along the respiratory
epithelium the symptoms are sneezing, coughing and asthmatic
reactions. If the interaction occurs in the digestive tract,
as in the case of food allergies, abdominal pain and diarrhea
are common. Systematic reactions, for example following a
bee sting, can be severe and often life threatening.
The preferred, but frequently impossible, method of
relieving allergies is allergen avoidance. Failing that,
there are two medical approaches to allergy control, both
with an approximate 60 to 85% efficacy rate (Aas K., Allergy
37:1-14 (1982)). The most common approach to the medical
treatment of allergies is to treat the symptom. nrugs known
t'>:1 92/00756 ~'CT>US91J02923
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to block the effects of the chemical mediators of the
allergic reactions, including antihistamines, are used to
control the severity of the allergic symptoms. These drugs,
however, do nothing to prevent the allergic reaction and the
liberation of the chemical mediators, and do nothing to
prevent or diminish allergic responses to subsequent allergen
exposure.
Another approach is to prevent the allergic reaction by
desensitizing the allergic host. This is accomplished by
giving repeated small doses of the reactive allergen. The
treatment usually involves injecting the allev~gens under the
skin. Treatment with reactive allergens, more appropriately
called immunotherapy, is believed to increase the
concentration of antibodies of the IgG type against the
allergen. The IgG antibody competes with the IgE antibody
for allergen binding, and this competitive antibody somehow
neutralizes, arrests, or blocks the action of the tissue
sensitizing IgE antibody, although a distinct correlation
between blocking antibody and amelioration of symptoms has
not been definitely proven (Mailing, H.J., (ed.),
Immunatherapy Position Paper, Allergy (Supp.J 6, X3:9-33
(1988)). This rationale, although generally accepted, is not
fully understood, and does not reflect the complex
interactions and events that accompany the IgG response. For
example, other effects of immunotherapy that may be involved
with relief of symptoms include suppression of IgE, increase
in blocking IgA and IgG in secretions, reduced basophil
reactivity/sensitivity, and reduced lymphocyte responsiveness
to allergens. All of these changes may oat occur in every
patient, and those related to actual symptom relief have not
been conclusively defined (Norman, P.S., J. Allergy Clin.
Immunol. 75:531-545 (1985)).
Immunotherapy using reactive allergen is dangerous
because the sensitized host is actually treated with the
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molecules capable of eliciting an allergic response. The
treatment is started with extremely low does to avoid
inducing a severe reaction. The antigen concentration
required for 5Ofo histamine release from peripheral basophils ,
of an allergic individual varies 10,000 fold from patient to
patient (Norman, P.S., J. Allergy Clin. Immunol. 75:531-545
(1985)). If no adverse reaction occurs, higher doses are
given. The injection may cause severe allergic reactions and
extreme care must be taken. Only experienced doctors can
administer this treatment because if a severe reaction occurs
immediate medical treatment must be given to control the
symptoms of the allergic reaction. Desensitization is an
expensive, painful, and time consuming process (Aas K.,
Allergy 37:1-14 (1982)), therefore, only the most severe
allergies are treated by this method (Mailing, H.J., ed.,
Immunotherapy Position Paper, Allergy (Supp.) 6, 43:9-33
(1988)).
Modifying allergens to eliminate their allergenicity, or
ability to induce an IgE-mediated response, while preserving
their immunogenicity, or their ability to elicit the
protective IgG response, has been under investigation for
years. Polymerized antigens have been evaluated with the
theory that they would display reduced antigenicity due to
concealed antigenic determinants, a lower molecular
concentration hence decreased bridging opportunity on a
weight basis, and slower diffusion through the tissues
(Patterson R., J. Allergy Clip. Immunol. 68:85-90 (1981)).
Allergen conjugates have been studied with a variety of
allergens in attempts to selectively inhibit the formation of
IgE antibodies while normal IgM and IgG responses to the
antigen occurred (Lee, ld.Y. et al., Imm. Rev. 41:200-217
(1978)). Conjugates with a glutamic acid/lysine copolymer
may be acting specifically on IgE lymphocytes, on suppressor
and/or helper T cells, or a combination of mechanisms may be
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921Q0756 PCTlUS91 /02923
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in effect (Liu, F.T. et al., Proc. Nat). Acad. Sci. IJSA
76:1430-1434 (1979)).
Modifications that decrease the size of the allergen in
efforts to eliminate their allergenicity have also been
attempted. Tt has been proposed that limited proteolysis may
actually reveal suppressor determinants on allergen molecules
(Mowatt A.M., Immunology 56:253-260 (1985)}.
Polypeptide fractions of allergens and the use of
enzymes to break down the parent proteins is not new (King,
T.P., Advances in Inununology 23:77 (1976)). U.S. patent
number 4,469,677 teaches the use of polypep~tide fractions
prepared from allergens for desensitization of allergic humans
and animals. The polypeptide fractions are prepared by
digesting allergens with proteolytic enzymes. Following the
enzymatic digestion procedure, the enzymes and residual parent
allergens must be removed from the polypeptide subfractions.
The specific structure of the polypeptide fractions is
dependent on the enzymes used for the digestive process.
However, this is a synthetic process that requires pre=
selection of the enzymes used for the digestive process. The
polypeptide fraction is produced by controlled proteolytic
digestion of the poiypeptide allergen. Although this
represents an improvement over using the reactive antigens,
the treatment still requires frequent and painful injections.
The function of the polypeptide fractions for suppressing
immune function are structure dependent. see Unanue, E.R..et
al., Science 236:551-557 (1977) for a review of the structure
dependence of polypeptide fractions of prateins in the immune
response.
The structure of the polypeptide fractions of Michael is
limited because the selection of enzymes is based on a
limited understanding of which enzymes are most important.
The development of oral "tolerance" is a normal
phenomenon, and a necessary 'function in response to the
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variety of foreign antigens consumed in the diet. Oral
tolerance is initiated by a special class of T lymphocytes
and their products, and it results in systematic suppression
of the IgE-mediated hypersensitivity reaction. Variable
responses of other components of the immune system have been
reported to occur during induction of the tolerized state, so
that the actual mechanism has not been succinctly defined.
In addition, animal studies have shown an actual
enhancement of IgE production in response to intragastric
administration of pollen extracts (Henderson, D.C. et al.,
Int. Archs. AIIergy Appl. Immun. 19:66-71 (1986)). Although
this may be a dose-related phenomenon, conflicting results
were reported from two studies where 20 mg ovalbumin was
administered orally to parenterally immunized mice, with IgE
increased in ane study (Handson, D.G. et al., Int. Arch.
Allergy Appl. Immun. 55:256-532 (1977)) and decreased in the
other (Lafont, S. et al., J. Exp. Med. 155:1573-1578
(1982)).
Some investigators have concluded that recognition of
antigenic determinants varies between parenterally and orally
induced suppressor T cells. Suppressor cells resulting from
feeding antigen were able to recognize different forms of the
antigen, while parenterally induced suppressor T cells were
specific for the molecular confirmation to which they
initially responded (Mowatt, A.M., Tmmunology 56:253-260
(1985)). If this phenomenon is exhibited by a majority of
antigens, it would further warrant the use of oral
immunotherapy.
The problem with administering allergens orally to
allergic subjects is that severe immune reactions may occur
following treatment by this route as well. Anaphylaxis,
relapse, urticaria, rectal bleeding, and anaphylactic shock
have all been reported following oral immunotherapy (Platts- '
Mills, T.A.E., J. Allergy Clin. Immunol. 50:129-132 (1987)).
!, 92/00?56 PCT/L1S91 /02923
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In addition, oral treatment requires significantly larger
doses than parenteral therapy, due to the action of the
digestive tract enzymes upon the allergens. Individual
responses to the ingested antigens may vary, however, so that
more or less intact allergen may be represented to the
gastrointestinal mucosa from the same dose given to different
subjects, increasing the risk of inappropriate dosing.
Nonallergenic fractions of allergens can be given orally
without concern for severe irtanune reactions; however, for
reasons that are not clearly understood, oral administration
of the polypeptide fractions of some allergens, including
those prepared in the Michaels patent (U.S. 4,469,677) are
not effective in causing desensitization. Studies of the
immune response to feeding other modified proteins are
limited, with varying results. Collagen-induced arthritis in
mice was suppressed by feeding native type II collagen but
not if the denatured molecule was fed (Nagler-Anderson, C.L.
et al., Proc. Natl. Acad. Sci. USA x3:7443 (1986)). In a
study of another autoimmune disease, both the disease-
producing and nondisease-producing fragments and decapeptides
of the myelin base protein were capable of eliciting immune
suppression following oral treatment in rats (Higgins, P.J. et
al., J. Immunology 140:440-445, (1988)). Results from a mouse
study evaluating induction of oral tolerance with both natural
and denatured ovalbumin identical suppression of delayed type
hypersensitivity response to each form (Mowat, A.M.,
Immunology 56:253-260 (I985)).
One factor that may be i nvol ved i n the fai 1 ure of some
molecular fragments to effectively induce immune tolerance is
the low pH of the stomach, which may further modify the
polypeptide fraction, abrogating its ability to induce
tolerance.
Another factor involved in the failure of orally dosed
allergen fragments to induce desensitization to future
iV0 92!00756 PCf/US91/029 ~;,,
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exposure may be the actual nature of the fragment itself. In
vitro synthesis of fragments requires specific enzymes and
conditions that may or may not result in the preparation of a
biologically optimal formulation. The fragment may be
effective when it is acutely exposed to the immune system
following intravenous administration, but may be so
susceptible to modification that any alterations occurring in
the gastrointestinal tract before its exposure to the
appropriate immune system components may destroy its
effectiveness.
A need exists, therefore, for an oral 'formulation of
allergen fractions that, when administered orally, is
sufficiently active to induce tolerance but is not
allergenic. A need also exists for methods of producing such
products.
SUMhLARY OF THE INVENTION
This invention embodies the discovery of a method of
manufacture, method of use, and product by process. The
generic form of the product, by process of this invention, is
milk that contains polypeptide fractions of allergens. The
specific product by process of this invention is the milk
polypeptide fraction that contains polypeptide fragments of
specific allergens.
Specifically, the invention is directed to a milk
produced by an immunized milk-producing animal, such milk
containing non-immunoglobulin polypeptide fragments of the
allergens used to immunize the animal.
The invention is further directed to a food product
containing the milk of the invention.or an active fraction of
said milk.
The invention is further directed to an oral vaccine for '
administration to an allergic subject for relief of the
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allergic response in such subjects, such vaccine containing
the milk of the invention or an active fraction thereof.
The invention is further directed to a method for the
production of an immune suppressive product comprising 'the
immunization of a milk-producing animal and the collection of
milk from such animal after such animal reaches an immune
state.
The invention is further directed to a rnethod for the
desensitization of a subject to an allergen which method
comprises administration of the milk of the invention, or an
active fraction there, to such subject, in an amount and for a
time sufficient to produce an immune suppressive effect.
The invention is further directed to pharmaceutical
compositions which provide to a subject who is in need of
desensitization to an allergen, the active, polypeptide
fragment-containing fractions of the invention at amounts
which are efficacious in the treatment and suppression of
such subject's allergic responses and allergic reactions.
DESCRIPTION OF THE PREFERRED EMBODI~9ENTS
The invention provides an oral formulation for
desensitization, an oral vaecine, which is of significant
medical benefit to the subject who ingests it. By subject is
meant a human or other animal in need of allergy desensitizing
according to the method of the invention. The oral vaccine of
the invention can be self administered, is less painful, and
less expensive then vaccines which must be injected. The
subject ingesting the oral vaccine of the invention is exposed
to the allergen in a derivatized, processed form which allows
such subject to acquire tolerance to such allergen.
The milk of the invention, or an active fraction thereof,
may be administered in any form which retains the allergic
response suppressive properties of the milk, either powdered
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or liquid. Any food product which incorporates the active
fractions of such milk may also be provided, far example,
skim milk, yogurt and cheese.
By an "active fraction" of the milk of the invention is
meant a preparation or composition which has been extracted
from- the milk of the invention and which retains the
beneficial, allergic reaction-suppression properties of the
unextracted milk of the invention which are due to the
presence of derivatized, processed forms of the allergen used
to immunize such animal providing such milk.
The derivatized, processed form of the allergen of the
invention is found in the milk of a milk-producing animal
which has been immunized against the allergen. The advantage
of administering the polypeptide fraction of the invention to
a subject rather than administering the unprocessed allergen
itself is that, unlike the unprocessed allergen, the allergic
response suppressive polypeptide fraction of the milk of the
invention is nonallergenic. Desensitization of the subject
who ingests the milk of the invention occurs but there is no
risk of severe allergic reactions in such subject because the
allergen has been processed to a non-allergic form by the
immune system in the milk-producing host.
The allergen which is used as a source of the immune
suppressant active polypeptides can be any allergen which is
capable of provoking an immune system response in the
mammalian animal which processes such allergen. If an
allergen is not capable of inducing an immune system response
in one mammalian species, another species may be used. In a
preferred embodiment, the milk-producer bovine dairy cow is
used to process the allergen. However, any mammalian species
may be used, including humans, and animals in the ovid,
poreine, and equine species, etc.
The allergen preparation may contain a mixture of
allergens or only one specific allergen. Examples of allergen
~,..,9210~756 PCT/US91 /02923
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mixtures which may be protected against by the administration
of the milk preparation of the invention include mixtures of
pollens of various trees and grasses, venoms from animals and
insects, and food allergens. Examples of specific allergens
which may be protected against by the administration of the
preparation of the invention include, for example, ragweed,
bee venom, and wheat protein in the case of a food allergy.
The method of manufacture of the polypeptide fraction of
this invention produces a unique subfraction of the parent
allergen, such subfraction exhibiting immune suppressive
activity when used to treat a host allergic to such allergen.
By "immune suppressive activity" is meant the ability of a
composition to lessen allergic symptoms or allergic responses
in an allergic subject who is administered such composition
By "allergic symptoms" is meant physiological reactions or
conditions which are induced in an individual in response to
that individual's being exposed to substances which are
allergic for that individual, such as, for example, allergy-
induced congestion, headaches, breathing, itching, swelling,
sneezing, wheezing, coughing, rhinorrhea, and decreased
ability to smell and taste.
The milk composition of the invention, and polypeptide
fractions produced therefrom according to the process of this
invention, lack the allergenicity of the parent allergen (the
allergen used to immunize the milk-producing animal) but
possess the ability to desensitize a host administered such
compositions.
It is not necessary that the milk-producing animal be
induced to, or maintained in, a hyperimmune state to produce
the milk of the invention. Further, it is not necessary that
the milk-producing animal be induced to an antibody-producing
state to produce the milk of the invention because the active
compositions of the invention do not rely on antibodies to ,
provide their protective and suppressive activity. It is only
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necessary that the allergen-administered animal be provided
with time sufficient to process such allergen into a form .
which is secreted in the milk of the animal.
Because immune cell processing of the allergen is one of a
the earliest steps in the immune response, milk containing the.
active polypeptides of the invention may be obtained
relatively soon after administering the allergen, for example,
24 hours to a week after allergen administration. Cows
administered allergen according to the method described herein
provide such active polypeptides in their milk 24 hours after
allergen administration.
Any amount of the allergen which results in the
appearance of the active polypeptides of the invention in the
milk of such animal may be administered to the milk-producing
animal. If the amount of such active polypeptides in the milk
of the allergen-administered animal is too low to provide
efficacious benefits to a subject in need of desensitization,
such active polypeptides may be provided to the subject in a
more concentrated form. Such concentration is obtainable using
techniques known in the art, for example, by salt
precipitation, evaporation or lyophilization of the
compositions of the invention.
Active polypeptide-containing preparations may be
combined from more than one allergen-administered animal, and
from more than one species of allergen-administered animal so
as to provide the subject who i.s in need of desensitization
with a composition containing a variety of polypeptide
subtractions from different genetic heritages.
Although inducing a milk-producing animal to an antibody
producing state or to a hyperimmune state is not necessary,
neither is it detrimental to 'the milk of the invention and
continuous production of milk containing the active
polypeptides of the invention may be achieved by repeatedly '
administering the allergen to the milk-producing host.
~92/OU755 P'(.'F/US9r/02923
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Without meaning to be held to this theory, since the
processing mechanism of the immune system is not fully
understood, it is thought that the foreign protein or
polypeptide is ingested or phagocytized by special cells of
the immune system, the most noteworthy of which are the
macrophage and neutrophil. After administration to a milk-
producing animal, enzymes within the immune cell break down
or derivatize the protein or polypeptide allergen into forms
herein interchangeably termed "processed" forms or
"subtractions" of the allergen.
The immune cell-produced subfractions are unique and
cannot be produced outside of the natural host. The
uniqueness is based first on the structure of the parent
allergen and second on the combination of enzymes contained in
the mammalian host's allergen processing cells. The enzyme
combination which is expressed in the mammalian allergen
processing cells are genetically determined; other species
each have a unique combination of enzymes. For example
peptide fractions of allergens processed by the immune cells
of the bovine species are similar, but not identical, to
polypeptide fractions processed by the immune cells of other
species. It is because of this genetic specificity that the
polypeptides produced by the process of this invention are
unique and different from polypeptides produced by any other
method.
Special immune cells called helper cells assist the
macrophage in presenting the processed antigens to T and B
lymphocytes. The passage of the allergen subtraction with
and/or from the macrophage through the blood and across the
mammary gland and into the milk may further modify the
structure of the allergen polypeptide found in milk. This
natural biological method of processing environmental antigens
and transporting natural immune suppressants into the milk
provides a mechanism whereby the dairy cow transfers immune
WO 92!0a756 PCT/US91/029
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protective factors to the calf through milk. These milk
I
immune factors protect the nursing young of such animals
against allergic reactions. Nature, through i:he process of
evolution, has found a method for' the mother of the species to ,
sample the environment for potential allergens. The immune
system then transforms the environmental allergens into the
factors that protect against allergic reactions. The mother
passes the immune protective factors to the nursing infant
through milk. The essence of the discovery of the invention
is that man can utilize this natural biological process to
transform known allergens into immune protective polypeptide
fractions that have utility for preventing and treating
allergic reactions.
The milk immune suppressive factor is a polypeptide
subfraction found in milk and not the antibodies found in
mi 1 k. A1 though it i s probabl a that anti bodi es are useful i n
treating allergies, milk antibody is not the product of this
invention.
The milk of animals treated by the process of this
invention contains specific and unique polypeptide
subfractions of the allergens used to treat the host. The
same milk lacks the intact or underivatized parent allergen.
The uniqueness of these polypeptide fractions results from
specific enzymatic breakdown by specialized immune cells of
the host. Blood and mammary tissue enzymes may also play a
role in modifying the chemistry of the allergens before entry
into the milk. Milk containing these unique nonreactive
polypeptides is useful as an oral immune treatment for
inducing protection and/or tolerance when fed to other species
including man. The milk allergen polypeptide fractions, when
isolated from the milk, are useful for suppressing allergic
reactions against the representative parent allergens.
The allergen may be administered to the milk-producing
animal by various routes of administration including oral,
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rect a , vaginal, intramuscular injection, subcutaneous
injection, intradermal injection, transcutaneous
administration, etc. Ylhile, any route of treatment can be
used for administering the allergen, the preferred route of
treatment is by intramuscular injection of the allergen into
the host species. The allergen, upon entry into the body of
the milk-producing animal, is processed by the immune system
of the milk-producing host.
Treatment of the allergic individual or animal with the
compositions of the invention effects protection in that
individual or animal against the parent allergen without
danger of side effects that may occur when the parent molecule
is used. Examples of protein or polypeptide allergens that
can be processed by the mammalian milk system of mammals
include pollen allergens of weeds, grasses and trees, animal
venoms and poisons, and various food allergens. In a
preferred embodiment, the bovine milk system is used.
Theoretically, any allergen can be processed by the
mammalian, and especially by the bovine, macrophage system to
produce immune suppressive polypeptide subfractions.
The process of this invention is advantageous because it
uses the full spectrum of processing enzymes available in the
allergen processing cell of the mammalian immune system. A
second advantage of the process of this invention is that the
full spectrum of polypeptide fractions is contained in the
immune suppressive milk product and the immune suppressant
allergen fractions, when contained in a natural milk vehicle,
are orally active. The process of this invention, does not
require selection of specific enzymes to degrade the allergen
in vitro or regulation of the enzymatic digestive process, nor
does the process of this invention require separation of the
enzymes and residual parent molecules from the reaction
system. The entire antigen processing occurs naturally within
the body of the milk-producing animal. The genetics of the
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mammal selects the enzymes and the natural metabolism of the
mammal maintains reaction conditions at an optimal level for
antigen processing.
The peptide fractions of the invention may be monitored,
purified and isolated by any technique or combination of
techniques wherein such peptides retain their bioactivity,
that is, wherein such peptides retain the ability to suppress
allergic responses. Such techniques are known in the art and
include, but are not limited to, chromatographic techniques,
including adsorption chromatography, gel chromatography and
especially, gel chromatography through a dextran,
polyacrylamide, or agarose matrix, ion-exchange
chromatography, for example, utilizing a strong cationic
exchanger such as, for example, SP-Sephadex*(which contains a
sulfopropyl functional group derivatized to a dextran), AG 50
(which contains a sulfonic acid derivatized to styrene-
divinyl-benzene), and Bio-Rex 40 (which contains a sulfonic
acid functional group derivatized to a phenolic matrix); weak
cationic exchangers, such as, for example, CM-Sepharose (which
contains a carboxymethyl functional group derivatized to a
dextran), Bio-ReX 70 (which contains a carboxylic functional
group derivatized to a phenolic matrix); strong anionic
exchangers, such as, for example, QAE-Sephadex~(which contains
a diethyl-(2-hydroxypropyl)-aminoethyl functional group
derivatized to a dextran), AG 1* (which contains a
tetramethylammonium ion functional group derivatized to
styrene-divinyl-benzene); weak anionic exchangers, such as,
far example, DEAE-Sephadex (which contains a diethylaminoethyl
functional group derivatized to a dextran, AG-3~ (which
contains a tertiary amino functional group derivatized to an
epoxyamine matrix); medium strength cationic exchangers, such
as, for example, CM-cellulose (which contains a carbaxymethyl
functional group derivatized to cellulose), P-cel* (which
contains a phospho- functional group derivatized to
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cellulose); and medium strength anionic exchangers, such as,
for example, DEAF-cellulose {which contains a
diethylaminoethyl functional group derivatized to cellulose),
PEI-cellulose (which contains a polyethyleneimine functional
group derivatized to cellulose, DEAE(BND)-cellulose {which
contains a benzoylated-naphthoylated, diethylaminoethyl
functional group derivatized to cellulose), PAB cellulose
(which contains a p-aminobenzyl functional group derivatized
to cellulose); and, exchangers which provide a mixture of
functional groups, such as, for example, AG501 {which
contains two functional groups, sulfonic acid and
tetramethylammonium ion derivatized to a styrene-divinyl
benzene matrix). In addition, high pressure liquid
chromatography, may be used to separate the peptides of the
invention.
Other compounds may be conjugated, either chemically or
by genetic engineering, to the active polypeptides of the
invention so as to enhance or provide additional properties to
such polypeptide-containing compositions, especially
properties which enhance the active polypeptide's ability to
promote relief of an allergen's effects in a subject in need
of immune suppressive treatment.
Amounts and regimens for the administration of the
compositions of the invention can be determined readily by
those with ordinary skill. Generally, the dosage of the
active peptide-containing composition will vary depending upon
considerations such as: type of allergen employed; age of the
subject being treated; health of the subject being treated;
type of allergy being treated; kind of concurrent treatment,
if any; physiological tolerance to the compositions of the
invention; frequency of treatment and the nature of the effect
desired; gender; duration of the symptoms; and,
counterindications, if any, and other variables as may be
adjusted, as desired, by the individual's physician. Dosage
y
WO 9210056 PCT/US91/029 '~~
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can be administered in one or more applications to obtain the
desired results.
The compositions of the invention can be administered in
any appropriate pharmacological carrier for administration.
They can be administered in any form that effects
prophylactic, palliative, preventative or curing conditions of
allergic reactions in humans and animals.
Preparations of the active polypeptide-containing
compositions of the invention for parenteral administration
includes sterile aqueous or non-aqueous solvents, suspensions
and emulsions. Examples of non-aqueous solvents are propylene
glycol, polyethylene glycol, vegetable oil, fish oil, and
injectable organic esters. Aqueous carriers include water,
water-alcohol solutions, emulsions or suspensions, including
saline and buffered medical parenteral vehicles including
sodium chloride solution, Ringer's dextrose solution, dextrose
plus sodium chloride solution, Ringer's solution containing
lactose, or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers, such as
those based upon Ringer's dextrose and the like.
The compositions of the invention may also be
administered by means of pumps, or in sustained-release form.
Administration in a sustained-release form is more
convenient for the patient when continuous repeated
administration for prolonged periods of time are indicated.
The active polypeptide-containing compositions of the
invention can be employed in dosage forms such as tablets,
capsules, powder packets, or liquid solutions for oral
administration.
The pharmaceutical compositions of the present invention
are manufactured in a manner which is in itself know, for
example, by means of conventional mixing, granulating, dragee-
making, dissolving, lyophilizing or similar processes.
'~ 92/~0756 PC.'T/US91/02923
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2~~~~.~~.
Having now generally described the 'invention, the
following examples further describe the materials and methods
used in carrying out the invention. The examples are not
intended to limit the invention in any manner.
EXAMPLES
Example 1
Immunization of Cows and Collection of Imrnune Milk
The immunogen for the preparation of the milk product for
allergy symptom alleviation comprises a mixture of commercial
allergen extracts purchased as 1:100 wt:vol preparations and
pooled in the following ratios:
ALLERGEN Volume (ml)
Alternaria 10
tenuis
Aspergillus 10
niger
Monilia 10
albicans
Hormodendrum 10
hordei
Lambsuarter 10
q
Houseust 10
d
Helminthosporium 10
Sat.
Russian 10
thistle
Careless 10
weed
Spiny 10
pigweed
Mugwort,.common 10
Ragweed, 10
slender
Ragweed, 10
southern
Ragweed, 10
false
Ragweed, 10
short
Ragweed, 10
giant
Pigweed, 10
redroot
Trichophyton 10
mentag
Goldenrod 10
Palmer's 10
ameranth
Sage, 10
prairie
Curvularia 30
spisifera
Pullularia 30
pullulans
Mucor 30
plumbeus
Fusarium 30
moniliforme
GS 30
Mold
Mix
#2
GS 30
Tree
Mix
Rhizopus 30
nigricans
..,
WU 92/00756 PCT/US91/029 3
....: _,
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The pooled mixture is diluted with an equal volume of
sterile saline. Cows are injected intramuscularly with 5 ml
of the preparation at 2 week intervals during normal
lactation, and the milk is collected and powdered. Milk is
collected beginning 24 hr. post treatment.
Examol a 2
Fractionation of Milk Fractions
Containing Processed Allergens
Twenty liters of fresh milk from hyperimmunized cows were run
through a cream separator (DeLaval Model 102) to remove the
fat.
The resulting sixteen liters of skimmed milk was
ultrafiltered to remove the high molecular weight species
(over 100,000 daltons) using a hollow fiber diafiltration/
concentrator. The concentrator is equipped with two 100,000
daltons molecular weight cut-off cartridges. The skimmed milk
was run at the pump speed of 80 on the meter and inlet and
outlet pressure of ~0 psi and 25 respectively.
Twelve liters of the filtrate (<100,000 daltons) coming
out of the cartridges at the flow rate of four liters per hour
was frozen or lyophilized for storage.
The same methods may be used to fractionate the milk from
non-hyperimmunized cows.
~~cJ 92/00796 PCT/US91/02923
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Exampl a 3
Desensitization of Alleroic Sub.iects
Human volunteers consumed approximately 1/2 cup of the
immune milk powder reconstituted with 8 oz of water. Two
hundred volunteers ranged in age from 4 to 87 years, and drank
the milk for 1 to 172 months. Of 168 subjects with allergies
who submitted responses indicating the status of their
condition, 138 (82.1fa) indicated that they had improved while
drinking the milk. Ninety-four percent of subjects reporting
benefits stated their improvement occurred within 1 month of
starting to drink the milk. Specific effects reported
included less congestion, fewer or no headaches, easier
breathing, less itching, swelling, sneezing, wheezing,
coughing, rhinorrhea, and an enhanced ability to smell and
taste. Many subjects reported a recurrence of symptoms of
milk consumption was stopped, and several subjects
discontinued allergy immunotherapy after drinking 'the milk.
Having now fully described the invention, it will be
understood by those with skill in the art that the scope may
be performed within a wide and equivalent range of
conditions, parameters and the like, without affecting the
spirit or scope of the invention or any embodiment thereof.
