Canadian Patents Database / Patent 2355168 Summary

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(12) Patent Application: (11) CA 2355168
(54) English Title: EGG ANTI-INFLAMMATORY COMPOSITION AND METHOD OF TREATING AND PREVENTING INFLAMMATION
(54) French Title: COMPOSITION ANTI-INFLAMMATOIRE A BASE D'OEUF ET METHODE DE TRAITEMENT ET DE PREVENTION DE L'INFLAMMATION
(51) International Patent Classification (IPC):
  • A61K 35/54 (2006.01)
  • A61P 29/00 (2006.01)
(72) Inventors :
  • FITZPATRICK-MCELLIGOTT, SANDRA (United States of America)
  • LEE, YOUNG-ZOON (United States of America)
  • BECK, LEE R. (United States of America)
  • ADALSTEINSSON, ORN (United States of America)
  • DALEY, MICHAEL J. (United States of America)
  • XING, RUYE (United States of America)
  • IYER, SUBRAMANIAN (United States of America)
  • HUNCHAR, JEFFREY G. (United States of America)
  • GREENBLATT, HELLEN CHAYA (United States of America)
(73) Owners :
  • ARKION LIFE SCIENCES (United States of America)
(71) Applicants :
  • DCV, INC. (United States of America)
(74) Agent: DIMOCK STRATTON LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 1999-12-28
(87) Open to Public Inspection: 2000-07-27
(30) Availability of licence: N/A
(30) Language of filing: English

(30) Application Priority Data:
Application No. Country/Territory Date
09/233,379 United States of America 1999-01-19

English Abstract




The invention is directed to a composition and method for the treatment and
prevention of inflammation in general. The composition is an egg product
obtained from an avian which has been hyperimmunized with no immunogenic
mixture. The invention is further directed to an anti-inflammatory composition
which has been partially purified from a hyperimmunized egg. The partially
purified anti-inflammatory composition, when administered to a subject animal
is effective in treating and preventing inflammation.


French Abstract

L'invention concerne une composition et une méthode de traitement et de prévention de l'inflammation en général. Ladite composition est un produit de l'oeuf, provenant d'un oiseau ayant été hyperimmunisé à l'aide d'un mélange immunogène. L'invention porte également sur une composition anti-inflammatoire ayant été partiellement purifiée, provenant d'un oeuf immunisé. La composition anti-inflammatoire partiellement purifiée, lorsqu'elle est administrée à un sujet animal, est efficace dans le traitement et la prévention de l'inflammation.


Note: Claims are shown in the official language in which they were submitted.




CLAIMS


WE CLAIM

1. An anti-inflammatory composition in partially purified form, wherein said
anti-inflammatory composition comprises the following characteristics:
a) being present in whole egg, egg white and egg yolk of an egg produced by
an egg-producing animal;
b) having a molecular weight of less than approximately 3000 daltons;
c) being heat stable;
d) being orally active; and
e) being resistant to degradation by digestive enzymes.
2. The anti-inflammatory composition of claim 1 wherein the egg-producing
animal has been hyperimmunized with an immunogenic or genetic vaccine.
3. The anti-inflammatory composition of claim 2 wherein the immunogenic
vaccine comprises at least one antigen selected from the group consisting of
bacterial, viral, protozoan, fungal and cellular immunogens and mixtures
thereof.
4. The anti-inflammatory composition of claim 2 wherein the genetic vaccine
comprises at least one immunogen coding DNA construct selected from the
group consisting of fragments of naked DNA, plasmid DNA, viral DNA,
bacterial DNA, DNA expression libraries, DNA-RNA immunogens, DNA-
protein conjugates and DNA liposome conjugates, and mixtures thereof.



50




5. The anti-inflammatory composition of claim 1 wherein the ante-inflammatory
composition is partially purified by the method comprising:
a) defatting the whole egg, egg yolk or egg white;
b) isolating a water soluble fraction from the de-fatted whole egg, egg yolk
or
egg white;
c) separating a less than approximately 3000 dalton permeate from the water
soluble fraction; and
d) fractionating said less than approximately 3000 dalton permeate to recover
said partially purified anti-inflammatory composition.
6. A method for treating and preventing inflammation in a subject animal
suffering from or susceptable to inflammation, the method comprising
administering to the subject animal an effective amount of an egg product.
7. The method of claim 6 wherein the egg product is selected from the group
consisting of whole egg, egg yolk, egg white and any fraction thereof.
8. The method of claim 7 wherein the egg product is obtained from an egg-
producing animal which has been hyperimmunized with an immunogenic or
genetic vaccine.
9. The method of claim 8 wherein the immunogenic vaccine comprises at least
one antigen selected from the group consisting of bacterial, viral, protozoan,
fungal and cellular immunogens and mixtures thereof.
10. The method of claim 8 wherein the genetic vaccine comprises at least one
immunogen coding DNA construct selected from the group consisting of
fragments of naked DNA, plasmid DNA, viral DNA, bacterial DNA, DNA
expression libraries, DNA-RNA immunogens, DNA-protein conjugates and
DNA liposome conjugates, and mixtures thereof.
11. The method of claim 7 wherein the egg product further comprises an anti-
inflammatory composition.



51




12. The method of claim 11 wherein the anti-inflammatory composition is
partially purified and comprises the following characteristics:
a) being present in whole egg, egg white and egg yolk of an egg produced by
an egg-producing animal;
b) having a molecular weight of less than approximately 3000 daltons;
c) being heat stable;
d) being orally active; and
e) being resistant to degradation by digestive enzymes.
13. The method of claim 12 wherein the anti-inflammatory composition is
partially purified by the method comprising:
e) defatting the whole egg, egg yolk or egg white;
f) isolating a water soluble fraction from the de-fatted whole egg, egg yolk
or
egg white;
g) separating a less than approximately 3000 dalton permeate from the water
soluble fraction; and
h) fractionating said less than approximately 3000 dalton permeate to recover
said partially purified anti-inflammatory composition.
14. The method of claim 6 wherein the inflammation comprises arthritis.
15. The method of claim 7 wherein the effective amount of egg product
administered is between approximately 100 milligrams and 10 grams of whole
egg per kilogram of subject weight, or the equivalent thereof for a fraction
of
the whole egg.
16. The method of claim 12 wherein the effective amount of partially purified
anti-inflammatory composition administered is between approximately 1
microgram and 400 milligrams per kilogram of subject weight.



52



17. A method of treating an autoimmune disease in a subject animal suffering
from or susceptable to said autoimmune disease, the method comprising
administering to the subject animal an effective amount of an egg product.
18. The method of claim 17 wherein the egg product is obtained from an egg-
producing animal which has been hyperimmunized with an immunogenic or
genetic vaccine.
19. The method of claim 18 wherein the egg product further comprises an anti-
inflammatory composition.
20. The method of claim 19 wherein the anti-inflammatory composition is
partially purified and comprises the following characteristics:
a) being present in whole egg, egg white and egg yolk of an egg produced by
an egg-producing animal;
b) having a molecular weight of less than approximately 3000 daltons;
c) being heat stable;
d) being orally active; and
e) being resistant to degradation by digestive enzymes.



53

Note: Descriptions are shown in the official language in which they were submitted.


CA 02355168 2001-06-13
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TITLE
EGG ANTI-INFLAMMATORY COMPOSITION AND METHOD OF
TREATING AND PREVENTING INFLAMMATION
BACKGROUND OF THE INVENTION
RELATED APPLICATIONS
This application is a continuation-in-part of copending U.S. Patent
Application
l0 Serial No. 08/814,187, filed March 10, 1997 and U.S. Patent Application
Serial
No. 09/008,728, filed January 19, 1998.
FIELD OF THE INVENTION
The invention generally relates to a composition and method for treating and
15 preventing inflammation. The invention specifically relates to an egg anti-
inflammatory composition, methods for its production in partially purified
form,
and methods for its use in the treatment of inflammation, (in particular
arthritis),
and autoimmune diseases.
2o Inflammation
Inflammation, as def ned in Dorland's Medical Dictionary, is "a localized
protective response, elicited by injury or destruction of tissues, which
serves to
destroy, dilute or wall off both the injurious agent and the injured tissue."
It is
characterized by fenestration of the microvasculature, leakage of the elements
of
25 blood into the interstitial spaces, and migration of leukocytes into the
inflamed
tissue. On a macroscopic level, this is usually accompanied by the familiar
clinical signs of erythema, edema, hyperalgesia (tenderness), and pain.
During this complex response, chemical mediators such as histamine, 5-
3o hydroxytryptamine, various chemotactic compositions, bradykinin,
leukotrienes,
and prostaglandins are liberated locally. Phagocytic cells migrate into the
area,
and cellular lysosomal membranes may be ruptured, releasing lytic enzymes. All
of these events can contribute to the inflammatory response.


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Inflammation resulting from rheumatoid arthritis likely involves the
combination
of an antigen with an antibody complement causing the local release of
chemotactic and chemoactivating compositions that attract leukocytes. The
leukocytes phagocytose the complexes of antigen-antibody and complement, and
also release the many enzymes contained in their lysosomes. These lysosomal
enzymes then cause injury to cartilage and other tissues, and this furthers
the
degree of inflammation. Cell-mediated immune reactions may also be involved.
Prostaglandins, which are key intracellular regulators of cellular function,
are also
1o released during this process.
The inflammatory response is any response characterized by inflammation as
defined above. It is well known, to those skilled in the medical arts, that
the
inflammatory response causes much of the physical discomfort (i.e., pain and
loss
of function) that has come to be associated with different diseases and
injuries.
Arthritis
Arthritis manifests itself in a variety of forms. Some of the more common
forms
include rheumatoid arthritis, osteoarthritis and generalized rheumatism.
Rheumatoid arthritis is an autoimmune disease characterized by pain, swelling
and
stiffness in the joints. Rheumatoid arthritis is a disease which afflicts
approximately 3% of Americans, and particularly women. Rheumatoid arthritis is
an extremely disabling disease and usually strikes adults between the ages of
30
and 40 years, while the occurrence of clinical illness is greatest among those
aged
40 - 60 years. Although drug therapy is somewhat effective, as many as 7% of
rheumatoid arthritis sufferers are disabled to some extent as quickly as 5
years
after disease onset, and within 10 years, as many as 50% are too disabled to
work
(Medical Sciences Bulletin, December 1994).
Osteoarthritis produces similar symptoms to rheumatoid arthritis. In
particular,
although osteoarthritis begins as a degeneration of articular cartilage
whereas
rheumatoid arthritis begins as inflammation in the synovium, each process
2


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WO 00/43020 PCTNS99/30969
approaches the other as the disease progresses. In osteoartliiitis;-as
cartilage
deteriorates and joint congruence is altered, a reactive synovitis often
develops.
Conversely, as rheumatoid arthritis erodes cartilage, secondary osteoarthritis
changes in bone and cartilage develop. At the end stages of both
osteoarthritis and
rheumatoid arthritis, the involved joints appear the same.
Some other forms of arthritis include Ankylosing Seronegative
Spondyloarthropathy (ankylosing spondylitis) and reactive arthritis. These
conditions are often referred to as the "B-27 associated diseases," and are
difficult
l0 to differentiate from rheumatoid arthritis. In some cases anlcylosing
spondylitis,
Reiters syndrome or psoriatic arthritis are present coincidingly with
Rheumatoid
Arthritis in the same patient. In many cases, these patients. are treated with
the
same disease modifying drugs as those suffering from progressive rheumatoid
arthritis.
Onset of arthritis generally occurs after the age of 30 in those who are
susceptable
to such disease. However, some forms of arthritis may be initiated by
different
causes, such as slow virus infections. Because there is great overlap, many
physicians consider these forms as "generalized rheumatism" and approach
2o management of the diseases in the same way. Some diseases which fall into
this
category include Chronic Fatigue Syndrome, fibromyalgia (fibrositis) and gout.
In
fact, for some patients, evidence is accumulating for superimposition of
rheumatoid arthritis and fibromyalgia. (Hams, Edward D. and W: B. Saunders,
Rheumatoid Arthritis, 1997).
Autoimmune Diseases
As stated above, rheumatoid arthritis is an autoimmune disease, and as such,
its
etiology is much the same as the etiology of any other autoimmune disease.
Generally, the body normally recognizes the difference between its own by-
3o products and foreign invaders (i.e. bacteria, viruses, fungi and
protozoans, to name
a few). When an immune cell (T or B lymphocyte) reacts to a "self protein"
during its development, that cell is deemed defective and usually destroyed or
inactivated. Sometimes, however, a "self reactive" immune cell will escape
3


CA 02355168 2001-06-13
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destruction. At a certain later time, that cell can be activated and trigger
an
immune response. Activation is thought to occur after infection with a common
bacteria or virus which contains a polypeptide having a stretch of amino acids
which match a stretch on the defective self protein. Several bacteria, such as
.
Streptococcus, Mycoplasma, and borrelia, have been implicated in the
initiation
of the disease, as well as certain viruses, namely retroviruses. In addition
to
Rheumatoid Arthritis, autoimmunity often results in such diseases as juvenile
diabetes, multiple sclerosis, Graves' disease, Meneri's disease, myasthenia
gravis,
lupus erythematosus and psoriasis. (Medical Sciences Bulletin, September,
1994).
Autoimmunity effects specific organs. For example, some autoimmune diseases
of liver bile ducts, and kidneys are: primary biliary cirrhosis, necrotizing
glomerulonephritis, "idiopathic" crescentic glomerulonephritis, virus-induced
liver and kidney disease, chronic hepatitis, autoimmune and drug-induced
15 hepatatis (Gershwin, Manns, and Mackay 1992). Immune destruction of the
islets
of Langerhans results in diabetes meillitus (Hagopian and Lernmark 1992) and
insulin autoantibodies have been described (Palmer 1987).
There are a large category of systemic vasculitides diseases in which
autoimmune
mechanisms have been suggested as the cause of the pathogenesis. Some of the
diseases are: leukocytoclastic angiitis, polyarteritis nodosa, Goodpasture's
syndrome, Kawasaki disease, Wegener's granulomatosis, Churg-Struass
syndrome, giant-cell arteritis, Takayasu arteritis, immune-complex-mediated,
lupus, rhuematoid, and cryoglobulinemic vasculitis, Henoch Schonlein purpura
(Kallenberg, 1996; Jennette, Jones, Falk, 1992).
There is also a body of evidence that autoimmunity may play a role in many
forms
of heart disease includings: postpericardiotomy and postmyocardial infarction
syndromes, myocarditis, and idiopahtic dilated cardiomyopathy. Autoimmunity
30 may be responsible for the progression of acute disease of heart muscle to
degenerative (Rose, Neumann, Burek, Herskowitz 1992).
4


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Symptomatic involvement of skeletal muscle is common iri many autoirrimune
diseases such as polymyositis or inflammatory myopathy (which may include
rheumatoid arthritis, polymyaigia rheumatica, myasthenia gravis, myasthenic
myopathy, neurogenic atrophy, motor neuron disease, fibromyalgia, fibrositis,
muscular dystrophy, endocrine, metabolic, and carcinomatous myopathy).
(Hollingsworth, Dawkins, Thomas 1992).
Other diseases with autoimmune origins may be uveitis, Vogt-Koyanagi-Harada
syndrome, (Detrick and Hooks 1992), and Sjbgren's syndrome, scleroderma,
to ankylosing spondylititis, dermatomyositis, psoriasis, psoriatic arthritis,
Reiter's
syndrome (N)H 1994).
Also evidence of autoantibodies has been found in Alzheimer's disease (Singh
et
al., 1992), dementia complex (Mastroianni et al., 1991) and autistic children
(Singh et al., 1993).
Several neurologic diseases such as Sydenham's Chorea, chronic obsessive-
compulsive disorders (OCD), attention deficit hyperactivity disorder (ADHD),
Tourette's Syndrome (TS) and some cases of schizophrenia may have an auto-
immune component and may be associated with anti-neuronal antibodies (Medical
Sciences Bulletin, Sept. 1994).
This summary is not all inclusive and those in the art are familiar with other
autoimmune diseases, such as, for example Guillain-Barre syndrome (idiopathic
polyneuritis).
Treatment
In order to treat inflammatory related disorders, it is a common medical
practice to
administer pharmacological agents that reduce the physical discomfort of the
inflammatory response. Agents having these properties are classified as anti-
inflammatory. Anti-inflammatory drugs are used for the treatment of a wide
spectrum of disorders, and the same drugs are often used to treat different
5


CA 02355168 2001-06-13
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diseases. Treatment with anti-inflammatory drugs is not for the disease,
but~most
often for the symptom (i.e., inflammation).
The anti-inflammatory, analgesic, and anti-pyretic drugs are a heterogeneous
group of compounds, often chemically unrelated, which nevertheless share
certain
therapeutic actions and side-effects. Corticosteroids represent the most
widely-
used class of compounds for the treatment of inflammation. Proteolytic enzymes
represent another class of compounds that are thought to have anti-
inflammatory
effects. Hormones that directly or indirectly cause the adrenal cortex to
produce
l0 and secrete steroids represent another class of anti-inflammatory
compounds.
Unfortunately, the natural and synthetic corticosteroid preparations cause a
number of severe side effects, including elevation of blood pressure, salt and
water retention, kidney damage and increased potassium and calcium excretion.
Moreover; corticosteroids may mask the signs of infection and enhance
15 dissemination of infectious microorganisms. These hormones are considered
unsafe for use in pregnant women, and long-term corticosteroid treatment has
been associated with gastric hyperactivity and/or peptic ulcers. Treatment
with
corticosteroids may also aggravate diabetes mellitus, requiring higher doses
of
insulin, and may produce psychotic disorders. Hormonal anti-inflammatory
20 agents which indirectly increase the production of endogenous
corticosteroids
have the same potential for adverse side-effects.
Another common treatment for inflammation, and in particular rheumatoid
arthritis, other arthritis and other autoimmune diseases, is drug therapy. In
25 general, patients are initially treated with "first-line" agents, usually
non-steroidal
anti-inflammatory drugs (NSAIDs) which primarily relieve the symptoms. The
patients are later treated with "second-line" or disease-modifying agents
(DMARDs) such as methotrexate, gold compounds, penicillamine, sulfasalazine,
and antimalarial drugs. However, all of the above drugs have serious side
effects,
3o especially when administered in elevated doses. For example aspirin, an
NSA)D,
may produce indigestion and stomach pain; phenyibutazone may produce stomach
ulcers and phenacetin may lead to kidney disease. Viethotrexate may cause oral
ulceration and gastrointestinal (G17 side effects.
6


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If a natural food product having anti-inflammatory effects could be obtained,
it
would provide an easily administratable, readily available, and safe
therapeutic
composition for the treatment of arthritis, autoimmune diseases and
inflammation
in general.
Various genera of the class Aves, such as chickens (callus domesticus),
turkeys,
and ducks, produce antibodies in blood and eggs against immunogens that cause
avian diseases, as well as against other immunogens. For example, LeBacq-
to Verheyden et al. (Immunology 27:683 (i974)) and Leslie, G.A., et al. (J.
Med.
130:1337 (1969)), have quantitatively analyzed immunoglobulins of the chicken.
Poison, A., et al. (Immunological Communications 9:495-514 (1980)) immunized
hens against several proteins and natural mixtures of proteins, and detected
IgY
antibodies in the yolks of the eggs. Fertel, R., et al. (Biochemical and
Biophysical
15 Research Communications 102:1028-1033 (1981)) immunized hens against
prostaglandins and detected antibodies in the egg yolk. Jensenius et al.
(Journal of
Immunological Methods 46:63-68 (1981)) provide a method of isolating egg yolk
IgG for use in immunodiagnostics. Poison et al. (Immunological Communications
9:475-493 (1980)) describe antibodies isolated from the yolk of hens that were
20 immunized with'a variety of plant viruses.
U.S. Patent No. 4,357,272 discloses the isolation of antibodies from the yolks
of
eggs derived from hyperimmunized hens. The hyperimmunization was elicited by
repetitive injections of immunogens derived from plant viruses, human IgG,
25 tetanus antitoxin, snake antivenins, and Serameba. U.S. Patent No.
4,550,019
discloses the isolation from egg yolks of antibodies raised in the hen by
hyperimmunization with immunogens having a molecular or particle weight of at
least 30,000. The immunogens used to hyperimmunize the chickens were selected
from among plant viruses, human immunoglobulins, tetanus toxin, and snake
30 venoms.
U.S. Patent No. 4,748,018 discloses a method of passive immunization of a
mammal that comprises parenterally administering purified antibody obtained


CA 02355168 2001-06-13
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from the eggs of an avian that has been immunized against the corresponding
antigen, and wherein the mammal has acquired immunity to the eggs.
U.S. Patent No. 5,772,999, assigned to DCV-Biologics, discloses a method of
preventing, countering or reducing chronic gastrointestinal disorders or Non-
Steroidal Anti-Inflammatory Drug-induced (NSAID-induced) gastrointestinal
damage in a subject by administering hyperimmunized egg and/or milk or
fractions thereof to the subject.
1o None of these references, however, discloses or suggests that eggs, when
administered to animals, have the capability to prevent or reduce
inflammation,
treat or prevent arthritis or treat or prevent autoimmune diseases. Nor do
these
references disclose or suggest a method providing a reasonable expectation
that
hyperimmunization of an avian could produce an avian which lays eggs having
15 such a capability. Finally, these references do not disclose or suggest the
presence
of an anti-inflammatory composition in an egg from an avian.
SUMMARY OF THE INVENTION
The invention is based on the inventors' discovery that there is anti-
inflammatory
2o activity in egg and egg products, and particularly in egg products obtained
from
hyperimmunized avians, which when administered to a subject animal, in
particular, mammals, prevents or reduces inflammation in that subject animal.
In particular, the invention is directed to a partially purified anti-
inflammatory
25 composition obtained from the eggs of an avian. The anti-inflammatory
composition was partially purified from fractions isolated from both egg yolk
and
egg white.
The invention is also directed to a hyperimmunization process which produces
30 supranotmal levels of the anti-inflammatory composition in an avian egg. It
is the
inventors' belief that the level of the anti-inflammatory composition can be
increased in both egg yolk and egg white by the process of hyperimmunization.


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The invention also encompasses a method of treating inflammation in a subject,
and especially mammals, which comprises administering to the subject the
partially pure anti-inflammatory composition or a composition comprising the
partially pure anti-inflammatory composition. This aspect encompasses
administering whole egg itself and/or fractions thereof.
The invention is further based on the inventors' discovery that there is
activity in
egg and egg products, and particularly in egg products obtained from
hyperimrnunized avians, which when administered to a subject animal, in
to particular, mammals, treats or prevents arthritis and/or autoimmune
diseases in the
subject animal.
The invention is finally directed to a method wherein the egg product is
administered to the subject animal in combination with a drug selected from
the
15 group consisting of non-steroidal, anti-inflammatory drugs and disease-
modifying,
anti-arthritic drugs.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 is a flow chart of the purification of the less than 3,000 molecular
weight
2o anti-inflammatory composition from powdered egg yolk.
Figure 2 is a flow chart of the purification of the less than 3,000 molecular
weight
anti-inflammatory composition from powdered egg white.
Figure 3 is our absorbance depiction of the separation of the partially
purified anti-
25 inflammatory composition by Sepharose Column.
Figure 4 is a chromatogram of the HPLC separation of the partially pruified
anti-
inflammatory composition from hyperimmune egg yolk.
3o Figure 5 is a graph showing the effect of hyperimmune egg on the incidence
of
arthritis using the Rat Type II Collagen Model.


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Figure 6 is a graph showing the effect of the hyperimmune egg; its fractions
and
the partially purified anti-inflammatory composition on the severity of
arthritis in
the Rat Type II Colagen Model.
DETAILED DESCRIPTION OF THE INVENTION
The invention generally relates to a composition and method for treatment and
prevention of inflammation in general, and particularly arthritis and
autoimmune
diseases. The composition is preferably a natural food product which comprises
hyperimmune egg or egg product. The food product, when administered by the
l0 method of the invention, not only provides relief from the pain and other
symptoms caused by arthritis and autoimmune diseases, but can delay, and even
prevent, the onset of such diseases. The preferred antigen mixture injected
into
the avians to produce the hyperimmune egg product does not contain specific
antigens which are known to cause inflammation or autoimmune diseases.
~s Therefore, it is surprising that administration of hyperimmune egg or egg
product
obtained from avians immunized against a mixed antigen vaccine is effective in
reducing the symptoms of and preventing inflammation and autoimmune diseases
when administered to a subject.
2o DEFINITIONS
The following definitions apply throughout:
The term "inflammation" is used in its art-recognized sense as a localized
protective response elicited by injury or destruction of tissues which serves
to
25 destroy, dilute or wall off both the injurious agent and the injured
tissue,
characterized in the inappropriate, uncontrolled form by the classical
sequence of
pain, heat, redness, swelling, and loss of function, and histologically
involving a
complex series of events, including dilation of the arterioles, capillaries,
and
venules with increased permeability and blood flow, exudation of fluids
including
30 plasma proteins, and leukocyte migration into the inflammatory focus.
The term "arthritis" means any of a variety of disorders marked by
inflammation
and degeneration of connective tissue structures, especially the joints and
related


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structures.. It may be attended by pain, stiffness, or limitation of'motion of
these
parts. Some forms of arthritis include rheumatoid arthritis, osteoarthritis,
ankylosing seronegative spondyloarthropathy, reactive arthritis, chronic
fatigue
syndrome, fibromyalgia (fibrositis) and gout.
5
The term "autoimmune disease" is applied the standard medical definition as
found in standard medical dictionaries such as Dorland's and Taber's. A
description of a variety of autoimmune diseases can be found in the Background
section of this document.
The term "hyperimmunization" means exposure to one or more antigens such that
an immune response is elevated and maintained above the natural unexposed
state.
The terms "egg" or "egg product" each mean any whole egg (table,
15 hyperimmunized or otherwise) or any product or fraction derived therefrom.
The terms "table egg" or "table egg product" each mean a whole egg, or any
product or fraction derived therefrom, obtained from egg-producing animals
which are not maintained in a hyperimmune state.
The terms "hyperimmune egg" or hyperimmune egg product" each mean whole
egg or any product or fraction derived therefrom, obtained from an egg
producing
animal maintained in a hyperimmune state.
25 The term "supranormal levels" means levels in excess of those found in eggs
of
egg-producing animals not maintained in a hyperimmune state.
The term "anti-inflammatory egg or egg fraction" means egg or egg fractions
containing the anti-inflammatory composition disclosed herein.
The term "anti-inflammatory composition" means the composition disclosed
herein which counteracts or suppresses the inflammatory process.
11


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The term "partially pure egg anti-inflammatory composition" means an anti-
inflammatory composition at least of the purity described in Example 4 and the
exemplary materials and figures.
The term "combinatorial derived immunogens" refers to a process of generating
molecular diversity among immunogens by way of combinatorial synthesis.
The term "bioengineered immunogens" refers to immunogens which are obtained
through the process of gene cloning technologies and genetic manipulation
which
allow the insertion and translation of proteins which have antigenic
properties.
The term "genetic vaccine" refers to a nucleic acid vaccine which is generally
produced by recombinant technologies and which may elicit an immune response.
15 The term "treatment" means that the onset of the symptoms (including pain)
of the
disorder and/or pathogenic origin of the disorder be delayed or completely
prevented, or, if present, the symptoms be ameliorated or completely
eliminated.
For example, the hyperimmune egg product treats arthritis and/or an autoimmune
disease not only by suppressing the symptoms of the disorder in humans and
other
2o mammals, but also by acting as a prophylactic agent to counteract the
presence of
the disorder in the recipient.
The term "prevention" means that the progression of the disease is reduced
and/or
eliminated, or that the onset of the disease is eliminated.
The term "administer" means any method of providing a subject with a
substance,
including orally, intranasally, parenterally (intravenously, intramuscularly,
or
subcutaneously), rectally, topically or intraocularly.
3o The term "animal" means the animal kingdom definition.
The term "target animal" refers to an animal which functions as the egg or egg
product producing animal.
12


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The term "subject animal" refers to the animal which is administered the egg
or
egg product produced by the target animal.
The term "immunogen" means a substance that is able to induce a humoral
antibody and/or cell-mediated immune response rather than immunological
tolerance. The term signifies the ability to stimulate an immune response as
well
as react with the products of it, e.g., antibody.
to THE INVENTION
The product and method of the invention relate particularly to the use of
hyperimmune egg, which is a natural food product, in the treatment and
prevention of inflammatory and autoimmune diseases. Being natural, this food
product can be used to treat and prevent such diseases without the fear of
side
effects, except, of course, for allergic reactions in those intolerant to
eggs.
In one embodiment, the invention comprises a hyperimmune egg or egg product
which is effective in treating and preventing arthritis and/or an autoimmune
disease in a subject animal. The hyperimmune egg is obtained from an egg-
producing animal, and more preferably, an avian, which has been hyperimmunized
with at least one immunogen. The hyperimmune egg product is one which is
preferably administered orally to the subject animal. The hyperimmune egg or
egg
product can be further separated into more potent fi-actions which can
subsequently be administered to a subject animal in a variety of forms.
In an alternate embodiment, the invention comprises an anti-inflammatory
composition obtained from avian eggs, its partial purification, and the
administration of the partially purified composition to a subject for the
treatment
of inflammation. The invention further comprises the same anti-inflammatory
3o composition partially purified from the egg of an avian that has been
hyperimmunized with one or more immunogens, and, in particular, bacterial
antigens, or their synthetic equivalent. The anti-inflammatory composition is
13


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present in hyperimmune eggs at supranorrnal levels which piovide anti-
inflammatory activity in subject animals.
The details of the invention are given below.
PARTIAL PURIFICATION PROCESS
The egg anti-inflammatory composition can be partially purified from
whole egg, egg yolk or egg white. An example of a preferred partial
purification
process is as follows:
l0 1. Preparation of a water-soluble fraction from an egg;
2. Ultrafiltration of the water-soluble fractions;
3. Separation of fractions by ion or anion exchange chromatography;
and
4. Bioassay of the separated fractions for anti-inflammatory activity.
The following is a more detailed description of this process:
Step 1:
The anti-inflammatory composition can be partially purified from whole egg,
egg
yolk or egg white. Iwa preferred embodiment, the composition is purified from
2o egg yolk. The lipid portion is removed from the whole egg or egg yolk by
methods well-known to those having skill in the art. For example, in the case
of
spray-dried egg yolk powder, defatting can be accomplished with solvents
(propane, butane or hexane or with binary solvents), supercritical COZ,
enzymes
and the like, and in the case of liquid egg yolk, defatting can be
accomplished by
the caprylic acid separation method (CAPS) disclosed by Lee (U.S. Pat. No.
5,367,054). No fat removal is necessary for egg white, and thus the liquid or
powdered form of the egg white can be either heated or dissolved directly by
conventional methods and as described in the examples listed below. The whole
egg, egg yolk or egg white is then preferably processed into either liquid or
powder form, and is further processed to obtain water soluble fractions. (See
Examples)
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Step 2":
The resulting water soluble fractions, from whole egg, egg yolk or egg white,
are
subjected to ultrafiltration using ultrafiltration systems equipped with a
3,000
molecular weight cut-off membrane. The ultrafiltration process separates
molecules having a molecular weight of more than approximately 3,000 daltons
from those having a molecular weight of less than approximately 3000 daltons.
Once filtered, the resulting ultra-filtrates contain molecules of less than
approximately 3,000 dalton molecular weight are then lyophilized, weighed, and
l0 prepared for bioassay testing and further separation.
Step 3:
Fractions from the less than 3,000 dalton ultra-filtrate can be separated by,
for
example, Reverse Phase High Performance Liquid Chromatography to further
15 purify the partially pure anti-inflammatory composition. As an alternate or
additional strategy, the egg anti-inflammatory composition in the ultra-
filtrate can
be further characterized by anion exchange DEAF-Sepharose chromatography. In
a specific disclosed embodiment, the preferred ultrafiltration is by Amicon
RA1000 (3K MWCO) and DEAF ion-exchange chromatography . It is
20 understood, however, that equivalent techniques and materials could be used
to
isolate the composition, given the information herein, and the knowledge
available
to the person of ordinary skill in the art.
Sten 4:
25 The anti-inflammatory activity of the composition can be tested by a
standard
bioassay which determines anti-inflammatory activity. Some examples include
inhibition of leukocyte migration, rat paw edema test, adjuvant-induced
arthritis,
collagen induced arthritis, and intra-vital microscopy among others.
Comparisons
of egg anti-inflammatory composition with known anti-inflammatory drugs such
30 as aspirin and indomethacin can also be done. And finally, clinical tests
for
rheumatoid arthritis, degenerative joint disease and injury induced arthritis
can
also be used to determine anti-inflammatory activity.


CA 02355168 2001-06-13
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The anti-inflammatory action of the partially pure egg anti-inflammatory
composition is measured by bio-assays. A preferred bio-assay is the rat type
II
collagen assay as set forth in Example 6.
Alternative bio-assays include the pleural leukocyte migration inhibition
assay as
described in Vinegar et al., "Some Quantitative Characteristics of Carrageenan
Induced Pleurisy in the Rat," Proc. Soc. Exp. Bio. Med. 143:711-714 (1973);
Ammendola, G. et al., "Leukocyte Migration and Lysozomal Enzymes Release in
Rat Carrageenan Pleurisy," Agents and Actions 5:250-255 (1975); Vinegar, R. et
to al., "Quantitative Studies of the Pathway to Acute Carrageenan
Inflammation,"
Fed. Proc. 35:2447-2456 {1976)
The leukocyte migration inhibition assay is another useful assay which is
generally performed as follows: Samples containing the anti-inflammatory
composition are administered to the artificially inflamed adult female rats
with 1%
carrageenan solution and then the anti-inflammatory effect of the samples at
each
dosage is determined (using Automated Image Analysis Technique) by the
reduction in the number of leukocytes in the pleural exudates of the treated
rats as
compared to those of control rats.
Alternatively, the anti-inflammatory action of the substantially pure anti-
inflammatory composition can be tested on edema caused by injecting
carrageenan into rat footpads (Winter, C.A., Risley, G.A., Nuss, A. W.,
"Carrageenan-Induced Edema in the Hind Paw of the Rat as an Assay for Anti-
Inflammatory Drugs," Proc. Soc. Exper. Biol. Med. 3:544 (1967)).
A variety of other bio-assays may also be used. (See Wetnick, A.S., and Sabin,
C.,
"The Effects of Clonixin and Bethaurethasone on Adjuvant-Induced Arthritis and
Experimental Allergic Encephalomyelitis in Rats," Jap. J. Pharm. 22:741 (
1972)).
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CHARACTERISTICS OF THE PARTIALLY PURIFIED ANTI-
INFLAMMATORY COMPOSITION
The partially pure anti-inflammatory composition has the following
characteristics:
1) has anti-inflammatory activity in a subject animal ;
2) is present in both the egg white and egg yolk of avian eggs;
to
3) when isolated from egg yolk, has greater anti-inflammatory activity than
when
isolated from egg white;
4) has a molecular weight of less than approximately 3000 daltons;
5) is non-proteinaceous.and non-steroidal;
6) is heat stable;
7) is orally active and is not degraded by digestive enzymes;
The 3,000 dalton molecular weight is deduced from the partial isolation and
purification of the composition wherein the isolation and purification process
uses
an ultra-filtration membrane that does not allow the passage of molecular
species
greater than 3,000 Dalton therethrough. The partially purified anti-
inflammatory
composition is determined to be non-proteinaceous and non-steroidal because it
is
small in size and is not degraded by enzymes which degrade proteins. Moreover,
the composition is orally active and is not degraded by digestive enzymes. The
small stable form of the partially purified anti-inflammatory composition (as
differentiated from proteins which are much larger) facilitates its absorption
from
the digestive tract. Finally, the partially purified anti-inflammatory
composition is
heat-stable.
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The partially purified egg anti-inflammatory composition can be isolated from
whole egg, egg yolk and egg white. The anti-inflammatory composition partially
purified from egg yolk shows higher anti-inflammatory activity than anti-
inflammatory composition partially purified from egg white.
It is the inventors' finding that supranormal levels of the partially purified
anti-
inflammatory composition can be isolated from whole egg, egg yolk and egg-
white from an egg-producing animal which has been hyperimmunized.
HYPERIM1~~IUNIZATION OF THE EGG-PRODUCING ANIMAL
As just described, it is the inventor's fording that the anti-inflammatory
composition is present in hyperimmune eggs at supranormal levels which provide
anti-inflammatory activity in subject animals. The hyperimmune egg or egg
product can be produced by any egg-producing animal. it is preferred that the
animal be a member of the class Aves or, in other words, an avian. Within the
class Aves, domesticated fowl are preferred, but other members of this class,
such
as turkeys, ducks, and geese, are a suitable source of hyperimmune egg
product.
When such egg-producing animals are brought to a specific state of
immunization
by means of, for example, periodic booster administrations of immunogens, the
animals will produce eggs that, when consumed by a subject, will have
beneficial
properties, including supranormal levels of the anti-inflammatory composition,
which are effective in the treatment and prevention of inflammatory related
diseases as well as autoimmune diseases in that subject.
The induction of immune sensitivity alone is insufficient to cause the
appearance
of supranormal levels the egg anti-inflammatory composition in eggs, as is
shown
by the fact that table eggs do not contain these supranormal levels, even
though
the avians have been sensitized against various immunogens during normal
3o immunization against avian diseases and during normal exposure to
environmental factors. It is the inventors' finding that it is only in the
specific
hyperimmune states that the eggs have the desired supranormal levels of the
anti-
inflammatory composition.
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This special state of hyperimmunization, in which the egg will contain higher
levels of the anti-inflammatory composition, is preferably achieved by
administering an initial immunization, followed by periodic boosters with
sufficiently high doses of specific immunogens or mixtures of immunogens. The
preferred dosage of booster should be equal to or greater than 50% of the
dosage
necessary to produce primary immunization of the avian. Thus, there is a
threshold booster dosage below which the properties are not produced in the
avian's egg, even though the avian is in what normally would be called an
10 immune state. Having knowledge of the requirement for developing and
maintaining a hyperimmune state, it is within the skill of the art to vary the
amount of immunogen administered, depending on the egg-producing animal
genera and strain employed, in order to maintain the animal in the hyperimmune
state.
15
The hyperimmune state is preferably produced by any immunogen or combination
of immunogens. Hyperimmunization is preferably achieved by multiple
exposures to multiple immunogens, multiple exposure to single immunogens, or
single exposures to libraries of immunogens. Nearly any immunogen can be used
2o to induce the hyperimmune state, including, but not limited to, bacterial,
viral,
protozoan, allergan, fungal or cellular substances.
Having knowledge of the requirement for developing and maintaining a
hyperimmune state, it is within the skill of the art to vary the amount of
25 immunogen administered, depending on the egg-producing animal genera and
strain employed, in order to maintain the animal in the hyperimmune state.
In addition to immunizations with naturally occurring immunogens, immunization
may also be accomplished using immunogens which are synthetically derived by
3o combinatorial chemistries. The basic strategy is to assemble multiple
combinations of chemical building blocks for producing a population of
molecules
with diversity. Several methods have recently been developed for solid and
solution phase combinatorial synthesis of libraries of oligomers (Fodor, S. et
al.,
19


CA 02355168 2001-06-13
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Science 251:767 (1991); Houghton, R. et al., Nature 354:82 (1991)) as well as
small organic molecules (Bunin, B. & Ellman, J., J. Am. Chem. Soc. 114:10997
(1992)). Rapid multiple peptide and oligomer synthesis can serve as a source
for
combinatorial derived immunogens. Furthermore, an alternative strategy would
allow the addition of organic building blocks in combinatorial fashion to a
backbone molecule for improved immunogenicity.
Alternative modes of hyperimmunizing egg producing animals can be used in
place of immunogenic vaccines and include the use of genetic vaccines. In
particular, any DNA construct (generally consisting of a promoter region and
an
1o antigen encoding sequence) will trigger an immune response. Genetic
vaccines
consist of antigen-coding vectors, fragments of naked DNA, plasmid DNA, DNA-
RNA antigens, DNA-protein conjugates, DNA-liposome conjugates, DNA
expression libraries, and viral and bacterial DNA delivered to produce an
immune
response. Methods of DNA delivery include particle bombardment, direct
15 injection, viral vectors, liposomes and jet injection, among others. When
applying
these delivery methods, much smaller quantities may be necessary and generally
result in more persistent immunogen production. When using such genetic
processes, the preferred method for introducing DNA into avians is through
intramuscular injection of the DNA into the breast muscle.
Methods of DNA delivery include, but are not limited to, particle bombardment,
direct injection, liposomes, jet injection (Fynan, E.F. et al., Proc. Natl.
Acad. Sci.
USA 90:11478-11482 (1993)). The nucleic acids that code for known or
unknown immunogens, promoter regions (notably CMV cauliflower mosaic virus)
and SV40 bacterial origin can be replicated in bacteria to produce plasmid DNA
for use in DNA injections. Although several routes of parenteral
administration of
the DNA are effective in chickens, the preferred method is intramuscular
injection
to the breast muscle. Vaccine trials are carried out in egg laying avians,
preferably
chickens. Repeated immunizations are given at one to two week intervals for up
3o to six months.
It is preferred that the amounts of DNA used are generally in the order of ~0-
300
p.g of DNA in saline for direct injection. For particle bombardment, 4-100 pg
of


CA 02355168 2001-06-13
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DNA co-precipitated onto gold beads by the addition of 2.5 M CaClz are
preferred. Repeated immunizations can be given intradermally by this method of
accelerating DNA coated particles into the live animal.
The following is a detailed description of a preferred procedure used to bring
an
egg-producing animal to a heightened state of immunity from which the
resultant
hyperimmunized egg or egg product can be administered to a subject:
1. Selecting one or more immunogens.
2. Eliciting an immune response in the egg-producing animal by
io primary immunization.
3. Administering booster vaccines of immunogens of appropriate
dosage to induce and maintain the hyperimmune state.
4. Testing the hyperimmune eggs for anti-inflammatory activity
levels.
5. Collecting and processing the eggs.
Below is a more detailed description of this procedure.
St_ ep 1: Any immunogen or combination of immunogens may be employed
as a vaccine. The immunogens can be bacterial, viral, protozoan, fungal,
cellular,
or any other substances to which the immune system of an egg-producing animal
will respond. The critical point in this step is that the immunogen(s) must be
capable of inducing immune and hyperimmune states in the egg-producing
animal. Although only a single immunogen may function as the vaccine for the
25 method of the invention, one preferred vaccine is a mixture of polyvalent
bacterial
and viral antigens selected from the following antigen families: the enteric
bacilli
and bacteroides, pneumococci, pseudomonas, salmonella, streptococci, bacilli,
staphylococci, neisseria, clostridia, mycobacteria, actinomycetes chlamydiae,
and
mycoplasma. Viral antigens are preferably selected from the following antigen
3o families: adenoviruses, picornaviruses and herpes viruses, although other
viral
antigen families will work.
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In a preferred embodiment, a polyvalent vaccine referred to as Series 100 (S-
100)
is used. The bacteria included in the S-100 vaccine are listed in Table 1 of
Example 1. This vaccine has been previously described in US patent Nos.
5,106,618 and 5,215,746, both assigned to Stolle Research and Development
Corporation.
Sten Z:The vaccine can be either a killed or live-attenuated vaccine and can
be
administered by any method that elicits an immune response. It is preferred
that
immunization be accomplished by administering the immunogens through
intramuscular injection. The preferred muscle for injection in an avian is the
breast muscle. Dosage is preferably 0.05-5 milligrams of the immunogenic
vaccine. Other methods of administration that can be used include intravenous
injection, intraperitoneal injection, intradermal, rectal suppository,
aerosal, oral,
topical or ocular administration. When DNA techniques are used for the
i5 hyperimmunization process, much smaller quantities are required, generally
300
micrograms.
It can be determined whether the vaccine has elicited an immune response in
the
egg-producing animal through a number of methods known to those having skill
in the art of immunology. Examples of these include enzyme-linked
immunosorbent assays (ELISA), tests for the presence of antibodies to the
stimulating antigens, and tests designed to evaluate the ability of immune
cells
from the host to respond to the antigen. The minimum dosage of antigen
necessary to induce an immune response depends on the vaccination procedure
used, including the type of adjuvants and formulation of antigens) used as
well as
the type of egg-producing animal used as the host.
Step 3:The hyperimmune state is preferably induced and maintained in the
target
animal by repeated booster administrations of an appropriate dosage at fixed
time
intervals. The time intervals are preferably 2-8 week intervals over a period
of 6-
12 months. However, it is essential that the booster administrations do not
lead to
immune tolerance. Such processes are well known in the art.
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It is possible to use other hyperimmunization maintenance' procedures or
combination of procedures, such as, for example, intramuscular injection for
primary immunization and intravenous injection for booster injections. Further
procedures include simultaneously administering microencapsulated and liquid
5 antigen, or intramuscular injection for primary immunization, and booster
dosages
by oral administration or parenteral administration by microencapsulation
means.
Several combinations of primary and hyperimmunization are known to those
skilled in the art.
1o St_ ep 4. It is necessary to test the eggs for anti-inflammatory activity
levels.
This can be accomplished by any clinical and pre-clinical evaluation that
tests the
effects of either the hyperimmune egg, or products derived therefrom, on
inflammation. Chemical-induced inflammation of the rat is a preferred standard
assay for anti-inflammatory drugs. (See Example 6)
Stea 5. This step involves the collection and processing of the eggs)
containing the anti-inflammatory composition. The egg can be collected by
conventional methods. Processing the egg can be accomplished in a variety of
ways described later in this document. The egg can also be further processed
to
purify the anti-inflammatory composition as described below.
PROCESSING AND ADMINISTRATION
It is preferred that the eggs or fractions thereof, including the partially
purified
anti-inflammatory composition, collected from hyperimmunized animals are
processed to produce a hyperimmune egg product, which can subsequently be
administered to a subject animal.
The egg itself or fractions thereof, including the partially purified anti-
inflammatory composition of the present invention, are administered to
a.subject
30 animal by any means that treats or prevents inflammation, including
arthritis,
and/or autoimmune disease in the subject animal. It is preferred that
administration occur by directly feeding the egg or any derivative of the egg
to the
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CA 02355168 2001-06-13
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subject animal. It is important to note that whole egg, egg yolk, and egg
white are
natural food ingredients which are non-toxic and safe.
In an alternative embodiment, the egg or any fraction thereof, including the
5 partially purified anti-inflammatory composition, is integrated into a
nutritional
supplement. One preferred method for preparing the egg or any fraction thereof
to
be incorporated into a nutritional supplement involves drying the egg into an
egg
powder. Although various methods are known for drying eggs, spray drying is a
preferred method. The process of spray drying eggs is well known in the art.
to
Such a dried egg powder can be incorporated into drinks in the form of, for
example, protein powders, power building drinks, protein supplements and any
other nutritional, athlete-associated products. In addition, the egg powder
can be
used in bake mixes, power bars, candies, cookies, etc. Other examples of egg
t5 processing include making an omelet, soft or hard-boiling the egg, baking
the egg,
or, if desired, the egg can be eaten raw or processed as liquid egg.
Finally, further separation and purification, such as in the case of the
partially
purified anti-inflammatory composition, will allow for other modes of
20 administration such as administering egg product parenterally,
subcutaneously,
intravenously, intramuscularly, intraperitoneally, intranasally, orally or
topically.
In addition, such further separation will provide for the ability to make
encapsulated products and pharmaceutical compositions with said egg or
fraction
thereof.
Preparations of the anti-inflammatory composition for parenteral
administration
include sterile aqueous or nonaqueous solutions, suspensions or emulsions.
Examples of nonaqueous solvents or vehicles are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil and injectable organic esters such as
ethyl
oleate.
«'ith particular reference to the partially pure anti-inflammatory
composition, oral
administration is preferably accomplished through solid dosage forms which
24


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include capsules, tablets, pills, powders and granules, among otliets. In
solid
dosage forms, the anti-inflammatory composition is admixed with at least one
inert diluent such as sucrose, lactose or starch. Such dosage forms can also
comprise, as is normal practice, additional substances other than inert
diluent. In
the case of capsules, tablets, and pills, the dosage forms may also comprise
buffering agents, pH sensitive polymers, or any other slow-releasing
encapsulants
which are typically used as encapsulating compositions in the food and drug
industry. Tablets and pills can additionally be prepared with an enteric
coating.
Liquid dosage forms of the anti-inflammatory composition for oral
administration
include pharmaceutically acceptable emulsions, solutions, suspensions, syrups
and
elixirs, containing inert diluents commonly used in the pharmaceutical art.
Besides inert diluents, compositions can also include wetting agents,
emulsifying,
and suspending , and sweetening agents.
The hyperimmune egg or egg product, including the partially purified anti-
inflammatory composition of the invention is effective in treating and
preventing
inflammation, and in particular, all forms of arthritis, including, but not
limited to,
rheumatoid arthritis, osteoarthritis, ankylosing seronegative
spondyloarthropathy,
reactive arthritis, chronic fatigue syndrome, fibromyalgia (fibrositis) and
gout.
The egg product of the invention is equally effective in treating autoimmune
diseases, such as rheumatoid arthritis, juvenile diabetes, multiple sclerosis,
Graves' disease, Meneri's disease, myasthenia gravis, lupus erythematosus,
psoriasis, systemic scleroderma, rheumatic fever and Sjogren syndrome among
others. {See Background section of this document)
When it comes to treatment and prevention of a particular disorder, whether it
be
inflammation in general or a form of arthritis or an autoimmune disease, the
hyperimmune egg product or any active fraction thereof, including the
partially
3o purified anti-inflammatory composition, is preferably administered to the
subject
in an amount that is immunologically effective in treating and preventing the
particular disorder. Duration and intensity of the treatment will depend upon
the
particular condition, whether it is present, and, if so, the advancement of
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CA 02355168 2001-06-13
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condition in the subject. The hyperimmune egg product or any active fraction
thereof, including the partially purified anti-inflammatory composition, are
also
provided in any amount that treats and/or prevents the condition and the
symptoms of the condition. For example, in some cases, daily amounts ranging
from less than one to several whole, hyperimmune eggs (or hyperimmune egg
products containing the equivalent of less than one to several whole,
hyperimmune eggs) can be administered to the subject depending on the
particular
circumstance of the condition. More potent fractions can be separated and
concentrated by methods described herein as well as other known methods in the
art.
In one embodiment, the egg product of the invention as well as the partially
purified anti-inflammatory composition were found to be effective in treating
rheumatoid arthritis in rats using a collagen-induced arthritis animal model
(see
Example 2). This animal model is well recognized by those in the art as one
which parallels the rheumatoid arthritis effect in humans. In addition to
treatment
of arthritis, it was determined that the egg product, when administered to
rats prior
to induction of arthritis, delayed, and in some cases, prevented the onset of
the
arthritic symptoms. Thus, the egg product of the invention is effective in not
only
treating the symptoms of the disease, but also delaying and/or preventing the
onset
or progression of the disease.
In an alternative embodiment, the egg product was tested in humans and showed
positive effects in treating various forms of arthritis in several humans
suffering
from such symptoms (see Example 9). The humans who were treated by the egg
product demonstrated a clinical reduction in such symptoms as pain in addition
to
a general reduction in swelling and stiffness.
Indicative of an effect on autoimmune disease is the surprising reduction of
Type
II collagen antibodies by the egg product as well as the partially purified
anti-
inflammatory composition of the invention in this disease model, which was
also
seen in rats (see Example 3). It is contemplated that the egg product of the
invention is effective in reducing antibodies involved with other autoimmune
26


CA 02355168 2001-06-13
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diseases such as juvenile diabetes, multiple sclerosis, Graves' disease,
Merieri''s'
disease, myasthenia gravis, lupus erythematosus, psoriasis, systemic
scleroderma,
rheumatic fever and Sjogren syndrome among others.
As described in the background of the invention, patients having arthritis are
presently treated with non-steroidal anti-inflammatory drugs (NSA)T7s) and/or
disease modifying anti-arthritic drugs (DMARDs) in order to relieve the
symptoms of the arthritic condition. As shown in the examples (See Example 9)
patients were administered the egg product of the invention concurrently with
the
to present drug therapy. The egg product worked synergistically with the drug
therapy to reduce the symptoms of the particular disorder. The egg product can
therefore be administered concurrently with alternative treatment for
arthritis and
autoimmune diseases to produce a synergistically better effect.
In addition, as is well known in the art, NSAIDs and DMARDs can cause severe
gastrointestinal damage. U.S. patent number 5,772,999, assigned to DCV, Inc.,
and incorporated herein by reference, discloses that hyperimmune egg product
is
effective in treating NSA1D-induce gastrointestinal damage. Therefore, an
alternative embodiment is contemplated for cases where the subject is taking
or is
preparing to take NSA)Ds for the treatment of an arthritic condition. In such
a
case; the subject can also be administered the egg product of the invention
to, not
only treat the arthritic condition, but also protect the gastrointestinal
system from
NSA1D-induced damage. In such treatments, a lower dose of hyperimmune egg
may be sufficient. Also, the subject animal suffering from the disorder can be
administered egg product prior to beginning its regimen of NSAms and
DMARDs, in order to better prepare the gastrointestinal system.
Examples of inflammatory conditions that may be treated by administration of
the
egg, egg product, and/or the anti-inflammatory composition of the present
3o invention include acute and subacute bursitis, acute non-specific
tendinitis,
. systemic lupus erythematosus, systemic detmatomyositis, acute rheumatic
carditis,
pemphigus, bullous dermatitis, herpeteformis, severe erythema, multiform
exfoliative dermatitis, cirrhosis, seasonal perennial rhinitis, bronchial
asthma,
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ectopic dermatitis, serum sickness, keratitis, opthahnicus iritis, diffuse
ureitis,
chorditis, optic neuritis, sympathetic ophthalmic, symptomatic sarcoidosis,
Loeffler's syndrome, berylliosis, hemolytic anemia, mastitis, mastoiditis,
contact
dermatitis, allergic conjunctivitis, psoriatic arthritis, ankyiosing
spondylitis, acute
gouty arthritis, herpes zoster rheumatoid arthritis, osteoarthritis, any other
degenerative joint diseases, and any other related autoimmune diseases.
Further,
the isolated and purified egg product may be used to treat individuals who are
exposed to potentially inflammatory agents such as allergans.
l0 EFFECTIVE AMOUNTS
With regard to administration to a subject of the hyperimmunized egg or egg
product, it has been determined, and is detailed in the following examples,
that the
preferred dose range of hyperimmunized egg or egg product to be given to a
subject is between 100 milligrams to 10 grams per kilogram of subject weight.
With regard to the partially purified anti-inflammatory composition itself, it
has
been determined that the preferred dose range of the partially purified
composition, purified and isolated from whole egg, egg yolk and egg white of a
hyperimmunized egg, is between 1 microgram and 400 milligrams per kilogram of
the anti-inflammatory composition.
The dosage of active ingredients may be varied; however it is necessary that
the
amount of the active ingredient shall be such that a suitable dosage form is
obtained. It will be recognized that the selected dosage form depends upon the
25 desired therapeutic effect, on the route of the administration and on the
duration of
the treatment.
The advantageous properties of this invention can be observed by reference to
the
following examples which illustrate the invention.
EXAMPLES
Example 1
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PREPARATION OF S-100 VACCTNE
The multivalent vaccine known as "Series 100" or "S-100," disclosed in U.S.
Pat.
No. 5,215,746 and containing the bacteria shown in Table 1 (obtained from the
American Type Culture Collection), was reconstituted with 15 ml of medium and
incubated overnight at 37C. Once good growth was obtained, approximately one-
half of the bacterial suspension was used to inoculate one liter of broth
which was
then incubated at 37C. The remaining suspension was transferred to sterile
glycol
tubes and stored at -20C for up to six months.
1o After good growth was visible in the culture, the bacteria were harvested
by
centrifugation. The bacterial pellet was resuspended in sterile physiological
saline
solution and the bacterial sample was centrifuged three times to wash the
cells.
After the third wash, the pellet obtained was resuspended in a small amount of
double distilled water.
The medium-free bacterial suspension was heat-killed by placing the suspension
in a glass flask in an 80C water bath overnight. The viability of the broth
culture
was tested with a small amount of heat-killed bacteria. Broth was inoculated
with
heat-killed bacteria, incubated at 37C for five days and checked daily for
growth,
as the bacteria have to be killed for use in the vaccine.
The heat-killed bacteria were lyophilized until dry. The dry bacteria were
then
mixed with sterile saline solution to a concentration of 2.2 x 10$ bacterial
cells/ml
saline ( 1.0 optical density reading at 660 nm).
TABLE 1
Antigens in the S-100 Vaccine
''~3~-_ w ; Name... IVX,ediaz , r ~ Name ~,~~"~'' Media
~ : , . .3~~-,~'o-.al.. v,
~_. ~'Tf _ 1 a:


Staphylococcus simulansBHI Pseudomonas aeruginosaBHI


Staphylococcus epidermidisBHI Klebsiella pneumoniae BHI


Streptococcus pyogenes,APT- Salmonella typhimuriumBHI
A Type 1


Streptococcus pyogenes,APT Haemophilus influenzaeBHI
A Type 3


Streptococcus pyogenes,APT Streptococcus mitis APT
A Type 5


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Streptococcus pyogenes,AT Proteus vulgaris BHI
A Type 8


Streptococcus pyogeries,APT Shigella dysenteriae BHI
A Type 12


Streptococcus pyogenes,APT Diplococcus pneumoniaeAPT
A Type 14


Streptococcus pyogenes,APT Propionibacter acnes Broth
A Type 18


Streptococcus pyogenes,APT Streptococcus sanguisAPT
A Type 22


Aerobacter aerogenes BHI Streptococcus salivarusAPT


Escherichia coli BHI Streptococcus mutans BHI


Salmonella enteritidisBHI Streptococcus agalactiaeAPT


Immunization Procedure For Hyperimmune Egg Product
A killed preparation of pathogens was prepared as described above. For
the first vaccination, the bacteria were mixed with complete Freund's
adjuvant, and 5.6 mg of bacterial material were injected into the breast
muscle of a chicken. For the remaining vaccines, the bacterial preparation
was mixed with the incomplete Freund's adjuvant and injected into the
chickens at two week intervals for six months.
Eggs were collected from the hyperimmunized chickens and then sprayed
dried into a powder form. During the spray drying procedure, inlet
temperatures did not exceed 320 Degrees F, exhaust temperatures were
maintained in accordance with producing powder in the range of 3.0 to 4.0
percent finished moisture, and pump pressure was maintained around 2500
to 4000 P.S.I. Lower temperatures ranging from 100 - 160 F were used,
and samples were monitored for moisture contend during the drying
process to obtain a final product having any consistency desired.
2o Example 2
PREFERRED METHOD FOR PREPARING A PARTLALLY PURg'IED ANTI-
INFLAMMATORY COMPOSITION FROM EGG
The following examples describe a'method (suitable for large scale
purification)
for obtaining the anti-inflammatory composition from avian eggs in a partially
pure, low molecular weight, non-aggregated form. Whole eggs, hyperimmunized
and control table eggs, were cracked and the egg white was separated from the


CA 02355168 2001-06-13
WO 00/43020 PCT/US99/30969
yolk and both were spray-dried. Hyperimmune eggs were obtained-as
described°iil~~
Example 1. Egg white powder was processed separately to obtain the aqueous
fraction for ultra-filtration.
All of the purification steps were performed so as to minimize possible
contamination with bacteria or pyrogens. Sterile water was used to prepare
solutions and all glassware was de-pyrogenated. In addition, the solution was
sterile filtered.
Example 2a
PREPARATION OF EGG ANTI-INFLAMMATORY COMPOSITION FROM
EGG YOLK POWDER
SOLVENT EXTRACTION
The dried egg yolk as prepared in Example 1, was subjected to liquid solvent
extraction with either propane, or butane to separate the lipids from the
aqueous
yolk fraction containing the anti-inflammatory composition. Briefly, 500 grams
of
dry egg yolk powder was placed in a column, to which was added 4 liters of
liquid
propane solvent. The solvent supernatant and extracted lipid were removed. Six
additional solvent extractions were performed for a total of six lipid
extractions.
ULTRA-FILTRATION
Four hundred grams of dry de-fatted egg yolk was diluted with 4 liters of
sterile
distilled water and homogenized with a Virtis (handishear). , The yolk mixture
was
either centrifuged at 24 RPM or allowed to stand refrigerated until the non-
dissolved yolk particles precipitated. The resulting aqueous fraction was
ultrafiltered using an Amicon RA1000 ultra-filtration system equipped with
3,000
dalton cut-off spiral-wound membrane. The pump speed was maintained at 20 psi
inlet pressure and 15 psi outlet pressure. The <3,000 daltons molecular weight
permeates was sterile filtered using a 0.45 pm Sterile disposable Nalgene
filter
3o and lyophilized or frozen, for storage, bioassay testing or further
purification.
Figure 1 shows a flow chart of the purification process.
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Molecular species below 3,000 daltons from egg yolk contain the anti-
inflammatory composition in a low molecular weight, non-aggregated form. From
400 grams of starting material, the yield of anti-inflammatory composition was
approximately I2 grams or 3% of the total. Bioassay testing of this 3KDa
fraction
for anti-inflammatory activity showed high levels of activity (Example 5).
Example 2b
PREPARATION OF EGG ANTI-INFLAMMATORY ACTIVITY FROM EGG
WHITE POWDER
Four hundred grams of egg white, isolated from both hyperimmunized egg as
1o described in Example 1 and control table egg, was diluted with four liter
of
deionized water. The mixture was homogenized and filtered through a 40 ~,m
filter and ultrafiitered through a 3KDa MW CO ultra-filtration system (Figure
2).
From 400 g of egg white powder, 8.6 g or 2.15 % of anti-inflammatory
composition was recovered.
This example shows the preparation of the active anti-inflammatory composition
from egg white material of both hyperimmune and control table eggs.
Example 3
DEAE ION EXCHANGE CHROMATOGRAPHY
The egg anti-inflammatory composition was also further characterized by anion
exchange DEAF chromatography.
3,000 Dalton ultra-filtration permeates from both delipidated egg yolk and egg
white were prepared in the same manner as described in Example 2. A DEAF
Sepharose column (2.5 x 40.0 cm) was previously equilibrated at room
temperature with 1,000 ml of 20 mM ammonium acetate buffer in sterile double
distilled pyrogen free water pH 8Ø 3,000 Dalton permeate of delipidated egg
yolk (15 grams) or egg white (18 grams) was dissolved in 200 ml of sterile
pyrogen free double distilled water and applied to the DEAF Sepharose colummn
(pharmacia Biotech., Fast flow) with a flow rate of 100 ml/h. Components of
3,000 Dalton permeate were eluted with following buffers to separated egg anti-

inflammatory composition and other molecules. With the same flow rate DEAE
Sepharose was washed with 200 ml of 20 mM ammonium acetate, pH 8.0 and 200
32

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ml of 20 mM ammonium acetate, pH 6.5, to remove unbound materials. 200 aril
of 150 ml ammonium acetate, pH 5.0, then was applied to elute the weakly bound
molecules. More strong bound molecules were eluted with 200 ml of 150 mM
ammonium acetate, pH 4Ø DEAF Sepharose column was finally regenerated
with 400 ml of 1.5 N NaCI in 20 mM ammonium acetate, pH 6.5 (Figure 3).
Figure 3 shows a typical chromatogram of DEAF Sepharose separation of 3Kda
permeate from delipidated egg yolk and egg white. Letters indicated the
elution
profile with buffers. Fractions were collected by every 15 ml elutent and
their
molecular absorbances at 280 nm and 220 nm were measured. Four fractions
were pooled and the water was removed by lyophilization to dryness. Table 2 is
the summary of the separation.
TABLE 2
DEAE separation of 3,000 Dalton permeates of delipidated egg yolk and egg
white.
Fraction Elute # Vol(ml) Weight (g) Recovery (%)
Egg yolk


I 1-22 294 11.79 78.6


II 23-40 252 0.40 2.7


III 41-56 224 0.44 2.9


IV 57-70 196 0.19 1.3


Egg white


I 2-26 350 15.33 85.2


II 27-46 280 0.23 1.3


3o III 47-60 196 0.32 1.8


IV 6I-73 182 0.44 2.4


Example 4
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HPLC ANALYSIS OF 3,000 DALTON PERMEATES"AND THEIR DEAF'
SEPHAROSE FRACTIONS
3,000 Dalton permeates of 3,000 Dalton permeates of egg yolks were analyzed
with reverse High Performance Liquid Chromatography (HPLC) (Shimadzu Unit,
SPD-M10A VP Diode array detector, LC-6AD liquid chromatography, SCL-l0A
VP Degassor, LPM-600 Low pressure mixing, and CTO-l0A VP Column oven).
All of the fractions were prepared as 5.0 mg/ml in super-pure water followed
by
filtration through a 0.2 um filter. 20 pl of each sample was applied on a
Water
Symmetry C18 column (3.9 x 150 mm) at 29°C and with a flow rate of
1.0
ml/min. A linear gradient of 0% - 60% acetonitril in dH20 that contained 0.1%
TFA was used as the mobile phase.
Figure 4 shows a Max plot chromatography of the separation of 3,000 Dalton
permeate from delipidated PL-100 egg yolk. By using the same analytic
condition, 3,000 Dalton permeate prepared from delipidated Table egg yolk was
also obtained (chromatography has not shown).
Table 3 summaries the result of HPLC analysis of 3,000 Dalton permeates. Peaks
that are greater than 0.5% of peak area were collected. The similarities of
peaks
were compared within +/- 0.5% variability in their retention times. Common
peaks mean that they are present in both PL-100 egg yolk and Table egg yolk
while PL-100 peaks indicates that they are only present in PL-100 egg.
Comparing two fractions, 3,000 Dalton permeate of Pl-100 egg yolk shows 15
more peaks than that of Table egg yolk, suggesting that hyperimmunization on
chicken could produce more biological components in its egg.
TABLE 3
Common PL-100Table
3KDapermeate peaks peaks peaks Total
PL-100 18 15 0 33
Table 18 0 5 23
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Preparative HPLC
To obtain large quantities of the anti-inflammatory factor after
ultrafiltration, the
preferred step is to separate the partially purified, less than approximately
3,000
daltons molecular weight, composition on a preparative HPLC column.
Accordingly, a 5.08 X 20 cm column was packed with Zorbax C8 packing
material and 4 grams of the less than 3,000 daltons MW ultrafiltrate was
purified
during each run. Using a mobile phase of 80/20 H20/methanol with 0.05% TFA,
at a flow rate of 90 ml per minute, the permeate was separated into the
partially
1o purified composition collected and lyophilize. Next the collected peak was
re-
tested on an analytical CS column. With the method described, large quantities
of
the ultra-pure peak were obtained by preparative HPLC and analyzed for
bioassay
activity, elemental composition and chemical structure. Mass Spectroscopy,
_ Elemental analysis, Infra-red spectroscopy and Nuclear Magnetic Resonance,
NMR are being used to determine the structure of the factor.
Example S
ANTI-ARTHRTTIC PROPERTIES OFHYPERIMMUNE EGG PRODUCT IN A
COLLAGEN-INDUCED ARTHRTTIS MODEL IN RATS
2o Collagen induced arthritis in rat is a popular experimental animal model of
Rheumatoid arthritis and has been used by several laboratories and research
institutions since 1977. Since its initial discovery, the animal model of
collagen-
induced arthritis has demonstrated many parallels to human rheumatoid
arthritis.
For example, Stuart and coworkers have demonstrated that several of the
clinical
signs and historological changes in the joints of arthritic rats resemble
those in
patients with Rheumatoid Arthritis (Stuart et al 1983). Of particular interest
is the
fact that many Rheumatoid Arthritis patients exhibit autoimmunity to Type II
collagen. Historically, this animal model has proven to be a useful predictive
model of clinical efficacy of new modalities with iznmunosuppressive
properties
and potential immune modifying substances in terms of their anti-arthritic
action.
The arthritis in mice and rats is generally induced by immunization with
heterologous type II collagen, which initiates a combined humoral and cellular


CA 02355168 2001-06-13
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immune response targeted to joint tissues. Current therapies are inadequate or
have side effects that limit their prolonged use.
The present example looks at the effect of the egg product and the partially
purified anti-inflammatory composition of the invention orally administered to
rats prior to and during arthritis induction with collagen II. The suppression
of the
incidence of arthritis was examined in a dose dependent manner when compared
to a control group.
1o Method
30 Sprague-Dawley female rats-VAF+ (Charles Rivers, Wilmington, MA),
weighing 100-125 gm, were randomized.to 3 groups (10 animals/group). The
experiment was repeated three times so that the final groups included 30
rats/treatment regimen with total of 90 rats for this study. Spray dried
hyperimmune egg product (as prepared in Example 1 ) was diluted for oral
gavage.
10% and 0.2% solutions of egg product for oral gavage were made every other
day. The second day solution was stored at 4°C until use. Egg Product
(3.5m1 of
the respective solutions) was orally gavaged into rats for 7 days prior to
initiation
of the type II collagen induced arthritis in rats and for 14 days after
induction.
The method of inducing and evaluating Arthritis in rats was according to
Trentham, et al. 1977. Briefly, Sprague-Dawley rats were immunized on the
lower back by intra-dermal inoculation of 400 pepsin-treated native type II
chick
collagen (CII) (Genzyme, Boston, MA), solubilized at 1 mg/ml in 0.1 M acetic
acid and emulsified 1:1 with Incomplete Freund's Adjuvant. Three groups:
Water gavage (Control), SOX hyperimmune egg product (High Dose) (3.Sml of a
10% solution of hyperimmune egg product) and 1X hyperimmune egg product
(Low Dose) (3.Sml of a 0.2% solution of hyperimmune egg product) with 10 rats
per group were used. Starting on day 10 following intradermal collagen II
immunization and daily thereafter until Day 21, the rats were clinically
evaluated
(blinded) for rat paw periarticular erythema ( 0 - 4+) and paw swelling (0 -
4+) for
each limb.
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The group arthritis index (AI) is a summation of paw scores based on the
degree
of incidence of arthritis and severity of arthritis as derived from the mean
AI. On
Day 21, all rats were sacrificed and bled. Serum were evaluated for antibody
titer
to type II collagen. Enzyme Linked Immunoassays (ELISA's) were used to
5 measure specific antibody titer to Collagen Type II (Trentham, et al. 1983).
The
animals were then sacrificed with metafane overdose.
Results
The DCV egg product showed definitive antiarthritic properties in a dose-
dependent fashion in the Type II collagen rat model.
Consumption of a high dose of the DCV egg product clearly demonstrated
antiinflammatory properties in this model by influencing both the cellular and
humoral arm of the immune system.
The compound appeared to be nontoxic at both the low and high doses. The high
dose group showed not only a statistically significant decrease in the
incidence of
arthritis, but also a delay in onset and a decrease in severity of arthritis
as
compared to the water control.
Figure S shows that the high dose of egg product, given prior to immunization
with Type II collagen, prevented the development of arthritic symptoms in more
animals than those not given egg. Also, within eleven days of treatment with
the
egg product and thereafter for the entire duration of the study, a significant
25 decrease in the incidence of arthritis was observed as compared to group
that did
not receive any egg.
Anti-arthritic effects were observed in the Type II collagen induced arthritis
model
with S-100 and Pl-100 whole eggs. Upon separation of the egg, the yolk showed
3o antiarthritic activity in the animal model but not the egg white. However,
delipidating the same egg yolk completely abrogated the antiarthritic
activity.
This delipidated egg yolk, was further purified by 3000 molecular weight cut
off
size exclusion column, and tested in the rat Type II collagen model. The
partially
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pure fraction showed high antiarthritic activity which was comparable to that
shown by the whole Pl-100 egg.
Another important observation made was that the rats fed with PL-100 eggs had
a
statistically significant reduction in the antibodies to Type II Collagen in
their
serum than in animals in the control group. This would indicate that the PL-
100
egg had an immunosuppressive effect in the animals and thereby aided in
alleviating their arthritic symptoms in the joints.
1o TABLE 4
Average Day to Maximum Incidence of Arthritis
Control Low Dose High Dose


Expt 1 14 19 20*


Expt 2 14 18 17


Expt 3 12 15 15


* Delay in onset of maximum arthritis after immunization with Collagen lI.
At day 21 the arthritic index which reflects the degree and severity of
arthritis was
significantly reduced in animals which received the high dose of hyperimmune
egg product as compared to the control group (2.46+Ø55 vs 4.167+ 0.51 ; p
>0.003, Student t-test). The effect of the low dose of egg product did not
differ
significantly from the controls. In addition, the incidence of arthritis was
2o significantly suppressed in the high-dose group compared to control (30%
vs.
70%; p< 0.03). This decrease in the percent incidence of arthritis seen with
the
high dose of hyperimmune egg product (Table 4) is as good as has been reported
for steroids, NSAIDS (non-steroidal anti-inflammatory drugs) methotrexate, and
minocycline.
This example also shows that the high dose of egg product, given prior to
inducing arthritis, prevented the development of arthritis in more animals
than
those not given egg. These results are demonstrated in Figure 6.
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Figure 6 shows the prevention of arthritis by the high dose of egg product. A
lower percentage of animals, orally gavaged with egg product, developed
arthritis
symptoms when compared to control animals, which did not receive egg.
Animals were orally gavaged for 21 days, seven days prior to immunization
(induction of arthritis) and fourteen days after immunization.
Conclusion
The data clearly shows a definitive anti-arthritic effect in rats for the egg
product
to in a dose-dependent manner in the type II collagen induced rat arthritis
model.
Statistically significant decreases in the incidence, delay in onset, and a
decrease
in the severity of arthritis were observed in the animals receiving a high
dose of
hyperimmune egg product. This data shows the preventative effects of the high
dose of the egg product on the incidence of arthritis. Most importantly, at
both the
high and low dose the hyperimmune egg product is nontoxic.
Example 6
REDUCTION OF COLLAGEN II ANTIBODIES
Rats from Example 5 were bled on Day 21 and serum samples were collected. A
2o standard ELISA assay was used to measure titers against Collagen II
autoimmune
antibodies. These results show that the animals fed either high or low doses
of
egg product had significantly lowered antibody titers to collagen II when
compared to controls (Table 5).
TABLE 5
Control Low Dose Hi h Dose


Antibody titers9.3 +_0.03 8.5 _+ 0.28 8.3 _+ 0.36
to


Colla en II
(-l0 2)


Statistical --- p < 0.004 p< 0.005


Si ificance


The decrease in autoimmune antibody titers to Collagen II in the serum of
animals
3o gavaged with egg product when compared to control animals (no egg product}
is
39


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highly significant. Because all groups of animals received 'the same level of
collagen II to induce antibodies to Collagen II and the resulting arthritic
symptoms, it was unexpected that the groups receiving egg product would have
lower titers of collagen II antibodies on Day 21. This decrease in antibody
titers to
collagen II indicates a positive influence in both the cellular and humoral
immune
response for the hyperimmune egg product.
Example 7
ANTI-INFLAMMATORY EFFECT OF HYPERIMMUlVIZED EGG
This example shows the anti-inflammatory effect of hyperimmunized egg (which
is prepared as described in Example I) on carrageenan induced skin edema in
dogs on a diet including hyperimmunized egg. These results demonstrate that
hyperimmune egg reduces inflammation. The effect on inflammation was
comparable to that obtained with the non-steroidal anti-inflammatory drug
ibuprofen at 10 mg /kg, ED So, dose in this test.
Twenty White Eagle Beagles (four groups of five dogs each) were fed a basal
diet
(350 grams) of commercially available lamb and rice dog food. As illustrated
in
table 2, two groups of five dogs (groups 1 & 2) received only the basal diet
during
the conditioning period of approximately 100 days. In addition, dogs from
group
2 were treated with the non-steroidal anti-inflammatory drug, ibuprofen, prior
to
inflammatory challenge with carrageenan. During this same period, the
remaining
two groups (3&4) were fed the basal diet along with hyperimmune egg (HIE).
Group 3 received 3.5 grams of hyperimmune egg, while group 4 received 35
grams of hyperimmune egg added to the diet. At the end of the conditioning
diet
treatment, the dogs were challenged with an infra-dermal injection of 2%
carrageenan and an inflammatory response was elicited.
The challenge process was as follows. One dog from each group was randomly
3o selected for challenge and testing each day. This procedure was repeated
daily for
five days until all dogs from all groups were tested. Fifteen minutes prior to
the
challenge procedure, dogs from group 2 were given lOmgikg ibuprofen orally.
All dogs were anesthetized by intravenous injection and shaved on the left
lateral


CA 02355168 2001-06-13
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side (approximately 6 X 8 inches). Two lines of 3 marks were made on th'e
shaved area and numbered 1 - 6. An intra-dermal injection of 0.1 ml saline was
given at mark 1 as a negative control. From mark 2 - 6 , a 0.1 ml intra-dermal
injection of a 2% solution of carrageenan was given. Injections were
administered
by the same person for the duration of the study. Intra-dermal injections of
this
concentration of carrageenan was previously determined to cause a measurable
swelling in 100% of the animals which could be reduced by 10 mg/kg ibuprofen.
At the completion of the last injection of carrageenan, measurements were made
of each swelling using a micrometer and the numbers were recorded. The
anesthetized animals were allowed to recover and six hours later the
measurements were repeated and recorded. The mean size of the inflammatory
response was determined for each group.
TABLE 6
Groups TreatmentNumber Mean Mean Significance
of


Animals InflammatoryDifference
Per


Group Response From ControlP value


1 Basal 5 4.41 - -
Diet


Control


2 Ibuprofen5 2.25 2.2 P<0.01


Drug { l Omg/kg)
+


Basal
Diet


3 HIE egg 5 2.8 l.6 P<0.05


Test 3 , 5
grams
+


Basal
Diet


4 HIE egg 5 2.38 2.0 P<0.05


Test 35 grams
+


Basal
Diet


These results showed a significant reduction in swelling in the animals fed
with
hyperimmune egg (3.5 grams plus basal diet and 35 grams plus a basal diet)
indicating a preventative effect of the hyperimmune egg on inflammation when
fed orally. Moreover these results show that the anti-inflammatory activity of
the
hyperimmune egg was comparable to the effect seen with the anti-inflammatory
41


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drug ibuprofen. Differences at the p<0.05 level of significance were seen
between"
the treated and control group.
Example 8
EFFECT OF EGG PRODUCT ON ARTHRITIC HUMAN PATIENTS
A nutritional drink supplement was obtained which comprised a high protein,
high
carbohydrate powder containing 30% of recommended daily allowance of
vitamins, and approximately 4.5 grams of hyperimmune powdered egg
(approximately equal to 0.4 eggs). The drink supplement was provided by DCV,
1o Inc., Wilmington, DE.
Three patients were administered the nutritional drink supplement for a period
of
two months. Clinical evaluations regarding tolerance to the product an
clinical
response, as evaluated by each patient's estimation of clinical status and a
detailed
15 clinical examination was performed prior to the study at one month, two
months
and three months (one month after ceasing the product). Clinical chemistry,
hematology and urinalysis profiles were performed. Described below are the
results of the treatments of each of these patients:
2o PATIENT #1
Patient #1 was a 29-year-old female, 5'3" 170 Ibs with a previous history of
rheumatoid arthritis (juvenile). On first examination the patient presented
with
joint pains, swelling, and tenderness of fingers, wrists, toes, feet, and
knees. She
complained of functional limitations such as inability to do knee bends or
run, and
25 had difficulty with kneeling. Her hematology tests had normal etythrocyte
sedimentation rate (ESR) and C-reactive protein (CRP) readings. Patient's
cholesterol was at 199mg/dl. She was being treated with Minocin 300mg/week,
Loestrin Fe 1.5/30 and used multivitamins. The diagnosis was rheumatoid
arthritis with uveitis.
30
After one month of consuming the Nutritional Drink Supplement on a daily
basis,
the patient's hand redness and swelling decreased, and the pain and swelling
in
her feet disappeared. Patient reported that she felt "less achy" and "felt 75%
42


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better". She reported that she had increased energy. Her ESR and'CRP readings
remained normal but her cholesterol dropped to 186 mg/dl. Patient continued
her
medications in conjunction with the egg-based drink.
s During the second month, the patient felt that her hands were worse and she
presented with swelling of fingers and wrists and reported numbness in hands
at
night. Her legs felt "better". Physician's global assessment was that patient
was
slightly worse. She still experienced pain when carrying shopping bags or upon
opening boxes. Her ESR and CRP readings remained normal and her cholesterol
remained at 189mg/dl. She continued medic ations and as scheduled, by the
protocol, discontinued use of product.
After one month without the product, swelling and tenderness returned to hands
and feet. Her function limitations remained as she first presented. Patient
still felt
75% "better". Her ESR and CRP readings remained normal as her cholesterol
dropped to 180mg/dl.
Scoring of the arthritic symptoms was determined from the patient's own
assessment of the symptoms. In particular, the patient was asked to indicate
the
amount of swelling, pain and/or tenderness of the joints. Based upon this
patient
assessment, numbers were generated showing the degree of the symptoms. The
numbers range from a high of 36 (greatest amount of swelling, pain and
tenderness) to a low of 0 (no swelling, pain or tenderness).
Scores generated for Patient #1's arthritic condition were as follows:
At presentation= 14
One month on~egg-based drink = 2
Two months on egg-based drink = 6
3o One month after discontinuing drink= 8
PATIE~1T #2
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Patient #2 was an 88-year-old female S'3" 104 lbs. with a previous history of
rheumatoid arthritis and chronic sinusitis. She presented with pain in the
lower
extremities and shoulders. Physical examinations revealed swelling, pain, and
tenderness of her fingers and ankles. Functionally she was only able to walk
in a
limited manner and had difficulty in bending her arms. Her hematology tests
indicated elevated ESR (36 mm/hr), CRP (1.64mg/dl) and a total cholesterol at
220 mg/dl. She was being treated with: Methotrexate Smg/week, Orudis KT
2/day, Ca 1500mg, Fosomax lOmg, Prednisone Smg q2d (every other day), and
took multivitamins. Again, the patient continued to take these medications in
addition to the egg-based drink.
One month after starting the egg-based drink, she still complained of swelling
of
hands, knee pain, and inability to sleep. The patient reported that she was
"SO%
better"-; "feeling generally better" and "walking better." She was still in
pain;
although her pain attacks were less frequent. Hematology results were ESR
(30mnn/hr) and CRP (1.64rng/dl), and an increase in cholesterol to 232 mg/dl
was
observed.
After two months of consumption of product, patient reported that "she felt
98%
2o better". Her fimctional limitations however, remained the same. Hematology
results indicated a drop in her ESR (2lmm/hr) and a return to normal in the
CRP
reading.(<O.Smg/dl). Her total cholesterol remained at 234 mg/dl. Patient
continued her medications but, as scheduled, discontinued the egg-based drink.
One month after discontinuing product patient complained of severe joint pains
in
her fingers and legs and exhibited swelling of wrists. She felt she was "50%
worse". Her ESR (27mm/hr) levels and cholesterol (257 mg/dl) started to climb,
but her CRP (<O.Smg/dl) levels remained normal.
Scoring of the arthritic symptoms was determined from the patient's own
assessment of the symptoms. In particular, the patient was asked to indicate
the
amount of swelling, pain and/or tenderness of the joints. Based upon this
patient
assessment, numbers were generated showing the degree of the symptoms. The
44


CA 02355168 2001-06-13
WO 00/43020 PCT/US99/30969
numbers range from a high of 36 (greatest amount of swelling, pain and
tenderness) to a low of 0 (no swelling, pain or tenderness).
Scores generated for Patient #2's arthritic condition were as follows:
At presentation= 10
One month on egg-based drink = 7
Two months on egg-based drink = 2
One month after discontinuing drink= 10
1o PATIENT #3
Patient #3 was a 79-year-old male 5' 10" 152 lbs with a history of
osteoarthritis
and atherosclerotic cardiovascular disease. Symptoms were pain in the lower
back, shoulders, knees and feet. Medications included: Nitroderm patch,
Digoxin
0.125, aspirin 80mg and Feldene 20mg. Function limitations were: difficulty in
is moving arms and exhaustion. Hematology results were normal ESR and CRP and
cholesterol readings at 217 mgldl.
After one month of drinking the egg-product, along with the above-mentioned
medication, the patient felt "slightly better (10-20%)". He still reported
shoulder
20 pain on right and left side, and reported that he was unstable on feet and
experienced buckling of his right knee. Function limitations remained as
previously reported. His ESR and CRP readings were normal and his cholesterol
level was 180 mgldl.
25 After two months of consuming product patient continued to feel "slightly
better".
Symptoms included right knee pain and difficulty in walking. Cholesterol
readings were not obtained but ESR and CRP remained at normal levels.
After one month without product, patient felt slightly better overall but
slightly
3o worse than the previous month. Symptoms included buckling of right leg,
shoulder pain and lower back pain. Joint assessment indicated pain in
shoulders,
and lower back. Functional limitations were difficulty in moving arms and
feeling


CA 02355168 2001-06-13
WO 00/43020 PCT/US99/30969
"exhausted". ESR and CRP. readings were normal amd chdlesteiolvremain~d
at°
180 mg/dl.
Scoring of the arthritic symptoms was determined from the patient's own
assessment of the symptoms. In particular, the patient was asked to indicate
the
amount of swelling, pain and/or tenderness of the joints. Based upon this
patient
assessment, numbers were generated showing the degree of the symptoms. The
numbers range from a high of 36 (greatest amount of swelling, pain and
tenderness) to a low of 0 (no swelling, pain or tenderness).
to
Scores generated for Patient #3's arthritic condition were as follows:
At presentation= 11.5
One month on egg-based drink = 2
Two months on egg-based drink = 3
One month after discontinuing drink = 8.5
Other case histories showed similar improvements and demonstrated the
effectiveness of the present invention in eliminating pain and inflammation in
arthritic joints without side effects.
The invention has been described with particular reference to certain
preferred
embodiments thereof, but it will be understood that variations and
modifications
can be effected within the spirit and scope of the invention as described
above and
as defined in the appended claims.
Example 9
SIALIC ACID ANALYSIS
Sialic Acid Release, Purification, And Analysis Flow Chart
Sample Dowex 1 XS Dowex AG 4X4 HPAEC/PAD
Preparation ----~ Strong Cation ----~ Weak Anion
Exchange Exchange
2M Acetic Acid 46
Hydrolysis, 80°C,
3 hrs.


CA 02355168 2001-06-13
WO 00/43020 PCT/US99/30969
to Release And Purification Of Sialic Acids From Glycoproteins
Sialic acid release with relatively low O-acetyl loss was achieved with lml of
2M
acetic acid at 80°C for 3 hours in a sealed, crimp-top vial. KDN is
added as a pre-
hydrolysis internal standard.
Following acid hydrolysis, the reaction mixture was cooled and the vial
centrifuged to remove any insoluble material. Transfer the supernatant to a
micron (4.5 ml), centricon (2 ml), or centriplus (15 ml) 10,000 molecular
weight
cut-off centrifugation device. The acetic acid in the filtrate was removed by
2o vacuum centrifugation and the sample was redissolved in 0.5 ml of water.
The sample was applied to a 1 ml column ofDowex 50 AG 1X8 (hydrogen form)
in water to remove cationic contaminants. The flow through and 4-5 ml of water
washings were collected in a thin walled centrifuge tube containing 40 ul of
1 M formic acid. The acidified washings were taken to dryness by vacuum
centrifugation to remove the weak acids generated by the column.
The sample was redissolved in 0.5 ml of 10 mM sodium formate, pH 5.50 and
applied to 1 ml column of Dowes AG 3X4 (formate form) weak anion exchange
3o resin that was buffered in IOmM sodium formate pH 5.50. The column was
immediately washed with 5m1 of lOmM formic acid. The flow through and
washings were discarded. The sialic acids were eluted into a thin walled
centrifuge tube with l Oml of 1 M formic acid and the eluate was taken to
dryness
and then resuspend in water.
Note: All steps in this procedure can be performed at room temperature if
preserving total O-acetylation is the only concern. If prevention of O-acetyl
47

CA 02355168 2001-06-13
WO 00/43020 PCT/US99/30969
migration is necessary than all steps should be perforined'at
4°C°vifh 1 IVl
pyridine acetate buffer, pH 5.5 in place of 1 M formic acid.
Column Instructions
Dowex 50 AG 1 X8, hydrogen form: 1 ml of resin was spooned into a mall,
disposable, plastic-fritted column. Column was primed with Sml of 1 M HCI
followed by washing with 5 ml aliquots of water until the pH rose above -4.
Dowes AG 3X4 (or 4X4), formate form: 1 ml of resin was spooned into a small,
disposable, plastic-fritted column, and converted to hydroxide form by washing
column with 5 mI of 1 M NaOH. Hydroxide form was then converted to formate
form by washing column with 3-5 ml of 1 M formic acid or until pH of the flow
t5 through was -1.5. The column was washed with double DI water. The column
was then buffered with IOmM sodium formate, pH 5.5.
Analysis Of Sialic Acids By Dionex HPAEC/PAD On The Carbopac PA-10
Column
Sialic acid samples were analyzed by HPAEC/PAD using the Dionex GP40
gradient pump, ED40 electrochemical detector and CarboPac PA-10 guard and
analytical columns. Sample injection was made by an AS3500 autosampler.
Sialic acids were eluted using 100mM NaOH (eluent C) and 100mM NaOH, 1M
NaAc (eluent D) by gradient method. The separation gradient for this method
was:
Time (min) A% B% C% D% INJECT TITLI


Initial - - 93.0 7.0


0 - - 93.0 7.0 X X


10 - - 70.0 30.0


11 - - 70.0 30.0


12 - - 93.0 7.0


27 - - 93.0 7.0


48


CA 02355168 2001-06-13
WO 00/43020 PCT/US99/30969
This method required 27 minutes at a flow rate of lml/min. Sialic acids were
quantitated using KDN as the internal standard. Sample peak areas were
normalized to adjust for KDN signal loss due to hydrolysis or ion exchange
cleanup.
Conclusion
Sialic Acid Analysis Of PL-100 Egg and its Components
Egg Fractions Free + Bound Sialic % Sialic Acid
acid
content er m of E


Table Egg Yolk 1.9 mg .19%


Table Egg White 1 mg .10%


3k fraction of PL-1007.7 mg .77%
Egg
Yolk


3k fraction of PL-1000.75 mg .075%
Egg
White


The above table shows the free and bound sialic acid content in various
fractions
of egg as analyzed by the Standard Dionex protocol and PAD detection. This
15 procedure performed along with preliminary clean up over Dowex resins,
results
in quantitative recovery and eliminates other contaminating peaks from that
position of the chromatogram.
Isolation of the 3k fraction of the egg yolk does not enrich the bound and
free
2o sialic acid content appreciably.
The biological activity of the 3k fraction of the egg yolk in the rat Type II
Collagen assay cannot be attributed to the sialic acid content as it is very
low
2.7mg/dose).
49

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Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 1999-12-28
(87) PCT Publication Date 2000-07-27
(85) National Entry 2001-06-13
Dead Application 2005-12-28

Abandonment History

Abandonment Date Reason Reinstatement Date
2004-12-29 FAILURE TO REQUEST EXAMINATION

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $300.00 2001-06-13
Maintenance Fee - Application - New Act 2 2001-12-28 $100.00 2001-06-13
Registration of a document - section 124 $100.00 2002-02-20
Registration of a document - section 124 $50.00 2002-07-12
Maintenance Fee - Application - New Act 3 2002-12-30 $100.00 2002-09-27
Maintenance Fee - Application - New Act 4 2003-12-29 $100.00 2003-09-23
Maintenance Fee - Application - New Act 5 2004-12-28 $200.00 2004-10-05
Maintenance Fee - Application - New Act 6 2005-12-28 $200.00 2005-12-28
Current owners on record shown in alphabetical order.
Current Owners on Record
ARKION LIFE SCIENCES
Past owners on record shown in alphabetical order.
Past Owners on Record
ADALSTEINSSON, ORN
BECK, LEE R.
DALEY, MICHAEL J.
DCV, INC.
FITZPATRICK-MCELLIGOTT, SANDRA
GREENBLATT, HELLEN CHAYA
HUNCHAR, JEFFREY G.
IYER, SUBRAMANIAN
LEE, YOUNG-ZOON
XING, RUYE
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.

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Cover Page 2001-10-11 2 37
Abstract 2001-06-13 1 59
Description 2001-06-13 49 2,336
Claims 2001-06-13 4 136
Drawings 2001-06-13 6 111
Correspondence 2001-09-05 1 25
Assignment 2001-06-13 4 154
PCT 2001-06-13 13 445
Assignment 2002-02-20 8 313
Assignment 2002-07-12 5 207
Fees 2003-09-23 1 37
Fees 2002-09-27 1 39
Fees 2004-10-05 1 35
Fees 2005-12-28 1 37