Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
GLUCOSAMINE AND EGG
FOR REDUCING INFLAMMATION
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/153,887, filed September 14, 1999 and U.S. Provisional Application No.
60/192,386, filed March 27, 2000.
BACKGROUND OF THE INVENTION
Field Of The Invention
The invention relates to a method of reducing inflammation in animals. More
particularly, the invention relates to the combination of egg product and
glucosamine to produce a synergistic effect in reducing inflammation, and
particularly arthritis, in animals.
Inflammation
Inflammation, as defined 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
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-
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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In the particular case of rheumatoid arthritis, the resulting inflammation
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 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.
Osteoarthritis produces similar symptoms to rheumatoid arthritis. In
particular,
although osteoarthritis begins as a degeneration of particular cartilage
whereas
rheumatoid arthritis begins as inflammation in the synovium, each process
approaches the other as the disease progresses. In osteoarthritis, as
cartilage
deteriorates and joint congruence is altered, a reactive synovitis often
develops.
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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
to differentiate from rheumatoid arthritis. In some cases ankylosing
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
susceptible
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
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.
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. The
body normally recognizes the difference between its own by-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 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
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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 affects 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
hepatitis (Gershwin, Manns, and Mackay 1992). Immune destruction of the islets
of Langerhans results in diabetes mellitus (Hagopian and Lernmark 1992) and
insulin autoantibodies have been described (Paliner 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 angitis, polyarteritis nodosa, Goodpasture's
syndrome, Kawasaki disease, Wegener's granulomatosis, Churg-Struass
syndrome, giant-cell arteritis, Takayasu arteritis, immune-complex-mediated,
lupus, rheumatoid, 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 including: postpericardiotomy and post myocardial infarction
syndromes, myocarditis, and idiopathic dilated cardiomyopathy. Autoimmunity
may be responsible for the progression of acute disease of heart muscle to
degenerative (Rose, Neumann, Burek, Herskowitz 1992).
Symptomatic involvement of skeletal muscle is common in many autoimmune
diseases such as polymyositis or inflammatory myopathy (which may include
rheumatoid arthritis, polymyalgia rheumatics, myasthenia gravis, myasthenic
myopathy, neurogenic atrophy, motor neuron disease, fibromyalgia, fibrositis,
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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 Sjogren's syndrome, scleroderma,
ankylosing spondylitis, dermatomyositis, psoriasis, psoriatic arthritis,
Reiter's
syndrome (NI~I 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
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
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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
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
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
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
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,
especially when administered in elevated doses. For example aspirin, an NSAm,
may produce indigestion and stomach pain; phenylbutazone may produce stomach
ulcers and phenacetin may lead to kidney disease. Methotrexate may cause oral
ulceration and gastrointestinal (Gn side effects.
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.
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Passive Immunization
Various genera of the class Aves, such as chickens (Gallus 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-
Verheyden et al. (Immunology 27:683 (1974)) and Leslie, G.A., et al. (J. Med.
130:1337 (1969)), have quantitatively analyzed immunoglobulins of the chicken.
Polson, 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
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. Polson et al. (Immunological Communications
9:475-493 (1980)) describe antibodies isolated from the yolk of hens that were
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,
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
venoms.
U.S. Patent No. 4,748,018 discloses a method of passive immunization of a
mammal that comprises parenterally administering purified antibody obtained
from the eggs of an avian that has been immunized against the corresponding
antigen, and wherein the mammal has acquired immunity to the eggs.
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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.
U.S. Patent Application Serial Number 09/233,379 discloses the existence of an
anti-inflammatory factor found in the egg of a hyperimmunized avian.
An immunized egg is an egg which comes from an avian which has been
immunized with, for example, a specific antigen or mixture of antigens. A
hyperimmunized egg is an egg which comes from an avian which has been
brought to a specific state of immunization by means of, for example, periodic
booster administrations of antigens. Hyperimmunized eggs, no matter the type
of
antigen their avian maker has been administered, have been found to have
various
beneficial factors, including, as mentioned above, the treatment of chronic
gastrointestinal disorders, NSAID-induces gastrointestinal damage (see U.S.
Application No. 08/688,576) and anti-inflammatory effects due to the presence
of
an anti-inflammatory composition (see U.S. Application No. 09/233,379).
Glucosamine
Glucosamine a chondroprotective agent which has been studies for its potential
beneficial effects in osteoarthritis. Chondroprotective agents are those
which, in
addition to relieving symptoms, appear to aid in balancing synthesis and
degradation of cartilage tissue. Glucosamine has been known to biologists for
several decades as an endogenous aminomonosaccharide synthesized from
glucose. Its importance in joint dysfunctions relates to its physiologic role
in the
synthesis of proteoglycans and glycosaminoglycans, which are cartilage
components. Research over the past four decades suggests that glucosamine is
effective in reducing the symptoms of joint dysfunction and is well-tolerated.
Glucosamine significantly reduces pain and tenderness and improves mobility.
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Researchers have proposed several mechanisms for glucosamine's putative
benefits in joint health. In vitro studies have suggested that glucosamine
affects
cartilage metabolism. One such effect may be the stimulation of proteoglycan
synthesis, as seen with SAMe. Another possibility is that glucosamine enhances
gene expression of the chondrocyte. In addition, glucosamine may act as an
anti-
inflammatory agent, though the effect is 50 to 300 times lower than NSA>Ds.
However, the NSA>D exert their anti-inflammatory effects via inhibition of the
cyclooxygenase enzyme system and thereby interfering with prostaglandins,
while
glucosamine does not. The anti-inflammatory effect may be the result of
stimulating proteoglycan synthesis, which would stabilize cell membranes. A
recent in vitro study demonstrated that glucosamine increased chondrocyte
adhesion to fibronectin, effectively reversing the abnormal reduction in
adhesion
which occurs in joint degeneration.
SUMMARY OF THE INVENTION
The invention is based on the inventors' discovery that there is anti-
inflammatory
activity when egg and egg products, and in partuicular, hyperimmune egg or egg
products, are combined with glucosamine and administered to a subject animal
in
that such administration results in the reduction, and sometime even
prevention of
inflammation and inflammatory related disorders in that subject animal.
In particular, the invention is directed to a composition comprising
glucosamine
and an egg product.
The invention is also directed to a method for reducing inflammation in a
subject,
the method comprising administering to the subject an effective amount of
glucosamine and an egg product.
The invention is further directed to a method for reducing serum fibrinogen
levels
the method comprising administering to the subject an effective amount of
glucosamine and an egg product.
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The invention further encompasses a method for reducing or preventing the
onset
of rheumatoid arthritis the method comprising administering to the subject an
effective amount of glucosamine and an egg product.
The invention finally covers a method for reducing or preventing the onset of
osteoarthritis the method comprising administering to the subject an effective
amount of glucosamine and an egg product.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a bar graph depicting the effect of hyperimmune egg and
glucosamine
in the rat adjuvant arthritis model.
Figure 2 is a bar graph depicting plasma fibrinogen levels in the rat adjuvant
arthritis model.
DESCRIPTION OF THE INVENTION
The invention relates to the discovery that the combination of egg-product
with
glucosamine works synergistically against inflammation and inflammatory
related
disorders.
The following definitions apply throughout:
Definitions
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
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
plasma proteins, and leukocyte migration into the inflammatory focus.
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The term "arthritis" means any of a variety of disorders marked by
inflammation
and degeneration of connective tissue structures, especially the joints and
related
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.
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 means any whole egg (table,
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, and containing the anti-inflammatory
composition as described in U.S. Serial Number 09/233,379.
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 composition" means the composition disclosed in
U.S. Serial Number 09/233,379 and herein, which counteracts or suppresses the
inflammatory process.
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The term "partially pure egg anti-inflammatory composition" means an anti-
inflammatory composition described as such in U.S. Serial Number 09/233,379.
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.
1 S 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 plus glucosamine product treats arthritis
and/or an autoimmune disease not only by suppressing the symptoms of the
disorder in humans and other 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 subj ect with a
substance,
including orally, intranasally, parenterally (intravenously, intramuscularly,
or
subcutaneously), rectally, topically or intraocularly.
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.
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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.
The Invention
The present invention comprises a combination of hyperimmunized egg product
and glucosamine. According to the principles of the invention, a combination
of
hyperimmunized egg product and glucosamine, in exogenous quantities, is
provided to a subject experiencing an inflammatory reaction in order to reduce
the
inflammation.
In a preferred embodiment, the invention comprises a hyperimmune egg or egg
product in combination with glucosamine which when administered to a subject
animal is effective in reducing inflammation and in treating and preventing
arthritis and/or an autoimmune disease in that subject animal. The hyperimmune
egg is preferably obtained from an egg-producing animal, and more preferably,
an
avian, which has been hyperimmunized with at least one immunogen. The
hyperimmune egg plus glucosamine product is one which is preferably
administered orally to the subject animal. The hyperimmune egg or egg product
and/or glucosamine can be further separated into more potent fractions which
can
subsequently be administered to a subject animal in a variety of forms. It is
further contemplated that the Glucosamine and the egg product can be
administered separately albeit preferably simultaneously. Glucosamine and
table
or hyperimmunized egg product work synergistically to reduce inflammation
when administered in combination to a subject.
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Glucosamine
The glucosamine provides the primary substrate for both collagen and
proteoglycan synthesis. Administration of glucosamine to a subject bypasses
the
glucose to glucosamine rate-limiting step in the subject's natural production
of
collagen and proteoglycans because production of additional quantities of
collagen
and proteoglycans become available for use by the subject's natural healing
processes to repair connective tissue.
The glucosamine is, preferably, in a salt form so as to facilitate its
delivery and
uptake by the subject. The preferred salt forms are glucosamine hydrochloride
and glucosamine sulfate, among others. The use of the non-salt glucosamine is
believed by many to fail to provide bioavailable glucosamine because the
compound is not absorbed in the gastrointestinal tract. It is noted that in
the case
of the glucosamine sulfate, the sulfate may be available for later use in
catalyzing
the conversion of glucosamine to glycosaminoglycans. The unsulfated form is
desired for the production of hyaluronic acid.
Regarding the administration of glucosamine, it is rapidly and almost
completely
absorbed into humans and other animals through oral administration and, as
such,
oral administration is most preferred. A significant portion of the ingested
glucosamine localizes to cartilage and joint tissues, where it remains for
long
periods of time. This indicates that oral administration of glucosamine
reaches
connective tissues, where the glucosamine gets incorporated into newly-
synthesized connective tissue.
E~~ Product
The egg product is any egg or fraction of egg obtained from an egg-producing
animal. In a preferred embodiment, the egg product of the invention is an egg
obtained from an avian which has been manipulated, such as by
hyperimmunization, to produce among other things supranormal levels of an anti-
inflammatory composition in the egg (see U.S. Application Serial No.
09/233,379).
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Hynerimmunization Of The E~~-Producing Animal
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
hyperimmur~e 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 when administered in combination with glucosamine 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
immunization against avian diseases and during normal exposure to
environmental factors. It is only in the specific hyperimmune states that the
eggs
have the desired supranormal levels of the anti-inflammatory composition.
This special state of hyperimmunization, in which the egg will become
effective,
in part because it 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 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
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amount of immunogen administered, depending on the egg-producing animal
genera and strain employed, in order to maintain the animal in the hyperimmune
state.
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
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
immunogeri 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 occurnng immunogens, immunization
may also be accomplished using immunogens which are synthetically derived by
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.,
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
antigen encoding sequence) will trigger an immune response. Genetic vaccines
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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
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
to six months.
It is preferred that the amounts of DNA used are generally in the order of 50-
300
pg of DNA in saline for direct injection. For particle bombardment, 4-100 p.g
of
DNA co-precipitated onto gold beads by the addition of 2.5 M CaCl2 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:
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1. Selecting one or more inununogens.
2. Eliciting an immune response in the egg-producing animal by
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.
S. 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
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
families: adenoviruses, picornaviruses and herpes viruses, although other
viral
antigen families will work.
In a preferred embodiment, a polyvalent vaccine referred to as PL 100 is used.
The bacteria included in the PL-100 vaccine are listed in Table 1 of Example
1.
This vaccine has been previously described in U.S. Patent Number 5,772,999 and
U.S. Patent Application Serial Number 09/233,379 both assigned to DCV, Inc.
Step 2: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
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intramuscular injection. The preferred muscle for injection in an avia-i 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
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.
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
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.
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Step 4. It is appropriate 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.
Sten 5. This step involves the collection and processing of the
hyperimmunized egg(s). 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 anti-inflammatory
compositions as described in U.S. Serial Number 09/233,379.
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 hyperlrnmune egg product, which can subsequently be
administered to a subject animal in combination with glucosamine to treat an
inflammatory disorder.
The egg itself or fractions thereof, including the partially purified anti-
inflammatory composition of the present invention, are administered to a
subject
animal in combination with glucosamine 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 in combination with glucosamine to the 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, glucosamine and the egg or any fraction thereof,
including the partially purified anti-inflammatory composition, are integrated
into
a nutritional supplement. With particular regard to the egg, 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
WO 01/19374 CA 02384246 2002-02-28 pCT/[JS00/24484
methods are known for drying eggs, spray drying is a preferred method. The
process of spray drying eggs is well known in the art.
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
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.
The inventors submit that the glucosamine and egg, although preferably
administered in combination, do not need to be administered in the same form
or
in one product. For instance, the glucosamine could be taken in pill form
while
the egg product is administered as a drink.
Should one prefer to combine the partially pure anti-inflammatory composition
with glucosamine, then the egg can even be further separated to purify the
partially
purified anti-inflammatory composition, as described earlier which will allow
for
other modes of administration such as separately 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.
With particular reference to the partially pure anti-inflammatory composition,
oral
administration is preferably accomplished through solid dosage forms which
include capsules, tablets, pills, powders and granules, among others. In solid
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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.
Combination of Glucosamine and Immunized E~~ Product
Glucosamine and egg product, as described above, work synergistically, when
administered concurrently to a subject, to reduce inflammation in that
subject.
With regard to the administration of both glucosamine and immunized egg
product, in one embodiment, an effective amount of glucosamine is administered
to the subject independently of an effective amount of immunized egg product.
In
an alternate embodiment, the subject is administered a composition comprising
an
effective amount of glucosamine and immunized egg product. Either method of
administration will effectuate a synergistic effect.
The administration to the subject should be carried out in an amount that is
effective for treating or preventing inflammation. It is preferred that the
subject is
administered a higher dosage of glucosamine mixed with a lower dosage of
immunized egg product. Depending on the particular disorder it may be more
preferred vice versa, then such unequal dosages are appropriate for treatment
and
prevention, and should be administered in those amounts. Due to the
synergistic
effect of these two compositions, administration may need to be adjusted
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accordingly. Those having skill in the art are familiar with determining
dosage
amounts that will best treat and prevent inflammation of interest.
The glucosamine and egg of the present invention may be administered by any
means that provide anti-inflammatory activity. For example, administration may
be parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,
intranasal, or oral.
Oral administration is preferably accomplished through solid dosage forms
which
include capsules, tablets, pills, powders and granules, among others. In solid
dosage forms, the glucosamine and egg product are 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 glucosamine and egg product combination 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.
Preparations of the glucosamine and egg product combination 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.
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
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desired therapeutic effect, on the route of the administration and on the
duration of
the treatment.
Administration dosage and frequency will depend on the age and general health
condition of the patient, taking into consideration the possibility of side
effects.
Administration will also be dependent on concurrent treatment with other drugs
and patients' tolerance of the administered drug.
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.
With regard to administration to a subject of the glucosamine, it has been
determined, and is detailed in the following examples, that the preferred dose
range of glucosamine to be given to a subject is between 10 milligrams to 5
grams
per kilogram of subject weight. And more preferably, 100 milligrams to 2.5
grams of subject weight.
In a preferred embodiment, a 50 1b subject suffering from inflammation should
be
administered a single tablet, twice a day, comprising 250 mg of glucosamine
HCI,
750 mg of hyperimmunized egg product and 320 mg of coated ester C. The
administration is continued as long as needed to reduce or prevent
inflammation.
Duration and intensity of the treatment will depend on the particular
condition and
the advancement of the subject's condition.
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Examples of inflammatory conditions that may be treated by administration of
the
glucosamine and egg product of the present invention include 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, Sjogren syndrome among others; acute and subacute bursitis,
acute non-specific tendonitis, systemic lupus erythematosus, systemic
dermatomyositis, acute rheumatic carditis, pemphigus, bullous dermatitis,
herpeteformis, severe erythema, multiform exfoliative dermatitis, cirrhosis,
seasonal perennial rhinitis, bronchial asthma, ectopic dermatitis, serum
sickness,
keratitis, opthalmicus iritis, diffuse ureitis, chorditis, optic neuritis,
sympathetic
ophthalmia, symptomatic sarcoidosis, Loeffler's syndrome, berylliosis,
hemolytic
anemia, mastitis, mastoiditis, contact dermatitis, allergic conjunctivitis,
psoriatic
arthritis, ankylosing spondylitis, acute gouty arthritis, herpes zoster
rheumatoid
arthritis, osteoarthritis, any other degenerative joint diseases, and any
other related
autoimmune diseases. Further, the glucosamine and egg product combination may
be used to treat individuals who are exposed to potentially inflammatory
agents
such as allergens.
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,
Glucosamine in combination with the hyperimmune egg product or any active
fraction thereof, including the partially 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 the condition in the subject. Glucosamine in
combination with 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
WO 01/19374 CA 02384246 2002-02-28 pCT/(jS00/244g4
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 in combination with an effective dose of glucosamine 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.
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
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
Preparation of PL-100 Vaccine
A bacterial culture containing the spectrum of bacteria shown in Table 1
below, as
obtained from the American Type Culture Collection, was reconstituted with 15
mL of media and incubated overnight at 37 C. Once good growth was obtained,
approximately one-half of the bacterial suspension was employed to inoculate
one
liter of broth with the inoculate being incubated at 37 C.
After good growth was visible in the culture, the bacterial cells were
harvested by
centrifugation of the suspension for 20 minutes to remove the media. The
bacterial pellet obtained was resuspended in sterile saline solution and the
bacterial sample was centrifuged three times to wash the media from the cells.
After the third sterile saline wash, the bacterial pellet was resuspended in a
small
amount of double distilled water.
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The media-free bacterial suspension was killed by placing the suspension in a
glass flask in an 80 C water bath overnight. The viability if the broth
culture was
tested with a small amount of killed bacteria, incubated at 37 C for five days
and
checked daily for growth to certify that the bacteria had been killed.
The killed bacteria were lyophilized until dry. The dry bacteria were then
mixed
with sterile saline solution to a concentration of 2.2 x 108 bacterial
cells/mL saline
(1.0 optical density reading at 660 nm). Bacteria contained in PL-100 vaccine
are
listed in Table 1 below.
TABLE 1
PL-100 Bacterial List
Escherichia coli Escherichia coli (Aerobacter)
Klebsiella pneumoniae Pseudomonas aeruginosa
Salmonella typhimurium Salmonella dysenteriae
Salmonella enteriditis Salmonella epidermis
Salmonella simulans Streptococcus pyogenes, type 1
Streptococcus pyogenes, type 3 Streptococcus pyogenes, type 5
Streptococcus pyogenes, type 8 Streptococcus pyogenes, type 12
Streptococcus pyogenes, type 14 Streptococcus pyogenes, type 18
Streptococcus pyogenes, type 22 Pseudomonas vulgaris
Streptococcus agalactiae Streptococcus mitis
Streptococcus mutans Streptococcus salavarius
Streptococcus sanguis Streptococcus pneumoniae
Propionibacterium acnes Haemophilia influenzae
Immunization Procedure for Hyperimmunized 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 was injected into the breast muscle of a chicken. For
the
remaining vaccines, the bacterial preparation was mixed with incomplete
Freund's
adjuvant and injected into the chickens at two week intervals for six months.
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EXAMPLE 2
Obi ective
To assess the synergistic antiarthritic effect of Glucosamine hydrochloride
and the
DCV hyperimmune egg in the established rat adjuvant arthritis, a chronic
animal
model of inflammation.
Results
Table 1 shows all the paw edema data on days 14, 22 and 30 of the study. The
results indicate that the PL-100 + Glucosamine HCl group showed the highest
percent inhibition of inflammation on days 14 and 30 of the disease. The PL-
100
egg and Glucosamine hydrochloride by itself showed inhibitory effect on days
14
and 22 of the study, while they both showed ineffectiveness on day 30 when the
disease has rapidly progressed to a severe polyarthritis.
Table 2 shows all the serum fibrinogen levels on days 16, 23 and 30 of the
study.
The results indicate that the Fibrinogen levels initially increase on day 16
followed
by a drop in levels on day 23 and 30. However, the increase in levels on day
16 is
least in PL-100 + Glucosamine HCl group as compared to PL-100 and
Glucosamine HCl alone, and also the inhibition of serum fibrinogen levels is
highest in PL-100 + Glucosamine HCl group as compared to the PL-100 and
Glucosamine HCl groups individually. Also, on day 30, the percent inhibition
of
fibrinogen levels in the PL-100 + Glucosamine HCl group is 32% as compared to
46% in the Indomethacin treated group.
Table 3 shows all the Histomorphologic observations on day 22 and 30 of the
study. The results show that the inflammation of periarticular/ soft tissue, (
which
indicates the infiltration of mononuclear and/or polymorphonuclear
inflammatory
cells into the soft tissues, including synovial membranes around the joint )
is least
affected in the PL-100 + Glucosamine group both on day 22 and 30 as compared
to the other groups.
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The Hyperostosis condition, ( which indicates increased and excessive amounts
of new bone growth, particularly in subperiosteal areas ) is least in the PL-
100 +
Glucosamine HCI group on day 22 and low on day 30 as compared to the other
groups.
The cartilage erosion on day 22 and 30 is least in the PL-100 + Glucosamine
HCI
group as compared to the other groups.
The pannus cartilage, ( an accumulation of inflammatory cells, exudate, fibrin
or
fibrous tissues on joint surfaces ) is least affected on day 22 and 30 in the
PL-100
+ Glucosamine HCl treated group as compared with the other groups.
The Ankylosis, ( which denotes enlarged and immobile joints due to the
formation
of fibrous and inflammatory tissues between joint surfaces, usually with
complete
loss o0f cartilage and replacement in this area with fibrous tissue that
results in
adhesions and joint cavities ) is minimal in the PL-100 + Glucosamine HCl
group
as compared with any other group.
Finally, the osteolysis, ( which indicates the necrosis and lysis of the bone
) is
absent in PL-100 + Glucosamine HCl treated group on day 22 as compared to the
other groups, where there is some degree of lysis present.
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Table 1
DCV Rat Adiuvant
Effect of Immune Egg Preparations, Glucosamine, singly and in
combinations, and CAF in the Rat Established Adjuvant Arthritis Model
GP Orala Day 14-Mean% Day 22-Mean% Day 30-Mean
TreatmentPaw Edeman Chg Paw Edema Chg Paw Edema Chg
mlSE,n (mlSE,n mlSE,n
1 PL-200 1.5 0.2, +15 1.7 0.1, -26 2.4 0.3, +14
+ n=8 n=7 n=5
GLU-250
2 PL-100 0.7 0.3, -46 1.2 0.2, -37 2.5 0.3, +19
n=5 n=5 n=5
3 PL-100 0.5 0.3, -61 1.4 0.2, -26 1.8 0.2, -14
+ n=6 n=6 n=6
GLU-250
4 GLU-250 1.1 0.3, -15 1.6 0.3, -16 3.1 0.2, +48*
n=7 n=7 n=5
5 CAF 0.8 0.2, -38 1.8 0.3, -5 2.3, n=2 +10
n=5 n=5
6 Whole 1.2 0.4, -8 2.2 0.5, 16 1.9 0.2, -10
E n=5 n=5 n=5
7 Indo 1.2 0.4, -8 1.2 0.1, -37 0.3 0.1, -86*
- n=6 n=6 n=5
lm
8 Control 1.3 0.3, - 1.9 0.2, - 2.1 0.2, -
Ad'uvantn=8 n=8 n=6
9 Normal Ob, n=3 - 0.2, n =3 - 0.3, n=2 -
a - Test articles were given in 1 mL of deionized water, when not in a
combination an extra 1 mL of water was given to each rat
b - Normal paw growth
*P<_0.05 from Control Adjuvant (Dunnett's Test)
W~ 01/19374 CA 02384246 2002-02-28 pCT/US00/24484
Table 2
Effects of Immune Egg Preparations, Glucosamine, Singly And In
Combinations with PL-100 and Chondroitin S04, And CAF in the Rat
Established. Adjuvant Arthritis Model
Mean
Plasma
Fibrino
en
Levels
m
/dl
SE
GP Treatment Oral n Day 16 %0 Day 23 %0 Day 30 %D
Dose from from from
ctrl ctrl ctrl
1 PL 100 2 ml 5 841 +35 707 -14 550 89 -19
34 70
2 Gluco HCI 2 ml 5 735 +18 803 -2 487 112 -28
44 39
3 Gluco HCI 2 ml 5 735 +7 710 -14 457 42 -32
+ PL 29 37
100
4 CAF 2 ml 5 788 +26 776 -6 560 59 -17
57 58
5 Gluco HCI 2 ml 5 658 +5 701 -15 574 109 -15
+ 84 51
Chond S04
6 Indomethacin4mg/kg 5 590 - 617 -28 366 48 -46*
86a 72
n=4 b
7 Control Ad'uvant2 ml 5 _625 - 822 - 672 72 -
73 40
8 Normal ~ ~5 186 - ~ 178 - 193 9 -
~ 5 4
a - Control fibrinogen levels since indomethacin was not dosed until Day 16.
b - One of the plasma samples clotted
~ p<_0.05 Dunnett's test
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Table 3
Incidence Of Histomorphologic Observations
Control IndomethacinGlucosamineTable PL-100 PL 100 Egg
E E &
Glucosamine
TibiotarsalDay 22 Day 22 Day 22 Day 22 Day 22 Day 22 /
Joint / 30 / 30 / 30 / / 30 30
&E 30
Inflammation,3.5 / 3.5 / 3.5 3.5 / 2.5 / 2 / 3.5 1.0 / 3.0
eriarticular3.5 4.0 4.0
H erostosis3.0/2.5 3.0/2.0 3.0/3.5 1.0/2.0 0.5/3.0 0.0/2.5
Erosion, 3.0/2.0 1.5/2.5 2.0/4.0 1.5/2.5 1.0/3.0 0.0/1.0
Cartlia
a
Pannus, 3.0/3.0 1.5/2.5 2.0/4.0 1.5/2.5 1.0/3.0 0.0/1.0
Cartlia
a
losis 2.0/3.0 1.5/1.5 3.5/4.0 1.5/2.0 1.0/3.0 0.0/1.5
Osteol 2.5/1.0 2.5/1.0 1.5/3.5 1.0/2.0 0.0/3.0 0.0/2.5
sis
Tibiotarsal
Joint
PAS
Erosion, 3.0/3.0 1.5/2.5 2.0/4.0 1.5/2.5 1.0/3.0 0.0/1.0
Cartila
a
Key: 0 Within normal limits
1 Minimal degree or amount of indicated change
2 Mild degree or amount of indicated change
3 Moderate degree or amount of indicated change
4 Severe degree or amount of indicated change
15
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Summary
In the rat adjuvant arthritis model, which is a chronic animal model for
inflammation, the PL-100 egg and Glucosamine HCl showed an additive effect in
a) reducing the left hind paw swelling of the animal, thereby showing anti-
inflammatory effect on days 14, 22 and 30,
b) reducing the serum fibrinogen levels ( which is an accepted serum marker
for
inflammation ) after day 22 of injection, and continued till day 30, which was
the end of the study,
c) minimizes the effect on the periarticular / soft tissues from the
infiltration of
mononuclear and polymorphonuclear cells on day 22, when the intensity of
rheumatoid arthritis is at its peak
d) completely protects from hyperostosis on day 22 of the study,
e) completely protects the erosion of the cartilage around the arthritic
joints, on
day 22 and minimizes the damage on day 30,
f) completely protects the cartilage in the pannus area on day 22 , and
minimizes
the damage on day 30,
g) completely protects the affected joints from ankylosis on day 22,
completely protects bones in the arthritic joint area from osteolysis on day
22.
h) figure 1 shows all the paw edema data on days 14, 22 of the study. The
results
indicate that the PL-100 + Glucosamine HCl group showed the highest percent
inhibition of inflammation on day 14 of the disease.
i) figure 2 shows all the serum fibrinogen levels on days 16, 23 and 30 of the
study. The results indicate that the Fibrinogen levels initially increase on
day
16 followed by a drop in levels on day 23 and 30. However, the increase in
levels on day 16 is least in PL-100 + Glucosamine HCl group as compared to
PL-100 and Glucosamine HCl alone, and also the inhibition of serum
fibrinogen levels is highest in PL-100 + Glucosamine HCl group as compared
to the PL-100 and Glucosamine HCl groups individually. Also, on day 30, the
percent inhibition of fibrinogen levels in the PL-100 + Glucosamine HCl
group is 32% as compared to 46% in the Indomethacin treated group. Thus
PL-100 + Glucosamine HCl not only restricts the severity of inflammation at
33
CA 02384246 2002-02-28
WO 01/19374 PCT/US00/24484
the beginning of the disease, but also inhibits it towards the end of the
study
(as indicated by the serum inflammatory marker levels).
34
