AU1248400A

AU1248400A - Egg anti-inflammatory composition and method of treating and preventing inflammation

Info

Publication number: AU1248400A
Application number: AU12484/00A
Authority: AU
Inventors: Lee R. Beck; Sandra G. Fitzpatrick-Mcelligott; Jeffrey G. Hunchar; Young-Zoon Lee
Original assignee: Arkion Life Sciences LLC
Current assignee: Arkion Life Sciences LLC
Priority date: 1996-03-26
Filing date: 2000-01-19
Publication date: 2000-07-20
Anticipated expiration: 2017-03-19
Also published as: JP2002535279A; NZ502421A; CA2355168A1; EP1143984A1; KR20010101565A; MY135972A; AU767570B2; WO2000043019A2; WO2000043020A1; AU2372400A; AU2389800A; KR100789488B1

Description

P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION

V.

FOR A STANDARD PATENT

ORIGINAL

TO BE COMPLETED BY APPLICANT Name of Applicant: DVC, INC.

Actual Inventor(s): Sandra G. Fitzpatrick-McElligott: Jeffrey G. Hunchar; Young-Zoon Lee; Lee R. Beck Address for Service: CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia Invention Title: EGG ANTI-INFLAMMATORY COMPOSITION AND METHOD OF TREATING AND PREVENTING INFLAMMATION The following statement is a full description of this invention, including the best method of performing it known to me:- 19/01/00,TDI 1057.CS.I la-

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 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 preventing inflammation. The invention specifically relates to an egg antiinflammatory composition, methods for its production in partially purified form, and methods for its use in the treatment of inflammation, (in particular 15 arthritis), and autoimmune diseases.

Inflammation Inflammation, as defined in Dorland's Medical Dictionary, is "a localized protective response, elicited by injury or destruction of tissues, which serves 20 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, 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.

19/01/00.td11057.spe.1 CV-0083A PCT 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 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 pain and loss 15 of function) that has come to be associated with different diseases and injuries.

9 Arthritis Arthritis manifests itself in a variety of forms. Some of the more common forms Io 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 and 40 years, while the occurrence of clinical illness is greatest among those aged 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 approaches the other as the disease progresses. In osteoarthritis, 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 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 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 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. (Harris, 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 byproducts and foreign invaders 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 CV-0083A PCT 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 Lemmark 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 Sch6nlein 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 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 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 Sj6gren's syndrome, scleroderma, ankylosing spondylititis, dermatomyositis, psoriasis, psoriatic arthritis, Reiter's syndrome (NIH 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 15 (Singh et al., 1993).

Several neurologic diseases such as Sydenham's Chorea, chronic obsessivecompulsive disorders (OCD), attention deficit hyperactivity disorder (ADHD), Tourette's Syndrome (TS) and some cases of schizophrenia may have an autoimmune component and may be associated with anti-neuronal antibodies (Medical Sciences Bulletin, Sept. 1994).

o This summary is not all inclusive and those in the art are familiar with other autoimmune diseases, such as, for example Guillain-Barr6 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 antiinflammatory. Anti-inflammatory drugs are used for the treatment of a wide spectrum of disorders, and the same drugs are often used to treat different CV-0083A PCT diseases. Treatment with anti-inflammatory drugs is not for the disease, but most often for the symptom 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 widelyused 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 NSAID, 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 (GI) 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.

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, et al. Med.

130:1337 (1969)), have quantitatively analyzed immunoglobulins of the chicken.

Poison, 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, 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 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 CV-0083A PCT 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.

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 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 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.

I In particular, the invention is directed to a partially purified anti-inflammatory 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 supranormal 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.

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 hyperimmunized avians, which when administered to a subject animal, in 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 S 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 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 antiinflammatory composition by Sepharose Column.

Figure 4 is a chromatogram of the HPLC separation of the partially pruified antiinflammatory composition from hyperimmune egg yolk.

Figure 5 is a graph showing the effect of hyperimmune egg on the incidence of arthritis using the Rat Type II Collagen Model.

CV-0083A PCT 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 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.

Therefore, it is surprising that administration of hyperimmune egg or egg product obtained from avians immunized against a mixed antigen vaccine is effective in S'reducing the symptoms of and preventing inflammation and autoimmune diseases when administered to a subject.

DEFINITIONS

The following definitions apply throughout: set.

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.

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 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.

a 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.

CV-0083A PCT The term "partially pure egg anti-inflammatory composition" means an antiinflammatory 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.

g *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 Sprevented, 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 20 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.

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.

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, antibody.

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 15 effects, except, of course, for allergic reactions in those intolerant to eggs.

In one embodiment, the invention comprises a hyperimmune egg or egg product S .which is effective in treating and preventing arthritis and/or an autoimmune disease in a subject animal. The hyperimmune egg is obtained from an eggproducing animal, and more preferably, an avian, which has been hyperimmunized with at least one immunogen. The hyperimmune egg product is one which is o*o.

preferably administered orally to the subject animal. The hyperimmune egg or egg S-product can be further separated into more potent fractions which can i 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 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 CV-0083A PCT s present in hyperimmune eggs at supranormal levels which provide antiinflammatory 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: 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. In-a preferred embodiment, the composition is purified from 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 CO 2 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 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) 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 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 DEAE-Sepharose chromatography. In S a specific disclosed embodiment, the preferred ultrafiltration is by Amicon S. RA 1000 (3K MWCO) and DEAE ion-exchange chromatography. It is 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.

Step 4: 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 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.

CV-0083A PCT 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 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 i" 15 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 S, 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 antiinflammatory composition can be tested on edema caused by injecting carrageenan into rat footpads (Winter, Risley, 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, 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)).

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; 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; 9.* 96) 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 25 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.

CV-0083A PCT 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 antiinflammatory composition partially purified from egg white.

It is the inventors' finding that supranormal levels of the partially purified antiinflammatory composition can be isolated from whole egg, egg yolk and eggwhite from an egg-producing animal which has been hyperimmunized.

HYPERIMMUNIZATION OF THE EGG-PRODUCING ANIMAL As just described, it is the inventor's finding 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 15 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 ofimmunogens, the Sanimals 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 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 antiinflammatory composition.

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 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.

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, i 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 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 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., CV-0083A PCT Science 251:767 (1991); Houghton, R. et al., Nature 354:82 (1991)) as well as small organic molecules (Bunin, B. Ellman, 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 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 oo 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 ofparenteral 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 jig of DNA in saline for direct injection. For particle bombardment, 4-100 jig of DNA co-precipitated onto gold beads by the addition of 2.5 M CaCl 2 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 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.

15 5. Collecting and processing the eggs.

.o Below is a more detailed description of this procedure.

Step 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.

CV-0083A PCT 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.

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 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 i 15 hyperimmunization process, much smaller quantities are required, generally 300 micrograms.

oooo• It can be determined whether the vaccine has elicited an immune response in the oo 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 S•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 antigen(s) 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.

Step 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) :i Step 5. This step involves the collection and processing of the egg(s) containing the anti-inflammatory composition. The egg can be collected by S 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 antiinflammatory composition of the present invention, are administered to a subject 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 CV-0083A PCT 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 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.

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 15 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 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 S"encapsulated products and pharmaceutical compositions with said egg or fraction ooooe S"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 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.

S:i !"Besides inert diluents, compositions can also include wetting agents, emulsifying, and suspending, and sweetening agents.

S.i The hyperimmune egg or egg product, including the partially purified antiinflammatory 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, 20 reactive arthritis, chronic fatigue syndrome, fibromyalgia (fibrositis) and gout.

S 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, thehyperimmune 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 CV-0083A PCT 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 15 Example 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 20 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 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 antiinflammatory composition of the invention in this disease model, which was also seen in rats (see Example It is contemplated that the egg product of the invention is effective in reducing antibodies involved with other autoimmune diseases such as juvenile diabetes, multiple sclerosis, Graves' disease, Meneri'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 (NSAIDs) 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 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 S•autoimmune diseases to produce a synergistically better effect.

15 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 NSAID-induce gastrointestinal damage. Therefore, an alternative embodiment is contemplated for cases where the subject is taking or is 20 preparing to take NSAIDs 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 SNSAID-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 NSAIDs 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 invention include acute and subacute bursitis, acute non-specific tendinitis, systemic lupus erythematosus, systemic dermatomyositis, acute rheumatic carditis, pemphigus, bullous dermatitis, herpeteformis, severe erythema, multiform exfoliative dermatitis, cirrhosis, seasonal perennial rhinitis, bronchial asthma, CV-0083A PCT 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 isolated and purified egg product may be used to treat individuals who are exposed to potentially inflammatory agents such as allergans.

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 1 subject is between 100 milligrams to 10 grams per kilogram of subject weight.

S*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 20 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 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 S-100 VACCINE 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 onehalf 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.

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

S

heat-killed bacteria, incubated at 37C for five days and checked daily for growth, 20 as the bacteria have to be killed for use in the vaccine.

S

The heat-killed bacteria were lyophilized until dry. The dry bacteria were then Smixed with sterile saline solution to a concentration of 2.2 x 108 bacterial cells/ml saline (1.0 optical density reading at 660 nm).

TABLE 1 Antigens in the S-100 Vaccine Name Media Name MediaNae edia Staphylococcus simulans BHI Pseudomonas aeruginosa BHI Staphylococcus epidermidis BHI Klebsiella pneumoniae BHI Streptococcus pyogenes, A Type 1 APT Salmonella typhimurium BHI Streptococcus pyogenes, A Type 3 APT Haemophilus influenzae BHI Streptococcus pyogenes, A Type 5 APT Streptococcus mitis APT CV-0083A PCT Streptococcus pyogenes, A Type 8 AT Proteus vulgaris BHI Streptococcus pyogenes, A Type 12 APT Shigella dysenteriae BHI Streptococcus pyogenes, A Type 14 APT Diplococcus pneumoniae APT Streptococcus pyogenes, A Type 18 APT Propionibacter acnes Broth Streptococcus pyogenes, A Type 22 APT Streptococcus sanguis APT Aerobacter aerogenes BHI Streptococcus salivarus APT Escherichia coli BHI Streptococcus mutans BHI Salmonella enteritidis BHI Streptococcus agalactiae APT 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 i* 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 15 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.

Example 2 PREFERRED METHOD FOR PREPARING A PARTIALLY PURIFIED 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 yolk and both were spray-dried. Hyperimmune eggs were obtained as described in Example 1. Egg white powder was processed separately to obtain the aqueous fraction for ultra-filtration.

s 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 s15 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 nondissolved 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 gm Sterile disposable Nalgene filter and lyophilized or frozen, for storage, bioassay testing or further purification.

Figure 1 shows a flow chart of the purification process.

CV-0083A PCT Molecular species below 3,000 daltons from egg yolk contain the antiinflammatory composition in a low molecular weight, non-aggregated form. From 400 grams of starting material, the yield of anti-inflammatory composition was approximately 12 grams or 3% of the total. Bioassay testing of this 3KDa fraction for anti-inflammatory activity showed high levels of activity (Example Example 2b PREPARATION OF EGG ANTI-INFLAMMATORY ACTIVITY FROM EGG WHITE POWDER Four hundred grams of egg white, isolated from both hyperimmunized egg as 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 Lim filter and ultrafiltered 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 DEAE 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 DEAE 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 8.0. 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 DEAE 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 antiinflammatory 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 ml of 20 mM ammonium acetate, pH 6.5, to remove unbound materials. 200 ml 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 4.0. DEAE Sepharose column was finally regenerated with 400 ml of 1.5 N NaCl in 20 mM ammonium acetate, pH 6.5 (Figure 3).

Figure 3 shows a typical chromatogram of DEAE 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 15 DEAE separation of 3,000 Dalton permeates of delipidated egg yolk and egg white.

o.

9 9 Fraction Elute Vol(ml) Weight Recovery Egg yolk I 1-22 294 11.79 78.6 II 23-40 252 0.40 2.7 111 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 Im 47-60 196 0.32 1.8 IV 61-73 182 0.44 2.4 Example 4 CV-0083A PCT HPLC ANALYSIS OF 3,000 DALTON PERMEATES AND THEIR DEAE 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-M 10A VP Diode array detector, LC-6AD liquid chromatography, SCL-1OA VP Degassor, LPM-600 Low pressure mixing, and CTO-10A 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 lm filter. 20 pl of each sample was applied on a Water Symmetry C18 column (3.9 x 150 mm) at 29 0 C and with a flow rate of 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).

oe 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 i 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 P1-100 egg yolk shows 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 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 H 2 0/methanol with 0.05% TFA, at a flow rate of 90 ml per minute, the permeate was separated into the partially purified composition collected and lyophilize. Next the collected peak was retested on an analytical C8 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, 15 NMR are being used to determine the structure of the factor.

Example ANTI-ARTHRITIC PROPERTIES OF HYPERIMMUNE EGG PRODUCT IN A COLLAGEN-INDUCED ARTHRITIS MODEL IN RATS 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 collageninduced 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 immunosuppressive 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 CV-0083A PCT 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.

Method 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 rats/treatment regimen with total of 90 rats for this study. Spray dried 15 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 0 C until use. Egg Product (3.5ml 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), 50X hyperimmune egg product (High Dose) (3.5ml of a solution of hyperimmune egg product) and IX hyperimmune egg product (Low Dose) (3.5ml 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 and paw swelling (0 for each limb.

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 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 dosedependent 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 5 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 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 PI-100 whole eggs. Upon separation of the egg, the yolk showed 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 CV-0083A PCT pure fraction showed high antiarthritic activity which was comparable to that shown by the whole P1-100 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.

TABLE 4 Average Day to Maximum Incidence of Arthritis 9* S* o*oo o4 °oo*o* *o oooo Control Low Dose High Dose Expt 1 14 19 Expt2 14 18 17 Expt3 12 15 Delay in onset of maximum arthritis after immunization with Collagen II.

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+.0.55 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 20 significantly suppressed in the high-dose group compared to control (30% vs.

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.

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 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 15 high and low dose the hyperimmune egg product is nontoxic.

Example 6 REDUCTION OF COLLAGEN n ANTIBODIES Rats from Example 5 were bled on Day 21 and serum samples were collected. A 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 25 TABLE Control Low Dose High Dose Antibody titers to 9.3 0.03 8.5 0.28 8.3 0.36 Collagen II (-log 2 Statistical p< 0.004 p< 0.005 Significance The decrease in autoimmune antibody titers to Collagen II in the serum of animals gavaged with egg product when compared to control animals (no egg product) is CV-0083A PCT 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 I indicates a positive influence in both the cellular and humoral immune response for the hyperimmune egg product.

Example 7 ANTI-INFLAMMATORY EFFECT OF HYPERIMMUNIZED EGG This example shows the anti-inflammatory effect of hyperimmunized egg (which is prepared as described in Example 1) 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 15 ibuprofen at 10 mg /kg, ED 50, dose in this test.

*i 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 *1**o o 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 were fed the basal diet along with hyperimmune egg (HIE).

S* Group 3 received 3.5 grams of hyperimmune egg, while group 4 received grams of hyperimmune egg added to the diet. At the end of the conditioning diet treatment, the dogs were challenged with an intra-dermal injection of 2% carrageenan and an inflammatory response was elicited.

The challenge process was as follows. One dog from each group was randomly 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 10mg/kg ibuprofen orally.

All dogs were anesthetized by intravenous injection and shaved on the left lateral side (approximately 6 X 8 inches). Two lines of 3 marks were made on the 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 Treatment Number of Mean Mean Significance Animals Per Inflammatory Difference Group Response From Control P value 1 Basal Diet 5 4.41 Control 2 Ibuprofen 5 2.25 2.2 P<0.01 Drug (10mg/kg) Basal Diet 3 HIE egg 5 2.8 1.6 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 CV-0083A PCT 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, 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 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 S"results of the treatments of each of these patients: PATIENT #1 Patient #1 was a 29-year-old female, 5'3" 170 lbs 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.

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 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/d. She continued medic ations and as scheduled, by the protocol, discontinued use of product.

After one month without the product, swelling and tendermess 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 20 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 ofO 0 (no swelling, pain or tenderness).

Scores generated for Patient #1 's arthritic condition were as follows: At presentation= 14 One month onegg-based drink 2 Two months on egg-based drink 6 One month after discontinuing drink= 8 PATIENT #2 CV-0083A PCT Patient #2 was an 88-year-old female 5'3" 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 5mg/week, Orudis KT 2/day, Ca 1500mg, Fosomax 10mg, Prednisone 5mg 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 better", "feeling generally better" and "walking better." She was still in pain, 15 although her pain attacks were less frequent. Hematology results were ESR (30mm/hr) and CRP (1.64mg/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% 20 better". Her functional limitations however, remained the same. Hematology results indicated a drop in her ESR (21 mm/hr) and a return to normal in the CRP •reading.(<0.5mg/dl). Her total cholesterol remained at 234 mg/dl. Patient continued her medications but, as scheduled, discontinued the egg-based drink.

25 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 worse". Her ESR (27mm/hr) levels and cholesterol (257 mg/dl) started to climb, but her CRP (<0.5mg/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 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= One month on egg-based drink 7 Two months on egg-based drink 2 One month after discontinuing drink= 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 o15 moving arms and exhaustion. Hematology results were normal ESR and CRP and -cholesterol readings at 217 mg/dl.

After one month of drinking the egg-product, along with the above-mentioned medication, the patient felt "slightly better 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 S °previously reported. His ESR and CRP readings were normal and his cholesterol level was 180 mg/dl.

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 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 CV-0083A PCT "exhausted". ESR and CRP. readings were normal and cholesterol remained at 180 mg/dl.

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 15 One month after discontinuing drink s.

*go 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 i 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 1X8 Dowex AG 4X4 HPAEC/PAD Preparation Strong Cation Weak Anion Exchange Exchange 1 2M Acetic Acid 46 Hydrolysis, 3 hrs.

Release And Purification Of Sialic Acids From Glycoproteins Sialic acid release with relatively low O-acetyl loss was achieved with 1ml of 2M acetic acid at 80 0 C for 3 hours in a sealed, crimp-top vial. KDN is added as a prehydrolysis 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 (0.5 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 20 vacuum centrifugation and the sample was redissolved in 0.5 ml of water.

oS The sample was applied to a 1 ml column of Dowex 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 tl 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 ofDowes AG 3X4 (formate form) weak anion exchange resin that was buffered in 10mM sodium formate pH 5.50. The column was immediately washed with 5ml of 10mM formic acid. The flow through and washings were discarded. The sialic acids were eluted into a thin walled centrifuge tube with 10ml 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 0-acetyl CV-0083A PCT migration is necessary than all steps should be performed at 4 0 C with 1 M pyridine acetate buffer, pH 5.5 in place of I M formic acid.

Column Instructions Dowex 50 AG 1X8, hydrogen form: 1 ml of resin was spooned into a mall, disposable, plastic-fritted column. Column was primed with 5ml 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 ml 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 15 through was The column was washed with double DI water. The column was then buffered with 10mM sodium formate, pH *fr "Analysis Of Sialic Acids By Dionex HPAEC/PAD On The Carbopac Column 20 Sialic acid samples were analyzed by HPAEC/PAD using the Dionex 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 25 was: Time (min) A% B% C% D% INJECT TITL1 Initial 93.0 0 93.0 7.0 X X 10 70.0 30.0 11 70.0 30.0 12 93.0 27 93.0 S. S

S

*5 This method required 27 minutes at a flow rate of Iml/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 acid Sialic Acid content per gm of Egg Table Egg Yolk 1.9 mg .19% Table Egg White 1 mg 3k fraction of PL-100 Egg 7.7 mg .77% Yolk 3k fraction of PL-100 Egg 0.75 mg .075% 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 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 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).

S. S 49a Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification, they are to be interpreted as specifying the presence of the stated features, integers, steps or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component or group thereof.

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19/01/00.tdl 1057.spe,49

Claims

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 15 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 20 mixtures thereof. S

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. 19/01/00,tdl 1057.spe.50 I I I a. a a a The anti-inflammatory composition of claim 1 wherein the anti-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- 20 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. 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. CV-0083A PCT

12. The method of claim 11 wherein the anti-inflammatory composition is partially purified and comprises the following characteristics: s 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: S. 15 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; S° 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. 5.5.5.

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. -53-

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 S. 15 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 20 daltons; c) being heat stable; d) being orally active; and e) being resistant to degradation by digestive enzymes. Dated this 19th day of January, 2000. DVC, INC. By their Patent Attorney~: CALLINAN LAWRIE J 19/01/00.tdl 1057.spe.53