KR20010101565A - Egg Anti-Inflammatory Composition and Method of Treating and Preventing Inflammation - Google Patents

Abstract

The present invention generally relates to compositions and methods for treating and preventing inflammation. The composition is an oval obtained from algae overimmunized with a non-immunogenic mixture. The invention also relates to an anti-inflammatory composition partially purified from overimmunized eggs. Partially purified anti-inflammatory compositions are effective in treating and preventing inflammation when administered to a subject.

Description

Egg Anti-Inflammatory Composition and Method of Treating and Preventing Inflammation

Related application

This application is a partial consecutive application of pending US patent application Ser. No. 08 / 814,187 filed March 10, 1997 and US patent application Ser. No. 09 / 008,728 filed January 19, 1998.

Inflammation

As defined in Dorland's Medical Dictionary, inflammation is caused by damage or destruction of tissue and is a topical protective reaction that acts to destroy, dilute, or separate both the damaging component and the damaged tissue. This is characterized by the fenestration of the microvascular system, the leakage of blood elements into the interstitial gap and the migration of leukocytes into the inflammatory tissues. Visually, this usually involves typical clinical signs of erythema, edema, hyperalgesia (pain) and pain.

Among these complex reactions, chemical mediators such as histamine, 5-hydroxytriphtamine, various chemotactic compositions, bradykidin, leukotriene and prostaglandin are topically released. Phagocytes can migrate into the region and rupture the cellular lysosomal membrane to release the soluble enzyme. All these phenomena can cause inflammatory reactions.

Inflammation due to rheumatoid mastitis includes binding of the antigen and antibody complement to cause local release of chemotactic and chemotactic compositions that attract leukocytes. White blood cells phagocytize complexes of antigen-antibody and complement and also release many of the enzymes contained in their lysosomes. These lysosomal enzymes then cause damage to cartilage and other tissues, which worsen the inflammation. Cell-mediated immune responses may also be involved. Prostaglandins, the major intracellular regulators of cellular function, are also liberated during this process.

Inflammatory response is any response characteristic of inflammation as defined above. It is well known to those skilled in the medical art that the inflammatory response causes most of the physical discomfort associated with various diseases and injuries (ie, loss of pain and function).

arthritis

Arthritis itself manifests in various forms. Some of the more common forms include rheumatoid arthritis, osteoarthritis and systemic rheumatism.

Rheumatoid arthritis is an autoimmune disease that is characteristic of joint pain, swelling and stiffness. Rheumatoid arthritis is a disease that affects about 3% of Americans, especially women. Rheumatoid arthritis is a very obstructive disease and typically attacks adults of age between 30 and 40 years, while the incidence of clinical disease is greatest in people aged 40-60 years. Although pharmacotherapy is somewhat effective, about 7% of people with rheumatoid arthritis die quickly after about 5 years of onset of the disease, and within 10 years, about 50% are unable to work. ( Medical Sciences Bulletin , December 1994).

Osteoarthritis presents symptoms similar to rheumatoid arthritis. In particular, osteoarthritis begins with degeneration of articular cartilage, whereas rheumatoid arthritis begins with inflammation in the synovial fluid, but each process becomes similar to each other as the disease progresses. In osteoarthritis, reactive synoviitis often occurs due to cartilage deterioration and joint changes. In contrast, rheumatoid arthritis develops secondary osteoarthritis in bone and cartilage because it corrodes cartilage. In the final stages of both osteoarthritis and rheumatoid arthritis the involved joints appear identical.

Some other forms of arthritis include ankylosing seropositive spondyloarthropathy (ankylosing spondylitis) and reactive arthritis. These diseases are often called "B-27 associated diseases" and are difficult to distinguish from rheumatoid arthritis. In some cases, ankylosing spondylitis, Reiter's syndrome or psoriatic arthritis are present with rheumatoid arthritis in the same patient. In most cases, these patients are treated with the same disease-modifying drug as in patients with advanced rheumatoid arthritis.

The onset of arthritis usually occurs after age 30 in people susceptible to this disease. However, some forms of arthritis may be initiated by other causes, such as slow virus infection. There is a great deal of overlap here, so most doctors regard these forms as "systemic rheumatism" and approach disease management in the same way. Some diseases in this category include chronic fatigue syndrome, fibromyalgia (fibromyalitis), and gout. Indeed, evidence accumulates in some patients with overlapping rheumatoid arthritis and fibromyalgia (Harris, Edward D. and wB Saunders, Rheumatoid Arthritis , 1997).

Autoimmune diseases

As mentioned above, rheumatoid arthritis is an autoimmune disease, and hence its etiology is largely the same as that of any other autoimmune disease. In general, the body normally recognizes the differences between its own by-products and heterologous invaders (ie, a few designated bacteria, viruses, fungi and protozoa). When immune cells (T or B lymphocytes) react with a "self-protein" during their development, the cells are considered defective and are usually destroyed or inactivated. However, sometimes "self-reactive" immune cells can avoid destruction. At some later point, the cells can be activated to elicit an immune response. Activation is believed to occur after infection with a common bacterium or virus that contains a polypeptide having a stretched portion of amino acids that conforms to the stretched portion on the defective self-protein. A particular virus, that is not only a retrovirus (retrovirus) of myeotjong bacteria such as Streptococcus (Streptococcus), mycoplasma (Mycoplasma) and borelri O (Borrelia) also relates sikineunde initiate disease. In addition to rheumatoid arthritis, autoimmunity often causes diseases such as juvenile diabetes, multiple sclerosis, Graves' disease, Meniere's disease, myasthenia gravis, lupus erythematosus and psoriasis ( Medical Sciences Bulletin , September, 1994).

Autoimmunity affects certain organs. For example, some autoimmune diseases of hepatobiliary and renal include primary biliary cirrhosis, necrotic glomerulonephritis, "idiopathic" sickle glomerulonephritis, virus-induced liver and kidney disease, chronic hepatitis, autoimmune and drug-induced Hepatitis (Gershwin, Manns, and Mackay 1992). Immunodestruction of Langerhans's islets causes diabetes mellitus (Hagopian and Lemmark 1992) and insulin autoantibodies have been described (Palmer 1987).

There is a large category of systemic vasculitis diseases that suggest that autoimmune mechanisms are the cause of etiology. Some of these diseases include leukocytosis, nodular polyarteritis, Goodpasture syndrome, Kawasaki disease, Wegner's granulomatosis, Chug-Strauss syndrome, giant cell arteritis, Dagaya's arteritis, immune-complex-mediated rheumatoid lupus, And Hans globulinemia vasculitis, Henoch-Schoene purpura (Kallenberg, 1996; Jennette, Jones, Falk, 1992).

There is also a source of evidence that autoimmunity plays a role in many forms of heart disease, including post-pericardiotomy and post-myocardial infarction syndrome, myocarditis, and idiopathic dilated myocardial disease. Autoimmunity can cause degeneration of acute diseases of the heart muscle (Rose, Neumann, Burek, Herskowitz 1992).

Symptomatic invasion of skeletal muscle may include polymyositis or inflammatory myopathy (rheumatic arthritis, rheumatoid polymyalgia, myasthenia gravis, myasthenia gravis, neurotrophic atrophy, motor neuropathy, fibromyalgia, fibromyalitis, muscular dystrophy, endocrine, metabolic and Common in many autoimmune diseases, such as cancerous myopathy) (Hollingsworth, Dawkins, Thomas 1992).

Other diseases of autoimmune origin include uveitis, Fogg-Koyagi-Harada syndrome (Detrick and Hooks 1992), and Sjogren's syndrome, scleroderma, ankylosing spondylitis, dermatitis, psoriasis, psoriatic arthritis, and Reiter syndrome (NIH 1994). Can be.

Evidence of autoantibodies can also be found in Alzheimer's disease (Singh et al., 1992), dementia complex (Mastroianni et al., 1991) and pediatric autism (Singh et al., 1993).

Some neurological disorders, such as side syndrome, chronic obsessive compulsive disease (OCD), attention deficit hyperactivity disorder (ADHD), Tourette's Syndrome (TS), and some cases of schizophrenia, can have autoimmune components and May be related to neurogenic antibodies (Medical Sciences Bulletin, Sept. 1994).

This summary is not absolute, and other autoimmune diseases such as, for example, Ulang-Barré syndrome (idiopathic polyneuritis) are well known to those skilled in the art.

cure

To treat inflammation-related diseases, it is a common medical practice to administer pharmacological agents that reduce the physical discomfort of the inflammatory response. Drugs with these properties are classified as anti-inflammatory agents. Anti-inflammatory drugs are used to treat a broad spectrum of diseases, and the same drugs are often used to treat different diseases. Treatment with anti-inflammatory drugs is not about the disease, but in most cases the symptoms (ie inflammation).

Anti-inflammatory, analgesic and antipyretic drugs are often a group of chemically unrelated heterogeneous compounds, but nevertheless they share certain therapeutic actions and side effects. Corticosteroids are a class of compounds that are most widely used for the treatment of inflammation. Proteolytic enzymes are another class of compounds that are thought to have anti-inflammatory effects. Hormones that directly or indirectly induce the adrenal cortex to produce and secrete steroids are another class of anti-inflammatory compounds. Unfortunately, natural and synthetic corticosteroid preparations cause a number of serious side effects, including elevated blood pressure, retention of salt and water, kidney damage and increased potassium and calcium excretion. In addition, corticosteroids can mask the symptoms of infection and enhance the spread of infectious microorganisms. These hormones are considered unsafe to use in pregnant women, and long-term corticosteroid treatment is associated with gastrointestinal hyperactivity and / or peptic ulcer. Treatment with corticosteroids can also aggravate diabetes mellitus, requiring higher doses of insulin and causing mental illness. Hormonal anti-inflammatory agents that indirectly increase the production of endogenous corticosteroids have the same potential for adverse side effects.

Another common treatment for inflammation, in particular rheumatoid arthritis, other arthritis and other autoimmune diseases, is pharmacotherapy. In general, patients are treated primarily with "first-line" medications, usually non-steroidal anti-inflammatory drugs (NSAIDs) that primarily relieve symptoms. Patients are then treated with "second-line" or disease-modifying agents (DMARDs) such as methotrexate, gold compounds, penicillamine, sulfasalazine, and antimalarial agents. However, all of these drugs have serious side effects, especially when administered at increased doses. For example, aspirin, an NSAID, can cause indigestion and stomach pain; Phenylbutazone can cause gastric ulcers, and phenacetin can also cause kidney disease. Methotrexate can cause oral ulceration and gastrointestinal (GI) side effects.

If a natural food product having an anti-inflammatory effect can be obtained, it can generally be easily administered for the treatment of arthritis, autoimmune diseases and inflammation, and can provide a readily available and safe therapeutic composition.

Various genera of birds such as chickens, turkeys, and ducks produce antibodies to immunogens and other immunogens that cause bird disease in the blood and eggs. For example, LeBacq-Verheyden et al., Immunology 27: 683 (1974); and Leslie, GA, et al., J. Med. 130: 1337 (1969) quantitatively analyze immunoglobulins in chickens. It was. Polson, A. et al. (Immunological Communications 9: 495-514 (1980)) immunized hens against several proteins and natural mixtures of proteins and detected IgY antibodies in egg yolks. Fertel, R. et al. (Biochemical and Biophysical Research Communications 102: 1028-1033 (1981)) immunized hens against prostaglandins and detected antibodies in egg yolk. Jensenius et al. (Journal of Immunological Methods 46: 63-68 (1981)) provide a method for isolating egg yolk IgG for use in immunodiagnosis. Polson et al. (Immunological Communications 9: 475-493 (1980)) describe antibodies isolated from egg yolk of hens immunized with various plant viruses.

US Pat. No. 4,357,272 describes the isolation of antibodies from egg yolks derived from hyperimmunized hens. Hyperimmunity is caused by repeated injections of immunogens derived from plant viruses, human IgG, tetanus antitoxins, snake antitoxins, and Serameba. US Pat. No. 4,550,019 describes isolating antibodies derived from hens from egg yolk by overimmunizing with an immunogen having a molecular weight or particle weight of at least 30,000. The immunogen used to hyperimmunize chickens was selected from plant viruses, human immunoglobulins, tetanus toxins and deadly venom.

U.S. Patent No. 4,748,018 describes a method for passive immunization of mammals comprising parenteral administration of purified antibodies obtained from eggs of birds immunized against the corresponding antigens, wherein the mammal is directed to eggs. Gain immunity.

U. S. Patent No. 5,772, 999 (assigned to DCV-Biologics) discloses chronic gastrointestinal disease or non-steroidal anti-inflammatory-derived (NSAID-derived) in a subject by administering to the subject a hyperimmunized egg and / or lactation or fraction thereof. It describes methods for preventing, countering, or reducing gastrointestinal damage.

However, none of these documents describe or imply that when administered to animals, eggs have the ability to prevent or lessen inflammation, treat or prevent arthritis, or treat or prevent autoimmune diseases. These documents also do not describe or imply how the overimmunization of algae provides the ideal expectation to produce algae that give rise to eggs having this ability. Finally, these documents do not describe or imply that anti-inflammatory compositions are present in eggs obtained from algae.

Summary of the Invention

The present invention is based on the findings of the present inventors that, when administered to a test animal, especially a mammal, in an egg product obtained from an egg and an egg product, especially an over-immune bird, the anti-inflammatory activity prevents or alleviates inflammation in the test animal. It is.

In particular, the present invention relates to partially purified anti-inflammatory compositions obtained from eggs of algae. The anti-inflammatory composition was partially purified from fractions isolated from both egg yolk and egg white.

The present invention also relates to a method of hyperimmunization which produces an anti-inflammatory composition at a level above normal in eggs of algae. We believe that the level of anti-inflammatory composition can be increased in both egg yolk and egg white by the superimmunization method.

The invention also includes a method of treating inflammation in a subject, particularly a mammal, characterized in that the subject is administered a composition comprising a partially pure anti-inflammatory composition or a partially pure anti-inflammatory composition. This aspect includes administering the whole egg itself and / or a fraction thereof.

The invention also provides activity for treating or preventing arthritis and / or autoimmune diseases in an animal when administered to a test animal, in particular a mammal, in an egg and egg product, in particular an egg obtained from an overimmunized bird. It is based on the findings of the inventors.

The present invention finally relates to a method of administering an oocyte to a subject in combination with a drug selected from the group consisting of non-steroidal anti-inflammatory and disease-modified anti-arthritis agents.

The present invention generally relates to compositions and methods for treating and preventing inflammation. The present invention particularly relates to egg anti-inflammatory compositions, methods for their preparation in partially purified form, and methods of using the compositions in the treatment of inflammation (in particular arthritis) and autoimmune diseases.

1 is a process diagram for purifying an anti-inflammatory composition having a molecular weight of less than 3,000 from powdered egg yolk.

FIG. 2 is a process diagram for purifying an anti-inflammatory composition having a molecular weight of less than 3,000 from powdered egg whites.

Figure 3 shows the absorbance of the separation of the partially purified anti-inflammatory composition by Sepharose column.

4 is a chromatogram of HPLC separation of partially purified anti-inflammatory composition obtained from hyperimmune egg yolk.

5 is a graph showing the effect of hyperimmunity eggs on the incidence of arthritis using a rat type II collagen model.

FIG. 6 is a graph showing the effect of hyperimmune eggs, fractions thereof and partially purified anti-inflammatory compositions on the severity of arthritis in a rat type II collagen model.

Detailed description of the invention

The present invention generally relates to compositions and methods for treating and preventing inflammation, in particular arthritis and autoimmune diseases. The composition is preferably a natural food product containing hyperimmune eggs or egg products. Administration of the food product by the method of the present invention not only alleviates the pain and other symptoms caused by arthritis and autoimmune diseases, but also delays and prevents the onset of such diseases. Preferred antigen mixtures injected into birds to produce hyperimmune oocytes do not contain specific antigens known to cause inflammation or autoimmune diseases. Therefore, it is surprising that administration of hyperimmune eggs or oocytes obtained from birds immunized against mixed antigen vaccines is effective in preventing and alleviating the symptoms of inflammation and autoimmune diseases when administered to a subject.

Justice

Throughout the specification, the following definitions apply.

The term “inflammation” is caused by damage or destruction of tissue and acts to destroy, dilute or separate both the damaging component and the damaged tissue, and in the classical sequence of pain, fever, redness, swelling and malfunction. Is characterized by an uncontrolled and uncontrolled form and histologically includes permeation of the small arteries, capillaries and venous veins, dilation with increased blood flow, exudation of body fluids containing plasma proteins, and migration of leukocytes to inflammatory sites. As a local protective reaction involving a series of complex phenomena, it is used as a concept recognized in the art.

The term "arthritis" refers to various diseases manifested by inflammation and degeneration of connective tissue structures, especially joints and related structures. Arthritis may involve pain, stiffness and limitation of movement in these parts. Some forms of arthritis include rheumatoid arthritis, osteoarthritis, ankylosing serum reactive spondyloarthropathies, reactive arthritis, chronic fatigue syndrome, fibromyalgia (fibromyalitis) and gout.

The term “autoimmune disease” is given in a standard medical dictionary, such as the dictionary of Dorland and Taber, to which standard medical definitions apply. Descriptions of various autoimmune diseases can be seen in the background section of this specification.

The term “hyperimmunization” refers to exposure to one or more antigens such that the immune response is elevated and maintained above the natural, unexposed state.

The term "egg" or "oval product" means an entire egg (table, hyperimmunized or otherwise) or a product or fraction derived therefrom, respectively.

The terms "table egg" or "table egg product" refer to whole eggs obtained from egg-producing animals, or products or fractions derived therefrom, that do not remain hyperimmune, respectively.

The terms “hyperimmune egg” or “hyperimmune egg product” refer to whole eggs obtained from egg-producing animals, or products or fractions derived therefrom, that each remain superimmune.

The term "normal level" means more levels than are present in eggs of egg-producing animals that do not remain hyperimmune.

The term "anti-inflammatory egg or egg fraction" means an egg or egg fraction containing the anti-inflammatory composition described herein.

The term "anti-inflammatory composition" means a composition described herein that counters or inhibits the inflammatory process.

The term "partially pure egg anti-inflammatory composition" means an anti-inflammatory composition represented by at least the purity described in Example 4 and the exemplified materials and numbers.

The term “combinatorial derived immunogens” relates to a method of generating molecular diversity in an immunogen in the manner of combinatorial synthesis.

The term "biological immunogen" relates to an immunogen obtained through genetic cloning techniques and methods of genetic engineering that allow insertion and translation of proteins with antigenic properties.

The term "genetic vaccine" generally refers to a nucleic acid vaccine produced by recombinant technology and capable of causing an immune response.

The term "treatment" refers to delaying or completely preventing the onset of the symptoms of a disease (including pain) and / or pathogenic origin of the disease, or to alleviate or eliminate the symptoms if there are symptoms. For example, hyperimmune oocytes not only suppress symptoms of the disease in humans and other mammals, but also treat arthritis and / or autoimmune diseases by acting as a preventive agent against the presence of the disease at the receptor.

The term "prevention" means reducing and / or eliminating disease progression or eliminating the onset of disease.

The term "administration" means a method of providing a substance to a subject, including oral, intranasal, parenteral (intravenous, intramuscular or subcutaneous), rectal, topical or intraocular administration.

The term "animal" means the definition of the animal kingdom.

The term "target animal" refers to an animal that acts as an animal that produces an egg or egg product.

The term "test animal" refers to an animal to which an egg or oval product produced by a target animal is administered.

The term "immunogen" refers to a substance capable of inducing humoral antibodies and / or cell-mediated immune responses that are not immunotolerant. The term refers to the ability to react not only with an immune response but also with a product thereof, for example an antibody.

The present invention

The products and methods of the invention relate to the use of hyperimmune eggs, which are natural food products, in particular in the treatment and prevention of inflammation and autoimmune diseases. Since this food product is a natural product, it can, of course, be used to treat and prevent these diseases without the risk of side effects, except for allergic reactions in cases of intolerance to eggs.

In one embodiment, the present invention includes hyperimmune eggs or oocytes that are effective for treating and preventing arthritis and / or autoimmune diseases in a test animal. Hyperimmune eggs are obtained from egg-producing animals, more preferably birds, which are hyperimmunized by at least one immunogen. The hyperimmune oocyte is preferably administered orally to the test animal. Hyperimmune eggs or oocytes may also be separated into more powerful fractions, which may then be administered to the subject in various forms.

In an alternative embodiment, the invention encompasses administering an anti-inflammatory composition obtained from an egg of an alga, a partial tablet thereof, and a partially purified composition to a subject for the treatment of inflammation. The present invention further encompasses the same anti-inflammatory composition partially purified from eggs of birds that are over-immunized by one or more immunogens, in particular bacterial antigens or synthetic equivalents thereof. Anti-inflammatory compositions are present in hyperimmune eggs at levels above normal that provide anti-inflammatory activity in the subject.

In the following a detailed description of the invention is presented.

Partial Purification Method

The egg anti-inflammatory composition may be partially purified from whole egg, egg yolk or egg white. Examples of preferred partial purification methods are as follows:

1. Preparation of an aqueous fraction from eggs;

2. ultrafiltration of aqueous fractions;

3. Separation of fractions by ion or anion exchange chromatography; And

4. Biological assay of separated fractions with respect to anti-inflammatory activity.

A more detailed description of this method is given below:

Step 1:

The anti-inflammatory composition may 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 skilled in the art. For example, degreasing in the case of spray-dried egg yolk powder can be achieved using solvents (using propane, butane or hexane or binary solvents), supercritical CO ₂ , enzymes, etc., in the case of liquid yolk Degreasing can be accomplished by the caprylic acid separation method (CAPS) described by Lee (US Pat. No. 5,367,054). No fat removal is required in the case of egg whites, so the liquid or powdered form of egg whites can be directly heated or dissolved by conventional methods and as described in the examples below. The whole egg, egg yolk or egg white can then be processed, preferably in liquid or powder form, and further processed to obtain a water soluble fraction (see, Examples).

Step 2:

The water soluble fraction generated from whole egg, egg yolk or egg white is subjected to ultrafiltration using an ultrafiltration system equipped with a 3,000 molecular weight cut-off membrane. The ultrafiltration method separates molecules having a molecular weight of at least about 3,000 Daltons from molecules having a molecular weight of less than about 3,000 Daltons. Once filtered, the resulting ultrafiltrate contains molecules having a molecular weight of less than about 3,000 Daltons, which are lyophilized and weighed for bioassay test methods and further separation.

Step 3:

Fractions from ultrafiltrations below 3,000 Daltons can be separated, for example, by reversed phase high performance liquid chromatography, to further purify partially pure anti-inflammatory compositions. Alternatively or additionally, the egg anti-inflammatory composition in the ultrafiltrate can be further characterized by anion exchange DEAE-Sepharose chromatography. In a particularly described embodiment, preferred ultrafiltration is performed by Amicon RA1000 (3K MWCO) and DEAE ion-exchange chromatography. However, it is understood that equivalent techniques and materials may be used to separate the compositions, given the information herein and the knowledge available to those of ordinary skill in the art.

Step 4:

The anti-inflammatory activity of the composition can be tested by standard biological assays measuring anti-inflammatory activity. Some examples include, in particular, inhibition of leukocyte migration, edema testing of rat paws, adjuvant-induced arthritis, collagen-induced arthritis, and biomicroscopy. Comparison of egg anti-inflammatory compositions with known anti-inflammatory agents such as aspirin and indomethacin can also be performed. Finally, clinical trials for rheumatoid arthritis, degenerative joint disease and injury-induced arthritis can also be used to determine anti-inflammatory activity.

Anti-inflammatory activity of partially pure egg anti-inflammatory compositions is determined by bioassay. Preferred bioassays are rat type II collagen test methods as described in Example 6.

Another alternative bioassay includes 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., "Qunatitative Studies of the Pathway to Acute Carrageenan Inflammation", Fed. Proc. 35: 2447-2456 (1976)).

Leukocyte migration inhibition test method is another useful test method generally performed as follows: A sample containing an anti-inflammatory composition is administered to a mature female rat artificially inflamed with 1% carrageenan solution, and then each dose The anti-inflammatory effect of the samples in is measured by comparing the reduction in white blood cell counts in pleural effusions of treated rats with that of control rats (using automated imaging techniques).

Alternatively, the anti-inflammatory action of a substantially pure anti-inflammatory composition can be tested for edema caused by injecting carrageenan into the footpad of the rat (Winter, CA, Risley, GA, Nuss, AW, " Carrageenan-Induced Edema in the Hind Paw of the Rats as an Assay for Anti-Inflammatory Drugs ", Proc. Soc. Exper. Biol. Med. 3: 544 (1967)).

Various other bioassay methods may also be used (Wetnick, AS, 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).

Features of Partially Purified Anti-inflammatory Composition

Partially pure anti-inflammatory compositions have the following characteristics:

1) has anti-inflammatory activity in the test animal;

2) present in both egg whites and egg yolks of eggs of algae;

3) separation from egg yolk has greater anti-inflammatory activity than that from egg white;

4) has a molecular weight of less than about 3,000 Daltons;

5) non-proteinaceous and non-steroidal;

6) it is thermally stable;

7) orally active and not degraded by digestive enzymes;

The 3,000 Dalton molecular weight is derived by partial separation and purification of the composition, where the separation and purification method uses an ultrafiltration membrane that does not pass molecular weight species above 3,000 Daltons. Partially purified anti-inflammatory compositions are considered non-proteinaceous and non-steroidal because they are small in size and not degraded by enzymes that degrade proteins. Moreover, the composition is orally active and is not degraded by digestive enzymes. Small stable forms of partially purified anti-inflammatory compositions (as distinguished from much larger proteins) facilitate their absorption from the digestive system. Finally, partially purified anti-inflammatory compositions are heat-stable.

The partially purified egg anti-inflammatory composition can be separated from the whole egg, egg yolk and egg white. Anti-inflammatory compositions partially purified from egg yolks exhibit greater anti-inflammatory activity compared to anti-inflammatory compositions partially purified from egg whites.

It has been found by the inventors that anti-inflammatory compositions partially purified from whole egg, egg yolk and egg white obtained from over-immune egg-producing animals can be separated to above normal levels.

Hyperimmunization of Egg-producing Animals

As described above, the inventors have found that the anti-inflammatory composition is present at a level above normal that provides anti-inflammatory activity in the test animal within the hyperimmune egg. Hyperimmune eggs or egg products can be produced by egg-producing animals. The animal is preferably a member of the algae, ie algae. Poultry is preferred among algae rivers, but other members of this river, such as turkeys, ducks and geese, are also suitable sources of hyperimmune oocytes.

When such an egg-producing animal is subjected to a particular immune state, for example, by the periodic additional administration of an immunogen, when the animal is consumed by the subject, the subject treats and prevents inflammation-related diseases and autoimmune diseases in the subject. It will produce eggs with beneficial properties that include an anti-inflammatory composition that is effective at above normal levels.

As can be seen from the fact that the table egg does not contain abnormal levels of anti-inflammatory composition, even if the algae are sensitized to various immunogens during normal immunization to algal disease and during normal exposure to environmental factors, Induction of only) is insufficient to induce egg anti-inflammatory composition to be present above normal levels in eggs. The inventors have found that this is only present in certain immune states in which the egg has an anti-inflammatory composition at a desired above normal level.

This particular state of hyperimmunization, in which the egg contains a higher level of anti-inflammatory composition, is preferably achieved after initial immunization followed by periodic boosting with a sufficiently high dose of specific immunogen or mixture of immunogens. Preferred doses of booster doses should be at least 50% of the dose required to provide algal primary immunization. In other words, the threshold booster dose here is an amount that does not exhibit its characteristics in the egg of the bird, even if the bird is commonly called an immune state. If you have knowledge of the requirements for developing and maintaining a hyperimmune condition, the amount of immunogen administered to maintain the animal in an over-immune condition within the scope of the art is dependent on the genus and species of the egg-producing animal used. Can be changed accordingly.

The hyperimmune state is preferably provided by an immunogen or combination of immunogens. Hyperimmunization is preferably achieved by multiple exposures to multiple immunogens, multiple exposures to a single immunogen, or single exposure to a library of immunogens. Almost all immunogens, including bacterial, viral, protozoal, allergen, fungal or cellular substances, can be used to induce hyperimmune conditions, but are not limited to these.

If you have knowledge of the requirements for developing and maintaining a hyperimmune condition, the amount of immunogen administered to maintain the animal in an over-immune condition within the scope of the art is dependent on the genus and species of the egg-producing animal used. Can be changed accordingly.

In addition to immunization with naturally occurring immunogens, immunization can also be accomplished using immunogens synthesized synthetically by combinatorial chemistry. The basic method is to assemble multiple blends of chemical building blocks that produce a flexible population of molecules. Recently, libraries of oligomers (Fodor, S. et al., Science 251: 767 (1991); Houghton, R. et al., Nature 354: 82 (1991)) and small organic molecules (Bunin, B. & Ellman, J., J. Am. Chem. Soc. 114: 10997 (1992)) have developed a number of methods for solid and solution phase combination synthesis. Synthesis of rapid multiple peptides and oligomers can serve as a source for combinated immunogens. Alternative methods also allow the addition of organic building blocks in combination to skeletal molecules for improved immunogenicity.

Alternative modes of overimmunizing egg-producing animals can be used in place of immunogenic vaccines, which methods include the use of genetic vaccines. In particular, any DNA construct (generally composed of a promoter site and antigen coding sequence) can initiate an immune response. Genetic vaccines are delivered to cause antigen-encoding vectors, fragments of naked DNA, plasmid DNA, DNA-RNA antigens, DNA-protein conjugates, DNA-liposomal conjugates, DNA-expression libraries, and immune responses. Composed of viral and bacterial DNA. Methods of delivering DNA include, in particular, particle bombardment, direct injection, viral vectors, liposomes and jet injection. If this delivery method is applied, much smaller amounts may be needed and generally lead to more sustained immunogen production. When using this genetic method, the preferred method of introducing DNA into algae is by intramuscular injection of DNA into the pectoral muscles.

DNA delivery methods include, but are not limited to, particle impact, direct injection, liposomes, jet injection (Fynan, EF et al., Proc. Natl. Acad. Sci. USA 90: 11478-11482 (1993)) . Known or unknown immunogens, promoter regions (especially CMV cauliflower mosaic virus) and nucleic acids encoding SV40 bacterial origin can be replicated in bacteria to produce plasmid DNA for use in DNA injection. Although several routes of parenteral administration of DNA are effective in chickens, the preferred method is intramuscular injection into the pectoral muscle. Vaccine experiments are carried out in egg-producing birds, preferably chickens. Repeated immunization is performed at 1-2 week intervals for a period of up to 6 months.

The amount of DNA used is generally about 50-300 μg of DNA in saline for direct injection. For particle impact, 4-100 μg of DNA co-precipitated on gold beads with 2.5 M CaCl ₂ is preferred. Repeated immunization can be provided by the intradermal route in living animals by this method of promoting DNA coated particles.

Below is a detailed description of the preferred method used to induce an o-producing animal to have a higher level of immunity, and the superimmunized egg or oocyte product generated from this animal may be administered to the subject:

1. Select one or more immunogens.

2. Primary immunization elicits an immune response in egg-producing animals.

3. Induce and maintain hyperimmunity by administering an additional dose of the immunogen at an appropriate dose.

4. Test anti-inflammatory activity against hyperimmune eggs.

5. Collect and process eggs.

A more detailed description of this method is given below.

Step 1 : As a vaccine, an immunogen or combination of immunogens can be used. The immunogen may be bacterial, viral, protozoan, fungal, cellular, or any other substance to which the immune system of an egg-producing animal responds. It is important at this stage that the immunogen (s) must be capable of inducing immunity and hyperimmunity in egg-producing animals. Although only a single immunogen may serve as a vaccine for the method of the invention, preferred vaccines are mixtures of multivalent bacterial and viral antigens selected from the following antigen classes: enteric Bacillus and Bacteroides, Pneumococcus, Pseudomonas, Salmonella, Streptococcus, Bacillus, Staphylococcus, Neisseria, Clostridium, Mycobacteria, Actinomycetes chlamydiae and Mycoplasma. Viral antigens are preferably selected from the following antigen classes, but other viral antigen classes may be used: adenoviruses, picornaviruses and herpesviruses.

In a preferred embodiment, a multivalent vaccine called Series 100 (S-100) is used. The bacteria included in the S-100 vaccine are listed in Table 1 of Example 1. These vaccines are already described in US Pat. Nos. 5,106,618 and 5,215,746, all assigned to Stolle Research and Development Corporation.

Step 2 : The vaccine can be either a vaccine or a live-attenuated vaccine and can be administered by any method that results in an immune response. Immunization is preferably achieved by administering an immunogen by intramuscular injection. Preferred muscle for injection in birds is the pectoral muscle. The dose is preferably 0.05-5 mg of the immunogenic vaccine. Other methods of administration that can be used include intravenous injection, intraperitoneal injection, intradermal, rectal suppositories, aerosols, oral, topical or intraocular administration. When DNA technology is used in the hyperimmunization process, much smaller amounts, typically 300 μg, are required.

A number of methods known to those skilled in the art of immunology can determine whether the vaccine caused an immune response in egg-producing animals. Examples of these include enzyme-linked immunosorbent assays (ELISA), tests for the presence of antibodies to stimulatory antigens, and tests designed to assess the ability of immune cells from a host to respond to antigens. The minimum dose of antigen required to elicit an immune response depends on the inoculation procedure used, including the type of adjuvant used and the preparation of the antigen (s), and the type of egg-producing animal used as a host.

Step 3 : The hyperimmunity is induced and maintained in the target animal, preferably by repeating additional doses of the appropriate dose at fixed time intervals. The time interval is preferably a 2-8 week interval over a period of 6-12 months. However, it is essential that further administration does not induce immune tolerance. Such methods are well known in the art.

It is also possible to use other methods of maintaining hyperimmunization, or a combination of methods such as intramuscular injection for primary immunization and intravenous injection for further injection. Additional methods include simultaneous administration of an additional dose by intramuscular injection for microencapsulated liquid antigen or primary immunization, and oral or parenteral administration by microencapsulation means. Many combinations of primary and hyperimmunization are known to those skilled in the art.

Step 4 : It is necessary to test eggs for anti-inflammatory activity levels. This can be done by clinical and preclinical evaluations testing the effects on inflammation of hyperimmune eggs or products derived therefrom. Chemically-induced inflammation in rats is the preferred standard test method for anti-inflammatory agents (see Example 6).

Step 5 : This step involves collecting and processing the egg (s) containing the anti-inflammatory composition. Eggs can be collected by conventional methods. The processing of eggs can be performed in various ways as described later in this specification. The eggs may also be further treated to purify the anti-inflammatory composition as described below.

Processing and Dosing

Eggs comprising a partially purified anti-inflammatory composition collected from a hyperimmunized animal, or fractions thereof, are preferably processed to produce a hyperimmune egg product that can be subsequently administered to the subject.

The eggs themselves or fractions thereof comprising the partially purified anti-inflammatory composition of the present invention may be administered to the subject by any method of treating or preventing arthritis and / or autoimmune disease in the subject. Administration is preferably carried out by directly supplying the egg or derivative of the egg to the subject. It is important to note that whole egg, egg yolk and egg white are non-toxic and safe natural food ingredients.

In an alternative embodiment, eggs or fractions thereof comprising partially purified anti-inflammatory compositions are incorporated into a nutritional supplement. One preferred method of preparing eggs or fractions thereof to be incorporated into a nutritional supplement includes drying the eggs with egg powder. Various methods of drying eggs are known, but spray drying is the preferred method. Methods of spray drying eggs are well known in the art.

This dried egg powder can be incorporated into the beverage in the form of, for example, protein powders, power building drinks, protein supplements and other nutritional exercise-related products. Egg powder can also be used in bake mixes, power bars, candy, cookies and the like. Other examples of egg processing include making omelets, ripening or ripening eggs, baking eggs, or, if necessary, eggs can be reproduced or processed into liquid eggs.

Finally, further isolation and purification, such as in the case of partially purified anti-inflammatory compositions, may be achieved by other methods such as parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, oral or topical administration. Consideration should be given to mode of administration. This additional separation also provides the ability to prepare encapsulated products and pharmaceutical compositions having the aforementioned eggs or fractions thereof.

Formulations of anti-inflammatory compositions for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.

Especially with regard to partially pure anti-inflammatory compositions, oral administration preferably takes place in particular by solid dosage forms, including capsules, tablets, pills, powders and granules. In solid dosage forms, the anti-inflammatory composition is mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also contain additional ingredients in addition to the inert diluent in conventional practice. In the case of capsules, tablets and pills, the dosage form may also contain buffers, pH sensitive polymers, or other slow-releasing encapsulants typically used to encapsulate compositions in the food and pharmaceutical industries. It may also include.

Liquid dosage forms of anti-inflammatory compositions for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the pharmaceutical art. In addition to inert diluents, the compositions may also include wetting agents, emulsifiers and suspending agents and sweetening agents.

Hyperimmune eggs or oocytes comprising the partially purified anti-inflammatory compositions of the present invention are characterized by inflammation, in particular rheumatoid arthritis, osteoarthritis, ankylosing seronegative spondyloarthropathies, reactive arthritis, chronic fatigue syndrome, fibromyalgia (fibromyalitis) and gout It is effective in the treatment and prevention of all forms of arthritis including, but not limited to. The oocytes of the present invention are particularly useful for treating autoimmune diseases such as rheumatoid arthritis, juvenile diabetes, multiple sclerosis, Graves' disease, Meniere's disease, myasthenia gravis, lupus erythematosus, psoriasis, systemic scleroderma, rheumatic fever and Sjogren's syndrome. Equally effective (see the Background section of this specification).

When treating and preventing a particular disease, whether it is general inflammation or in the form of arthritis or autoimmune disease, a hyperimmune oocyte product or a fraction of its actives, including a partially purified anti-inflammatory composition, is preferably used. Is administered to a subject in an immunologically effective amount for treating and preventing The duration and intensity of treatment is determined by the presence or absence of a particular condition and, if present, by the progress of the condition in the subject. Hyperimmune oocytes or active fractions thereof, including partially purified anti-inflammatory compositions, are also provided in amounts to treat and / or prevent the condition and symptoms of the condition. For example, in some cases a hyperimmune egg containing less than one or several whole hyperimmune eggs with daily doses ranging from less than one to several depending on the particular circumstances of the condition. Product) can be administered to the subject. Stronger fractions can be separated and concentrated by the methods described herein and other methods known in the art.

In one embodiment, egg products and partially purified anti-inflammatory compositions of the present invention have been found to be effective in treating rheumatoid arthritis in rats using a collagen-induced arthritis animal model (cf. Example 2). This animal model is well recognized by those skilled in the art to correspond to the effects of rheumatoid arthritis in humans. In addition to treating arthritis, oocytes have been shown to delay and, in some cases, prevent the development of arthritis symptoms when administered to rats before inducing arthritis. Thus, the egg product of the present invention is effective not only in treating the symptoms of a disease but also in delaying and / or preventing the onset or progression of the disease.

In alternative embodiments, oocytes have been tested in humans and have shown positive effects in treating these arthritis symptoms in several humans suffering from various forms of arthritis (see, Example 9). In addition to the overall reduction in swelling and stiffness, humans treated with oocytes have demonstrated a clinical reduction in symptoms such as pain.

The effect on autoimmune diseases has been shown to be a surprising reduction in type II collagen antibodies by partially purified anti-inflammatory compositions and oocytes of the present invention in this disease model, which was also seen in rats (see, Example 3). . The oocyte product of the present invention is particularly effective in reducing antibodies associated with other autoimmune diseases such as combustible diabetes, multiple sclerosis, Graves' disease, Meniere's disease, myasthenia gravis, lupus erythematosus, psoriasis, systemic scleroderma, rheumatic fever and Sjogren's syndrome. It is thought to be effective.

As described in the background of the present invention, patients with arthritis are treated with non-steroidal anti-inflammatory drugs (NSAIDs) and / or disease-modified anti-arthritis agents (DMARDs) to alleviate the symptoms of current arthritis conditions. As shown in Examples (see Example 9), patients were administered the oocyte product of the present invention in parallel with current pharmacotherapy. Ovarian products have been synergistic with drug therapy to reduce symptoms of certain diseases. Thus, oocytes can be administered in combination with alternative treatments for arthritis and autoimmune diseases to provide synergistically better effects.

In addition, as is well known in the art, NSAIDs and DMARDs can cause severe gastrointestinal damage. U.S. Patent 5,772,999, assigned to DCV, Inc. and incorporated herein by reference, states that hyperimmune oocytes are effective in treating NSAID-induced gastrointestinal damage. Thus, alternative embodiments are contemplated when the subject is taking or preparing to take NSAIDs for the treatment of arthritis conditions. In such cases, the subject may also be administered the oocyte product of the invention to treat the arthritis condition as well as to protect the gastrointestinal tract from NSAID-induced damage. Lower doses of hyperimmune eggs may be sufficient in these treatments. In addition, oocytes may be administered prior to the start of their regimen with NSAIDs and DMARDs in order to better prepare the gastrointestinal tract in a subject suffering from this disease.

Examples of inflammatory conditions that can be treated by administering an egg, oocyte, and / or anti-inflammatory composition of the invention include acute and subacute bursitis, acute nonspecific tendinitis, systemic lupus erythematosus, systemic dermatitis, acute rheumatic heartitis, Celtic swelling, bullous dermatitis, herpeteformis, severe erythema, polymorphic dermatitis, sclerosis, seasonal perennial rhinitis, bronchial asthma, ectopic dermatitis, serum disease, keratitis, eye iris, erosive euretis (diffuse ureitis) ), Vocalitis, optic neuritis, sympathetic ophthalmitis, symptomatic granulomatosis, leuupler syndrome, beryllium poisoning, hemolytic anemia, mastitis, mastitis, contact dermatitis, allergic conjunctivitis, psoriatic arthritis, ankylosing spondylitis, acute gouty arthritis Rheumatoid arthritis, osteoarthritis, other degenerative joint diseases, and other related autoimmune diseases. Isolated and purified oocytes may also be used to treat individuals exposed to potential inflammatory agents such as allergan.

Effective capacity

With regard to the administration of the hyperimmunized egg or egg product to the subject, the preferred dosage range of the hyperimmunized egg or egg product to be administered to the subject has been determined to be 100 mg to 10 g, per kg body weight of the subject, It was described in detail.

With regard to the partially purified anti-inflammatory composition itself, the preferred dosage range of the partially purified composition, purified and isolated from the whole egg, egg yolk and egg white of the over-immunized egg is from 1 μg to 400 mg per kg of anti-inflammatory composition. It was determined to be between.

Although the dosage of the active ingredient can be varied, it is necessary to select the amount of active ingredient so that a suitable dosage form is obtained. It will be appreciated that the dosage form chosen will depend upon the desired therapeutic effect, route of administration and duration of treatment.

Advantageous features of the invention may be identified with reference to the following examples illustrating the invention.

Example 1

Preparation of the S-100 Vaccine

A multivalent vaccine known as "Series 100" or "S-100", described in US Pat. No. 5,215,746 and containing the bacteria shown in Table 1 (obtained from the American Type Culture Collection), was reprepared in 16 ml of medium 37 Incubated overnight at ° C. Once good growth was obtained, approximately half of the bacterial suspension was used to inoculate 1 liter of broth and then incubated at 37 ° C. The remaining suspension was transferred to sterile glycol tubes and stored at −20 ° C. for up to 6 months.

The bacteria were harvested by centrifugation after good growth was seen in the culture. The bacterial pellet was resuspended in sterile physiological saline solution and the bacterial samples were centrifuged three times to wash the cells. After the third wash, the obtained pellet was resuspended in a small amount of distilled water.

The medium-free bacterial suspension was heat-killed by placing the suspension in a glass flask in an 80 ° C. water bath overnight. Survival in the broth was tested using small amounts of heat-killed bacteria. The juice was inoculated with heat-killed bacteria and incubated at 37 ° C. for 5 days and their growth checked daily because the bacteria had to be killed for use in the vaccine.

Heat-killed bacteria were lyophilized until dry. Dry bacteria were then mixed with sterile saline solution at a concentration of 2.2 × 10 ⁸ bacterial cells / ml saline (1.0 absorbance reading at 660 nm).

Antigens in the S-100 Vaccine designation badge designation badge 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 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 Juicy 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 against Hyperimmune Oocytes

Killed formulations of pathogens were prepared as described above. For primary inoculation, the bacteria were mixed with complete Freund's supplement and 5.6 mg of the bacterial substance was injected into the breast muscle of the chicken. For the rest of the vaccine, bacterial formulations were mixed with incomplete Freund's supplement and injected into chickens at two week intervals for six months.

Eggs were collected from the hyperimmunized chickens and then spray dried in powder form. During spray drying, the inlet temperature did not exceed 320 ° F, the outlet temperature was maintained as the powder was prepared in the final moisture range of 3.0 to 4.0%, and the pump pressure was maintained at approximately 2500 to 4000 PSI. I was. Lower temperatures in the range of 100-160 ° F. were used and the samples were monitored for moisture content during the drying process to obtain the final product with the desired consistency.

Example 2

Preferred method for preparing partially purified anti-inflammatory composition from eggs

The following examples describe a method (suitable for large-scale purification) to obtain an anti-inflammatory composition from the egg of the algae in partially pure, low molecular weight, non-aggregated form. The whole egg, the superimmunized and control table egg, was broken, egg whites were separated from egg yolk, and both were spray dried. Hyperimmune eggs were obtained as described in Example 1. The egg white powder was processed separately to obtain an aqueous fraction for ultrafiltration.

All purification steps were performed to minimize the possibility of contamination by bacteria or pyrogenic material. The solution was prepared using sterile water, and all glass containers removed pyrogenic material. The solution was also 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 liquid solvent extracted with propane or butane to separate the liquid from the egg yolk fraction containing the anti-inflammatory composition. Briefly, 500 g of dry yolk powder was placed in a column, and 4 liter of liquid propane solvent was added thereto. Solvent supernatant and extracted lipids were removed. Six additional solvent extractions were performed for a total of six lipid extractions.

Ultrafiltration

400 g of degreased dry egg yolk were diluted with 4 L of sterile distilled water and homogenized with Virtis (handishear). The egg yolk mixture was centrifuged at 24 RPM, or kept in the freezer until non-dissolved yolk particles precipitated. The resulting aqueous fractions were ultrafiltered using an Amicon RA1000 ultrafiltration system equipped with a 3,000 Dalton cut-off membrane spiral-wound. Pump speed was maintained at 20 psi inlet pressure and 15 psi outlet pressure. <3,000 Dalton molecular weight permeate was sterilized using a 0.45 μm sterile disposable Nalgene filter and lyophilized or frozen for storage, bioassay or further purification. 1 shows a working flowchart of the purification process.

Molecular weight species up to 3,000 Daltons from egg yolk contain the anti-inflammatory composition in low molecular weight, non-aggregated form. The yield of anti-inflammatory composition from 400 g of starting material was about 12 g or 3% of the total. The bioassay of this 3 KDa fraction on anti-inflammatory activity showed a high level of activity (Example 5).

Example 2b

Preparation of Egg Anti-inflammatory Activity from Egg White Powder

400 g of egg white isolated from both hyperimmune eggs and control table eggs as described in Example 1 were diluted with 4 liters of deionized water. The mixture was homogenized and filtered through a 40 μm filter followed by ultrafiltration through a 3 KDa MW Co ultrafiltration system (FIG. 2). From 400 g of egg white powder, 8.6 g or 2.15% of an anti-inflammatory composition was recovered.

This example shows the preparation of an active anti-inflammatory composition from egg whites of both hyperimmune and control table eggs.

Example 3

DEAE ion exchange chromatography

Egg anti-inflammatory compositions have also been further characterized by anion exchange DEAE chromatography.

3,000 Dalton ultrafiltration permeate from both degreased egg yolk and egg white were prepared in the same manner as described in Example 2. The DEAE Sepharose column (2.5 × 40.0 cm) was previously equilibrated with 1,000 ml of 20 mM ammonium acetate buffer (pH 8.0) in sterile double distilled water containing no pyrogenic material at room temperature. 3,000 Dalton permeate of degreased egg yolk (15 g) or egg white (18 g) was dissolved in 200 mL of sterile double distilled water containing no pyrogenic material and DEAE Sepharose column (Pharmacia Biotech.,) At a flow rate of 100 mL / h. High speed flow). The components of the 3,000 Dalton permeate were separated by the following buffer to separate the anti-inflammatory composition and other molecules. At the same flow rate, DEAE Sepharose was washed with 200 ml 20 mM ammonium acetate (pH 8.0) and 200 ml 20 mM ammonium acetate (pH 6.5) to remove unbound material. Thereafter, 200 ml of 150 mM ammonium acetate, pH 5.0 was applied to elute the weakly bound molecules. More strongly bound molecules were eluted with 200 ml of 150 mM ammonium acetate, pH 4.0. Finally, the DEAE Sepharose column was regenerated with 400 mL of 1.5 N NaCl in 20 mM ammonium acetate, pH 6.5 (FIG. 3).

FIG. 3 shows a representative chromatogram of DEAE Sepharose separation of 3 Kda permeate from degreased egg yolk and egg white. Letters indicate the elution profile by the buffer. Fractions were collected by every 15 mL of eluate and the absorbance of those molecules measured at 280 nm and 220 nm. Four fractions were combined and lyophilized to remove water and dried. Table 2 summarizes the separation methods.

DEAE Separation of 3,000 Dalton Permeate of Degreased Yolk and Egg White Fraction Eluent # Capacity (ml) Weight (g) Recovery rate (%) Egg yolk IIIIIIIV 1-2223-4041-5657-70 294252224196 11.790.400.440.19 78.62.72.91.3 Egg white IIIIIIIV 2-2627-4647-6061-73 350280196182 15.330.230.320.44 85.21.31.82.4

Example 4

HPLC analysis of 3,000 Dalton permeate and their DEAE Sepharose fraction

Reverse phase high performance liquid chromatography (HPLC) (Shimadzu Unit, SPD-M10A VP diode array detector, LC-6AD liquid chromatography, SCL-10A VP Degassor, LPM-600 low pressure mixture) CTO-10A VP column oven). All fractions were prepared as 5.0 mg / ml in ultrapure water and then filtered through a 0.2 μm filter. 20 μl of each sample was applied on a Water Symmetry C18 column (3.9 × 150 mm) at 29 ° C. with a flow rate of 1.0 mL / min. A linear gradient of 0% -60% acetonitrile in dH ₂ O containing 0.1% TFA was used as the mobile phase.

FIG. 4 shows Max plot chromatography for the separation of 3,000 Dalton permeate from degreased PL-100 egg yolk. By using the same assay conditions, 3,000 Dalton permeates prepared from degreasing table egg yolk were also obtained (chromatography is not shown).

Table 3 summarizes the results of HPLC analysis of 3,000 Dalton permeate. Peaks with more than 0.5% of the peak area were collected. The similarity of the peaks was compared within +/- 0.5% variability in their retention time. Common peaks mean that they are present in both PL-100 yolk and table yolk, whereas PL-100 peaks suggest that they are only present in PL-100 eggs. Comparing the two fractions, the 3,000 Dalton permeate of PL-100 egg yolk showed 15 more peaks than that of table yolk, indicating that over-immunization for chickens could produce more biological components in their eggs. Is to suggest.

3KDa Permeate Common peak PL-100 peak Table peak count PL-100 18 15 0 33 table 18 0 5 23

Preparative HPLC

In order to obtain a large amount of anti-inflammatory factor after ultrafiltration, a preferred step is to separate the partially purified composition having a molecular weight of less than about 3,000 Daltons on a preparative HPLC column. Thus, a 5.08 × 20 cm column was packed with Zorbax C8 fill and 4 g of ultrafiltration less than 3,000 Daltons of MW was purified at each treatment. Permeate was collected, separated and lyophilized into partially purified compositions at a flow rate of 90 mL per minute using 80/20 H ₂ O / methanol containing 0.05% TFA as the mobile phase. The collected peaks were then retested on an analytical C8 column. According to the method described, a large amount of ultra high purity peaks were obtained by preparative HPLC and analyzed for biological activity, elemental composition and chemical structure. Mass spectrometry, elemental analysis, infrared spectroscopy and nuclear magnetic resonance NMR are used to determine the structure of the factor.

Example 5

Anti-arthritis Characteristics of Hyperimmune Oocytes in Collagen-Induced Arthritis Model in Rats

Collagen-induced arthritis in rats is a common experimental animal model of rheumatoid arthritis and has been used in various laboratories and research institutions since 1977. Since its initial discovery, animal models of collagen-induced arthritis have proven to correspond mostly to human rheumatoid arthritis. For example, Stuart and co-workers have demonstrated that some clinical signs and histological changes in the joints of arthritis rats are similar to those in patients with rheumatoid arthritis (Stuart et al., 1983). Of particular importance is the fact that most patients with rheumatoid arthritis show autoimmunity against type II collagen. Historically, this animal model has proven to be a useful model for predicting the clinical efficacy of potential immunomodulators in terms of new classes of immunosuppressive properties and their anti-arthritic activity. Arthritis in mice and rats is generally induced by immunizing with heterologous type II collagen, which initiates a targeted humoral and cellular immune response against articular tissue. Current therapies are inadequate and have side effects that limit their long-term use.

This example is intended to show the effect of the oocyte product and partially purified anti-inflammatory composition of the invention administered orally before and during the induction of arthritis with collagen II. Inhibition of arthritis incidence was examined in a dose-dependent manner compared to the control group.

Way

Thirty Sprague-Dawley female rat-VAF + (Charles Rivers, Wilmington, Mass.) Weighing 100-125 g were randomly divided into three groups (10 / group). The experiment was repeated three times so that the final group included 30 rats per treatment regimen and a total of 90 rats were included in the trial. Spray dried hyperimmune oocytes (prepared in Example 1) were diluted for orla gavaz. 10% and 0.2% solutions of oocytes for oral gabaz were made every other day. The solution on day 2 was stored at 4 ° C. until use. Oocytes (3.5 ml of each solution) were orally gabbed in rats for 7 days prior to initiating type II collagen induced arthritis in rats and for 14 days after induction.

Methods for inducing and evaluating arthritis in rats were made according to Trentham, et al., 1977. Briefly, 400 pepsin-treated natural type II chick collagen (CII) in which Sprague-Dawley rats were dissolved in 0.1 M acetic acid at a concentration of 1 mg / ml and 1: 1 emulsified with incomplete Freund's auxiliary solution. (Genzyme, Boston, Mass.) Was immunized on the lower back by inoculation. Three groups: water gabaz (control), 50x hyperimmune oocytes (high dose) (3.5 ml of 10% solution of hyperimmune oocytes) and 1x hyperimmune oocytes (low dose) (0.2 of hyperimmune oocytes 3.5 ml% solution) was used for 10 rats in each group. From day 10 after intradermal immunization with collagen II until the next 21 days, rats were clinically evaluated for periarticular erythema (0-4 +) and swelling (0-4 +) of the rat foot in each limb ( Blind).

The arthritis index (AI) in the group is derived from the mean AI and is the sum of the scores of the feet based on the incidence of arthritis and the severity of arthritis. On day 21 all rats were killed and bled. Serum was evaluated for antibody titers against type II collagen. Specific antibody titers against collagen type II were determined using enzyme-linked immunoassay (ELISA) (Trentham, et al., 1983). Animals were then killed by administering an excess of metafane.

result

DCV oocytes showed deterministic antiarthritis properties in a dose-dependent manner in type II collagen rat models.

Consumption of high doses of DCV oocytes has clearly demonstrated anti-inflammatory properties in this model by affecting both cellular and humoral pathways of the immune system.

The compound has been shown to be nontoxic at both low and high doses. The high dose group not only showed a statistically significant decrease in the incidence of arthritis, but also a delay in the expression of arthritis and a moderate decrease in arthritis compared to the water control group.

5 shows that high doses of oocytes administered prior to immunization with type II collagen prevented the development of arthritis symptoms in more animals than did not receive eggs. In addition, within 11 days of treatment with oocytes and subsequently over the entire duration of the test, a significant reduction in the incidence of arthritis was observed compared to the group that did not receive any eggs at all.

The antiarthritis effect was observed by S-100 and PL-100 whole eggs in a type II collagen induced arthritis model. After egg separation, egg yolk showed antiarthritis activity in animal models, but egg white did not. However, degreasing the same egg yolk completely lost the antiarthritis activity. This degreased egg yolk was further purified by a 3000 molecular weight cutoff size exclusion column and tested in a rat type II collagen model. The partially pure fraction showed high antiarthritis activity equivalent to that shown by the whole PL-100 eggs.

Another important observation is that rats fed PL-100 eggs show a statistically significant decrease in antibodies to type II collagen in their serum compared to animals in the control group. This suggests that PL-100 eggs have an immunosuppressive effect in animals and thus help to alleviate their arthritis symptoms in the joints.

Average number of days until the maximum incidence of arthritis Control Low dose High capacity Expt 1 14 19 20 * Expt 2 14 18 17 Expt 3 12 15 15 Delayed manifestation of maximal arthritis after immunization with collagen II

At day 21, the arthritis index, which reflects the severity and severity of arthritis, was significantly reduced in animals administered high doses of hyperimmune oocytes compared to controls (2.46 ± 0.55 vs 4.167 ± 0.51; p ≥0.003, Student's t -exam). The effect of low dose oocytes was not significantly different compared to the control. In addition, the incidence of arthritis was significantly suppressed in the high dose group compared to the control group (30% vs. 70%; p ≦ 0.03). This reduction in the incidence of arthritis caused by high doses of hyperimmune oocytes (Table 4) is as good as reported for steroids, NSAIDS (non-steroidal anti-inflammatory drugs) methotrexate and minocycline.

This example also shows that the higher doses of oocytes administered prior to inducing arthritis prevented the development of arthritis in a greater number of animals than when no eggs were administered. These results are shown in FIG.

Figure 6 shows the prevention of arthritis due to high dose of egg product. Compared to the control animals that did not receive eggs, animals that orally gabbed oocytes showed symptoms of arthritis at a smaller rate. Animals were orally gabbed for 7 days before immunization (induction of arthritis) and 21 days after immunization.

Review

The data clearly shows the deterministic antiarthritis effect in rats of oocytes in a dose-dependent manner in a type II collagen induced rat arthritis model. A statistically significant decrease in the incidence of arthritis, a delay in expression and a decrease in severity were observed in animals administered high doses of hyperimmune oocytes. This data shows the prophylactic effect of high dose oocytes on the incidence of arthritis. Most importantly, hyperimmune oocytes are nontoxic at both high and low doses.

Example 6

Reduction of Collagen II Antibodies

Rats from Example 5 were bled on day 21 and serum samples were collected. Titers to collagen II autoimmune antibodies were measured using standard ELISA test methods. These results show that animals fed high or low doses of oocytes had significantly lower antibody titers against collagen II compared to controls (Table 5).

Control Low dose High capacity Antibody titer against collagen II (-log ₂ ) 9.3 ± 0.03 8.5 ± 0.28 8.3 ± 0.36 Statistical significance - p≤0.004 p≤0.005

The decrease in autoimmune antibody titers against collagen II in the serum of the oocyte-produced animal is very significant compared to the control animal (no oocyte administration). Since all groups of animals were administered the same level of collagen II to induce antibodies against collagen II and the resulting arthritis symptoms, it was noted that the oocyte-administered group had lower titers of collagen II antibodies on day 21 It was unexpected. This decrease in antibody titer against collagen II suggests a positive effect on both cellular and humoral immune responses to hyperimmune oocytes.

Example 7

Anti-inflammatory Effects of Hyperimmune Eggs

This example demonstrates the anti-inflammatory effect of hyperimmunized eggs (prepared as described in Example 1) against carrageenan induced skin edema in dogs fed a meal comprising hyperimmunized eggs. These results demonstrate that hyperimmune eggs reduce inflammation. The effect on inflammation was equivalent to the effect obtained when using the non-steroidal anti-inflammatory ibuprofen at the dose of 10 mg / kg ED ₅₀ in this test.

Twenty White Eagle Beagles (four groups of five dogs per group) were fed a basal diet of commercially available lamb and rice dog food. As described in Table 6, the two five groups (Groups 1 and 2) were fed only basic meals during a conditioning period of approximately 100 days. Dogs in Group 2 were also treated with ibuprofen, a non-steroidal anti-inflammatory agent, before an inflammatory attack with carrageenan. During the same period, the other two groups (groups 3 and 4) were fed a basic meal with a hyperimmune egg (HIE). Group 3 was fed 3.5 g of hyperimmune eggs, while Group 4 was fed 35 g of hyperimmune eggs added to the meal. At the end of the conditioned meal treatment, the dog was attacked by intradermal injection of 2% carrageenan causing an inflammatory response.

The attack process was as follows. One dog from each group was randomly selected for daily challenge and testing. This procedure was repeated daily for 5 days until all dogs in all groups were tested. Fifteen minutes before the challenge procedure, group 2 dogs were orally administered 10 mg / kg of ibuprofen. All dogs were anesthetized by intravenous injection and the left side (about 6 × 8 inches) was shaved. Two lines of three marks were made in the shaved area and numbered 1-6. As a negative control, 0.1 ml of saline was injected intravenously in Mark 1. In Mark 2-6, 0.1 ml of carrageenan 2% solution was injected subcutaneously. Injections were administered by the same person during the trial. Intradermal injections of carrageenan at this concentration were predetermined to cause measurable swelling that could have been reduced by 10 mg / kg ibuprofen at 100% of the animals. At the end of the last injection of carrageenan, each swell was measured using a micrometer and the number was recorded. Anesthetized animals were restored and the measurements were repeated and recorded after 6 hours. The average size of the inflammatory responses for each group was determined.

group process Number of animals per group Average inflammatory response Mean Difference for Control Significance P value 1 control group Basic meal 5 4.41 - - 2 drugs Ibuprofen (10 mg / kg) + Basic Meal 5 2.25 2.2 P <0.01 3 test group HIE I 3.5 g + basic meal 5 2.8 1.6 P <0.05 4test group HIE Egg 35g + Basic Meal 5 2.38 2.0 P <0.05

These results showed a significant reduction in swelling in animals fed the hyperimmune eggs (3.5 g + basal and 35 g + basal), suggesting a prophylactic effect on inflammation of orally fed hyperimmune eggs. will be. In addition, these results show that the anti-inflammatory activity of hyperimmune eggs was equivalent to the effect exhibited by the anti-inflammatory ibuprofen. Differences in significance p <0.05 levels were seen between treatment and control groups.

Example 8

Effects of Ovarian Products on Human Arthritis Patients

A nutritional drink supplement consisting of a high protein, high carbohydrate powder containing 30% of the daily recommended amount of vitamins and about 4.5 g (approximately 0.4 eggs) of hyperimmune powdered eggs was obtained. This beverage supplement was provided by DCV Inc. (DCV, Inc., Wilmington, DE).

Three patients received a nutritional beverage supplement for a period of two months. The clinical assessment of the tolerance to the product, which is a clinical response, as assessed by the assessment of the clinical status of each patient and by detailed clinical examinations, was determined at 1, 2, and 3 months before the test. 1 month after suspension). Clinical chemistry, hematology and urine analysis profiles were performed. The treatment results for each of these patients are described below:

Patient # 1

Patient # 1 is a 29-year-old woman with a 5'3 "170 lbs history of rheumatoid arthritis (combustibility). At the first examination, the patient had arthralgia, swelling, and fingers, wrists, toes, feet and The patient complained of functional limitations, such as the inability to bend or run the knee, had difficulty kneeling, and the patient's hematological examination showed normal erythrocyte sedimentation rate (ESR) and C- Reactive protein (CRP) readings were obtained Patients had cholesterol levels of 199 mg / dL Patients were treated with minocin 300 mg / week, Loestrin Fe 1.5 / 30 and multivitamins were used. Was rheumatoid arthritis with uveitis.

After consuming the nutritional supplement for one month on a daily basis, redness and swelling of the hand of the patient was reduced and foot pain and swelling were lost. The patient reported "less painful" and reported "75% better". The patient reported an increase in energy. The patient's ESR and CRP readings remained normal but cholesterol dropped to 186 mg / dl. The patient continued his own medication with the egg-base drink.

During the second month, the patient felt his hands worse, the patient showed swelling of the fingers and wrists and reported numbness in the hands at night. The patient's leg felt "better." The overall evaluation of the specialist showed that the patient was slightly worse. The patient still experienced pain when carrying the shopping bag or opening the box. The patient's ESR and CRP readings remained normal and cholesterol was maintained at 189 mg / dl. The patient continued with medical treatment and stopped using the product according to the protocol as planned.

After one month without using the product, swelling and tenderness appeared again on the hands and feet. The functional limits of the patient were maintained as initially shown. The patient still felt 75% "better". The patient's ESR and CRP readings remained normal but cholesterol dropped to 180 mg / dl.

The score of arthritis 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 joint. Based on the evaluation of these patients, numbers representing the extent of symptoms were prepared. The numbers range from the highest 36 (maximum amount of swelling, pain and tenderness) to the lowest zero (no swelling, pain and tenderness).

The scores for arthritis in Patient # 1 were as follows:

At start = 14

I-one month as a basic drink = 2

I-2 months for a basic drink = 6

1 month after stopping drink = 8

Patient # 2

Patient # 2 was a 88-year-old woman, 5'3 "104 lbs, with a past history of rheumatoid arthritis and chronic sinusitis. The patient had pain in the lower extremities and shoulders. And functionally, the patient could only walk in a limited manner and had difficulty bending his arms.The patient's hematological tests included elevated ESR (36 mm / hr), CRP (1.64 mg / dl) and A total cholesterol of 220 mg / dL was displayed Patients treated with methotrexate 5 mg / week, Orudis KT 2 / day, Ca 1500 mg, Fosomax 10 mg, Prednisone 5 mg q2d (every other day) Multivitamins were taken and patients continued to use these medications in addition to egg-base beverages.

One month after starting the nin-base drink, the patient still complained of swelling of the hands, knee pain and inability to sleep. The patient reported "50% better", "overall feeling better" and "walking better". Pain attacks in patients were less frequent, but patients were still in pain. Hematological results were ESR (30 mm / hr) and CRP (1.64 mg / dL), and an increase in cholesterol to 232 mg / dL was observed.

Two months after taking the product, the patient reported that she felt 98% better. However, the functional limits of the patient remained the same. Hematological results suggested a decrease in ESR (21 mm / hr) and a recovery of normal CRP readings (<0.5 mg / dL). The total cholesterol of the patient was maintained at 234 mg / dl. The patient continued with medicinal treatment but stopped using the egg-base beverage as planned.

After one month without using the product, the patient complained of severe arthralgia in the fingers and legs and showed swelling of the wrist. The patient felt "50% worse". The patient's ESR (27 mm / hr) level and cholesterol (257 mg / dL) began to rise, but the CRP (<0.5 mg / dL) level remained normal.

The score of arthritis 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 joint. Based on the evaluation of these patients, numbers representing the extent of symptoms were prepared. The numbers range from the highest 36 (maximum amount of swelling, pain and tenderness) to the lowest zero (no swelling, pain and tenderness).

The scores for arthritis in Patient # 2 were as follows:

At start = 10

I-one month as a basic drink = 7

I-2 months for a basic drink = 2

1 month after stopping drink = 10

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. Nitroderm patch, digoxin 0.125, aspirin 80 mg and Feldene 20 mg.Functional limitations were difficulty in arm movement and extreme fatigue.Hematologic results were normal ESR and CRP and 217 mg / Cholesterol readings are shown.

After one month of drinking the egg product with the aforementioned medical treatment, the patient felt "slightly better (10-20%)". The patient still reported shoulder pain in the right and left, and the patient reported that the foot was unstable and experienced buckling of the right knee. Functional limitations remained as previously reported. The patient's ESR and CRP readings were normal and the cholesterol level was 180 mg / dl.

Two months after using the product, the patient continued to feel “a little better”. Symptoms included pain in the right knee and difficulty walking. Cholesterol readings were not obtained, but ESR and CRP remained normal.

After one month without using the product, the patient felt slightly better overall, but slightly worse than the previous month. Symptoms included pain in the buckling of the right leg, shoulder pain and lower back. Evaluation of the joints suggested pain in the shoulder and lower back. Functional limitations included difficulty in moving the arm and a feeling of "extremely tired". ESR and CRP readings were normal and cholesterol was maintained at 180 mg / dl.

The score of arthritis 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 joint. Based on the evaluation of these patients, numbers representing the extent of symptoms were prepared. The numbers range from the highest 36 (maximum amount of swelling, pain and tenderness) to the lowest zero (no swelling, pain and tenderness).

The scores for arthritis in Patient # 3 were as follows:

At the start = 11.5

I-one month as a basic drink = 2

I-2 months for a basic drink = 3

1 month after stopping drink = 8.5

The history of other cases showed similar improvements and demonstrated the effectiveness of the present invention in the loss of pain and inflammation in arthritic joints without side effects.

Although the present invention has been described with reference to certain preferred embodiments thereof, it is to be understood that changes and modifications may be made within the spirit and scope of the invention as defined above and in the appended claims.

Example 9

Sialic acid analysis

Workflow diagram of sialic acid glass, purification and analysis

(Insert scheme at the bottom of page 46 in English specification)

Sample Manufacturing → Dow X 1X8 Kangyang Ion Exchange → Dow X AG 4X4 Weak Ion Exchange

→ HPAEC / PAD; (bottom) 2 M acetic acid hydrolysis, 80 ° C., 3 hours

Freeing and Purification of Sialic Acid from Glycoprotein

Sialic acid glass with relatively low O-acetyl loss was achieved using 1 ml of 2 M acetic acid at 80 ° C. for 3 hours in a sealed crimp-top vial. KDN is added as a pre-hydrolysis internal standard.

Following acid hydrolysis, the reaction mixture was cooled and the vial was centrifuged to remove insoluble matter. The supernatant was transferred to a micron (0.5 mL), centricon (2 mL) or centriplus (15 mL) 10,000 molecular weight cut-off centrifuge. Acetic acid in the filtrate was removed by vacuum centrifugation and the sample was redissolved in 0.5 ml of water.

Samples were applied to a 1 ml column of Dowex 50 AG 1X8 (in hydrogen form) in water to remove cationic contaminants. 4-5 mL of flow through and water washes were collected in a thin walled centrifuge tube containing 40 μl of 1 M formic acid. The acidified wash was dried by vacuum centrifugation to remove the weak acid produced by the column.

The sample was redissolved in 0.5 ml of 10 mM sodium formate, pH 5.50 and applied to a 1 ml column of Dowex AG 3X4 (format) weak anion exchange resin buffered in 10 mM sodium formate, pH 5.50. It was. The column was immediately washed with 5 ml of 10 mM formic acid. The throughflow fluid and wash fluid were discarded. Sialic acid was eluted in a thin-walled centrifuge tube containing 10 ml of 1 M formic acid, the eluate was dried and then resuspended in water.

Note: All steps in this process can be performed at room temperature only if the total O-acetylation is taken into account. If prevention of O-acetyl transfer is necessary, all steps should be performed at 4 ° C. using 1 M pyridine acetate buffer (pH 5.5) instead of 1 M formic acid.

Column description

Dowex 50 AG 1X8, hydrogen form: 1 ml of resin was placed in a small disposable plastic-fritification column. The column was started with 5 ml of 1 M HCl and then washed with a 5 ml aliquot of water until the pH rose above -4.

Dowex AG 3X4 (or 4X4), formate form: 1 ml of resin was placed in a small disposable plastic-fritizing column and converted to hydroxide form by washing the column with 5 ml of 1 M NaOH. The hydroxide form was then converted to the formate form by washing the column with 3-5 mL of 1 M formic acid or until the pH of the flow through was -1.5. The column was washed with double DI water. The column was then buffered with 10 mM sodium formate, pH 5.5.

Analysis of Sialic Acid by Dionex HPAEC / PAD on Carbopac PA-10 Columns

Sialic acid samples were analyzed by HPAEC / PAD using a Dionex GP40 gradient pump, ED40 electrochemical detector and CarboPac PA-10 guard and analytical columns. Sample injection was done using an AS3500 autosampler. Sialic acid was eluted using 100 mM NaOH (eluent C) and 100 mM NaOH, 1 M NaAc (eluent D) by the gradient method. The separation gradient for this method was as follows:

Time (min) A% B% C% D% INJECT TITL1

Initial--93.0 7.0

0--93.0 7.0 X X

10--70.0 30.0

11--70.0 30.0

12--93.0 7.0

27--93.0 7.0

This method took 27 minutes at a flow rate of 1 ml / minute. Sialic acid was quantified using KDN as an internal standard. Sample peak areas were specified to adjust for KDN signal loss due to hydrolysis or ion exchange cleanup.

Review

Sialic acid analysis of PL-100 l and its components I fraction Per g egg, free + bound sialic acid content Sialic acid% Table yolk 1.9 mg 0.19% Table egg whites 1 mg 0.10% 3k fraction of PL-100 egg yolk 7.7 mg 0.77% 3k fraction of PL-100 egg white 0.75 mg 0.075%

The table shows free and bound sialic acid content in various fractions of eggs as analyzed by standard Dionex protocol and PAD detection. This procedure, with pre-cleaning on Dow X resin, resulted in quantitative recovery and removes other contaminant peaks from that location in the chromatogram.

Separation of the 3k fraction of egg yolk does not make it rich enough to detect binding and free sialic acid content.

The biological activity of the 3k fraction of egg yolk in the rat type II collagen test method cannot be attributed to the sialic acid content because the sialic acid content (2.7 mg / dose) is very low.

Claims (20)

a) is present in the whole egg, egg white and egg yolk of an egg produced by an egg-producing animal; b) has a molecular weight of less than about 3000 Daltons; c) thermal stability; d) orally active; e) an anti-inflammatory composition in partially purified form, characterized by being resistant to degradation by digestive enzymes. The anti-inflammatory composition of claim 1 wherein the egg-producing animal is overimmunized with an immunogenic vaccine or a gene vaccine. The anti-inflammatory composition of claim 2 wherein the immunogenic vaccine comprises at least one antigen selected from the group consisting of bacterial, viral, protozoal, fungal and cellular immunogens and mixtures thereof. The method of claim 2, wherein the genetic vaccine is exposed DNA, plasmid DNA, viral DNA, bacterial DNA, DNA-expression library, DNA-RNA immunogen, DNA-protein conjugate and fragment of DNA-liposomal conjugate, and An anti-inflammatory composition comprising at least one immunogen encoded DNA construct selected from the group consisting of a mixture. The method of claim 1, further comprising: a) degreasing the whole egg, egg yolk or egg white; b) isolating an aqueous fraction from degreased whole egg, egg yolk or egg white; c) separating less than about 3000 Daltons permeate from the aqueous fraction; d) partially purified by a method comprising fractionating permeate less than about 3000 Daltons to recover the partially purified anti-inflammatory composition. A method for treating and preventing inflammation in a subject suffering from or prone to inflammation, characterized by administering an effective amount of an egg product to the subject. 7. The method of claim 6, wherein the egg product is selected from the group consisting of whole egg, egg yolk, egg white and fractions thereof. 8. The method of claim 7, wherein the oocyte product is obtained from an egg-producing animal overimmunized with an immunogenic vaccine or a gene vaccine. The method of claim 8, wherein the immunogenic vaccine comprises at least one antigen selected from the group consisting of bacterial, viral, protozoal, fungal and cellular immunogens and mixtures thereof. The method of claim 8, wherein the genetic vaccine is exposed DNA, plasmid DNA, viral DNA, bacterial DNA, DNA-expression library, DNA-RNA immunogen, DNA-protein conjugate and fragment of DNA-liposomal conjugate, and At least one immunogen encoded DNA construct selected from the group consisting of mixtures. 8. The method of claim 7, wherein the egg product further contains an anti-inflammatory composition. 12. The method of claim 11, wherein the anti-inflammatory composition is partially purified and a) is present in the whole egg, egg white and egg yolk of the egg produced by the egg-producing animal; b) has a molecular weight of less than about 3000 Daltons; c) thermal stability; d) orally active; e) a method characterized by being resistant to degradation by digestive enzymes. 13. The composition of claim 12, wherein the anti-inflammatory composition comprises a) degreasing the whole egg, egg yolk or egg white; b) isolating an aqueous fraction from degreased whole egg, egg yolk or egg white; c) separating less than about 3000 Daltons permeate from the aqueous fraction; d) fractionating the permeate of less than about 3000 Daltons to recover the partially purified anti-inflammatory composition. The method of claim 6, wherein the inflammation is arthritis. 8. The method of claim 7, wherein the effective amount of the oocyte product administered is about 100 mg to 10 g total eggs, or an equivalent amount thereof, per kg body weight of the subject. The method of claim 12, wherein the effective amount of the partially purified anti-inflammatory composition administered is between about 1 μg and 400 mg per kg of body weight of the subject. A method of treating an autoimmune disease in a test animal suffering from or susceptible to autoimmune disease, characterized by administering an effective amount of an egg product to the test animal. 18. The method of claim 17, wherein the egg product is obtained from an egg-producing animal overimmunized with an immunogenic vaccine or a gene vaccine. 19. The method of claim 18, wherein the egg product further comprises an anti-inflammatory composition. The method of claim 19, wherein the anti-inflammatory composition is partially purified and a) is present in the whole egg, egg white and egg yolk of the egg produced by the egg-producing animal; b) has a molecular weight of less than about 3000 Daltons; c) thermal stability; d) orally active; e) a method characterized by being resistant to degradation by digestive enzymes.