WO1999036077A1

WO1999036077A1 - Composition and method for treatment and prevention of arthritis and/or autoimmune diseases

Info

Publication number: WO1999036077A1
Application number: PCT/US1999/000749
Authority: WO
Inventors: Orn Adalsteinsson; Michael J. Daley; Sandra G. Fitzpatrick-Mcelligott; Hellen Chaya Greenblatt
Original assignee: Dcv, Inc.
Priority date: 1998-01-19
Filing date: 1999-01-14
Publication date: 1999-07-22
Also published as: CN1304312A; AU2115799A; JP2002509109A; CA2317813A1

Abstract

A natural composition for arthritis prevention and treatment. The composition comprising an effective amount of an egg or fraction thereof. The egg is obtained from an egg-producing animal which as been hyperimmunized with at least one antigen. The egg or fraction thereof is also effective in the prevention and treatment of autoimmune diseases. A method for treating and preventing arthritis and/or autoimmune disease through administration of the egg or a fraction thereof is also disclosed.

Description

TITLE COMPOSITION AND METHOD FOR TREATMENT AND PREVENTION OF ARTHRITIS AND/OR AUTOIMMUNE DISEASES

FIELD OF THE INVENTION This invention relates to the treatment and prevention of arthritis and autoimmune diseases. More particularly, this invention relates to a natural food product and its use in preventing, countering, or reducing arthritis and/or an autoimmune disease.

BACKGROUND OF THE INVENTION Arthritis

Arthritis manifests itself in a variety of forms. Some of the more common forms include rheumatoid arthritis, osteoarthritis and generalized rheumatism.

Rheumatoid arthritis is an autoimmune disease characterized by pain, swelling and stiffness in the joints. Rheumatoid arthritis is a disease which afflicts approximately 3% of Americans, and particularly women. Rheumatoid arthritis is an extremely disabling disease and usually strikes adults between the ages of 30 and 40 years, while the occurrence of clinical illness is greatest among those aged 40 - 60 years. Although drug therapy is somewhat effective, as many as 7% of rheumatoid arthritis sufferers are disabled to some extent as quickly as 5 years after disease onset, and within 10 years, as many as 50% are too disabled to work (Medical Sciences Bulletin, December 1994).

Osteoarthritis produces similar symptoms to rheumatoid arthritis. In particular, although osteoarthritis begins as a degeneration of articular cartilage whereas rheumatoid arthritis begins as inflammation in the synovium, each process approaches the other as the disease progresses. In osteoarthritis, as cartilage deteriorates and joint congruence is altered, a reactive synovitis often develops. Conversely, as rheumatoid arthritis erodes cartilage, secondary osteoarthritis changes in bone and cartilage develop. At the end stages of both osteoarthritis and rheumatoid arthritis, the involved joints appear the same.

Some other forms of arthritis include Ankylosing Seronegative Spondyloarthropathy (ankylosing spondylitis) and reactive arthritis. These conditions are often referred to as the "B-27 associated diseases," and are difficult to differentiate from rheumatoid arthritis. In some cases ankylosing spondylitis, Reiters syndrome or psoriatic arthritis are present coincidingly with RA in the same patient. In many cases, these patients are treated with the same disease modifying drugs as those suffering from progressive rheumatoid arthritis.

Onset of arthritis generally occurs after the age of 30 in those who are susceptable to such disease. However, some forms of arthritis may be initiated by different causes, such as slow virus infections. Because there is great overlap, many physicians consider these forms as "generalized rheumatism" and approach management of the diseases in the same way. Some diseases which fall into this category include Chronic Fatigue Syndrome, fibromyalgia (fibrositis) and gout. In fact, for some patients, evidence is accumulating for superimposition of rheumatoid arthritis and fibromyalgia. (Harris, Edward D. and W. B. Saunders, Rheumatoid Arthritis, 1997).

Autoimmune Diseases

As stated above, rheumatoid arthritis is an autoimmune disease, and as such, its etiology is much the same as the etiology of any other autoimmune disease. Generally, the body normally recognizes the difference between its own by-products and foreign invaders (i.e. bacteria, viruses, fungi and protozoans, to name a few). When an immune cell (T or B lymphocyte) reacts to a "self-protein" during its development, that cell is deemed defective and usually destroyed or inactivated. Sometimes, however, a "self-reactive" immune cell will escape destruction. At a certain later time, that cell can be activated and trigger an immune response. Activation is thought to occur after infection with a common bacteria or virus which contains a polypeptide having a stretch of amino acids which match a stretch on the defective self-protein. Several bacteria, such as Streptococcus. Mycoplasma, and borrelia, have been implicated in the initiation of the disease, as well as certain viruses, namely retroviruses. In addition to RA, autoimmunity often results in such diseases as juvenile diabetes, multiple sclerosis. Graves' disease, Meneri's disease, myasthenia gravis, lupus erythematosus and psoriasis. (Medical Sciences Bulletin,

September, 1994).

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

Presently, treatment for rheumatoid arthritis, other arthritis and other autoimmune diseases is drug therapy. In general, patients are initially treated with "first-line" agents, usually non-steroidal anti-inflammatory drugs (NSAIDs) which primarily relieve the symptoms. The patients are later treated with "second-line" or disease- modifying agents (DMARDs) such as methotrexate, gold compounds, penicillamine, sulfasalazine, and antimalarial drugs. However, all of the above drugs have serious side effects, especially when administered in elevated doses. For example aspirin may produce indigestion and stomach pain; phenylbutazone may produce stomach ulcers and phenacetin may lead to kidney disease. Methotrexate may cause oral ulceration and gastrointestinal (GI) side effects.

Accordingly, in spite of the large number of pharmaceutical drugs that are currently available for treatment of the various forms of arthritis and autoimmune disease, there is still need for a simple, effective treatment that can alleviate as well as prevent the onset of such diseases, without the complication of side effects. If a natural food product effective in treating and preventing such diseases could be obtained, it would provide an easily administered, readily available, and safe therapeutic composition.

Related Art:

U.S. Patent No. 4,357,272 discloses the isolation of antibodies from the yolks of eggs derived from hyperimmunized hens. The hyperimmunization was elicited by repetitive injections of antigens derived from plant viruses, human IgG. tetanus antitoxin, snake antivenoms, and Serameba. U.S. Patent No. 4,550,019 discloses the isolation from egg yolks of antibodies raised in the hen by hyperimmunization with immunogens having a molecular or particle weight of at least 30,000. The antigens used to hyperimmunize the chickens were selected from among plant viruses, human immunoglobulins, tetanus toxin, and snake venoms.

U.S. Patent No. 4,748,018 discloses a method of passive immunization of a mammal that comprises parenterally administering purified antibody obtained from the eggs of an avian that has been immunized against the corresponding antigen, and wherein the mammal has acquired immunity to the eggs.

Eggs from chickens immunized against specific bacterial antigens were disclosed as providing anti-atherosclerotic effects in mammals in U.S. Patent No. 5,215,746.

None of these references, however, discloses or suggests that hyperimmune eggs can be administered to animals to treat arthritis and/or an autoimmune diseases. Nor do any of these references disclose or suggest a method providing a reasonable expectation that hyperimmunization of an avian could produce an avian which lays eggs having such a capability.

SUMMARY OF THE INVENTION The invention is based on the inventors' discovery that there is activity in egg and egg products, and particularly in egg products obtained from hyperimmunized avians, which when administered to a subject animal, in particular, mammals, prevents or reduces arthritis and/or autoimmune diseases in the subject animal.

In particular, the invention is directed to a composition for the treatment and prevention of arthritis and an autoimmune disease, the composition comprising an egg product obtained from an avian which has been hyperimmunized with an antigenic or genetic vaccine.

The invention is also directed to a method of treating at least one of the following disorders in a subject animal: arthritis and an autoimmune disease, the method comprising administering to the subject an effective amount of an egg product.

The invention is additionally directed to a method wherein the egg product is administered to the subject animal in combination with a drug selected from the group consisting of non-steroidal, anti-inflammatory drugs and disease-modifying, anti- arthritic drugs.

The invention is finally directed to a composition for reducing antibodies against autoimmune diseases in a subject animal, the composition comprising an effective amount of an egg product derived from an avian which has been hyperimmunized with an antigenic or genetic vaccine.

DETAILED DESCRIPTION OF THE INVENTION The invention generally relates to a composition and method for treatment and prevention of arthritis and autoimmune diseases. The composition is preferably a natural food product which comprises hyperimmune egg or egg product. The food product, when administered by the method of the invention, not only provides relief from the pain and other symptoms caused by arthritis and autoimmune diseases, but can delay, and even prevent, the onset of such diseases. The preferred antigen mixture injected into the avians to produce the hyperimmune egg product does not contain specific antigens which are known to cause arthritis or autoimmune diseases. Therefore, it is surprising that administration of the egg product obtained from avians immunized against a mixed antigen vaccine is effective in reducing the symptoms of and preventing arthritis and autoimmune diseases when administered to a subject.

Definitions:

The following definitions apply throughout:

The term "arthritis" means any of a variety of disorders marked by inflammation and degeneration of connective tissue structures, especially the joints and related structures. It may be attended by pain, stiffness, or limitation of motion of these parts. Some forms of arthritis include rheumatoid arthritis, osteoarthritis, ankylosing seronegative spondyloarthropathy, reactive arthritis, chronic fatigue syndrome, fibromyalgia (fibrositis) and gout.

The term "autoimmune disease" is applied the standard medical definition as found in standard medical dictionaries such as Dorland's and Taber's. The following disorders are considered autoimmune diseases: rheumatoid arthritis, juvenile diabetes, multiple sclerosis, Graves' disease, Meneri's disease, myasthenia gravis, lupus erythematosus, psoriasis, systemic scleroderma, rheumatic fever and Sjogren syndrome.

The term "hyperimmunization" means exposure to one or more antigens such that an immune response is elevated and maintained above the natural unexposed state.

The terms "egg" or "egg product" each mean any whole egg (table, hyperimmunized or otherwise) or any product or fraction derived therefrom.

The terms "table egg" or "table egg product" each mean a whole egg, or any product or fraction derived therefrom, obtained from egg-producing animals which are not maintained in a hyperimmune state. The terms "hyperimmune egg^'* or hyperimmune egg product^" each mean whole egg or any product or fraction derived therefrom, obtained from an egg producing animal maintained in a hyperimmune state.

The term "supranormal levels'^" means levels in excess of those found in eggs of egg- producing animals not maintained in a hyperimmune state.

The term "combinatorial derived antigens" refers to a novel process of generating molecular diversity among antigens by way of combinatorial synthesis.

The term "bioengineered antigens" refers to antigens which are obtained through the process of gene cloning technologies and genetic rearrangements which allow the insertion of encoding nucleotides which can give rise to molecules having antigenic properties.

The term "genetic vaccine" refers to a nucleic acid vaccine which is generally produced by recombinant technologies and which may elicit an immune response.

The term "treatment" means that the onset of the symptoms (including pain) of the disorder and/or pathogenic origin of the disorder be delayed or completely prevented, or, if present, the symptoms be ameliorated or completely eliminated. For example, the hyperimmune egg product treats arthritis and/or an autoimmune disease not only by suppressing the symptoms of the disorder in humans and other mammals, but also by acting as a prophylactic agent to counteract the presence of the disorder in the recipient.

The term "prevention" means that the progression of the disease is reduced and/or eliminated, or that the onset of the disease is eliminated. The term "administer" means any method of providing a subject with a substance, including orally, intranasally, parenterally (intravenously, intramuscularly, or subcutaneously), rectally or topically.

The term "animal" means the animal kingdom definition.

The term "target animal" refers to an animal which functions as the egg or egg product producing animal.

The term "subject animal" refers to the animal which is administered the egg or egg product produced by the target animal.

The Invention

The product and method of the invention relate particularly to the use of hyperimmune egg, which is a natural food product, in the treatment and prevention of autoimmune diseases and arthritis. Being natural, this food product can be used to treat and prevent such diseases without the fear of side effects, except, of course, for allergic reactions in those intolerant to eggs.

In a preferred embodiment, the invention comprises a hyperimmune egg or egg product which is effective in treating and preventing arthritis and/or an autoimmune disease in a subject animal. The hyperimmune egg is obtained from an egg-producing animal, and more preferably, an avian, which has been hyperimmunized with at least one antigen. The hyperimmune egg product is one which is preferably administered orally to the subject animal. The hyperimmune egg or egg product can be further separated into more potent fractions which can subsequently be administered to a subject animal in a variety of forms.

The hyperimmune egg or egg product of the invention is effective in treating and preventing all forms of arthritis, including, but not limited to, rheumatoid arthritis, osteoarthritis, ankylosing seronegative spondyloarthropathy, reactive arthritis, chronic fatigue syndrome, fibromyalgia (fibrositis) and gout. The egg product of the invention is equally effective in treating autoimmune diseases, such as rheumatoid arthritis, juvenile diabetes, multiple sclerosis, Graves' disease, Meneri^'s disease, myasthenia gravis, lupus erythematosus, psoriasis, systemic scleroderma, rheumatic fever and Sjogren syndrome among others.

Hyperimmune egg Product

The hyperimmune egg product can be produced by any egg-producing animal. It is preferred that the animal be a member of the class Aves or, in other words, an avian. Within the class Aves, domesticated fowl are preferred, but other members of this class, such as turkeys, ducks, and geese, are a suitable source of hyperimmune egg product.

When such egg-producing animals are brought to a specific state of immunization by means of, for example, periodic booster administrations of antigens, the animals will produce eggs that, when consumed by a subject, will have beneficial properties in the treatment and prevention of arthritis and autoimmune diseases in that subject.

Having knowledge of the requirement for developing and maintaining a hyperimmune state, it is within the skill of the art to vary the amount of antigen administered, depending on the egg-producing animal genera and strain employed, in order to maintain the animal in the hyperimmune state.

Alternative modes of hyperimmunizing egg producing animals can be used in place of antigenic vaccines and include the use of genetic vaccines. In particular, any DNA construct (generally consisting of a promoter region and an antigen encoding sequence) will trigger an immune response. Genetic vaccines consist of antigen- coding vectors, fragments of naked DNA, plasmid DNA, DNA-RNA antigens, DNA- protein conjugates, DNA-liposome conjugates, DNA expression libraries, and viral and bacterial DNA delivered to produce an immune response. Methods of DNA delivery include particle bombardment, direct injection, viral vectors, liposomes and jet injection, among others. When applying these delivery methods, much smaller quantities may be necessary and generally result in more persistent antigen production. When using such genetic processes, the preferred method for introducing

DNA into avians is through intramuscular injection of the DNA into the breast muscle.

Hyperimmunization Procedure

The following list of steps is an example of a preferred procedure used to bring an egg-producing animal to a heightened state of immunity:

1. Selecting one or more antigens.

2. Eliciting an immune response in the egg-producing animal by primary immunization.

3. Administering booster vaccines of antigens of appropriate dosage to induce and maintain the hyperimmune state.

Step 1: Any antigen or combination of antigens may be employed as a vaccine.

The antigens can be bacterial, viral, protozoan, fungal, cellular, or any other substances to which the immune system of an egg-producing animal will respond. The critical point in this step is that the antigen(s) must be capable of inducing immune and hyperimmune states in the egg-producing animal. Although only a single antigen may function as the vaccine for the method of the invention, one preferred vaccine is a mixture of polyvalent bacterial and viral antigens selected from the following antigen families: the enteric bacilli and bacteroides, pneumococci, pseudomonas, salmonella, streptococci, bacilli, staphylococci, neisseria, clostridia, mycobacteria, actinomycetes chlamydiae, and mycoplasma. Viral antigens are preferably selected from the following antigen families: adenoviruses, picornaviruses and herpes viruses, although other viral antigen families will work.

In an alternative embodiment, a polyvalent vaccine referred to as Series 100 (S-100) is used. The bacteria included in the S-100 vaccine are listed in table 1 of Example 1. This vaccine has been previously described in US patent Nos. 5.106,618 and

5,215,746, both assigned to Stolle Research and Development Corporation. Another preferred vaccine for use is the EB-100E vaccine, the details of which are also described in Example 1.

Step 2: The vaccine can be either a killed or live-attenuated vaccine and can be administered by any method that elicits an immune response. It is preferred that immunization be accomplished by administering the antigens through intramuscular injection. The preferred muscle for injection in an avian is the breast muscle. Dosage is preferably 0.05-5 milligrams of the antigenic vaccine. Other methods of administration that can be used include intravenous injection, intraperitoneal injection, intradermal, rectal suppository, aerosal or oral administration. When DNA techniques are used for the hyperimmunization process, much smaller quantities are required, generally 1-100 micrograms.

It can be determined whether the vaccine has elicited an immune response in the egg- producing animal through a number of methods known to those having skill in the art of immunology. Examples of these include enzyme-linked immunosorbent assays (ELIS A), tests for the presence of antibodies to the stimulating antigens, and tests designed to evaluate the ability of immune cells from the host to respond to the antigen. The minimum dosage of antigen necessary to induce an immune response depends on the vaccination procedure used, including the type of adjuvants and formulation of antigen(s) used as well as the type of egg-producing animal used as the host.

Step 3: The hyperimmune state is preferably induced and maintained in the target animal by repeated booster administrations of an appropriate dosage at fixed time intervals. The time intervals are preferably 2-8 week intervals over a period of 6-12 months. However, it is essential that the booster administrations do not lead to immune tolerance. Such processes are well known in the art. It is possible to use other hyperimmunization maintenance procedures or combination of procedures, such as, for example, intramuscular injection for primary immunization and intravenous injection for booster injections. Further procedures include simultaneously administering microencapsulated and liquid antigen, or intramuscular injection for primary immunization, and booster dosages by oral administration or parenteral administration by microencapsulation means. Several combinations of primary and hyperimmunization are known to those skilled in the art.

Processing and Administration Once the egg-producing animals have been sufficiently hyperimmunized, it is preferred that the eggs from these animals are collected and processed to produce a hyperimmune egg product. Subsequently, the hyperimmune egg product can be administered to the subject.

The egg and/or egg product of the present invention is administered to a subject animal by any means that treats or prevents arthritis and/or autoimmune disease in the subject animal. It is preferred that administration occur by directly feeding the egg or any derivative of the egg. Egg and egg yolk are natural food ingredients and are non- toxic and safe.

In an alternative embodiment, the egg is integrated into a nutritional supplement. One preferred method for preparing the egg to be incorporated into a nutritional supplement involves drying the egg into an egg powder. Although various methods are known for drying eggs, spray drying is a preferred method. The process of spray drying eggs is well known in the art.

The dried egg powder can be incorporated into drinks in the form of, for example, protein powders, power building drinks, protein supplements and any other nutritional, athlete-associated products. In addition, the egg powder can be used in bake mixes, power bars, candies, cookies, etc. Other examples of egg processing include making an omelet, soft or hard-boiling the egg, baking the egg, or, if desired. the egg can be eaten raw or processed as liquid egg.

Finally, it is generally known in the art that the yolk and/or white fractions contain the agent or agents responsible for the beneficial properties observed and referred to above. Those having ordinary skill in the art would clearly recognize that further separation could provide more potent fractions or elimination of undesirable components, and would allow for other modes of administration such as administering egg product parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intranasally, orally or topically. Such further separation will provide for the ability to make encapsulated products and pharmaceutical compositions with said egg or fraction thereof.

When it comes to treatment and prevention of the disorder, whether it be a form of arthritis or an autoimmune disease, the hyperimmune egg product is preferably administered to the subject in an amount that is immunologically effective in treating and preventing the particular disorder. Duration and intensity of the treatment will depend upon the particular condition, whether it is present, and, if so, the advancement of the condition in the subject. The hyperimmune egg product is provided in any amount that treats and/or prevents the condition and the symptoms of the condition. For example, in some cases, daily amounts ranging from less than one to several whole, hyperimmune eggs (or hyperimmune egg products containing the equivalent of less than one to several whole, hyperimmune eggs) can be administered to the subject depending on the particular circumstance of the condition. More potent fractions can be separated and concentrated by methods well-known in the art, from several hundred eggs.

In one embodiment, the egg product of the invention was found to be effective in treating rheumatoid arthritis in rats using a collagen-induced arthritis animal model (see Example 2). This animal model is well recognized by those in the art as one which parallels the rheumatoid arthritis effect in humans. In addition to treatment of arthritis, it was determined that the egg product, when administered to rats prior to induction of arthritis, delayed, and in some cases, prevented the onset of the arthritic symptoms. Thus, the egg product of the invention is effective in not only treating the symptoms of the disease, but also delaying and/or preventing the onset or progression of the disease.

In an alternative embodiment, the egg product was tested in humans and showed positive effects in treating various forms of arthritis in several humans suffering from such symptoms (see Examples 2A-2C). The humans who were treated by the egg product demonstrated a clinical reduction in such symptoms as pain in addition to a general reduction in swelling and stiffness.

Indicative of an effect on autoimmune disease is the surprising reduction of Type II collagen antibodies by the egg product of the invention in this disease model, which was also seen in rats (see Example 3). It is contemplated that the egg product of the invention is effective in reducing antibodies involved with other autoimmune diseases such as juvenile diabetes, multiple sclerosis, Graves' disease, Meneri's disease, myasthenia gravis, lupus erythematosus, psoriasis, systemic scleroderma, rheumatic fever and Sjogren syndrome among others.

As described in the background of the invention, patients having arthritis are presently treated with non-steroidal anti-inflammatory drugs (NSAIDs) and/or disease modifying anti-arthritic drugs (DMARDs) in order to relieve the symptoms of the arthritic condition. As shown in the examples (see Example 4) patients were administered the egg product of the invention concurrently with the present drug therapy. The egg product worked synergistically with the drug therapy to reduce the symptoms of the particular disorder. The egg product can therefore be administered concurrently with alternative treatment for arthritis and autoimmune diseases to produce a synergistically better effect. In addition, as is well known in the art, NSAIDs and DMARDs can cause severe gastrointestinal damage. U.S. patent application, serial number 08/688,576, assigned to DCV, Inc., discloses that hyperimmune egg product is effective in treating NSAID- induce gastrointestinal damage. Therefore, an alternative embodiment is contemplated for cases where the subject is taking or is preparing to take NSAIDs for the treatment of an arthritic condition. In such a case, the subject can also be administered the egg product of the invention to, not only treat the arthritic condition, but also protect the gastrointestinal system from NSAID-induced damage. In such treatments, a lower dose of hyperimmune egg may be sufficient. Also, the subject animal suffering from the disorder can be administered egg product prior to beginning its regimen of NSAIDs and DMARDs, in order to better prepare the gastrointestinal system.

It is of significant importance to point out that the egg product of this invention has been shown to be safe, non-toxic, ideal for long term use and has no side effects other than on humans allergic to eggs. The egg product can be orally administered either alone or in combination with drug therapy, for long term use for arthritic and autoimmune diseases.

The advantageous properties of this invention can be observed by reference to the following examples which illustrate the invention.

EXAMPLES

EXAMPLE 1

Preparation of S-100 Vaccine

A bacterial culture containing the spectrum of bacteria shown in Table 1 below, as obtained from the American Type Culture Collection, was reconstituted with 15 mL of media and incubated overnight at 37 C. Once good growth was obtained, approximately one-half of the bacterial suspension was employed to inoculate one liter of broth with the inoculate being incubated at 37 C. After good growth was visible in the culture, the bacterial cells were harvested by centrifugation of the suspension for 20 minutes to remove the media. The bacterial pellet obtained was resuspended in sterile saline solution and the bacterial sample was centrifuged three times to wash the media from the cells. After the third sterile saline wash, the bacterial pellet was resuspended in a small amount of double distilled water.

The media-free bacterial suspension was killed by placing the suspension in a glass flask in an 80 C water bath overnight. The viability if the broth culture was tested with a small amount of killed bacteria, incubated at 37 C for five days and checked daily for growth to certify that the bacteria had been killed.

The killed bacteria were lyophilized until dry. The dry bacteria were then mixed with sterile saline solution to a concentration of 2.2 x 10° bacterial cells/mL saline (1.0 optical density reading at 660 nm). Bacteria contained in S-100 vaccine are listed in Table 1 below.

TABLE 1 S-100 Bacterial List Escherichia coli Escherichia coli (Aerobacter) Klebsiella pneumoniae Pseudomonas aeruginosa

Salmonella typhimurium Salmonella dysenteriae

Salmonella enteriditis Salmonella epidermis

Salmonella simulans Streptococcus pyogenes, type 1

Streptococcus pyogenes, type 3 Streptococcus pyogenes, type 5 Streptococcus pyogenes, type 8 Streptococcus pyogenes, type 12

Streptococcus pyogenes, type 14 Streptococcus pyogenes, type 18 Streptococcus pyogenes, type 22 Pseudomonas vulgaris Streptococcus agalactiae Streptococcus mitis

Streptococcus mutans Streptococcus salavarius Streptococcus sanguis Streptococcus pneumoniae

Propionibacterium acnes Haemophilis influenzae EB-100E Vaccine The EB-100E vaccine is known by the trade name of Scourmune®-CRT, manufactured by Schering-Plough Animal Health, of Kenilworth, New Jersey, USA. The vaccine consists of Clostridium perfringens, type C, Escherichia coli, porcine rotavirus, and transmissible gastroenteritis.

Immunization Procedure for Hyperimmune egg Product

A killed preparation of pathogens was prepared as described above. For the first vaccination, the bacteria were mixed with complete Freund's adjuvant, and 5.6 mg of bacterial material were injected into the breast muscle of a chicken. For the remaining vaccines, the bacterial preparation was mixed with incomplete Freund's adjuvant and injected into the chickens at two week intervals for six months.

Eggs were collected from the hyperimmunized chickens and then spray dried into a powder form. During the spray drying procedure, inlet temperatures did not exceed 320 Degrees F, exhaust temperatures were maintained in accordance with producing powder in the range of 3.0 to 4.0 percent finished moisture, and pump pressure was maintained around 2500 to 4000 P.S.I. Lower temperatures ranging from 100 - 160 F were used, and samples were monitored for moisture content during the drying process to obtain a final product having any consistency desired.

EXAMPLE 2

Anti-Arthritic Properties of Hyperimmune egg Product in a Collagen-Induced Arthritis Model In Rats.

Collagen-induced arthritis is an experimental animal model of Rheumatoid arthritis and autoimmune diseases, which has been explored by scientific investigators since 1977 (Trentham et al 1977). Rheumatoid arthritis is a classical autoimmune disease in which elevated immune responses to collagen in patients have been reported (Stuart et al 1983). Since its initial discovery, the animal model of collagen-induced arthritis has demonstrated many parallels to human rheumatoid arthritis. For example, Stuart et al., 1982, has demonstrated that several of the histological changes observed in the joints of arthritic rats resemble those in patients with rheumatoid arthritis. The arthritis in mice and rats is generally induced by immunization with heterologous type II collagen, which initiates a combined humoral and cellular immune response targeted to joint tissues. Current therapies are inadequate or have side effects that limit their prolonged use.

The present example looks at the effect of the egg product of the invention orally administered to rats prior to and during arthritis induction with collagen II. The suppression of the incidence of arthritis was examined in a dose dependent manner when compared to a control group.

NOT FURNISHED UPON FILING

30 Sprague-Dawley female rats-VAF+ (Charles Rivers, Wilmington. MA), weighing 100-

125 gm, were randomized to 3 groups (10 animals/group). The experiment was repeated three times so that the final groups included 30 rats/treatment regimen with total of 90 rats for this study. Spray dried hyperimmune egg product (as described in Example 1 ) was diluted for oral gavage. 10% and 0.2% solutions of egg product for oral gavage were made every other day. The second day solution was stored at 4°C until use. Egg Product (3.5ml of the respective solutions) was orally gavaged into rats for 7 days prior to initiation of the type II collagen induced arthritis in rats and for 14 days after induction.

The method of inducing and evaluating Arthritis in rats was according to Trentham. et al. 1977. Briefly, Sprague-Dawley rats were immunized on the lower back by intra-dermal inoculation of 400 pepsin-treated native type II chick collagen (CII) (Genzyme, Boston, MA), solubilized at 1 mg/ml in 0.1 M acetic acid and emulsified 1 :1 with IFA (23). Three groups: Water gavage (Control), 50X hyperimmune egg product (High Dose) (3.5ml of a 10% solution of hyperimmune egg product) and IX hyperimmune egg product (Low Dose) (3.5ml of a 0.2% solution of hyperimmune egg product) with 10 rats per group were used. Starting on day 10 following intradermal collagen II immunization and daily thereafter until Day 21, the rats were clinically evaluated (blinded) for rat paw periarticular erythema ( 0 - 4+) and paw swelling (0 - 4+) for each limb.

The group arthritis index (Al) is a summation of paw scores based on the degree of incidence of arthritis and severity of arthritis as derived from the mean Al. On Day 21, all rats were sacrificed and bled. Serum were evaluated for antibody titer to type II collagen. Enzyme Linked Immunoassays (ELISA's) were used to measure specific antibody titer to Collagen Type II (Trentham, et al. 1983). The animals were then sacrificed with metafane overdose.

Results

Administration of egg product resulted in anti-arthritic properties in a dose-dependent fashion. The high dose of egg product produced the most effective reduction in the incidence and severity of arthritis symptoms (Table 1). Scores in Table 1 indicate a reduction in the number of swollen joints and a decrease in the severity of the disease with the high dose of egg product. Moreover, both the low and high dose of egg product caused a delay in the onset of the arthritic condition after collagen II injection (Table 2). Table 1

Egg Product in Collagen- Induced Animal Model of Arthritis

Group

* Incidence of arthritis ^Λ This level of control is historically as good as steroids, NSAIDS, methotrexate/minocycline

Average Day to Maximum Incidence of Arthritis

Table 2

Delay in onset of maximum arthritis after immunization with Collagen II.

At day 21 the arthritic index which reflects the degree and severity of arthritis was significantly reduced in animals which received the high dose of hyperimmune egg product as compared to the control group (2.46+.0.55 vs 4.167+_0.51 ; p >0.003, Student t-test). The effect of the low dose of egg product did not differ significantly from the controls. In addition, the incidence of arthritis was significantly suppressed in the high-dose group compared to control (54% vs. 83%; p< 0.03). This decrease in the percent incidence of arthritis seen with the high dose of hyperimmune egg product (Table 1 ) is as good as has been reported for steroids, NSAIDS (non-steroidal anti- inflammatory drugs) methotrexate, and minocycline.

This example 2 also shows that the high dose of egg product, given prior to inducing arthritis, prevented the development of arthritis in more animals than those not given egg. These results are demonstrated in Figure 1 below.

Figure 1

Rats with Rheumatoid Arthritis

Days After Immunization with Type 11 Collagen

Figure 1 shows the prevention of arthritis by the high dose of egg product. A lower percentage of animals, orally gavaged with egg product, developed arthritis symptoms when compared to control animals, which did not receive egg. Animals were orally gavaged for 21 days, seven days prior to immunization (induction of arthritis) and fourteen days after immunization.

Conclusion

The data clearly shows a definitive anti-arthritic effect in rats for the egg product in a dose-dependent manner in the type II collagen induced rat arthritis model. Statistically significant decreases in the incidence, delay in onset, and a decrease in the severity of arthritis were observed in the animals receiving a high dose of hyperimmune egg product. This data shows the preventative effects of the high dose of the egg product on the incidence of arthritis. Most importantly, at both the high and low dose the hyperimmune egg product is nontoxic.

EXAMPLE 3 Reduction of Collagen II Antibodies

Rats from Example 2 were bled on Day 21 and serum samples were collected. A standard ELISA assay was used to measure titers against Collagen II autoimmune antibodies. These results show that the animals fed either high or low doses of egg product had significantly lowered antibody titers to collagen II when compared to controls (Table 3).

The decrease in autoimmune antibody titers to Collagen II in the serum of animals gavaged with egg product when compared to control animals (no egg product) is highly significant. Because all groups of animals received the same level of collagen II to induce antibodies to Collagen II and the resulting arthritic symptoms, it was unexpected that the groups receiving egg product would have lower titers of collagen II antibodies on Day 21. This decrease in antibody titers to collagen II indicates a positive influence in both the cellular and humoral immune response for the hyperimmune egg product.

EXAMPLE 4 Effect of Egg Product on Arthritic Human Patients A nutritional drink supplement was obtained which comprised a high protein, high carbohydrate powder containing 30% of recommended daily allowance of vitamins, and approximately 4.5 grams of hyperimmune powdered egg (approximately equal to 0.4 eggs). The drink supplement was provided by DCV, Inc., Wilmington, DE. Three patients were administered the nutritional drink supplement for a period of two months. Clinical evaluations regarding tolerance to the product an clinical response, as evaluated by each patient's estimation of clinical status and a detailed clinical examination was performed prior to the study at one month, two months and three months (one month after ceasing the product). Clinical chemistry, hematology and urinalysis profiles were performed. Described below are the results of the treatments of each of these patients:

EXAMPLE 4A

Patient #1 Patient #1 was a 29-year-old female, 5'3" 170 lbs with a previous history of rheumatoid arthritis (juvenile). On first examination the patient presented with joint pains, swelling, and tenderness of fingers, wrists, toes, feet, and knees. She complained of functional limitations such as inability to do knee bends or run, and had difficulty with kneeling. Her hematology tests had normal etythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) readings. Patient's cholesterol was at 199mg/dl. She was being treated with Minocin 300mg/week, Loestrin Fe 1.5/30 and used multi vitamins. The diagnosis was rheumatoid arthritis with uveitis. After one month of consuming the Nutritional Drink Supplement on a daily basis, the patient's hand redness and swelling decreased, and the pain and swelling in her feet disappeared. Patient reported that she felt "less achy" and "felt 75% better". She reported that she had increased energy. Her ESR and CRP readings remained normal but her cholesterol dropped to 186 mg/dl. Patient continued her medications in conjunction with the egg-based drink.

During the second month, the patient felt that her hands were worse and she presented with swelling of fingers and wrists and reported numbness in hands at night. Her legs felt "better". Physician's global assessment was that patient was slightly worse. She still experienced pain when carrying shopping bags or upon opening boxes. Her ESR and CRP readings remained normal and her cholesterol remained at 189mg/dl. She continued medications and as scheduled, by the protocol, discontinued use of product.

After one month without the product, swelling and tenderness returned to hands and feet. Her function limitations remained as she first presented. Patient still felt 75% "better". Her ESR and CRP readings remained normal as her cholesterol dropped to 180mg/dl.

Scoring of the arthritic symptoms was determined from the patient's own assessment of the symptoms. In particular, the patient was asked to indicate the amount of swelling, pain and/or tenderness of the joints. Based upon this patient assessment, numbers were generated showing the degree of the symptoms. The numbers range from a high of 36 (greatest amount of swelling, pain and tenderness) to a low of 0 (no swelling, pain or tenderness).

Scores generated for Patient #l 's arthritic condition were as follows:

At presentation^ 14

One month on egg-based drink = 2

Two months on egg-based drink = 6 One month after discontinuing drink= 8

EXAMPLE 4B Patient #2

Patient #2 was an 88-year-old female 5'3" 104 lbs. with a previous history of rheumatoid arthritis and chronic sinusitis. She presented with pain in the lower extremities and shoulders. Physical examinations revealed swelling, pain, and tenderness of her fingers and ankles. Functionally she was only able to walk in a limited manner and had difficulty in bending her arms. Her hematology tests indicated elevated ESR (36 mm/hr), CRP (1.64mg/dl) and a total cholesterol at 220 mg/dl. She was being treated with: Methotrexate 5mg/week, Orudis KT 2/day, Ca 1500mg, Fosomax lOmg, Prednisone 5mg q2d (every other day), and took multi vitamins. Again, the patient continued to take these medications in addition to the egg-based drink.

One month after starting the egg-based drink, she still complained of swelling of hands, knee pain, and inability to sleep. The patient reported that she was "50% better", "feeling generally better" and "walking better." She was still in pain, although her pain attacks were less frequent. Hematology results were ESR (30mm/hr) and CRP (1.64mg/dl), and an increase in cholesterol to 232 mg/dl was observed.

After two months of consumption of product, patient reported that "she felt 98% better". Her functional limitations however, remained the same. Hematology results indicated a drop in her ESR (21mm/hr) and a return to normal in the CRP reading. (<0.5mg/dl). Her total cholesterol remained at 234 mg/dl. Patient continued her medications but, as scheduled, discontinued the egg-based drink.

One month after discontinuing product patient complained of severe joint pains in her fingers and legs and exhibited swelling of wrists. She felt she was "50% worse". Her ESR (27mm/hr) levels and cholesterol (257 mg/dl) started to climb, but her CRP (<0.5mg/dl) levels remained normal.

Scores generated for Patient #2's arthritic condition were as follows: At presentation= 10

One month on egg-based drink = 7 Two months on egg-based drink = 2

One month after discontinuing drink= 10

EXAMPLE 4C Patient #3

Patient #3 was a 79-year-old male 5' 10" 152 lbs with a history of osteoarthritis and atherosclerotic cardiovascular disease. Symptoms were pain in the lower back, shoulders, knees and feet. Medications included: Nitroderm patch, Digoxin 0.125, aspirin 80mg and Feldene 20mg. Function limitations were: difficulty in moving arms and exhaustion. Hematology results were normal ESR and CRP and cholesterol readings at 217 mg/dl.

After one month of drinking the egg-product, along with the above-mentioned medication, the patient felt "slightly better (10-20%)". He still reported shoulder pain on right and left side, and reported that he was unstable on feet and experienced buckling of his right knee. Function limitations remained as previously reported. His ESR and CRP readings were normal and his cholesterol level was 180 mg/dl.

After two months of consuming product patient continued to feel "slightly better". Symptoms included right knee pain and difficulty in walking. Cholesterol readings were not obtained but ESR and CRP remained at normal levels.

After one month without product, patient felt slightly better overall but slightly worse than the previous month. Symptoms included buckling of right leg, shoulder pain and lower back pain. Joint assessment indicated pain in shoulders, and lower back.

Functional limitations were difficulty in moving arms and feeling "exhausted". ESR and CRP. readings were normal and cholesterol remained at 180 mg/dl.

Scores generated for Patient #3's arthritic condition were as follows: At presentation= 11.5

One month on egg-based drink = 2

Two months on egg-based drink = 3

One month after discontinuing drink = 8.5

Other case histories showed similar improvements and demonstrated the effectiveness of the present invention in eliminating pain and inflammation in arthritic joints without side effects.

The invention has been described with particular reference to certain preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention as described above and as defined in the appended claims.

Claims

TITLE COMPOSITION AND METHOD FOR TREATMENT AND PREVENTION OF ARTHRITIS AND/OR AUTOIMMUNE DISEASESCLAIMS What is claimed is:

1. A composition for the treatment and prevention of arthritis in a subject animal, the composition comprising an egg, or fraction thereof, obtained from an avian which has been hyperimmunized with an antigenic or genetic vaccine.

2. The composition of claim 1 wherein the avian has been hyperimmunized with an antigenic vaccine comprising at least one antigen selected from the group consisting of bacterial, viral, protozoan, fungal, and cellular antigens and mixtures thereof.

3. The composition of claim 1 wherein the avian has been hyperimmunized with a genetic vaccine comprising at least one antigen coding DNA construct selected from the group consisting of fragments of naked DNA, plasmid DNA, viral DNA, bacterial DNA, DNA expression libraries, DNA-RNA antigens, DNA-protein conjugates and DNA liposome conjugates, and mixtures thereof.

4. A method of treating and preventing arthritis in a subject animal suffering from or susceptable to arthritis, the method comprising administering to the subject animal an effective amount of an egg product.

5. The method of claim 4 wherein the egg product is obtained from an avian which has been hyperimmunized with an antigenic or genetic vaccine.

6. The method of claim 5 wherein the antigenic vaccine comprises at least one antigen selected from the group consisting of bacterial, viral, protozoan, fungal, and cellular antigens and mixtures thereof.

7. The method of claim 5 wherein the genetic vaccine comprises at least one antigen coding DNA construct selected from the group consisting of fragments of naked DNA, plasmid DNA, viral DNA, bacterial DNA, DNA expression libraries, DNA-RNA antigens, DNA-protein conjugates and DNA liposome conjugates, and mixtures thereof.

8. The method of claim 4 wherein the egg product comprises an egg or fraction thereof.

9. The method of claim 8 wherein the daily effective amount of egg product administered to the animal is equivalent to 0.25 to 100 eggs.

10. The method of claim 4 wherein the antibody is administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intranasally, orally or topically.

11. The method of claim 4 wherein the egg product is administered to the subject animal in combination with a drug selected from the group consisting of non- steroidal, anti-inflammatory drugs and disease-modifying, anti-arthritic drugs.

12. The method of claim 11 wherein the subject animal has been administered a regimen of said drug for a predetermined time prior to administration of the egg product.

13. A composition for the treatment and prevention of an autoimmune disease in a subject animal, the composition comprising an egg, or fraction thereof, obtained from an avian which has been hyperimmunized with an antigenic or genetic vaccine.

14. The composition of claim 13 wherein the avian has been hyperimmunized with an antigenic vaccine comprising at least one antigen selected from the group consisting of bacterial, viral, protozoan, fungal, and cellular antigens and mixtures thereof.

15. The composition of claim 13 wherein the avian has been hyperimmunized with a genetic vaccine comprising at least one antigen coding DNA construct selected from the group consisting of fragments of naked DNA, plasmid DNA, viral DNA, bacterial DNA, DNA expression libraries, DNA-RNA antigens, DNA-protein conjugates and DNA liposome conjugates, and mixtures thereof.

16. A method of treating an autoimmune disease in a subject animal suffering from or suscepatable to said autoimmune disease, the method comprising administering to the subject animal an effective amount of an egg product.

17. The method of claim 16 wherein the egg product is obtained from an avian which has been hyperimmunized with an antigenic or genetic vaccine.

18. The method of claim 17 wherein the antigenic vaccine comprises at least one antigen selected from the group consisting of bacterial, viral, protozoan, fungal, and cellular antigens and mixtures thereof.

19. The method of claim 17 wherein the genetic vaccine comprises at least one antigen coding DNA construct selected from the group consisting of fragments of naked DNA, plasmid DNA, viral DNA, bacterial DNA, DNA expression libraries, DNA-RNA antigens, DNA-protein conjugates and DNA liposome conjugates, and mixtures thereof.

20. The method of claim 16 wherein the egg product comprises an egg or fraction thereof.

21. The method of claim 20 wherein the daily effective amount of egg product administered to the animal is equivalent to 0.25 to 100 eggs.

22. The method of claim 16 wherein the antibody is administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, intranasally, orally or topically.

23. The method of claim 16 wherein the egg product is administered to the subject animal in combination with a drug selected from the group consisting of non- steroidal, anti-inflammatory drugs and disease-modifying, anti-arthritic drugs.

24. The method of claim 22 wherein the subject animal has been administered a regimen of said drug for a predetermined time prior to administration of the egg product.

25. A composition for reducing antibodies against autoimmune diseases in a subject animal, the composition comprising an effective amount of an egg, or fraction thereof, derived from an avian which has been hyperimmunized with an antigenic or genetic vaccine.

26. The composition of claim 25 wherein the avian has been hyperimmunized with an antigenic vaccine comprising at least one antigen selected from the group consisting of bacterial, viral, protozoan, fungal, and cellular antigens and combinations thereof.

27. The composition of claim 25 wherein the avian has been hyperimmunized with a genetic vaccine comprising at least one antigen coding DNA construct selected from the group consisting of fragments of naked DNA, plasmid DNA, viral DNA, bacterial DNA, DNA expression libraries, DNA-RNA antigens,

DNA-protein conjugates and DNA liposome conjugates, and mixtures thereof.

28. The composition of claim 25 wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile diabetes, multiple sclerosis, Graves' disease, Meneri's disease, myasthenia gravis, lupus erythematosus, psoriasis, systemic scleroderma, rheumatic fever and Sjogren syndrome.