HK1080749B

HK1080749B - Purified cytokine inhibitory factor

Info

Publication number: HK1080749B
Application number: HK06100902.1A
Authority: HK
Inventors: Subramanian Iyer; William L. Johnson; Lance Nguyen; Steven C. Ross; Ruye Xing
Original assignee: Arkion Life Sciences, Llc
Priority date: 2002-02-11
Filing date: 2003-02-11
Publication date: 2007-05-25

Description

Purified cytokine inhibitory factor

Cross Reference to Related Applications

This application claims priority from 35 of U.S. provisional application serial No.60/356,038 entitled "highly purified cytokine inhibitory factor and methods of use" filed on 11/2/2002 in accordance with u.s.c. § 119 (e). The disclosure of U.S. provisional application serial No.60/356,038 is incorporated herein by reference in its entirety.

Technical Field

The present invention relates generally to biological factors and methods for modulating the immune system. The invention specifically relates to substantially purified cytokine inhibitory factors, methods of purifying them, and methods of using them to modulate the immune system, particularly directed to methods of reducing inflammation.

Background

Biological factors, such as coenzymes, cofactors, vitamins, etc., play an important role in the bioconversion process. Their production is usually very small in intact cells (1X 10)^-5-1×10^-6). However, determining the presence of these biological factors from normal cells and then isolating and purifying them is a difficult project.

Hyperimmunized eggs have been developed and have been shown to contain overproduced antibodies and to produce certain biological factors as set forth in U.S. patent No.6,420,337. It is difficult to determine what biological agents are present in eggs, and it seems easier to use hyperimmunization methods. Hyperimmunization is achieved by injecting multivalent bacterial antigens into a target animal. It has been found that the amount of biological agents present in the eggs of these hyperimmunized animals is increased, but not by more than an order of magnitude. Thus, the small titer of biological agents in hyperimmunized eggs makes localization and subsequent purification of the biological agents difficult. Thus, there is a need for efficient methods to identify, isolate, purify, or otherwise produce such biological agents.

Cytokine

The normal immune system is in the balance of pro-inflammatory and anti-inflammatory cells and finely regulates molecules to promote normal host immune defenses without destroying host tissues. Once this finely regulated balance is disturbed, non-specific stimulation and activation can lead to an increased amount of immune and inflammatory molecules being produced and released with powerful destructive power. Thus, overproduction of pro-inflammatory cytokines or cytokine production in the wrong biological context is associated with lethality and pathology in a wide range of diseases such as malaria, sepsis related, rheumatoid arthritis, inflammatory bowel disease, cancer and AIDS.

Cytokines are pluripotent polypeptides that function in an autocrine/paracrine fashion by binding to specific cellular receptors. Their secretion is important in determining the duration and intensity of the immune response. For example, in mice, different subtypes of the CD4+ T helper cell (Th) clone secrete cytokines traditionally referred to as Th1 and Th 2. Th1 cells produce interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) and promote the immune response of the cells. Th2 cells produce IL-4, IL-5, IL-6 and IL-10 and support activation of immunoglobulin-secreting cells. In the inflammatory process, cytokines such as IL-1 β, IL-2, IL-6, and tumor necrosis factor- α (TNF- α) are released at the site of inflammation. These cytokines have pleiotropic effects and mediate a variety of symptoms associated with inflammation.

The key cytokine, TNF- α, also known as cachectin, is a 17KD protein consisting of 157 amino acids and is produced primarily by monocytes and activated macrophages. TNF- α has been shown to have tumoricidal activity and to have various physiological effects on most major organ systems. In the central nervous system, TNF- α is associated with fever, anorexia and changes in pituitary hormone release. In the cardiovascular system, TNF- α plays a role in shock, acute respiratory distress and the capillary leak syndrome (procoagulant). TNF-alpha plays a role in acute tubular necrosis of the kidney and nephritis as well as ischemia, colitis and hepatic necrosis of the gastrointestinal system. It is also a key cytokine involved in the inflammatory process.

Inflammatory disorders develop through multiple pathogenic processes, including several key molecules. For example, TNF-alpha, IL-1 beta, interleukin-2 (IL-2) and prostaglandin E₂(PGE₂) Is the main contributor to the development of inflammation. Inflammation can lead to disease states such as rheumatoid arthritis, septic arthritis and juvenile osteoarthritis.

TNF- α is produced primarily by T cells in response to inflammatory stimuli. TNF- α is a potent paracrine endocrine intermediate of inflammation and immune function, and it regulates endothelial cell function. For example, high concentrations of TNF- α can be detected in the serum and synovial fluid of patients with active rheumatoid arthritis, and it is presumed that TNF- α plays a major role in the pathogenesis of rheumatoid arthritis.

IL-1 β is involved in a variety of biological pathways and is a remarkably effective molecule capable of inducing its effects by triggering only 1 or 2 receptors per cell. As a signaling agent, IL-1. beta. is effective at an extremely low concentration, even in the femtomolar range. IL-1. beta. was first noted for induction of fever, enhancement of lymphocyte responses, and stimulation of acute phase responses. The induction of the inflammatory response in response to infection is primarily due to the signaling by IL-1 β.

Another T cell-derived cytokine, IL-2, induces growth, differentiation and functional activation of a variety of cells. For example, IL-2 promotes the growth and differentiation of T-and B-cells, the secretion of immunoglobulins by B cells, the growth and activity of NK cells, and the production of other cytokines. IL-2 also plays an important role in many inflammatory processes associated with the growth and proliferation of immune cells.

Prostaglandin E₂(PGE₂)

Prostaglandin E₂(PGE₂) Is the "predominant prostaglandin" found in many cells throughout the body. It is a major arachidonic acid metabolite and plays an important role in regulating the osmotic potential of blood vessels and the general conditions associated with body temperature, pain, immunosuppression and inflammation. PGE₂Is a major component of the inflammatory pathway of chronic inflammatory disease states.

There are multiple enzymes that determine PGE₂And several of the same enzymes produce other prostaglandins, thromboxanes and inflammatory intermediates. For example, PGE₂Produced by constitutively expressed and inducible forms of cyclooxygenase, COX-1 and COX-2, respectively. When cells are exposed to certain mitogens (e.g., LPS)) Or cytokines (IL-1), provide cytokine signaling and PGE₂In the context of production, COX-2 is strongly upregulated.

Because of TNF-alpha, IL-1 beta, IL-2 and PGE₂Are key inflammatory modulators, which play a crucial role in the development of inflammation.

Lipopolysaccharide (LPS) analogues

Various classes of Lipopolysaccharide (LPS) analogs interfere with cytokine expression in vitro and in vivo. For example, incubation of Solid Lipid Nanoparticles (SLNs) with murine peritoneal macrophages results in a concentration-dependent decrease in IL-6 production. However, TNF- α and IL-12 were not inhibited. The synthetic lipid a analogue SDZ MRL 953 prevented endotoxic shock and bacterial infection, as shown by studies in which 20 cancer patients were treated intravenously with increasing doses of SDZ MRL 953 followed by intravenous administration of endotoxin. Pretreatment with lipid A analogs significantly reduced the release of TNF- α, IL-1 β, IL-8, IL-6 and G-CSF, suggesting that pretreatment with SDZ MRL 953 may help prevent the complications of gram-negative sepsis in critically ill patients.

LPS-induced TNF- α production was inhibited in macrophages and human monocytes and primary mononuclear U937 cells. Lipid A analogues of monosaccharides, such as DY-9973, inhibit LPS-induced TNF- α and IL-1 β mRNA expression in U937 cells. In contrast, DY-9973 did not inhibit IL-1 β -induced TNF- α production in U937 cells. Thus, the monosaccharide lipid A analogs such as DY-9973 inhibit LPS-induced macrophage activation and reduce the lethal toxicity of LPS. Furthermore, early endotoxin tolerance was induced by the non-toxic LPS derivative monophosphoryl lipid a (mpl) against LPS infection.

In addition to lipid analogs, anti-LPS antibodies may act as neutralizers to prevent LPS infection. Monoclonal antibodies to lipid A inhibit the ability of lipid A and LPS from various gram-negative bacteria to induce TNF- α (36-67%) and IL-1 (30-98%) in murine peritoneal macrophages. These mabs also inhibited lipid a-induced TNF- α (87%) in mice.

LPS analogs also upregulate cytokine expression. Different lipid A analogs (DT-5461a) induced TNF- α, IL-1 β, IL-6 and GM-CSF in murine macrophages. In addition, DT-5461a increases the production of various cytokines in cells through enhanced transcription.

Hyperimmunized egg

Several genera of the class avidae, such as chickens (gallous domestica), turkeys and ducks, produce antibodies in the blood and eggs against immunogens which cause avian disease, as well as against other immunogens. For example, LeBacq-Verheyden et al (Immunology 27: 683(974)) and Leslie, G.A. et al (J.Med.130: 1337(1969)) have quantified chicken immunoglobulins. Polson et al (Immunological Communications 9: 495-514(1980)) immunized hens with several proteins and natural mixtures of proteins and detected IgY antibodies in the yolk of eggs. Fertel et al (Biochemical and biological Research Communications 102: 1028: 1033(1981)) immunized hens with prostaglandins and detected antibodies in the egg yolk of eggs. Jensenius et al (Journal of Immunological Methods 46: 63-68(1981)) provide a method for the isolation of egg yolk IgG for immunodiagnosis. Poison et al (Immunological Communications 9: 475-493(1980)) describe antibodies isolated from the egg yolk of hens immunized with various plant viruses.

U.S. patent No.4,357,272 discloses the isolation of antibodies derived from the yolk of the eggs of hyperimmunized hens. Antibody responses were initiated by repeated injections of immunogens derived from plant viruses, human IgG, tetanus antitoxin, snake antitoxin serum and Serameba.

U.S. Pat. No.4,550,019 discloses the isolation of antibodies produced in hens hyperimmunized with an immunogen having a molecular weight or particle weight of at least 30,000 from egg yolk. The immunogen for hyperimmunized chickens is selected from the group consisting of plant viruses, human immunoglobulins, tetanus toxin, and snake venom.

Us patent No.4,748,018 discloses a method of passively immunizing a mammal comprising parenterally administering purified antibodies obtained from eggs of birds that have been immunized with a corresponding antigen, and wherein the mammal has acquired immunity from immunization to the eggs.

U.S. patent No.5,772,999 discloses a method of preventing, arresting or slowing chronic gastrointestinal dysfunction or non-steroidal anti-inflammatory drug induced (NSAID-induced) gastrointestinal damage in a subject by administering hyperimmunized egg and/or milk or fractions thereof to the subject.

U.S. patent No.6,420,337 discloses a biological factor isolated from the eggs of birds hyperimmunized with a mixture of immunogens. This biological factor has been purified and sequenced and has been determined to activate certain pro-inflammatory cytokines and is therefore referred to as an activator of cytokines.

European patent application No.99967649.7 discloses an anti-inflammatory composition also obtained from eggs of birds hyperimmunized with a mixture of immunogens. The anti-inflammatory composition has been partially purified from eggs and has proven effective in the treatment and prevention of inflammation.

Unsaturated fatty acids, such as omega-3 and-6 fatty acids (naturally present in egg yolk), have been reported to play an important role in anti-inflammatory processes. Recently, many researchers have focused on the anti-inflammatory effects of fish oils enriched in omega-3 and-6 fatty acids. These studies showed that omega fatty acids are effective in the prevention of arthritis in fact. However, this study does not elucidate or suggest any demonstration of the effect of omega fatty acids on pro-inflammatory cytokines or cytokines in general.

Some of the properties of omega fatty acids are that they are partially soluble in water, that they have a maximum absorbance at 210-230nm and that due to their structure are long chain fatty acids, their melting points are generally very low (-49 ℃ to 13.4 ℃).

There is no report in the literature that egg yolk phospholipids inhibit proinflammatory cytokines and/or PGE₂And (4) synthesizing. Neither is the egg yolk reported to contain compounds that can inhibit proinflammatory cytokines and PGE in competition with LPS₂Any known lipopolysaccharide of (a)Analogues (LPS analogues).

The key to the present invention is the discovery by the present inventors that proinflammatory cytokines and PGE are naturally present in eggs and are capable of inhibiting₂New factors are synthesized.

Summary of The Invention

The present invention relates to a composition comprising a substantially purified cytokine inhibitory factor, wherein the cytokine inhibitory factor: has a molecular weight of less than 6,000 Da; naturally occurring in the lipid component of the yolk of avian eggs; has a maximum ultraviolet absorbance at a wavelength of 205 nm; a material of non-protein origin; has a melting point of about 39 ℃ to 42 ℃; and inhibiting RNA transcription of tumor necrosis factor alpha (TNF-alpha), interleukin-1-beta (IL-1 beta), and interleukin-2 (IL-2).

The invention further relates to a method for producing a purified cytokine inhibitory factor comprising: separating a water-insoluble fraction (WIF) from a water-soluble fraction (WSF) of egg yolk; separating the water-insoluble fraction into a neutral lipid fraction and a polar lipid fraction; purifying the polar lipid fraction by high performance liquid chromatography to form a cytokine inhibitory factor fraction.

Finally, the invention relates to a method of modulating the immune system of an animal comprising administering to the animal a composition comprising a cytokine inhibitory factor.

Brief Description of Drawings

FIG. 1 is an HPLC chromatogram of PL-100 ethanol extract.

FIG. 2 is a digitized image showing the effect of LPS stimulation on TNF-. alpha.and IL-. beta.in an in vitro assay.

FIG. 3 is a digitized image showing the inhibitory effect of lipid of hyperimmunized eggs on TNF-. alpha.and IL-. beta.stimulated by LPS in an in vitro assay.

FIG. 4 is a digitized image showing the effect of acetone and ethanol extracts of lipids of hyperimmunized eggs on LPS stimulated IL-2 in an in vitro study.

FIG. 5 is a digitized image showing the effect of HPLC purified fractions of ethanol extracts of lipids of hyperimmunized eggs on LPS stimulated TNF-. alpha.and IL-1. beta.

Figure 6 shows the effect of hyperimmunized egg yolk and defatted egg yolk on collagen-induced arthritis in a rat model study of collagen-induced arthritis.

Detailed Description

The present invention relates generally to novel Cytokine Inhibitory Factors (CIFs), compositions comprising Cytokines (CIFs) and methods of modulating the immune system using the compositions. The composition of the invention may comprise a composition comprising purified CIF and/or the composition of the invention may be a natural food product comprising CIF and preferably comprises a natural food product or a component thereof enriched in CIF, e.g. by a selection process or by the production of concentrated fractions. Such natural food products include hyperimmunized egg products, including components of CIF-enriched hyperimmunized egg products. The present inventors have found that novel CIF inhibits the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2) and interleukin-1 beta (IL-1 beta), and down-regulates prostaglandin E₂(PGE₂) And (4) synthesizing. Thus, the CIFs of the present invention typically modulate (i.e., regulate) the immune system by modulating the expression and production of, or cells affected by, these cytokines. Furthermore, because TNF- α, IL-1 β and IL-2 are generally considered to be pro-inflammatory cytokines, the CIFs of the invention are useful in the treatment of conditions where inhibition of the immune response and cells responding to pro-inflammatory cytokines is desired, including but not limited to: arthritis in general, and more specifically, osteoarthritis deformans, septic arthritis, and juvenile osteoarthritis. As expected, CIF is effective in treating and/or preventing most inflammatory conditions or responses, e.g., most in TNF- α -1 beta and/or IL-2 in one or another of the points involved in such disorders or responses.

The CIF of the invention is a component that is produced and/or enhanced in hyperimmunized egg products. The preferred immunogenic mixture administered, preferably by injection into an egg-laying animal to induce an immune response and produce a hyperimmunized egg product comprising the cytokine inhibitory factor of the invention, does not require the inclusion of specific immunogens known to modulate the immune system by activating specific cytokines or by inducing the production of factors capable of eliciting specific cytokines. Thus, inhibitors of this cytokine have surprisingly been found in hyperimmunized egg products obtained from animals immunized with a multivalent vaccine, which is expected to be effective in the modulation of the immune system when administered to a subject.

Certain properties have been determined for this novel cytokine inhibitory factor (see example 3). Initially, Cytokine Inhibitory Factor (CIF) is located in the lipid component of the egg yolk of eggs. Because it is present in the lipid component, CIF is of non-protein origin. Through basic knowledge of the lipid component of eggs, CIF sizes have been determined to be less than 6000 daltons. The absorbance of these new factors has also been studied and it has been determined that the maximum wavelength absorbance of CIF is at 205 nm. The melting point has been found to be in the range of about 39 deg.C to 42 deg.C. Finally, as noted above and as described in great detail in these applications, CIF inhibits certain cytokines. In particular, CIF inhibits at least the following pro-inflammatory cytokines: tumor necrosis factor alpha (TNF-alpha), interleukin-1-beta (IL-1 beta), and interleukin-2 (IL-2). Finally, the substantially purified CIF of the invention also inhibits prostaglandin E₂(PGE₂) The biosynthesis of (3).

Prior to the present invention, it was not known that hyperimmunization of egg-laying animals would result in the production of the novel cytokine inhibitory factor of the present invention, which would have the above-described properties and be capable of modulating cytokine production and differentiation of immune system cells in the animal. To the best of the inventors' knowledge, the Cytokine Inhibitory Factor (CIF) described herein has never been identified, purified or characterized prior to the present invention.

Definition of

Unless otherwise indicated, the following definitions apply throughout this application:

the term "hyperimmunization" refers to exposure to one or more immunogens (e.g., antigens) such that the immune response is enhanced and maintained in the natural unexposed state described above.

The term "immunogen" refers to a substance that induces a humoral antibody and/or cell-mediated immune response without conferring immunological tolerance. The term means capable of stimulating an immune response as well as reacting with its products, such as antibodies.

The term "combinatorial derived immunogens" refers to methods that generate molecular diversity among immunogens by way of combinatorial synthesis.

The term "bioengineered immunogens" refers to immunogens obtained by gene cloning techniques and methods of genetic manipulation or chemical synthesis.

The term "genetic vaccine" refers to a nucleic acid vaccine that is typically produced by recombinant techniques and can elicit an immune response.

The terms "egg" or "egg product" each refer to any whole egg (consumed, hyperimmunized or otherwise) or any product or component derived therefrom.

The term "table egg" or "table egg product" each refers to whole eggs obtained from egg-laying animals that are not maintained in a hyperimmune state or any product or component derived therefrom.

The term "hyperimmunized egg" or "hyperimmunized egg product" each refers to whole eggs obtained from eggs or any product or component derived from whole eggs.

The term "cytokine inhibitory factor" or "CIF" is used generically to refer to a biological factor of the present invention at any stage of purification (e.g., including as a component of an egg, as a substantially pure lipid fraction of an egg, or as a highly purified lipid fraction of an egg) and having the biochemical, physical, structural and/or functional characteristics of the cytokine inhibitory factor described herein.

The term "substantially pure cytokine inhibitory factor" refers to an inhibitory factor that has been purified to at least the levels set forth in example 2.

The term "supranormal level" refers to a level in excess of that determined in eggs from egg-laying animals that are not maintained in a hyperimmune state.

The term "immunomodulatory egg" or "immunomodulatory egg product" refers to an egg or a component of an egg that comprises a cytokine activating factor disclosed herein.

The term "immune response" generally refers to a cell-mediated response or cellular immune response (i.e., a cell-mediated immune response by the immune system including T lymphocytes, B lymphocytes, and macrophages) and/or a humoral immune response (i.e., an antibody-mediated immune response).

The term "animal" refers to any species of the kingdom animalia. Preferred animals to be immunized according to the invention include any animal of the vertebrate class, the class avia, including but not limited to chickens, turkeys and ducks. Preferred animals to be treated according to the present invention include any of the vertebrate, avian and mammalian classes, including but not limited to primates, rodents, farm animals and domestic pets. Livestock includes mammals that eat or produce useful products (e.g., sheep for wool production). Mammals that are preferably protected from a disease or disorder include humans, dogs, cats, mice, rats, sheep, cattle, horses and pigs, with humans being particularly preferred.

The term "target animal" refers to an animal to which an immunogenic mixture has been administered.

The term "subject animal" refers to an animal to which a composition comprising CIF of the present invention is administered, wherein the composition comprises a composition comprising purified CIF, or CIF-enriched eggs or egg products produced by the target animal. The subject animal may also be referred to as a patient.

The phrase "biologically active" or "biological activity" in reference to CIF of the present invention refers to any functional activity of CIF, as well as the functional activity of naturally occurring CIF in general. In particular, biological activity with respect to CIF preferably means that CIF is capable of down-regulating (inhibiting, reducing, suppressing) the expression of cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and/or interleukin-2 (IL-2). It is noted that the naturally occurring CIF as detailed herein may down-regulate the expression of each of TNF- α, IL-1 β and IL-2. CIF biological activity may also include down-regulation (inhibition, reduction, suppression) of prostaglandin E₂The ability to express. Biological activity of CIF may also include, but is not limited to, the ability of CIF to bind to receptors, proteins, DNA, carbohydrate components, or lipid components to cause or promote one of the same biological activities described above.

The term "maximum wavelength absorption" refers to the absorption of electromagnetic radiation in the ultraviolet and visible regions of the spectrum by compounds that differ in their ability to reflect at individual wavelengths. This radiation absorption is due to the difference in radiation beam energy when electrons on a low energy orbit are excited onto a high energy orbit. The absorption depends on the wavelength of the radiation and the structure of the compound. The wavelength of highest absorption is the maximum wavelength absorption of the compound to be tested.

The term "modulate" or a derivative of that term, means to change, modulate or alter from one state to another, and includes a measurable or observable increase or decrease in any measurable characteristic and/or change from one characteristic to another.

The phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable excipient, formulation and/or pharmaceutically acceptable delivery vehicle, wherein the delivery vehicle is suitable for administering the composition of the invention to a suitable site in vitro, ex vivo or in vivo. Pharmaceutically acceptable carriers may enable the compositions of the present invention to be manufactured/provided in any suitable form suitable for use, including, but not limited to, liquids, aerosols, capsules, tablets, pills, powders, gels, and granules. Some pharmaceutically acceptable carriers include cells, membranes, lipid formulations (including liquids that form solids or gels in situ when administered to a patient), antibody formulations, food products (e.g., any edible product or preparation), and recombinant viruses. Preferred carriers are also biodegradable (i.e., bioerodible).

"pharmaceutically acceptable excipient" includes excipients or formulations that transport or aid in transport, but do not specifically target the composition to cells (also referred to herein as non-targeting carriers). Examples of pharmaceutically acceptable excipients include, but are not limited to, water, phosphate buffered saline, ringer's solution, dextrose solution, serum containing solution, Hank's solution, other physiologically balanced aqueous solutions, oils, esters, and glycols. Aqueous carriers may contain suitable auxiliary substances required to approximate the physiological condition of the subject, for example by improving chemical stability and isotonicity. Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride and other substances used for the production of phosphate buffers, Tris buffers and bicarbonate buffers. Auxiliary substances may also include preservatives, such as thimerosal or o-cresol, formaldehyde solution and benzyl alcohol.

A "controlled release carrier" is a type of pharmaceutically acceptable carrier that is capable of releasing the compositions of the present invention in a controlled manner into a patient or culture. Controlled release formulations include a compound of the invention (e.g., a CIF composition, antibody or mimetic) in a controlled release carrier. Suitable controlled release carriers include, but are not limited to, biocompatible polymers, other polymer matrices, capsules, microcapsules, microparticles, pills, osmotic pumps, diffusion devices, liposomes, lipid spheres, and transdermal delivery systems.

A "pharmaceutically acceptable delivery vehicle" is a pharmaceutically acceptable vehicle capable of delivering the composition of the invention to a target site. Preferred pharmaceutically acceptable delivery vehicles are capable of directing (i.e., directing, selectively delivering) the composition to the target site. "target site" refers to a site in a patient to which delivery of a composition is desired. For example, the target site may be any cell or tissue targeted by direct injection or using artificial and natural lipid-containing delivery vehicles (e.g., liposomes), antibodies, viral vectors, or other delivery vehicles including ribozymes. Delivery vehicles that naturally contain lipids include cells and cell membranes. Artificial lipid-containing delivery vehicles include liposomes and micelles.

The term "administering" refers to any method of providing a substance to a subject (e.g., introducing a substance into a subject), including in vivo or ex vivo administration. Methods of in vivo administration include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, coronary intravenous administration, intraarterial administration (e.g., introduction into the carotid artery), subcutaneous administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), brain, nose, oral, ocular, pulmonary administration, catheter infusion, by suppository, and direct injection into a tissue.

The term "therapeutic benefit" does not necessarily mean a cure for a particular disease or condition, but preferably includes a treatment that may include alleviation of the disease or condition, elimination of the disease or condition, alleviation of symptoms associated with the disease or condition, prevention or alleviation of secondary symptoms or conditions resulting from the onset of the disease or condition, and/or prevention of the disease or condition.

The term "protect" with respect to a disease or disorder refers to reducing the symptoms of the disease; reduce the incidence of the disease, and/or reduce the severity of the disease. Protecting a patient or subject means that the composition of the invention, when administered to a patient, is capable of preventing the onset of disease and/or curing or alleviating the symptoms, signs or causes of disease. Thus, to protect patients from disease, both prevention of disease development (prophylactic treatment) and treatment of patients with disease (therapeutic treatment) are included.

The term "prevention" refers to reducing and/or eliminating the development of a disease, or eliminating the onset of a disease (prophylactic treatment).

The term "treating" refers to delaying, reducing, or completely preventing the onset of symptoms of a disease (including pain) and/or the etiology of the disease, or if present, the symptoms are ameliorated or completely eliminated. For example, CIF compositions treat arthritis not only by inhibiting the symptoms of the disease in humans and other mammals, but also by inhibiting the appearance of the disease in the recipient as a prophylactic agent.

The term "disease" refers to any condition that deviates from the standard health of a mammal, including conditions in which a symptom exists, as well as conditions in which a deviation (e.g., infection, genetic mutation, genetic defect, etc.) has occurred but the symptoms are not yet apparent.

The terms "a" or "an" entity refer to one or more entities. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

The terms "comprising", "including" and "having" are used interchangeably.

Hyperimmunization

While it is preferred that the compositions of the present invention be purified from hyperimmunized eggs, it is also contemplated that the cytokine inhibitory factor may be purified from edible eggs. A detailed description of a preferred method for placing an egg-laying animal in an immunopotentiating state and allowing hyperimmunized eggs or egg products obtained therefrom to be administered to a subject is disclosed in U.S. Pat. No.5,772,999, which is incorporated herein by reference in its entirety. Briefly, the following are examples of methods for placing an egg-laying animal in an immunopotentiating state for administration to a subject individual for the purpose of purifying CIF or producing a CIF-enriched food product. It is understood that this method may be altered as described above to select for or screen for enhanced production of CIF. Generally, the hyperimmunization method comprises the following steps:

1. one or more antigens are selected.

2. An immune response is elicited in egg-laying animals by primary immunization.

3. An enhanced vaccine of the appropriate dose of antigen is administered to induce and maintain the hyperimmune state.

4. Eggs are collected and processed from egg-laying animals maintained in a hyperimmunized state to produce hyperimmunized egg products.

Step 1: any antigen or combination of antigens may be used. The antigen may be bacterial, viral, protozoal, fungal, cellular or any other substance to which the immune system of an egg-laying animal will respond. The key point in this step is that the antigen must be able to induce an immune and hyperimmune state in the egg-laying animal. One preferred vaccine is a mixture of multivalent bacterial antigens known as the PL-100 vaccine. The bacteria included in the PL-100 vaccine are listed in Table 1 of U.S. Pat. No.5,772,999.

Step 2: the vaccine may be a killed or live attenuated vaccine and may be administered by any method of eliciting an immune response. Immunization is preferably accomplished by administering the antigen intramuscularly. The preferred muscle for injection in birds is the breast muscle. The dosage is preferably 0.5-5 mg of antigen vaccine. Other methods of administration that may be used include intravenous injection, intraperitoneal injection, rectal suppository or oral administration. When DNA technology is used in the hyperimmunization process, much smaller quantities, typically 1-100. mu.g, may be required. Whether the vaccine has elicited an immune response in an egg-laying animal can be determined by a number of methods well known to those skilled in the art of immunology. Examples of such methods include enzyme-linked immunosorbent assays (ELISAs), tests that test for the presence of antibodies to stimulatory antigens, and tests designed to assess the ability of immune cells of a host to respond to an antigen. Typically, the presence of egg antibodies following immunization with a vaccine indicates an immune response. The minimum dose of antigen necessary to induce an immune response depends on the vaccination method used, including the type of antigen used and the type of egg-laying animal used as the host.

And step 3: the hyperimmune state is preferably induced and maintained at regular intervals by repeated booster administrations of appropriate doses. The time interval is preferably a two week interval and lasts for a period of six months. However, the booster dose must not lead to immune tolerance. It is possible to use other hyperimmune maintenance methods or combinations of methods, such as, for example, intramuscular injection for primary immunization and intravenous injection for booster injections. Other methods include simultaneous administration of microencapsulated antigen and liquid antigen, or intramuscular injection for primary immunization, and microencapsulation to enhance the dosage by oral or parenteral administration. Several combinations of primary and hyperimmunization are known to those skilled in the art.

And 4, step 4: hyperimmunized eggs can be processed for administration to a subject or purified for use in a variety of methods. These methods include preparing a composition (e.g., in a capsule) comprising substantially independently a hyperimmunized egg product and incorporating the hyperimmunized egg product into a food product to be administered to a subject, or according to purification methods for cytokine inhibitory factors as described elsewhere herein.

Purification of cytokine inhibitory factor

The cytokine inhibitory factor of the present invention can be isolated and substantially purified from eggs using solvent extraction and normal phase HPLC techniques (see examples 1 and 2). In vitro cytokine expression studies and bioassay data have demonstrated the purification of biologically active cytokine inhibitory factors.

Preferably, the egg yolk lipids are extracted from the hyperimmunized eggs using solvent extraction. The phospholipid fraction is subsequently separated from the neutral lipid fraction by selective solvent extraction. The phospholipid fraction contains multiple immunomodulators and inhibits, for example, TNF-alpha, IL-1 beta, IL-2 and PGE in vitro₂Is a pro-inflammatory molecule of (a).

The cytokine inhibitory factor of the invention is present in the phospholipid fraction of hyperimmunized egg yolk. The phospholipid fraction inhibits mRNA of cytokines, as shown by comparative analysis of mRNA transcripts in cells that have been treated with the cytokine stimulator Lipopolysaccharide (LPS), and this activity is due to the action of cytokine inhibitors.

Monocytes produce minimal amounts of TNF α and IL-1 β under normal growth conditions. However, the expression of cytokine mRNA of cells was induced to a higher level by treatment with LPS. As shown in the examples below, stimulation of cells by LPS enhanced cytokine expression. The enhanced cytokine expression was subsequently suppressed by the hyperimmunized egg yolk phospholipid fraction. Additional LPS was removed from the cells, and as a result, mRNA for TNF α and IL-1 β was removed from the cells by the yolk phospholipid fraction. Thus, the phospholipid fraction comprises at least one cytokine inhibitory factor. The results presented below demonstrate that the egg yolk phospholipid fraction does not react with LPS and that its effect counteracts the induction of cytokine expression by LPS. The egg yolk phospholipid fraction specifically inhibited transcription of cytokine mRNA.

As illustrated in detail in the examples below, 4 proinflammatory modulators TNF α, IL-1 β, IL-2 and PGE were tested via LPS induction of proinflammatory modulators in monocytes (human and mouse)₂. Measurement of cytokine expression at mRNA level and PGE₂Production at the protein level. The yolk phospholipid fraction inhibits TNF-alpha, IL-1 beta, IL-2 and PGE₂Expression of (2).

Extraction of yolk phospholipid fraction

Preferably, the cytokine inhibitory factor of the invention is substantially purified from the lipid component of whole eggs, egg yolks or egg yolks of hyperimmunized birds. During the purification process, the egg yolk is finally subjected to solvent extraction. Solvent extraction can be applied to the egg yolk itself or any other form of egg yolk, like for example, spray dried egg yolk. Different solvents can be used for this purpose. Non-polar solvents, such as hexane, are useful for removing neutral lipids from egg yolk or the lipid component of egg yolk. Subsequently, polar solvents, such as methanol and ethanol, are useful for extracting the phospholipid component. Alternatively, several solvents are suitable for the various extraction methods, such as acetone and chloroform. A wide variety of solvents can be selected for purification applications depending on the requirements and optimization of the process, scale-up, and applicable rules.

Another method for isolating and purifying the cytokine inhibitory factor of the present invention is by Supercritical Fluid Extraction (SFE). SFE removes the neutral lipid fraction from egg yolk lipids containing neutral lipids such as triglycerides and cholesterol. Neutral lipids make up about 70% of the total egg yolk lipids. For example, carbon dioxide extraction in supercritical state can be an effective method for removing most of cholesterol and fat of egg yolk without destroying the function of phospholipid component. Phospholipids are not removed during SFE over carbon dioxide and carbon dioxide can be beneficial in maintaining the emulsifying properties. However the phospholipids may be extracted by other solvents such as propane.

Administration of drugs

In other embodiments, the compositions of the invention may comprise CIF in purified, recombinant, chemically synthesized, substantially purified, or any other enriched form. For administration, any suitable form of CIF may be administered in combination with any suitable pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers according to the present invention include pharmaceutically acceptable excipients, controlled release carriers and pharmaceutically acceptable delivery vehicles as described above. The composition may be in any form suitable for delivery, including but not limited to liquids, aerosols, capsules, tablets, pills, powders, gels, and granules. CIF formulations particularly suitable for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or carriers are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.

In solid dosage forms, the CIF protein may be mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also contain additional substances in addition to the inert diluent, as is conventionally practiced. In the case of capsules, tablets and pills, the dosage forms may also contain buffers, pH-sensitive polymers or any other slow-release encapsulating agent (i.e., a controlled-release carrier) or any other controlled-release formulation typically used in the food and pharmaceutical industries to encapsulate compositions. Tablets and pills can also be prepared with an enteric coating.

Liquid dosage forms of cytokine inhibitory factor 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 can also include wetting agents, emulsifying and suspending agents, and sweetening agents.

In one embodiment, the composition is in the form of a food product. In one aspect of this embodiment, the immunomodulatory egg, or any CIF-enriched component thereof, is incorporated into a nutritional supplement. A preferred method of preparing the eggs or any of its components for incorporation into the nutritional supplement includes drying the eggs into a powder. Although a variety of methods are known for drying eggs, spray drying is the preferred method. Methods of spray drying eggs are well known in the art. Such dried egg powder may be ingested only as a regular egg powder or incorporated into beverages in the form of, for example, protein powder, energy-constituting beverages, protein supplements, and any other nutritional, sports-related products. In addition, the egg powder can be used for baking mixes, energy bars, candies, cookies, and the like. Other examples of egg processing include preparing omelettes, gently or vigorously boiling eggs, baking eggs or, if desired, eggs that can be eaten raw or processed as de-shelled eggs. Preferred components of the present immunomodulatory egg product include, but are not limited to: the lipid component of the de-shelled egg yolk, the yolk powder and/or the hyperimmunized egg product.

The compositions of the invention may be delivered (i.e., administered) to a cell culture (e.g., in a cytokine assay) or to a patient via any suitable method. The choice of such a method will vary with the type of compound (i.e., purified fraction, protein, nucleic acid, mimetic) administered or delivered, the mode of delivery (i.e., in vitro, in vivo, in vitro), and the purpose to be achieved by the administration/delivery of the compound or composition. In accordance with the present invention, methods of effective administration (i.e., administration of the composition in an effective manner) include appropriate dosage parameters and modes of administration that result in delivery of the composition to the desired site (i.e., to the desired cells) and/or in modulation of the immune response in the subject. Preferably, the compositions of the present invention are administered to a subject by any means that modulates the immune system of the subject animal.

Routes of administration include in vivo, in vitro and ex vivo. In vivo routes include, but are not limited to, oral, nasal, intratracheal, inhalation, transdermal, rectal, catheter-infused, suppository, direct injection into tissue, and parenteral routes. Preferred parenteral routes may include, but are not limited to, subcutaneous, intradermal, intravenous, intramuscular, and intraperitoneal routes. In one embodiment of the invention, the composition comprising a CIF component, protein, antibody, mimetic or nucleic acid molecule of the invention is administered parenterally. Intravenous, intraperitoneal, intradermal, subcutaneous and intramuscular administration can be carried out using methods standard in the art. Aerosol (inhalation) delivery can also be carried out using methods standard in the art (see, e.g., Strib1ing et al, Proc. Nat1.Acad. Sci. USA 189: 11277-11281, 1992, which is incorporated herein by reference in its entirety). In another embodiment, the compositions of the invention comprising a CIF component are administered orally. Oral delivery can be carried out by compounding the composition of the present invention onto a carrier capable of undergoing digestive enzyme degradation in the intestinal tract of an animal. It has been noted that the CIF component of the invention is particularly resistant to digestive enzymes, and therefore the vectors described above may not be of particular importance. Examples of such carriers include, for example, plastic capsules or tablets as known in the art. The above approach may include the use of a pharmaceutically acceptable carrier as described above. By in vitro is meant that a partial regulatory step is accomplished in vitro in a patient, for example by transfecting a population of cells removed from the patient with a recombinant molecule comprising a nucleic acid sequence encoding a protein according to the invention under conditions such that the recombinant molecule is subsequently expressed by the transfected cells, and returning the transfected cells to the patient. Routes of administering the compositions to a culture of host cells, both in vitro and in vitro, can be performed by methods including, but not limited to, transfection, transformation, electroporation, microinjection, lipofection, adsorption, protoplast fusion, use of protein carrier agents, use of ionophore agents, use of detergents for cell permeabilization, and simple mixing (e.g., binding) of the compound in culture to the target cells.

In one embodiment, the immunomodulatory eggs of the invention, or fractions thereof, are processed to produce a hyperimmunized egg product that is subsequently administered to a subject animal. In one embodiment, administration is carried out by feeding the egg or any derivative thereof directly to the subject animal. It is important to note that eggs are non-toxic and safe natural food ingredients. Similarly, in another embodiment, substantially purified cytokine inhibitory factor is preferably prepared (e.g., formulated) in a manner that can be subsequently administered to an animal.

When modulating the immune system, the compositions of the invention preferably inhibit cytokines (i.e., TNF- α, IL-1 β, IL2, and/or PGE)₂) Expression, preferably inhibition of cytokines and modulation of immunity, is administered to the subject in an immunologically effective amount. The duration and intensity of treatment will depend on the particular subject and the condition of the subject, as well as whether it is present and, if present, the progression of the disorder in the subject. The composition is also provided in any amount that treats and/or prevents the disorder and symptoms of the disorder.

For example, in the case of administration of immunomodulatory eggs or products produced therefrom, the daily amount may range from less than one to several complete hyperimmunized eggs (or hyperimmunized egg preparations comprising less than one to several complete hyperimmunized eggs), and may be administered to a subject depending on the particular condition of the disorder. More potent fractions can be isolated and concentrated by the methods described herein as well as other methods known in the art. With respect to administering an immunomodulatory egg or egg product to a subject, it has been determined that a preferred dosage range for hyperimmunized egg or egg product administered to a subject is from 100 milligrams to 10 grams per kilogram of the subject's weight.

With respect to the isolated cytokine inhibitory factors of the present invention, including substantially purified CIF, recombinant CIF and/or chemically synthesized CIF, it has been determined that the dosage range of the substantially purified composition is from 1 nanogram to 400 milligrams per kilogram of weight of subject. In a preferred embodiment, the preferred dosage range is from about 0.01 micrograms to 100 milligrams per kilogram of the subject's weight. In another embodiment, the amount of protein or antibody administered is about 0.1: g to about 10mg, and more preferably about 0.1: g-about 100: g.

it will be apparent to those skilled in the art that the dosage administered to a patient will depend on the purpose for which it is administered (e.g., the extent of the inflammatory condition and the individual patient's response to treatment). Thus, suitable dosage amounts include those required to modulate the immune response in an animal or expected to be mediated by down-regulation of pro-inflammatory cytokines (TNF- α, IL-1 β, IL-2) and/or by down-regulation of PGE₂And any amount required to treat or prevent a condition or disorder is within the scope of the invention. Dosage parameters effective in vivo can be determined using standard methods in the art. Such methods include, for example, determining survival, side effects (i.e., toxicity), determining the effect of cellular and humoral immune responses, and/or the effect on conditions associated with the effect of the immune response.

One embodiment of the invention provides a method for modulating an immune response in an animal. This embodiment includes the step of administering to the animal a composition comprising a substantially purified cytokine inhibitory factor of the invention as described above. In a preferred embodiment, the composition comprises a pharmaceutically acceptable carrier as described above. In this embodiment, the compositions of the invention may be used as local or systemic stimulators of the immune system. It can also prevent and/or treat local and systemic bacterial infections and can be used as a general anticancer agent. It may also be labeled with a specific delivery reagent, such as a tissue-specific antibody, for delivery by the intravenous route to a specific site of bacterial infection or tumor formation. It may also be mixed with specific liposomes or delivery vehicles that are commercially available for delivery through the cytoplasmic and nuclear membranes of cells to promote inhibition of TNF-alpha, IL-1 beta and/or IL-6 expression at the RNA level. Suitable modes of administration, including preferred routes and dosages as aboveThe method is as follows. Preferably, by increasing TNF-alpha, IL-1 beta, IL-2 and/or PGE₂Is suitable for modulating the route and dosage of the immune response.

The advantageous properties of the invention can be observed by reference to the examples which follow, which illustrate the invention. These examples are provided for illustrative purposes and are not meant to limit the scope of the present invention.

Examples

Example 1: fractionation of PL-100 egg yolk lipids

PL-100 eggs were defatted using a buffer prepared by dissolving glacial acetic acid (0.44ml), sodium acetate (3.06g) and sodium chloride (5.26g) in 1,300ml of ultrapure water. The buffer had a pH of about 5.0. Egg material (100g of spray dried egg or 200ml shelled egg) was added to the buffer and the egg mixture was further homogenized by mixing at 24,000rpm for 5 minutes at room temperature. The homogenized egg mixture was stirred at 4 ℃ for 4 hours to provide maximum solubility. Caprylic acid (15ml) was added to the egg mixture and homogenized by mixing at 24,000rpm for 3 minutes at room temperature. The egg mixture was then left at room temperature for 2 hours for the separation stage. The floc and the foam were then mixed with the water phase and centrifuged at 2,190 Xg for 10 minutes at room temperature. The supernatant was filtered using a filter paper with a porosity of 40 μm and the filtrate was freeze-dried. The particles containing lipids and water-insoluble proteins were collected for further lipid extraction. This component is referred to as the water-insoluble component (WIF).

The egg lipids can be extracted from the Water Insoluble Fraction (WIF) by the above-described acid extraction method, or from the yolk of the shell-removed egg as a source material. For solvent extraction of egg lipids, 800 grams of liquid egg yolk or WIF particles were mixed with 1,000ml of acetone in a sealed bottle. Neutral lipids, such as triglycerides and cholesterol, were extracted by 3 times vigorous mixing with acetone or 3 minutes at 24,000 rpm. The acetone extract was filtered through 40 micron Whatman pleated filter paper. The acetone insoluble residue/acetone extract residue on filter paper was further mixed with 1,000ml of methanol and the polar lipids and cytokine inhibitory factor were extracted three times for 3 minutes each. Methanol was removed by evaporation at 50 ℃ using a rotary evaporator under reduced pressure of 5 pounds Per Square Inch (PSI). Traces of methanol and water in the extract were removed by lyophilization. The dried methanol extract was stored as a yellow paste at-20 ℃. For further use, the methanol extract is partially soluble in methanol, ethanol, dichloromethane and chloroform and partially soluble in water. Alternatively, ethanol may be used instead of methanol in the extraction of the egg lipids according to the above-described method.

Egg components can be separated according to solubility in various solvents. Egg yolk contains approximately 62% lipid and 30% protein. Of the lipid components, neutral lipids, mostly triglycerides, are important components of the total lipids, constituting approximately 65% of the total lipids. Neutral lipids are non-polar and highly soluble in hydrocarbon solvents such as hexane or benzene. However, they are completely insoluble in most polar solvents.

In contrast, phospholipids constitute about 30% of the total lipid or 18% of the total egg yolk mass. Phospholipids are very polar as a result of the phosphorylated choline or ethanolamine molecules and are therefore easily extracted by polar solvents such as methanol and ethanol.

The yield of phospholipid extract by solvent extraction in liquid egg yolk and dry egg yolk powder was about 10% of the egg yolk mass (see table 1). Some phospholipids are extracted and some are discarded during the acetone extraction process because acetone is moderately polar and it can extract a portion of the phospholipids containing neutral lipids. The yield of phospholipids can be increased using non-polar solvents such as hexane to extract neutral lipids.

TABLE 1 methanol extraction of egg phospholipids

Egg yolk¹	Yield of phospholipid (%)²
Egg yolk¹	Yield of phospholipid (%)²	PL-100 spray dried Whole egg PL-100 spray dried egg yolk SPAFAS³Spray dried yolk PL-100 shelled whole egg PL-100 shelled egg yolk	3.38.010.44.812.4

¹Whole eggs and egg yolks contain 34% and 51% dry weight, respectively.

²The yield of methanol extract from the in-shell eggs was calculated from the dry weight of all in-shell eggs.

³SPAFAS eggs are non-immunized eggs.

Example 2: HPLC purification

The polar or phospholipid extract from the extraction process described in example 1 can be further purified to CIF fractions using normal phase HPLC. HPLC chromatography (see fig. 1) showed 5 components of the phospholipid fraction. Of these 5 components, 2 components were particularly effective in inhibiting the expression of TNF-. alpha.and IL-1. beta. in vitro.

HPLC separations were carried out on normal phase HPLC columns (25X 2.12cm Zorbax PRO 1/150CN) in an Americhrome HPLC system. The separation was performed using 4 solvents: solvent a is 99.50% heptane plus 0.05% ethanol; solvent B is 94% heptane plus 6% ethanol; solvent C is 90% heptane plus 10% ethanol; and solvent D is 100% ethanol.

For isolation, approximately 4.0-4.5 grams of phospholipid extract was dissolved in 150ml of solvent A and filtered through a 0.2 μm pore size membrane to remove insoluble material. A clear yellow solution was obtained.

The clear yellow solution was then separated using HPLC. The multi-step gradient on the column was solvent A (0-2 min), solvent B (2.01-18.0 min), solvent C (18.01-30.0 min), solvent D (30.01-40.0 min) and solvent A (40.01-60.0 min). Thereby collecting 5 fractions as shown in fig. 1. The detection wavelength was 254nm, and fractions 1(F1) and 5(F5) showed the highest detection readings. HPLC separation was performed over multiple injections and the pool of fractions was collected. The solvent was removed by evaporation using a rotary evaporator.

The HPLC separation improves the purity of Cytokine Inhibitory Factor (CIF). Each fraction had a yellow color of different intensity. As shown by the chromatogram in fig. 1, most of the material in the phospholipid component was separated into fractions 1(F1) and 5(F5), while fractions 2-4(F2, F3, and F4) were more highly purified materials.

Example 3: characterization of cytokine inhibitory factor

Cytokine Inhibitory Factor (CIF), purified to essentially the levels set forth in example 2 above, was further characterized to determine specific characteristics. Briefly, the following basic study was conducted and relevant characteristics were determined.

The maximum wavelength of the substantially purified cytokine inhibitory factor was measured by a spectrophotometer equipped with a shimadzu hplc diode array detector and determined to be 205 nm.

The melting point of substantially purified Cytokine Inhibitory Factor (CIF) was measured by visual observation of cold CIF in a continuously warmed warm water bath and was determined to be in the range of 39 ℃ -42 ℃.

The molecular weight of substantially purified CIF was estimated from existing research data. It is known in the art that most compounds in the phospholipid fraction of egg yolk are small molecules (200-.

The solubility of the substantially purified CIF fraction was measured by adding 100mg of CIF to 1.0ml of solvent at room temperature. Solubility was verified by visual inspection.

It was finally determined that CIF cannot be a protein, since the protein is insoluble in methanol, ethanol and/or chloroform.

Example 4: in vitro cytokine inhibition

Cytokine inhibition assays were performed by culturing human monocytes THP1 followed by measuring cytokine mRNA expression by RT-PCR. The whole analysis consists of 4 steps: cytokine inhibition, RNA extraction, cDNA synthesis and PCR reactions.

Inhibition of cytokines in vitro: for cytokine inhibition assays, approximately 1X 10⁵Concentration of individual cells THP1 cells were cultured in 10mLRPMI 1640 medium with LPS (usually 0.2. mu.g/ml) to induce cytokines in the cells. The cells were also incubated with the yolk lipid fraction (5. mu.g/ml) for 4 hours at 37 ℃. Cells were harvested and mixed with 750ml Trizol (GIBCO BRL).

Extraction of RNA: after incubating the cell mixture at room temperature for 10 minutes, 250. mu.l of chloroform was added and the mixture was further shaken vigorously by hand for 20 seconds. The mixture was left at room temperature for 10 minutes and centrifuged at 12,000 Xg for 5 minutes. Carefully remove the upper clear aqueous layer into a new microcentrifuge tube and add 500. mu.l of isopropanol. The RNA mixture was incubated at room temperature for 10 minutes and centrifuged at 12,000 Xg at room temperature. The pellet containing RNA was washed with 1.0ml cold 70% ethanol and subsequently centrifuged at 12,000 × g for 5 minutes at room temperature. The RNA was dissolved in 40. mu.l of sterile distilled water (RNase free), mixed by gentle vortexing, and the lid was opened and incubated at 65 ℃ to evaporate the remaining 70% ethanol. The quality of the RNA was checked by loading 1.0. mu.l onto a 1% agarose gel in 1 XTBE running buffer, using lambda DNA digested with HindIII enzyme as standard RNA. Finally, RNA20 min was stained with 100mL of 1 XTBE buffer containing 10. mu.l of gold-colored nucleic acid gel stain (molecular probe) and the gel was visualized under UV light.

Synthesis of CDNA: mu.l of the RNA sample and 1.0. mu.l of OligodT primer were added to the PCR tube. RNA10 min was incubated at 65 ℃ and then 2 min in a PCR instrument at room temperature. The PCR tube was centrifuged to spin the sample to the bottom and 8. mu.l of the master mix was added to the tube. The master mix contained 1.0. mu.l of ribonuclease inhibitor, 4. mu.l of 5 XTT buffer, 1.0. mu.l of 100mM dNTPs, 1.0. mu.l of 80mM sodium pyrophosphate and 1.0. mu.l of AMV reverse transcriptase. The tubes were incubated at 42 ℃ for 1 hour in a PCR instrument and then at 95 ℃ for 2 minutes. After brief rotation of the tube, 1.0. mu.l of 0.5M EDTA, pH8.0 and 20. mu.l of phenol-chloroform were added and the sample was mixed by vortexing and then rotated for 2-3 minutes. The upper aqueous layer was transferred to a new tube (approximately 18. mu.l) and 20. mu.l ammonium acetate and 125. mu.l isopropanol were added. The sample was stirred by vortexing for 10 seconds and incubated in a dry ice/ethanol slurry (-20 ℃) for 1 hour. The supernatant was discarded by rotating the tube at 12,000 Xg for 10 minutes at room temperature and washing the precipitated particles with 1ml 70% ethanol. The RNA mixture was spun at 12,000 Xg for 5 minutes at room temperature and the ethanol was removed.

And (3) PCR reaction: GAPDH primers were used initially to ensure that cDNA levels were the same in all samples. 100 μ l of PCR reaction mixture contained 10 XPCR buffer (GibcoBRL, MgCl-free)₂)、2.5μM dNTP(GibcoBRL)、3.0μl MgCl₂2.0ml primer set for each cytokine, 1.0. mu.l Taq DNA polymerase and 63. mu.l sterile PCR water (Gibco BRL). Mu.l of cDNA and 4.0. mu.l of sterile distilled water were added to each PCR tube. The PCR reaction was set up according to standard procedures.

Once the PCR was complete, 50. mu.l of chloroform was added to each tube. Vortex with lid closed and centrifuge at 12,000 × g for 1 min. The upper aqueous layer was transferred to a newly labeled tube. Thereafter, 15. mu.l of each sample was applied to a 1.5% agarose gel in 1 XTBE electrophoresis buffer, and the cytokine DNA was run through the gel using a standard ladder of 100bp as a control. The gel was stained with 100ml of 1 XTBE buffer (molecular probe) containing 10. mu.l of gold-colored nucleic acid gel stain for 20 minutes and visualized under an ultraviolet detector.

Results

LPS stimulates the production of TNF α and IL-1 β by human monocytes (6). This result was constant for our study. LPS induced THP1 cells to express two cytokines (fig. 2). The maximum induction of TNF α was about 0.2mg/ml of medium. High concentrations of LPS in the medium showed weak induction as low concentrations of LPS. IL-1. beta. was induced by LPS at very low concentrations (0.008mg/ml), indicating that IL-1. beta. is a cytokine that is more sensitive to LPS-induced responses.

By using LPS induction assays, it was determined that TNF α, IL-1 β and IL-2 expression in human monocytes was inhibited by the phospholipid fraction of egg yolk. When the cells were incubated with 0.2mg/ml LPS and egg yolk phospholipid fraction, the TNF α and IL-1 β of the cells showed complete inhibition (FIG. 3). Cytokine inhibition was also determined by a modified assay to confirm specific inhibition by the phospholipid fraction of egg yolk. In this assay, cells were incubated with 0.2mg/ml LPS to induce TNF α and IL-1 β. After 2 hours of incubation, LPS in the medium was removed by washing the cells. The cells were then incubated with the egg yolk phospholipid fraction for an additional 2 hours. Consistently, both TNF α and IL-1 β were completely inhibited. In contrast, cells incubated with LPS for 2 hours without the addition of the egg yolk phospholipid fraction showed high expression of TNF α and IL-1 β.

Example 5: inhibition of IL-2

It was also found that the other proinflammatory cytokine IL-2 is inhibited by the lipid fraction of egg yolk. IL-2 was partially inhibited by acetone and ethanol extracts (FIG. 4). Due to the impure composition of neutral lipids, the acetone extract may be mixed with some fraction of the phospholipid fraction during solvent extraction. By the same assay, the yolk phospholipid fraction did not show any effect on the expression of IL-6, IL-12 and TGF β in the cells.

Example 6: inhibition of PGE2 biosynthesis

Inhibitory effect of the yolk lipid fraction on PGE2 biosynthesis in mouse monocyte macrophages.

Experiment of

Many cell types are capable of expressing COX-1 and COX-2 simultaneously; the other expression is one of them. RAW 264.7 cells used in these assays expressed two isoforms of cyclooxygenase (demonstrated in our laboratory by immunofluorescence analysis). The assay started overnight from the induction of RAW 264.7 monolayers of cells stimulating COX-2 via mitogenic (LPS). The single cell layer is then exposed to the test article (e.g., indomethacin) for 30 minutes at 37 deg.C, and the system is then provided with exogenous arachidonic acid. COX1 and COX2 (and PGE 2-specific synthetases) convert exogenous arachidonic acid into PGE 2. Prostaglandin E2 in the medium was then quantified by a commercial EIA system. When less PGE2 was recovered from the medium containing the test article than PGE2 recovered from the medium alone, this condition was inhibition of the marker. The specific mechanism of inhibition of PGE2 biosynthesis that exists in these assays cannot be deduced and further investigation is required.

The analysis method comprises the following steps: will be 5X 10⁶The cells of (2) were seeded in DMEM medium and incubated at 37 ℃ with 5% CO₂Was incubated overnight in an incubator. After approximately 24 hours of growth (typically 90-100% confluency) of the seeded cells on day 1; the growth medium was decanted in a sterile manner and discarded. Transfer 10ml of sterile PBSA gently with a pipette into a culture flask relative to the non-growth surface; and the cells were washed twice. 10ml DMEM containing LPS (2ng/ml) was added to the culture flasks tested. The flask was placed in an incubator at 37 ℃ with 5% CO2 for 16 hours overnight.

16 hours after the start of exposure to LPS, the growth medium was removed aseptically (aspiration) and discarded. Gently pipette 10ml of sterile PBSA into the flask against the non-growth surface; and the cells were washed twice. Cells were cultured at 37 ℃ in the presence of 2ml PBSA, 2ml positive control or 2ml egg yolk lipid sample (1.0 mg/ml). After 30 minutes of incubation, 222. mu.l of 300. mu.M arachidonic acid was added to each flask. The cells were incubated in the presence of 30. mu.M arachidonic acid for an additional 15 minutes at 37 ℃. After 15 minutes of incubation, 50. mu.l of 1mg/ml indomethacin was added to the contents of the flask to stop the reaction. All the liquid in the flask was collected in appropriately labeled polypropylene tubes.

Prostaglandin E2 was analyzed using a commercial ELISA kit (Amersham RPN222) at room temperature according to standard methods.

Results

Biosynthesis of PGE2 was inhibited in this model by various NSAIDs, e.g., indomethacin, aspirin and ibuprofen (data not shown). 3 NSAIDs have been reported to be non-selective inhibitors of the COX-1 and COX-2 enzymes. They are considered to be non-specific drugs, and in most cases, they produce side effects when used clinically to treat patients.

In our study, PGE2 biosynthesis in cells was inhibited by different levels in the egg yolk fraction (table 2). Among the raw materials, egg yolk demonstrated a stronger inhibitory effect on PGE2 biosynthesis than whole egg. However, the inhibitory potency of egg yolk and whole egg was relatively low compared to the lipid fraction (average 17%). Acetone and methanol extracts of egg yolk lipids inhibited PGE2 biosynthesis to 31% and 54%, respectively. In contrast, the two protein concentrated fractions, the water-soluble protein (filtrate) and the water-insoluble protein (residue), showed no inhibitory effect on PGE2 biosynthesis in the cells. It is suggested that the active inhibitor of egg yolk to PGE2 biosynthesis is located in the phospholipid component and that it is likely that the active ingredient is a non-peptidic molecule that is insoluble in water. These molecules may be distinct molecules from the active ingredients that inhibit cytokine expression. The yolk phospholipid fraction was shown to have multiple modulators that inhibit different proinflammatory molecules.

TABLE 2 composition of PL-100 eggs in vitro vs PGE₂Inhibition of biosynthesis

Components	Spray-dried		Fresh and fresh		Average
	Spray-dried		Fresh and fresh			Whole egg	Egg yolk	Whole egg	Egg yolk
	Starting egg filtrate acetone extract MeOH extract residue	-3-51648-11	30-2826112	2113-1955-21		Whole egg	Egg yolk	Whole egg	Egg yolk	18-643520	1703154-6

1.n＝5

2. The numbers show in vitro PGE₂Inhibition of biosynthesis.

3. The concentration of the egg component in the cell cultures tested was 1.0mg/ml of medium.

Example 7: collagen-induced arthritis model

Measurement of anti-inflammatory Effect of PL-100 egg yolk in a rat collagen-induced arthritis model.

Experiment of

Methods for the induction and assessment of collagen-induced arthritis in rats were developed according to Trentham et al, 1977 (8). The PL-100 egg yolk and defatted fractions were fed to rats for 7 days before initiation of type II collagen-induced arthritis and 16 days after induction in rats. A total of 30 Sprague-Dawley (VAF +), 6-8 week old (about 125g) female rats were obtained from Charles River-VAF +. 3 groups of rats (10 rats per group) were tested. Their controls were (gavage), 3.5ml of 5% egg yolk solution and 3.5ml of 5% defatted egg yolk fraction. The rats were fed with the samples every other day. The next day the solution was stored at 4 ℃ until use. After 2 days, the excess solution was discarded. Rats (blindly) were clinically evaluated for paw erythema (0-4+) and paw swelling (0-4+) starting on day 10 after intradermal immunization and up to day 21 thereafter. On day 21, all rats were sacrificed and bled.

Results

Hyperimmunized egg yolk inhibited collagen-induced arthritis model rats with approximately 40% paw erythema and paw swelling (figure 6). The defatted egg yolk and control samples showed no effect on the damage produced compared to the egg yolk fraction. Even in the absence of experiments in the yolk lipid group in test studies showing a specific effect on inflammation inhibition, indirect results also indicate that the yolk phospholipid fraction contains an anti-inflammatory component.

Although the invention is illustrated and described herein with reference to specific embodiments, the invention is not intended to be limited to the details shown. Rather, various changes may be made in the details within the scope and range of equivalents of the claims and without departing from the invention.

Claims

1.A composition comprising a substantially purified cytokine inhibitory factor, wherein the cytokine inhibitory factor:

a) has a molecular weight of less than 6,000 Da;

b) naturally occurring in the lipid component of the egg yolk of avian eggs;

c) has a maximum wavelength absorbance at 205 nm;

d) a material of non-protein origin;

e) has a melting point of about 39 ℃ to 42 ℃; and

f) inhibit RNA transcription of tumor necrosis factor alpha (TNF-alpha), interleukin-1-beta (IL-1 beta), and interleukin-2 (IL-2).

2. The composition of claim 1, wherein the cytokine inhibitory factor is soluble in a solvent selected from the group consisting of chloroform, ethanol and methanol.

3. The composition of claim 1, wherein 100mg of the cytokine inhibitory factor is soluble in 1.0ml of solvent.

4. The composition of claim 1, wherein said cytokine inhibitory factor is insoluble in a solvent selected from the group consisting of acetone, DMSO, hexane, diethyl ether, vegetable oil, and water.

5. The composition of claim 1, wherein said cytokine inhibitory factor inhibits prostaglandin E₂(PGE₂) The biosynthesis of (3).

6. The composition of claim 1, wherein said cytokine inhibitory factor has an anti-inflammatory effect in the treatment of arthritis.

7. The composition of claim 1, wherein said composition comprises a pharmaceutically acceptable carrier.

8. The composition of claim 7, wherein said pharmaceutically acceptable carrier is selected from the group consisting of: water, phosphate buffered saline solution, ringer's solution, dextrose solution, serum containing solution, Hank's solution, other physiologically balanced aqueous solutions, oils, esters, glycols, biocompatible polymers, polymer matrices, capsules, microcapsules, microparticles, pellets, osmotic pumps, diffusion devices, liposomes, lipid globules, cells, and cell membranes.

9. The composition of claim 7, wherein said pharmaceutically acceptable carrier is a product of a hyperimmunized egg selected for enrichment of said cytokine inhibitory factor.

10. The composition of claim 7, wherein said pharmaceutically acceptable carrier is a food product produced from at least a portion of a hyperimmunized egg product, wherein said portion comprises an enriched amount of said cytokine inhibitory factor as compared to said hyperimmunized egg product.

11. The composition of claim 7, wherein said pharmaceutically acceptable carrier comprises a fraction of a hyperimmunized egg product, wherein said fraction comprises an enriched amount of said cytokine inhibitory factor as compared to said hyperimmunized egg product.

12. The composition of claim 11, wherein said moiety is selected from the group consisting of: liquid egg yolk, egg yolk powder and water soluble components of said hyperimmunized egg product.

13. The composition of claim 7, wherein said composition is present in a dosage form selected from the group consisting of: liquid, aerosol, capsule, tablet, pill, powder, gel, and granule.

14. The composition of claim 7, wherein said pharmaceutically acceptable carrier comprises a controlled release formulation.

15. A purified cytokine inhibitory factor produced by a process comprising the steps of:

a) separating a water-insoluble fraction (WIF) from the water-soluble fraction (WSF) of the egg yolk;

b) separating the water-insoluble fraction into a neutral lipid fraction and a polar lipid fraction;

c) purifying the polar lipid fraction by high performance liquid chromatography to form a cytokine inhibitory factor fraction.

16. The purified cytokine inhibitory factor of claim 15 wherein in step b) the neutral lipid fraction is extracted using an organic solvent selected from the group consisting of acetone and hexane.

17. The purified cytokine inhibitory factor of claim 15, wherein in step b) the polar lipid fraction is extracted using an organic solvent selected from the group consisting of ethanol, chloroform and methanol.

18. A method for purifying a cytokine inhibitory factor from an avian egg comprising egg yolk and egg white, the method comprising:

a) separating said egg into a Water Insoluble Fraction (WIF) and a Water Soluble Fraction (WSF);

b) extracting the polar lipid fraction from the water-insoluble fraction;

c) the polar lipid fraction was purified by high performance liquid chromatography into cytokine inhibitory factor fractions.

19. The method of claim 18, further comprising extracting a neutral lipid fraction from the water insoluble fraction of the egg.

20. The method of claim 18, wherein step b) is performed by supercritical fluid extraction.

21. The method of claim 18, further comprising separating egg yolk from said egg white, wherein in step a) the egg yolk is separated into a Water Insoluble Fraction (WIF) and a Water Soluble Fraction (WSF).

22. A medicament for inhibiting the expression of a cytokine in an animal comprising the composition of claim 1, wherein the cytokine comprises a proinflammatory cytokine selected from the group consisting of tumor necrosis factor alpha (TNF- α), interleukin-1- β (IL-1 β), and interleukin-2 (IL-2).

23. The medicament of claim 22, wherein the composition comprises a pharmaceutically acceptable carrier.

24. The medicament of claim 23, wherein the pharmaceutically acceptable carrier is selected from the group consisting of: water, phosphate buffered saline solutions, ringer's solutions, dextrose solutions, serum-containing solutions, Hank's solutions, other physiologically balanced aqueous solutions, oils, esters, glycols, biocompatible polymers, polymer matrices, capsules, microcapsules, microparticles, pills, osmotic pumps, diffusion devices, liposomes, lipid globules, cells, and cell membranes.

25. The medicament of claim 22, wherein said medicament is for administration to an animal by a route selected from the group consisting of oral, intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal, intratracheal, inhalation, catheter injection, by suppository, and direct injection into a tissue.

26. The medicament of claim 22, wherein said medicament inhibits the expression of tumor necrosis factor-alpha (TNF- α), interleukin-1 β (IL-1 β), or interleukin-6 by cells of said animal.

27. The medicament of claim 22, wherein the medicament down-regulates prostaglandin E by cells of the animal₂(PGE₂) And (4) synthesizing.

28. The medicament of claim 22, wherein the animal is a mammal.

29. A medicament for treating arthritis in an animal comprising a composition comprising a cytokine inhibitory factor, wherein the cytokine inhibitory factor:

a) has a molecular weight of less than 6,000 Da;

b) naturally occurring in the lipid component of the egg yolk of avian eggs;

c) has a maximum wavelength absorbance at 205 nm;

d) a material of non-protein origin;

e) has a melting point of about 39 ℃ to 42 ℃; and

30. The medicament of claim 29, wherein the medicament is for administration to an animal by a route selected from the group consisting of oral, intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal, intratracheal, inhalation, catheter injection, by suppository, and direct injection into a tissue.

31. The medicament of claim 29, wherein said composition comprises a food product comprising said cytokine inhibitory factor.

32. The medicament of claim 29, wherein administration of the medicament results in an administration selected from the group consisting of: reduce arthritic symptoms, reduce inflammation associated with arthritis, and prevent the consequences of arthritis.