MXPA98007754A - Anti-inflammatory composition based on egg, method of insulation and - Google Patents
Anti-inflammatory composition based on egg, method of insulation andInfo
- Publication number
- MXPA98007754A MXPA98007754A MXPA/A/1998/007754A MX9807754A MXPA98007754A MX PA98007754 A MXPA98007754 A MX PA98007754A MX 9807754 A MX9807754 A MX 9807754A MX PA98007754 A MXPA98007754 A MX PA98007754A
- Authority
- MX
- Mexico
- Prior art keywords
- egg
- inflammatory
- dna
- inflammatory composition
- composition
- Prior art date
Links
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Abstract
The invention is directed to an anti-inflammatory composition obtained from the eggs of a bird that has been hyperimmunized with one or more antigens, and, in particular, bacterial antigens. The invention further relates to an egg or an egg product comprising effective amounts of the anti-inflammatory composition. The invention also relates to a method for isolating the anti-inflammatory composition. The invention finally relates to methods of treating inflammation using the anti-inflammatory composition and / or the egg or egg products comprising effective amounts of the anti-inflammatory composition.
Description
ANTI-INFLAMMATORY COMPOSITION OF EGG, METHOD OF ISOLATION AND USE
BACKGROUND OF THE INVENTION
Field of Invention:
The invention relates generally to an anti-inflammatory composition produced in a natural food product. The invention relates specifically to an anti-inflammatory composition of egg, methods for its production in substantially purified and highly purified forms, and methods for its use in the treatment of inflammation. Inflammation, as defined in the Dorland Medical Dictionary, is "a protective, localized response, produced by the injury or destruction of tissues, which serves to destroy, dilute or separate both the harmful agent and the injured tissue." The leakage of the blood elements within the interstitial spaces is characterized by the fenestration of the leakage of the microvasculature, and the migration of the leukocytes within the inflamed tissue. On one level
REF: 028209 macroscopic, this is usually accompanied by the clinical, familiar signs of erythema, edema, hyperalgesia (sensitivity), and pain. During this complex response, chemical mediators such as histamine, 5-hydroxytryptamine, various chemotactic compositions, bradykinin, leukotrienes, and prostaglandins are released locally. The phagocytic cells migrate within the area and the lysosomal, cellular membranes can break down and release the lytic enzymes. All of these events can contribute to the inflammatory response. Inflammation in patients with rheumatoid arthritis probably involves the combination of an antigen with an antibody complement that causes the local release of chemotactic and chemoactivating compositions that attract leukocytes. Leukocytes consume antigen-antibody and complement complexes by phagositosis, and also release many enzymes contained in their lysosomes. These lysosomal enzymes then cause damage to the cartilage and other tissues, and this promotes the degree of inflammation. The immune reactions mediated by the cells may also be involved. Prostaglandins, which are intracellular regulators, keys to cell function, are also released during this process. The Arthus response is an inflammatory response caused by the formation of immune complexes in subcutaneous locations where an antigen forms complexes with an antibody to that antigen. Neutrophils characteristically bind to the Fc portion of the immunoglobulin complex that is formed at the location of the subcutaneous injection where they release digestive enzymes, which causes acute, visible inflammation. In this way, the response is mediated mainly by neutrophils and the agents that effect the development of the response do so by means of an effect on these cells. There are several pathways by which an agent could interfere with the migration of neutrophils from blood vessels to an inflammatory location. One possible route is the inhibition of the marginalization, adhesion or reversible "stickiness" of the inflammatory cells to the lining of the endothelial cells of the blood vessel wall. This adhesion is controlled precisely by the quality and quantity of the adhesion and the integrin molecules that act as a "sailboat" for the white blood cells and the lining of the blood vessels. In the normal state approximately 50% neutrophils adhere reversibly, but during an acute inflammatory response, adhesion becomes much stronger and is a key step in the process of neutrophil migration. While prostaglandins are unlikely to be directly involved in the chemotactic response, another product of the metabolism of arachidonic acid, leukotriene, is a very potent chemotactic substance. The inflammatory response is any response characterized by inflammation as defined above. It is well known, for those experts in medical techniques, that the inflammatory response causes much of the physical discomfort (ie, pain and loss of function) that has come to be associated with different diseases and injuries. Therefore, it is a medical practice, common to administer pharmacological agents that reduce the physical discomfort of the inflammatory response. Agents that have these properties are classified as anti-inflammatory.
Anti-inflammatory drugs are used to treat a broad spectrum of disorders, and the same drugs are often used to treat different diseases. Treatment with anti-inflammatory drugs is not for the disease, but more frequently for the symptoms (ie, inflammation). The anti-inflammatory, analgesic and anti-pyretic drugs are a heterogeneous group of compounds, often not chemically related, which nevertheless share certain therapeutic actions and side effects. Corticosteroids represent the most widely used class of compounds for the treatment of inflammation. Proteolytic enzymes represent another class of compounds that are thought to have anti-inflammatory effects. • Hormones, which directly or indirectly cause the adrenal cortex. produce and secrete steroids, represent another class of anti-inflammatory compounds. Unfortunately, natural and synthetic corticosteroid preparations cause a number of serious side effects, including elevated blood pressure, salt and water retention, kidney damage and increased potassium and calcium excretion. On the other hand, corticosteroids can mask the signs of infection and increase the spread of infectious microorganisms. These hormones are considered unsafe for use in pregnant women, and treatment of corticosteroids over the long term has been associated with gastric hyperactivity and / or peptic ulcers. Treatment with corticosteroids can also aggravate diabetes mellitus, which requires higher doses of insulin, and can produce psychotic disorders. Anti-inflammatory, hormonal agents which indirectly increase the production of endogenous corticosteroids have the same potential for side effects, adverse. A number of anti-inflammatory, non-steroidal (NSAID's) agents have been described. Among those, the most widely used are salicylates. Acetylsalicylic acid or aspirin is the most widely prescribed analgesic-antipyretic and anti-inflammatory agent. Examples of the anti-inflammatory, steroidal and nonsteroidal agents are listed in the Reference for the Work of the Physician or Physician. The NSAIDs are biochemical, synthetic compounds which can be toxic in high doses due to their wide spectrum of undesirable side effects. For example, salicylates contribute to the serious alterations in acid-base balance that characterize poisoning by this class of compounds. Salicylates stimulate breathing directly and indirectly. The toxic doses of the salicylates cause a central respiratory paralysis as well as a circulatory collapse, secondary to the depression of the vasomotor system. Ingestion of salicylate can result in an epigastric danger, nausea and vomiting. The gastric blood flow induced by salicylate is well known. Salicylates can cause liver damage and a prolonged clotting time. Therefore, aspirin should be avoided in patients with severe liver damage, hypoprothrombinemia, vitamin K deficiency or hemophilia, because the inhibition of platelet hemostasis by the silicates can result in hemorrhage. Salicylate intoxication is common, and more than 10,000 serious cases of salicylate poisoning are observed in the United States each year, some of them fatal, and several occur in children. See The Pharmacological Basis of Therapeutics by Goodman and Gilman, 7a. edition, 1985. Therefore, despite the large number of anti-inflammatory agents that are currently available, there is still a need for an effective anti-inflammatory product that is free from side effects and adverse reactions. If a natural, food product having anti-inflammatory effects could be obtained, it would provide a readily administrable therapeutic composition, without difficulty of availability, and safe.
Related Technique:
Milks have been reported that have a variety of therapeutic effects. U.S. Patent No. 4,324,782 discloses a milk containing an antibody to mutans Streptococcus that has dental caries inhibition effects. Milk is obtained by immunizing a cow with the antigen of S. mutans in two stages. U.S. Patent No. 4,284,623 publishes a milk that has anti-inflammatory properties. U.S. Patent No. 4,879,110 publishes a milk that has anti-hypertensive properties. U.S. Patent No. 3,128,230 publishes a milk containing globulins of the alpha, beta and gamma components obtained by inoculating a cow with antigenic mixtures. U.S. Patent No. 3,376,198 and Canadian Patent No. 587,849 and British Patent No. 1,211,876 also disclose milk containing antibodies. U.S. Patent No. 4,897,265 and U.S. Patent No. 4,636,384 (Reissued No. 33,403) disclose a method for reducing blood lipid concentrations when feeding animals with milk containing antibodies derived from cows maintained in a hyperimmune state by injections of bacterial antigens, polyvalent. Several genera of bird classes, such as chickens (Gallus domesticus), turkeys, and ducks, produce antibodies in the blood and in the eggs against the antigens that cause bird diseases, as well as against other antigens.
For example, LeBacq-Verheyden et al. (Immunology 27: 683 (1974)) and Leslie, G.A. and coworkers (J. Med, 130: 1337 (1969)), have quantitatively analyzed chicken immunoglobulins. Polson A. et al. (Immunology Communications 9: 495-514 (1980)) immunized chickens against several proteins and natural protein mixtures, and detected IgY antibodies in the egg yolks. Fertel R. et al. (Biochemical and Biophysical Research Communications 102: 1028-1033 (1981)) immunized chickens against prostaglandins and detected antibodies in the egg yolk. Jensenius et al. (Journal of Immunological Methods 46: 63-68 (1981)) provide a method for the isolation of egg yolk IgG for use in immunodiagnostics. Polson et al. (Immunological Communications 9: 475-493 (1980)) describe antibodies isolated from the yolk of chickens that were immunized with a variety of plant viruses. US Patent No. 4,357,272 discloses the isolation of antibodies from egg yolks derived from hyperimmunized hens. Hyperimmunization was caused by repeated injections of antigens derived from plant viruses, human IgG, tetanus antitoxin, snake antivenom and Serameba. U.S. Patent No. 4,550,019 discloses the isolation of egg yolks from antibodies produced 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. Additionally, a field of study and a controlled infection experiment showed the protective effect of the lyophilized product of the egg yolk of antibodies and the lyophilized product of the whole egg, immune against enterotoxic E. coli (Wiedermann et al., J. Vet. Med. B. 38: 283-291 (1991)). Egg yolk powder, orally administered, prepared from hens vaccinated with heat-killed antigens from protected neonatal calves, enterotoxigenic E. coli, K99-pili, against diarrhea induced by E. Coli (Ikemori et al., Am. J. Vet. Res. 53: 2005-2008 (1992)).
It has also been shown that the IgY antirotavirus administered to mice infected with rotavirus provides complete protection (Ebina et al., Microbiol.Immunol.34: 617-629 (1990)). further, the egg yolk containing an anti-Edwardsiella tarda antibody was found effective in the prevention of ed ardyllosis in Japanese eel (Gutiérrez et al., J. Fish Diseases 16: 113-122 (1993)). U.S. Patent No. 4,748,018 discloses a method of passive immunization of a mammal comprising parenterally administering a purified antibody obtained from the eggs of a bird that has been immunized against the corresponding antigen, and wherein the mammal has a history of egg consumption. . The invention published in the US Patent No. 4,748,018 is extended in the concepts published in the US Patent No. 4,748,018 in which the administration of the antibody of the egg can be by any appropriate route, not only parenterally. US patent application Serial No. 08 / 688,576, assigned to DCV-Biologics, publishes a method of prevention, combat or reduction of gastrointestinal disorders, chronic or gastrointestinal damage induced by Anti-Inflammatory, Non-steroidal Drugs (induced by NSAIDs) in a subject when administering the hyperimmunized egg and / or milk or, fractions thereof to the subject. The eggs of chickens immunized against specific bacterial antigens were published as providing anti-atherosclerotic effects in mammals in US Pat. No. 5,215,746. Eggs containing an antibody to S. mutans were obtained from chickens immunized with S. mutans (Otake et al., J. Dental Research 70: 162-166 (1991)) and inhibited the development of dental caries. However, none of. those references, publish or suggest that the eggs may produce a composition (s) and / or a factor (s) that can be administered to animals to prevent or reduce inflammation. None publishes or suggests a method that provides reasonable hope that any treatment of a bird could produce such. composition and / or factor in the eggs. None of the references suggests or publishes the identity of an anti-inflammatory component of the eggs which produces the desired therapeutic effects.
BRIEF DESCRIPTION OF THE INVENTION
The invention is based on the discovery of the inventor that there is anti-inflammatory activity in an egg and in egg products and particularly in egg products obtained from hyperimmunized birds, which when administered to a subject, in particular mammals, prevents or reduces inflammation in the subject. In particular, the invention relates to a highly pure, anti-inflammatory composition obtained from the eggs of a bird. The anti-inflammatory activity was found in fractions isolated from both the egg yolk and the egg white. The invention is also directed to a highly pure anti-inflammatory composition that is produced by a process comprising the hyperimmunization of a bird by administering to the bird one or more antigens, and especially bacterial antigens, collecting the eggs of the hyperimmunized bird, and isolating the anti-inflammatory, highly pure composition thereof.
The process of hyperimmunization produces supranormal levels of an anti-inflammatory, highly pure composition of a bird egg. Surprisingly, the level of the anti-inflammatory composition can be increased in both the egg yolk and the egg white by the hyperimmunization process. The invention also includes a method of treating inflammation in a subject, and especially in mammals, which comprises administering to the subject the anti-inflammatory composition, substantially pure or a composition comprising the anti-inflammatory, substantially pure composition. This aspect includes administering the anti-inflammatory egg, complete and / or fractions thereof. The invention is further directed to methods for using eggs containing the anti-inflammatory composition and / or the highly purified anti-inflammatory factor itself to prevent lymphocytes and neutrophils from adhering to the endothelium of the venules or (to detach lymphocytes and lymphocytes). neutrophils which have already adhered to the endothelial cells that line the walls of the venules) and to reduce the migration of lymphocytes and neutrophils and thus reduce excessive edema and fluid increase. In this way, the composition is used to reduce tissue damage associated with the inflammatory response. Finally, the invention includes an anti-inflammatory, substantially pure composition that exhibits anti-inflammatory activity in mammals.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a chromatogram showing the separation of the anti-inflammatory composition of egg, molecular weight of less than 30,000 by the ion exchange chromatogram of DEAE Sepharose. Figure 2 is a graph which shows the inhibition of the migration of pleural leukocytes by the anti-inflammatory composition of egg, molecular weight of less than 30,000. Figure 3 is a graph which shows the inhibition of migration of pleural leukocytes of the rat by the anti-inflammatory, endotoxin-free composition of the egg yolk degreased with hexane-ethanol.
Figure 4 is a flow diagram of the preparation of the anti-inflammatory composition, with a molecular weight of less than 30,000 yolk. Figure 5 is a flow chart of the preparation of the anti-inflammatory composition, of molecular weight of less than 30,000 of the liquid egg white. Figure 6 is a graph which shows the inhibition of rat pleural leukocytes by anti-inflammatory composition, molecular weight less than 30,000, endotoxin-free from egg yolk S-100 degreased by extraction with Supercritical C02. Figure 7 is a flow chart of the preparation of the egg anti-inflammatory composition, molecular weight of less than 30,000 dry egg yolk powder. Figure 8 is a flow chart of the purification of the anti-inflammatory composition, of molecular weight of less than 3,000 of the powdered egg yolk. Figure 9, is a flow diagram of the purification of the anti-inflammatory composition, molecular weight of less than 3,000 of the powdered egg white. Figure 10 is a chromatogram of the separation of the anti-inflammatory composition of egg, molecular weight of less than 3,000 by ion exchange chromatography with DEAE Poros. Figure 11 is a graph which shows the inhibition of the migration of the leukocytes by the highly purified fractions, with a molecular weight of less than 3,000 of the egg yolk and the egg white. Figure 12 is a graph which shows the migration of the leukocytes by the DEAE ultrafiltered products of peak 2, molecular weight of less than 3,000 and molecular weight of less than 10,000 of the hyperimmune egg yolk. Figure 13 is a graph which compares the anti-inflammatory, molecular weight composition of less than 3,000, highly purified eggs with anti-inflammatory drugs (NSAIDS)
(Indomethacin and aspirin). Figure 14 is a chromatogram of the separation by CLAP of the anti-inflammatory composition, of molecular weight of less than 3,000 of the hyperimmune egg yolk.
Figure 15 is a chromatogram of the separation by CLAP of the DEAE fraction of the hyperimmune egg yolk.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term "egg product" means any product as described herein which contains an egg or an anti-inflammatory egg fraction. The term "egg or anti-inflammatory egg fraction" means the egg or egg fractions containing the anti-inflammatory composition published herein and specially produced, by the processes disclosed herein. The term "anti-inflammatory composition" means a composition that counteracts or suppresses the inflammatory process. The term "substantially pure egg anti-inflammatory composition" means an anti-inflammatory composition at least of the purity described in Example 3 and the exemplary materials and figures.
The term "highly pure egg anti-inflammatory composition" means an anti-inflammatory composition at least of the purity described in Example 4 and the exemplary materials and figures. The term "anti-inflammatory factor" means a substantially pure agent that counteracts or suppresses the inflammatory process which occurs in the anti-inflammatory composition at least of the purity described in Example 7. The term "combinatorial, derived antigens" is refers to a new process of generation of molecular diversity among antigens by means of a 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 and translation of proteins which have antigenic properties. The term "genetic vaccine" refers to a nucleic acid vaccine which is produced in general by recombinant technologies and which can produce an immune response. The term "hyperimmunized egg" means a whole egg or products derived therefrom, obtained from animals that produce eggs maintained in a hyperimmune state. The term "supranormal levels" means levels in excess of those found in the eggs of animals that produce eggs not maintained in a hyperimmune state. The term "table egg" means a non-hyperimmunized egg. The term "inflammation" is used in its recognized sense in the art as a protective, localized response, caused by injury or destruction of tissues which serves to destroy, dilute or separate both the harmful agent and the injured tissue, characterized in the inappropriate form, uncontrolled by the classical sequence of pain, heat, redness, swelling and loss of function, and histologically involves a series of complexes and events, including dilation of the arteries, capillary vessels and venules with permeability and blood flow increased, exudation of fluids that include plasma proteins and the migration of leukocytes within the inflammatory focus.
The term "treatment" means that the symptoms of the disorder and / or pathogenic origin of the disorder are prevented, improved or eliminated completely. For example, the anti-inflammatory composition treats inflammation not only by suppressing the symptoms of inflammation in humans and other mammals, but also by acting as a prophylactic agent to counteract the anticipated presence of inflammatory agents in the recipient. The term "administer" means any method for providing a subject with a substance that includes orally, intranasally, parenterally (intravenously, intramuscularly, or subcutaneously) or rectally. The term "subject" means any living animal that can mount an inflammatory response and is subject to inflammatory disorders and damage, including humans and other animals.
The invention
In one embodiment, the invention comprises an anti-inflammatory, highly pure composition obtained from the eggs of birds, their isolation and purification and the administration of the composition to a subject for the treatment of inflammation. The invention further comprises an anti-inflammatory composition, highly purified, obtained from the egg of a bird that has been hyperimmunized with one or more antigens and, in particular, bacterial antigens or their synthetic equivalent. The anti-inflammatory, highly pure composition is present in hyperimmunized eggs at supranormal levels which provide anti-inflammatory activity in the subjects. Although the highly pure anti-inflammatory composition is found in the water-soluble fraction of egg yolk and egg white of any table egg, when it is isolated from hyperimmunized eggs, the anti-inflammatory composition is at supranormal levels. which are effective in the treatment of inflammation in a subject. The invention also comprises an anti-inflammatory composition, substantially pure, obtained from poultry eggs, their isolation and purification and the administration of the composition to a subject for the treatment of inflammation. The invention further comprises an anti-inflammatory composition, substantially purified, obtained from the egg of a bird that has been hyperimmunized with one or more antigens and, in particular, bacterial antigens or their synthetic equivalent. The substantially pure anti-inflammatory composition is present in hyperimmunized eggs at supranormal levels which provide anti-inflammatory activity in the subjects. The anti-inflammatory composition (s) of the present invention can be administered by any means that provides an anti-inflammatory activity. For example, administration can be parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal or oral. Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. The invention further comprises an anti-inflammatory factor, substantially pure which is within the anti-inflammatory composition. The factor can be administered to a subject by the same means as described above for the anti-inflammatory composition.
The details of the invention are given below.
Characteristics of the anti-inflammatory, highly purified composition: the anti-inflammatory, highly pure composition has the following characteristics: 1) it has an anti-inflammatory activity in a subject, and in particular, in mammals; 2) is present in both the egg white and the egg yolk of the bird eggs; 3) when it is isolated from the egg yolk, it has an anti-inflammatory activity, larger than when it is isolated from the egg white; 4) has a molecular weight of less than about 3,000 daltons; 5) it is not degraded by enzymes that degrade proteins; 6) is not proteinaceous or spheroidal; 7) has a negative charge under prevailing conditions; 8) is stable to heat at 100 ° C for at least 30 minutes; 9) is active orally and is not degraded by digestive enzymes;
) and which is resistant to acid and base by returning to an active form even after treatment with conditions of pH 4 to pH 9.0. The molecular weight of 3,000 daltons is derived from the isolation and purification of the composition where the isolation and purification process uses an ultrafiltration membrane that does not allow the passage of molecular species larger than 3,000 Daltons through it. The composition is determined to be neither proteinaceous nor spheroidal because it is small in size and is not degraded by the enzymes which degrade the proteins. On the other hand, the composition is active orally and is not degraded by digestive enzymes. The stable, small form of the composition (as it is differentiated from the proteins which are much larger) facilitates its absorption from the digestive system. The composition is resistant to acid and base, returning to an active form even after treatment with pH 9.0 conditions. In addition, the composition is heat stable at 100 ° C for at least 30 minutes and has a negative charge at a neutral pH.
The highly purified anti-inflammatory egg composition can be isolated from the whole egg, egg yolk and egg white. The anti-inflammatory composition isolated from the egg yolk shows a higher anti-inflammatory activity, than the purified anti-inflammatory composition of the egg white. The supranormal levels of the highly purified anti-inflammatory composition can be isolated from the whole egg, the egg yolk and the hyperimmunized egg white of a bird.
Characteristics of the anti-inflammatory composition, substantially purified: The anti-inflammatory, substantially pure composition exhibits anti-inflammatory activity in mammals. The substantially pure anti-inflammatory composition is found in the water-soluble fraction of the egg yolk and the egg white in the fraction containing molecules of molecular weight of less than about 30,000 daltons. The composition has a negative charge, is heat stable at 90 ° C for at least one hour, and has the egg elution profile shown in Figure 1. In addition, the isolated composition of the egg yolk has an anti-inflammatory activity larger in mammals than in the factor isolated from egg white. The molecular weight of the egg anti-inflammatory composition prepared from chicken eggs by the method described above is less than 30,000 daltons. This was inferred from the fact that the first step in isolating the egg yolk and egg white factor was by ultrafiltration using a membrane that does not allow the passage of species of molecular weight larger than 30,000 daltons. The composition has a negative charge under prevailing conditions, as determined by applying an ultrafiltered 30K egg product to an ion exchange column of DEAE Sepharose. The anti-inflammatory composition does not elute with water from the column. The change of the means of elution to a solution of NH4OAc causes the anti-inflammatory composition of the egg to elute. When the ultrafiltered product is applied to a DEAE Sepharose column, several proteins and salts integrate the unbound fraction.
Hyperimmunization of the egg producing animal: The invention is based in part on the discovery that when an egg-producing animal such as a bird is brought to a specific state of hyperimmunization, the animal will produce eggs having supranormal levels of the anti-animal composition. inflammatory, highly beneficial and in this way a higher anti-inflammatory effect is provided. The product of the hyperimmunized egg can be produced by any egg-producing animal. It is preferred that the animal be a member of the class of birds. Within the class of birds, domestic birds are preferred, but other members of this class, such as turkeys, ducks and geese are a suitable source of the hyperimmunized egg product. When such egg producing animals are brought to a specific state of hyperimmunization by means of, for example, the periodic, reinforcement administration of antigens, the animals will produce eggs containing supranormal levels of the anti-inflammatory composition which, when administered to a subject, it will have beneficial properties in the treatment of inflammation. The induction of the immune sensitivity alone is insufficient to cause the appearance of the supranormal levels of the anti-inflammatory composition of egg in the eggs, as shown by the fact that the eggs of the normal birds, otherwise referred to as "table eggs" ", do not contain these supranormal levels, although birds have been sensitized against several antigens during normal immunization against bird diseases and during normal exposure to environmental factors. This is only in the hyperimmune, specific states that the eggs have the desired supranormal levels. This special state of hyperimmunization, in which the egg will contain higher levels of the anti-inflammatory composition, is achieved by administering an initial immunization, followed by periodic reinforcement with sufficiently high doses of specific antigens or mixtures of antigens. The preferred dosage of the boost must be equal to or greater than 50% of the dosage necessary to produce the primary immunization of the bird. In this way, there is a dosage of the reinforcement, initially below which the properties are not produced in the egg of the bird, although nevertheless the bird is in what would normally be called an immune state. Having the knowledge of the requirement to develop and maintain a hyperimmune state, it is within the skill of the technique to vary the amount of antigen administered, depending on the genus of the egg-producing animal and the breed used, in order to keep the animal in the hyperimmune state. It should be understood that if the levels of the anti-inflammatory composition are increased as described above, then it naturally follows that the anti-inflammatory factor levels will increase correspondingly at the same time. The hyperimmune composition can be produced by any antigen or a combination of antigens. Hyperimmunization can be achieved by multiple exposures to multiple antigens, multiple exposures to individual antigens, or individual exposures to collections of immunogens. Almost any antigen can be used to induce the hyperimmune state, including but not limited to, bacterial, viral, protozoan, allergen, fungal or cellular substances. In addition to immunizations with naturally occurring antigens, immunization can also be performed using antigens which are synthetically derived by combinatorial chemistries. The basic strategy is to join multiple combinations of chemical, component blocks to produce a population of molecules with a diversity. Several methods have been recently developed for the combinatorial synthesis of solid phase and solution of the oligomer collections (Fodor, S. et al., Science 251: 767 (1991): Houghton, R. et al., Nature 354: 82 (1991 )) as well as small, organic molecules (Bunin, B. &; Ellman, J., J, Am. Chem. Soc. 114: 10997 (1992)). The synthesis of fast, multiple peptides and oligomers can serve as a source for combinatorial, derived antigens. In addition, an alternative strategy would allow the addition of the component, organic blocks in a combinatorial manner for a molecule of the structure to improve antigenicity.
Alternative modes for hyperimmunization of egg-producing animals include the use of genetic vaccines or biodesigned antigens. In particular, any DNA construct (which generally consists of a promoter region and an antigen coding sequence) will elicit the release of antibodies. Genetic vaccines consist of antigen-coding vectors, single-DNA fragments, plasmid DNA, DNA-RNA antigens, DNA-protein conjugates, DNA-liposome conjugates, collections of DNA expression and viral and bacterial DNA supplied to produce an immune reaction. In particular, there is now evidence that DNA is immunomodulatory as well as immunogenic. The delivery of the antigen coding vectors, the fragments of the simple DNA, plasmid DNA, DNA-RNA antigens and DNA expression collections can in fact be similar to the thermo-killed and live viral and bacterial vaccines. One of the benefits of DNA vaccines is the production and release of antigens from individual cells that provide free antigens for the induction of a humoral response as well as an immune, cytotoxic response. The cytotoxic reaction is responsible for the increased efficiency (Ada, G.L., Lancet
335: 523-526 (1990)). An additional advantage is the ability of genetic vaccines to produce foreign proteins with an appropriate three-dimensional conformation. Genetic immunization is a new approach to the production of the vaccine that has many of the advantages of living / diminished pathogens but without risk of infection. A collection of expression comprised of DNA from various sources can be used as vaccines to produce the hyperimmune state. Methods of DNA delivery include, but are not limited to, particle bombardment, direct injection, liposomes, injection under pressure (Fynan, EF et al., Proc. Nati, Acad. Sci. USA 90: 11478-11482 (1993 )). Nucleic acids that are encoded for known or unknown immunogens, promoter regions (notably CMV cauliflower mosaic virus) and bacterial origin SV40 can be duplicated in bacteria to produce plasmid DNA for use in DNA injections. Although several routes of parenteral administration of DNA in chickens are effective, the preferred method is intramuscular injection into the breast muscle. The experiments of the vaccines are carried out in egg-laying birds, preferably in chickens. Duplicate immunizations are given at one to two week intervals for up to 6 months. It is preferred that the amounts of DNA used in general be in the order of 50-300 μg of DNA in a saline solution for direct injection. For the bombardment of particles, 4-100 μg of DNA co-precipitated in gold beds is preferred for the addition of 2.5 M CaCl 2. Repeated immunizations can be given intradermally by this method of accelerating the DNA-coated particles within the living animal. The following is a detailed description of a preferred method used to bring an egg-producing animal to an enhanced state of immunity from which the resultant egg or hyperimmunized egg product can be administered to a subject: 1. Select one or more antigens 2. Produce an immune reaction in the egg producing animal by primary immunization. 3. Administer antigen-reinforcing vaccines of appropriate dosage to induce and maintain the hyperimmune state. 4. Analyze hyperimmunized eggs for levels of anti-inflammatory activity. 5. Collect and process the eggs.
The following is a more detailed description of this procedure.
Step 1: Any of the antigens or combination of antigens can be used. The antigens can be bacterial, viral, protozoan, fungal, cellular, allergens or any other substance for which the immune system of an egg-producing animal will respond. The critical point in this step is that the antigen (s) must be able (ees) not only to induce the immune and hyperimmune states in the egg-producing bird, but also to induce the production of the Anti-inflammatory composition in the egg. A preferred vaccine is a mixture of bacterial, polyvalent antigens, referred to as a Series 100 vaccine (S-100). Bacteria included in the S-100 vaccine are listed in Table 1 of Example 1. This vaccine has been previously described in U.S. Patent Nos. 5,106,618 and 5,215,746, both assigned to Stolle Research and Development Corporation.
Step 2: The vaccine can be administered by any method that produces an immune response. It is preferred that the immunization be carried out by administering the antigens through intramuscular injection. The preferred muscle for injection in a bird is the muscle of the breast. The dosage is preferably 0.5-5 milligrams of the antigen vaccine (s). Other methods of administration that can be used include intravenous injection, intraperitoneal injection, rectal suppository, and oral administration, among others. When DNA techniques are used for the hyperimmunization process, much smaller amounts are required, generally 1-300 micrograms.
It can be determined if the vaccine has produced an immune reaction in the egg producing animal through a number of methods known to those skilled in the art of immunology. Examples of these include enzyme-linked immunosorbent assays (ELISAs), analyzes for the presence of antibodies for antigen stimulation, and assays designed to assess the ability of the host immune cells to respond to the antigen. In general, the appearance of the egg antibodies after immunization with the vaccine is indicative of an immune reaction. The minimum dosage of the antigen, necessary to induce an immune response, depends on the vaccination procedure used, which includes the type of antigen (s) used (s) as well as the type of egg-producing animal used as the host.
Step 3: The hyperimmune state is preferentially induced and maintained by the repeated administration of the boost of an appropriate dosage at fixed time intervals. The time intervals are preferably two weeks intervals over a period of six months. It is essential that booster administrations do not lead to immune tolerance. It is also possible to use other hyperimmunization maintenance methods or combinations of procedures, such as, for example, intramuscular injection for primary immunization and intravenous injection for booster injections. Additional methods include simultaneously administering the 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 immunization and hyperimmunization are known to those skilled in the art.
Step 4: It is necessary to analyze eggs for levels of anti-inflammatory activity. This can be done by any clinical or preclinical evaluation that analyzes the effects of either the hyperimmune egg, or the products derived from it, in inflammation. The inflammation induced by chemicals from the rat is a normal analysis for anti-inflammatory drugs. (See Example 6a, b, c).
Step 5: This step involves the collection and processing of the egg (s) containing the anti-inflammatory composition. The egg can be collected by conventional methods. The processing of the egg to isolate and purify the anti-inflammatory composition is described below.
Isolation and Purification:
The isolation and purification of the anti-inflammatory composition of egg can be carried out using the whole egg, the egg yolk or the egg white. An example of a preferred process is as follows: 1. Preparation of the water-soluble fraction of an egg; 2. Ultrafiltration of the water-soluble fractions;
3. Separation of fractions by reverse phase High Pressure Liquid Chromatography; and 4. Bioassay of fractions separated by anti-inflammatory activity.
The following is a more detailed description of this process: Step 1: The anti-inflammatory composition can be isolated from the whole egg, the egg yolk or the egg white. In a preferred embodiment, the composition is isolated by this method from egg white. In an especially preferred embodiment, the composition is isolated from the egg yolk. The lipid portion is removed from the whole egg or the yolk by methods well known to those skilled in the art. For example, in the case of spray dried egg yolk powder, the degreasing can be carried out with solvents (propane, butane or hexane or with binary solvents), supercritical CO 2, enzymes and the like, and in the case of the yolk liquid egg, degreasing can be done by the caprylic acid separation method (CAPS) published by Lee (US Patent No. 5,367,054). It is not necessary to remove the fat from the egg white, and in this way the liquid or pulverized form of the egg white can be either heated or dissolved directly by conventional methods and as described in the examples listed below. The whole egg, the egg yolk or the egg white are then preferentially processed in either a liquid or pulverized form, and further processed to obtain the water-soluble fractions, (See Examples)
Step 2: The resulting water soluble fractions of the whole egg, egg yolk or egg white are subjected to ultrafiltration using ultrafiltration systems equipped with a 3,000 molecular weight intercept membrane. The ultrafiltration process separates molecules that have a molecular weight of more than 3,000 daltons from those that have a molecular weight of less than 3,000 daltons. Once filtered, the resulting ultrafiltered products containing molecular weight molecules of less than 3,000 daltons are then lyophilized, weighed and prepared for bioassay analysis. In a described, specific embodiment, the preferred ultrafiltration is by Amicon RAIOOO ion exchange chromatography (3K molecular weight CO) and DEAE. However, it is understood that equivalent techniques and materials can be used to isolate the composition, given the information herein, and the knowledge available to the person of ordinary skill in the art.
Step 3: Fractions of the ultrafiltered product of less than 3,000 daltons can be separated by, for example, Reverse Phase High Pressure Liquid Chromatography to isolate the highly pure anti-inflammatory composition. As an alternative or additional strategy, the anti-inflammatory composition of egg in the ultrafiltered product can further be characterized by ion exchange chromatography with DEAE-Sepharose. Such separation methods are well known to those of ordinary skill in the art.
Step 4: The anti-inflammatory activity of the composition can be analyzed by any bioassay which determines the anti-inflammatory activity. Some examples include the inhibition of leukocyte migration, the analysis of rat paw edema, adjuvant-induced arthritis, collagen-induced arthritis and intravital microscopy among others. Comparisons of the anti-inflammatory composition of egg can also be made with anti-inflammatory drugs, known such as aspirin and indomethacin. And finally, clinical analyzes can also be used for rheumatoid arthritis, degenerative joint disease and arthritis-induced injury to determine anti-inflammatory activity. The anti-inflammatory action of the highly pure anti-inflammatory composition of egg is measured by the bioassays. A preferred bioassay is the analysis of the inhibition of the migration of pleural leukocytes as described in Vinegar et al., "Some Quantitative Characteristics of Carrageenan Induced Pleurisy in the Rat", Proc. Soc. Exp. Bio. Med. 143: 711-714 (1973); Ammendola, G. et al., "Leukocyte
Migration and Lysozomal Enzymes Reléase in Rat
Carrageenan Pleurisy ", Agents and Actions 5: 250-255
(1975): Vinegar, R. and collaborators, "Quantitative Studies of the Pathway to Acute Carrageenan
Inflammation ", Fed. Proc. 35: 2447-2456 (1976) (see
Examples 7, 7a, 7b, 7c and 9, Figures 3, 5 and 6). The analysis of the inhibition of leukocyte migration is generally carried out as follows: The samples containing the anti-inflammatory composition are administered to the female, adult, artificially inflamed rats with a 1% carrageenan solution and then the anti-inflammatory effect of the samples in each dosage (using an Automated Image Analysis Technique) by the reduction in the number of leukocytes in the pleural exudates of the treated rats when compared by those control rats. Alternatively, the anti-inflammatory action of the substantially pure anti-inflammatory composition can be analyzed in an edema caused by injecting carrageenan into the legs of the rat (Winter, CA, Risley, GA, Nuss, AW, "Carrageenan-Induced Edema in the Hind Pa of the
Rat as an Assay for Anti-inflammatory Drugs, "Proc. Soc. Exper. Biol. Med. 3: 544 (1967).) A variety of other assays can be used. (See Wetnick, AS and Sabin, C," The Effects of Clonixin and Bethaurethasone on Adjuvant-Induced Arthritis and Experimental Allergic Encephalomyelitis in Rats, "Jap J. Pharm. 22: 741 (1972)).
Administration to a subject for treatment: The invention is also directed to a method for the treatment of inflammation in a subject which comprises administering to the subject the egg, the egg product and / or the anti-inflammatory composition of this invention. The anti-inflammatory composition of the present invention can be administered by any means that provides an anti-inflammatory activity. For example, administration can be parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal or oral. Oral administration is preferably carried out through solid dosage forms which include capsules, tablets, pills, powders and granules among others. In solid dosage forms, the anti-inflammatory composition is mixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than the inert diluent. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents, pH sensitive polymers, or any other slow release encapsulants, which are typically used as encapsulation compositions in the food industry. and drugs. The tablets and pills can be further prepared with an enteric coating. The liquid, dosage forms of the anti-inflammatory composition for oral administration include the pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs, which contain inert diluents commonly used in the pharmaceutical art. In addition to the inert diluents, the compositions may also include wetting, emulsifying and suspending agents, and sweetening agents. Preparations of the anti-inflammatory composition for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. The dosage of the active ingredients can be varied: however it is necessary that the amount of the active ingredient should be such that a suitable dosage form is obtained. It will be recognized that the selected dosage form depends on the desired therapeutic effect, the route of administration and the duration of the treatment. It is preferred that the egg itself, which contains the anti-inflammatory composition, be incorporated into a food product to form the egg product. A preferred method for preparing the egg to be incorporated into a food product involves dehydrating the egg in an egg powder. Although several methods are known for dehydrating eggs, spray drying is a preferred method. A temperature of not more than 60 C (140 F) is preferably used. The samples are monitored for the moisture content during the dehydration process to obtain a final product that has any desired consistency. Egg powder, dehydrated can be used in beverages, protein supplements and any other products associated with athletes, nutritional. In addition, egg powder can be used in baking mixes, powder bars, candies, cookies, etc. Other examples of egg processing include, making an omelet, baking the egg soft or hard, baking the egg, or if desired, the egg can be eaten raw. The dose of administration and frequency will depend on the age and in general the health condition of the patient, taking into account the possibility of side effects. The administration will also be dependent on concurrent treatment with other drugs and the patient's tolerance of the drug administered. The preferred mode of storage for the preparations after the ion exchange step is as a lyophilized powder. The filtered product, collected in the first purification step, can be stored in refrigeration until its use. The activity of the anti-inflammatory composition resulting from the purification is determined using the migration analysis of the pleural leukocytes of the rat described above. Examples of inflammatory conditions that can be treated by the administration of the egg, egg product, and / or the anti-inflammatory composition of the present invention, include acute and subacute bursitis, nonspecific tendonitis, acute, systemic lupus erythematosus, dermatomyositis systemic, acute rheumatic carditis, pemphigus, bullous dermatitis, herpeteformis, severe erythema, multiform exfoliative dermatitis, cirrhosis, seasonal perennial rhinitis, bronchial asthma, ectopic demartitis, serum sickness, keratitis, ophthalmic iritis, diffuse ureitis, corditis, optic neuritis, sympathetic ophthalmia, symptomatic sarcoidosis, Loeffler syndrome, berylliosis, hemolytic anemia, mastitis, mastoiditis, contact dermatitis, allergic conjunctivitis, psoriatic arthritis, ankylosin spondylitis, acute gouty arthritis, rheumatoid arthritis herpes zoster, osteoarthritis, any of the other degenerative diseases of joints, and any other related autoimmune diseases. In addition, the isolated and purified egg product can be used to treat individuals who are exposed to potentially inflammatory agents such as allergens.
Effective Amounts: With respect to the administration to a subject of the egg or the hyperimmunized egg product, it has been determined and detailed in the following examples, that the preferred dose range of the egg or product of the hyperimmunized egg to be given to a subject is between 1 gram to 40 grams per kilogram of subject's weight. With respect to the same, highly purified anti-inflammatory composition it has been determined that the preferred dose range of the highly purified, purified and isolated composition of the whole egg, the egg yolk and the egg white of a hyperimmunized egg, is between 1 microgram and 400 milligrams of the anti-inflammatory composition.
Anti-inflammatory Factor: Once the anti-inflammatory composition is isolated and purified, the active fractions can be further purified and examined, with respect to both structure and activity to determine the characteristics of an active anti-inflammatory compound, more specific . In particular, using high pressure liquid chromatography (CLAP), an individual ultrapure peak has been identified which shows an anti-inflammatory activity. It is believed that this ultrapure, individual peak represents an individual compound having an elementary chemical structure. This compound has been labeled as the "anti-inflammatory factor" of the egg. Having now generally described this invention, it will be further described by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
EXAMPLES
Example 1 Preparation of S-100 Vaccine
The multivalent vaccine known as "Series 100" or "S-100", published in U.S. Patent No. 5,215,746 and containing the bacteria shown in Table 1 (obtained from the American Type Culture Collection), was reconstituted with 15 ml of one medium and incubated overnight at 37 ° C. Once good growth was obtained, approximately one-half of the bacterial suspension was used to inoculate one liter of broth which was then incubated at 37 ° C. The remaining suspension was transferred to sterile glycol tubes and stored at -20 ° C for up to 6 months. After good growth in the culture was visible, the bacteria were harvested by centrifugation. The bacterial pellet was resuspended in saline, sterile and the bacterial sample was centrifuged 3 times to wash the cells. After the third wash, the pellet obtained was resuspended in a small amount of double distilled water. The bacterial suspension, free of the medium, was removed by placing the suspension in a glass flask in a water bath at 80 ° C overnight. The viability of the broth culture was analyzed with a small amount of the thermoblasted bacteria. The broth was inoculated with the thermo-killed bacteria, incubated at 37 ° C for 5 days and checked daily for growth, as the bacteria had to be removed for use in the vaccine. The thermo-killed bacteria were lyophilized until dried. The dried bacteria were then mixed with sterile saline at a concentration of 2.2 x 10 8 bacterial cells / ml saline solution (optical density reading 1.0 at 660 nm).
TABLE 1
Antigens in the S-100 Vaccine
EXAMPLE 2 Anti-Inflammatory Effect of Hyperimmunized Egg This example shows the anti-inflammatory effect of the hyperimmunized egg, as described in Example 1, on an edema in the skin induced by carrageenan in dogs in a diet that includes hyperimmunized eggs. These results show that the hyperimmune egg reduces inflammation. The effect on inflammation was comparable to that obtained with the anti-inflammatory drug, non-spheroidal ibuprofen at a dose of DE5o 10 mg / kg, in this analysis.
Twenty small White Eagle dogs (four groups of five dogs each) were fed a basic diet (350 grams) of commercially available mutton and rice dog food. As illustrated in Table 2, two groups of five dogs (group 1 and 2) received only the basic diet during the conditioning period of approximately 100 days. In addition, group 2 dogs were treated with the anti-inflammatory, non-spheroidal drug, ibuprofrene, before inflammatory stimulation with carrageenan. During this same period, the two remaining groups (3 and 4) were fed the fundamental diet along with the hyperimmune egg (HIE). Group 3 received 3.5 grams of the hyperimmune egg, while group 4 received 35 grams of the hyperimmune egg added to the diet. At the end of the conditioning diet treatment, the dogs were stimulated with an intradermal injection of 2% carrageenan and an inflammatory reaction occurred. The stimulation process was as follows. One dog from each group was randomly selected to stimulate and analyze it each day. This procedure was repeated daily for five days until all dogs of all groups were analyzed. Fifteen minutes before the stimulation procedure, dogs of group 2 were given orally ibuprofen 10 mg / kg. All dogs were anesthetized by intravenous injection and shaved on the left lateral side (approximately 15.24 x 20.32 (6 x 8 inches)). Two lines of 3 marks were made in the shaved area and numbered 1-6. An intradermal injection of 0.1 ml of saline was given at label 1 as a negative control. From label 2-6, an intradermal injection of 0.1 ml of a 2% carrageenan solution was given. The injections were administered by the same person for the duration of the study. Intradermal injections of this carrageenan concentration were previously determined to cause a measurable swelling in 100% of the animals which could be reduced by 10 mg / kg ibuprofen. At the completion of the last injection of carrageenan, measurements of each swelling were made using a micrometer and the numbers were recorded. The anesthetized animals were allowed to recover and six hours later the measurements were repeated and recorded. The average size of the inflammatory response was determined for each group.
TABLE 2
These results showed a significant reduction in swelling in the animals fed the hyperimmune egg (3.5 grams plus the fundamental diet and 35 grams plus a fundamental diet) indicating a preventive effect of the hyperimmune egg on inflammation when fed orally. On the other hand, these results show that the inflammatory activity of the hyperimmune egg was comparable to the effect observed with the anti-inflammatory drug, ibuprofen. Differences in the level P < 0.05 of the significance between the treated and control group.
Example 3
Isolation of the Anti-inflammatory Egg Composition, < 30,000 daltons of the Hyperimmune Bird Egg Step 1: Preparation of the Product Soluble Filtration in Egg Water The liquid egg yolk, the liquid egg white and the egg yolk powder were processed to obtain the water soluble fraction. The liquid egg yolk was degreased by the phase separation method of caprylic acid (CAPS) published by Lee (U.S. Patent No. 5,367,054) as described in these Examples. The liquid egg white was heated for a specific length of time as described in those Examples. The yolk powder was degreased with a binary solvent, supercritical C02 or enzymes, as described in those Examples.
Step 2: Ultrafriltration of Water-Soluble Fractions The water-soluble fractions were concentrated in an Amicon DC10L Ultrafiltration System with a coiled spiral 30K molecular weight ultrafiltration CO membrane. The ultrafiltration cartridge has a surface area of the membrane of 305 cm2 (10 ft2), and was conducted near an inlet pressure of 2,808 kg / cm2 (40 psi) and an outlet pressure of 2.317 kg / cm2 ( 33 psi)
(transmembrane pressure of 0.491 kg / cm2 (7 psi)) with a recirculation flow rate of 10.4 L / minute and flow velocity of 2.3 L / minute.
Step 3: Ion Exchange Chromatography The filtrate from the ultrafiltration with DC10L of the water soluble fractions was further processed by ion exchange chromatography. In this procedure, the DEAE-Sepharose rapid flow gel (Pharmacia) was used to pack a 3 x 15 cm glass column which was equilibrated with double distilled, sterile water, pH 7.0. Five hundred ml of the filtered product (<30,000 daltons), prefiltered through a 0.2 micron filter, was applied to the column which was washed with 500 ml of double-distilled, sterile water, pH 7.0, at the flow rate of 10 ml per minute. The bound fraction, suspected of containing the anti-inflammatory composition of egg, was eluted with 250 ml of 0.15M NH4OAc. The fractions were collected and monitored at 280 mm on an LVB Uvicord 2138 monitor with an optical density printed on a connected recorder. The elution profile of the substantially pure, endotoxin-free egg anti-inflammatory composition is shown in Figure 1. The fraction of the egg anti-inflammatory composition was lyophilized by further processing.
Preparation of the Anti-inflammatory Composition of
Egg, < 30,000 Daltons of the Liquid Egg Yolk and the Liquid Egg Clara Two liters of the hyperimmune egg yolk was degreased by the phase separation method of caprylic acid (CAPS) published by Lee (U.S. Patent No. 5,367,054). In summary, egg yolk S-100 was diluted 15 times in a buffered solution of 1% caprylic acid and 0.06M sodium chloride in 0.02M acetate, at a pH of 5.0. The aqueous phase was separated by filtration of the flocculation phase, and adjusted to pH 7 with 0.5M NaOH. The aqueous phase was ultrafiltered until 2L of the retained product was left. The ultrafiltered product was applied to the DEAE-Sepharose column, 46.6 g of the 2L yolk egg yolk anti-inflammatory composition was obtained after the DEAE column chromatography (see Figure 4). One liter of the hyperimmune egg white was diluted with 3L of deionized water. The mixture was heated at 90 ° C for 1 hour, cooled and centrifuged. The supernatant was filtered through a 40 micron paper filter and ultrafiltered until about 64 ml of the retentate was obtained. Five hundred ml of the ultrafiltered product was applied to the DEAE-Sepharose column. From 1 L of egg white, 4.4 g of the anti-inflammatory composition was obtained after chromatography on DEAE Sepharose column (see Figure 5).
Preparation of the Anti-inflammatory Composition of
Egg, < 30,000 Daltons of Egg Yolk Powder Three procedures were used to degrease the egg yolk powder and then isolate the anti-inflammatory egg composition from the powder of the defatted yolk (see Figure
7).
Procedure 1: Binary Solvent Removal The hyperimmune egg yolk powder was combined with a mixture consisting of absolute ethanol and hexane to remove lipids from the yolk, followed by aqueous separation of the fraction of the anti-inflammatory composition. of egg. Briefly, 400 g of the hyperimmune yolk powder was combined with 2 L of the binary solvent (25% pure ethanol-75% hexane or 25% isopropanol-75% hexane) and mixed. The supernatant of the solvent was removed and two extra solvent extractions were made for a total of three lipid extractions. The final extraction mixture was centrifuged to aid in the final separation. The defatted bud material was dried, and combined with 4,000 ml of deionized water, and the pH was adjusted to 7 with 0.5M NaOH. It was then concentrated to 500 ml in an Amicon DC10L ultrafiltration system. The anti-inflammatory composition of egg in the ultrafiltered product (<30,000 daltons) was further isolated by DEAE-Sepharose chromatography, and the fractions of the egg anti-inflammatory composition were lyophilized by further processing. After chromatography of DEAE, 0.45 g of the anti-inflammatory composition of the ultrafiltered product was recovered and analyzed for its activity (Figure 3).
Procedure 2: Super Critical C02 Extraction Hyperimmune, dehydrated yolk powder was placed under supercritical conditions to remove a portion of the lipids from the yolk, followed by aqueous separation of the fraction of the anti-inflammatory egg composition. In summary, approximately 400 g of the yolk powder, immune, dehydrated were placed in the Autoclave Engineers® SCE unit, and were left to be subjected to static C02 extraction at -379,166 kg / cm2 (-5400 psi) to 32.2 C for 20 hours. The partially defatted bud was homogenized with 4,000 ml of deionized water and centrifuged at 10,000 rpm for 30 minutes. The aqueous supernatant was separated and then concentrated to 500 ml in an Amicon DC10L ultrafiltration system. The anti-inflammatory egg composition in the infiltration (<30,000 daltons) was further isolated by ion exchange chromatography of DEAE-Sepharose. The fractions of the egg anti-inflammatory composition were lyophilized by further processing, and 1.86 g of the anti-inflammatory composition of egg was obtained from the ultrafiltered product after DEAE chromatography. Figure 6 shows the bioassay activity of the substantially pure composition after degreasing by supercritical CO 2 extraction, ultrafiltration and DEAE chromatography.
Procedure 3: Extraction of the Enzyme Treatment The hyperimmune egg yolk powder was incubated with a mixture consisting of Newlase F in a lactate buffer to separate the lipids from the yolk, followed by the aqueous separation of the fraction from the yolk. anti-inflammatory composition. In summary, 300 grams of dehydrated Y-100 egg yolk powder was mixed with 6 L of 0.05M lactate buffer, pH 4.0, and 27 g of Newlase F (Sigma # P-5027) and homogenized. The mixture was incubated at 50 ° C for 1 hour and then the lower aqueous phase was filtered. The resulting aqueous phase was neutralized with 0.5M NaOH and then concentrated in an Amicon DC10L (30K molecular weight CO) until 3L of the infiltrated product was collected. 276 ml of the infiltrated product were applied to the DEAE Sepharose column. After chromatography of DEAE, the 30,000 molecular weight infiltrated product was analyzed for anti-inflammatory activity.
Depyrogenation and Endotoxin Content Analysis of products isolated from the
Anti-inflammatory Egg Composition of < 30,000 Daltons Depyrogenation The isolated products of the anti-inflammatory composition of egg were depyrogenated to eliminate the endotoxin which mimics the activity of the anti-inflammatory composition. Briefly, 60 mg of the egg anti-inflammatory powder was dissolved in 15 ml of physiological, sterile saline. The mixture was depyrogenated by ultracentrifugation in the Concentrators CO of molecular weight 3K Centriplus (Amicon). The Centriplus concentrators were previously depyrogenated with PyroCLEAN (AlerCHECK, Inc.) using aseptic techniques under a laminar flow. Endotoxin can also be removed by normal procedures, available to those skilled in the art and does not need to be based on molecular weight separation.
Analysis For verification purposes, the depyrogenated egg anti-inflammatory composition was analyzed for the endotoxin content by the assay. of enzyme-linked immunosorbent (ELISA) of Tumor Necrosis Factor (TNF). Briefly, an aliquot of the anti-inflammatory composition of egg diluted with saline, physiological was added to the plates containing the J774 macrofagos cultured in Roswell Park Memorial Institute (RPMI) media with 10% calf serum, fetal . After 4 hours of incubation at 37 ° C, the supernatant was added to a plate coated with a monoclonal anti-TNF antibody and blocked with 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS). . After incubation overnight plate at room temperature, the biotinylated antibody and the streptavidin peroxidase were added. A substrate of 3, 3 ', 5, 5' -tetramethyl benzidine (TMB) was added to the plate for color development. The absorbance of the contents of the wells of the plate was read at 450 nm. The concentration of TNF in the anti-inflammatory egg sample was determined by the interpolation of the normal curve. The TNF content of the products isolated from the anti-inflammatory composition of egg after depyrogenation was negligible. (See Table 3).
TABLE 3
Antiphlammatory Composition of Egg Yeast Not Hyperimmunized 66.43a
Anti-inflammatory composition of egg white Not hyperimmunized 52.90a
Anti-inflammatory Composition of Bud, Dehydrated, Extracted with Hexane: Ethanol 92.70 °
Anti-inflammatory Composition of Yolk, Extracted with Supercritical CO2 < 18D
aThe media had only 46.5 pg TNF / ml bThe media did not have TNF
Effect of Anti-inflammatory, Endotoxin-Free Composition, < 30,000 Daltons on Leukocyte Migration The anti-inflammatory effect of the anti-inflammatory egg composition, of < 30,000 daltons, substantially pure in each dosage was determined by the reduction in the number of leukocytes in the pleural exudates of the rats treated with the anti-inflammatory composition of egg, substantially pure when compared to those, of the control rats. (See Table 4,
Figure 2). Figure 2 and Table 4 compare the effect of the fractions of egg white and egg yolk, hyperimmunized, substantially pure with those obtained from regular table eggs. All doses of the anti-inflammatory, hyperimmune, substantially pure composition resulted in a decrease in the number of leukocytes migrating into the pleural exudate. The greatest effect was observed in those subjects who were given 2 mg of the substantially pure composition of the egg yolk.
TABLE 4 Inhibition of Migration of Leukocytes from Pleural Exudates in Different Doses of the Anti-inflammatory Egg Composition
Characterization of the Anti-inflammatory Egg Composition The molecular weight of the substantially pure egg anti-inflammatory composition prepared by the method described above was found to be less than 30,000 daltons. This was inferred from the fact that the first step in the isolation of the composition of the egg white and the egg yolk was by ultrafiltration using a membrane that does not allow the passage of the molecular weight species > 30,000 daltons The composition has a negative charge under predominant conditions. The pKa value of this gel is 9.5. This was determined by applying the ultrafiltered egg product to an ion exchange column of DEAE Sepharose. The anti-inflammatory composition was not eluted. with water from the column. By changing the elution media to the NH4OAc solution, or any saline or buffer with a pH higher than 9.5, it caused the anti-inflammatory composition of the egg to elute. When the ultrafiltered product was applied to the DEAE column, several proteins and salts constituted the unbound fraction (Peak 1, Figure 1). The peaks were recorded when the column was washed with a solution of NH4OAc (Pico 2) and then the NaCl solution (Pico 3). The isolated fraction of Peak 2 exhibited an anti-inflammatory activity based on a rat study. The third peak showed negligible activity. The anti-inflammatory composition of egg white is thermostable at 90 for 1 hour. On the other hand, the anti-inflammatory composition of egg can be isolated from the egg yolk and the egg white. The egg anti-inflammatory composition of the egg yolk shows a higher activity than the anti-inflammatory composition of the egg white (Figure 2, Table 4).
Example 4
Preferred Method for the Preparation of an Anti-inflammatory, Highly Purified Egg Composition The following Examples describe a method
(suitable for large-scale purification) to obtain the anti-inflammatory composition of bird eggs in its non-aggregated, lower molecular weight, highly pure form. The complete eggs, the hyperimmunized and control table eggs, were fractured and the egg white separated from the yolk and both were dehydrated by spraying. The hyperimmunized eggs were obtained as described in Example 1. The powder of the egg white was processed separately to obtain the aqueous fraction for ultrafiltration. All the purification steps were carried out in order to minimize possible contamination with bacteria or pyrogens. The sterile water was used to prepare the solutions and all the glassware was depyrogenated. In addition, the sterile solution was filtered.
Example 4a
Preparation of the Egg Anti-inflammatory Egg Egg Powder Composition Solvent Removal The dehydrated egg yolk was subjected to a liquid solvent extraction with either propane or butane to separate the lipids from the fraction of the aqueous bud contains the anti-inflammatory composition. In summary, 500 grams of egg yolk powder, dehydrated were placed in a column, to which 4 liters of propane solvent, liquid was added. The supernatant of solvent and the extracted lipid were removed. Six extra solvent extractions were made for a total of six lipid extractions.
Ultrafiltration Four hundred grams of dehydrated, dehydrated egg yolk were diluted with 4 liters of distilled, sterile water and homogenized with a Virtis (handishear). The yolk mixture was either centrifuged at 24 RPM or allowed to cool * at rest until the undissolved yolk particles precipitated. The resulting aqueous fraction was ultrafiltered using an Amicon RAIOOO ultrafiltration system equipped with a coiled-coiled membrane, 3,000 molecular weight interceptor. The pumping speed was maintained at an inlet pressure of 1,404 kg / cm2 (20 psi) and an outlet pressure of 1053 kg / cm2 (15 psi). The infiltrated products of molecular weight < 3,000 were sterile filtered using a disposable, sterile 0.45 μm Nalgene filter and lyophilized or frozen for storage, bioassay analysis or further purification. Figure 8 shows a flow chart of the purification process. The molecular species below 3,000 daltons of the egg yolk contain the anti-inflammatory composition in a non-aggregated, low molecular weight form. From 400 grams of the starting material, the product of the anti-inflammatory composition was about 12 grams or 3% of the total. The bioassay analysis of this 3K fraction for the anti-inflammatory activity showed high levels of activity (Example 6a).
Example 4b
Preparation of the Anti-inflammatory Activity of the Egg of the Egg White Powder Four grams of the egg white, isolated from both a hyperimmunized egg as described in Example 1 and a control table egg, were diluted with a liter of deionized water. The mixture was homogenized and filtered through a
40 μm and ultrafiltered through a 3K molecular weight CO ultrafiltration system (Figure 9). From 400 g of the egg white powder, 8.6 g or 2.15% of the anti-inflammatory composition was recovered. This material showed a bioassay activity in the inhibition of migration of rat leukocytes to an induced site of the lesion (Example 6a). This example shows the preparation of the anti-inflammatory, active composition of the egg white material of both hyperimmune and control table eggs.
Example 5
Ion exchange chromatography of DEAE. The anti-inflammatory composition of the egg was also further characterized by the ion exchange chromatography of DEAE. This example demonstrated that the anti-inflammatory composition has a negative charge at a neutral pH. The 10K ultrafiltered product from both egg white and egg yolk material was obtained in the same manner as described in Example 4a, except that a 10,000 molecular weight membrane was used in place of the weight membrane 3,000 molecular
A column of DEAE-Poros 50 (10/50 millimeters) was equilibrated with pyrogen-free, double-distilled, sterile water (Sigma), pH 7.0. Ten milligrams of the ultrafiltered product (molecular weight <10,000) dissolved in 100 ml of dually distilled, pyrogen-free, sterile water was applied to the DEAE Poros column in BioCad 700
(Perseptives Biosystems). At a flow rate of 10 ml / min, the material was applied to the column. The initial elution of the material not bound to the column was removed with distilled water and collected. A linear gradient of ammonium acetate (0% -100%) was used to elute the material bound to the column and the fractions were monitored with UV absorption at 280 nm and 254 nm and 10 ml of fractions collected by a harvester. Avantec fractions. NaCl (0.15M) was used to remove the remaining material attached to the column. The column was washed with distilled water until the absorbance of 280 nm and 254 nm returned to the baseline. Figure 10 shows a typical chromatogram. The arrow indicates the anti-inflammatory fraction, active (Peak 2). An elution profile, typical with NH Acetate, is demonstrated with changes in the elution buffer indicated by the arrowheads. The eluate was sterile filtered with a sterile 0.45 μm filter, lyophilized to dryness and weighed. The bioassay of the individual peaks showed that the peak containing the activity was found in Peak 2 (Example 6b). The fraction isolated by the ion exchange chromatography of DEAE showed that the anti-inflammatory composition was negatively charged.
Example 6
Analysis of Anti-inflammatory Activity Blind analyzes of substantially purified fractions of egg yolk and egg white, table eggs and hyperimmune eggs were analyzed for anti-inflammatory activity. The test analysis measured the inhibition of the migration of leukocytes from the venules to a site of injury induced in the pleural cavity of the rats. Female rats (Charles River, MA) weighing 120 to 150 grams were injected into the pleural cavity with 2 ml of 1% carrageenan (an irritant) in saline, physiological to induce the migration of leukocytes into the pleural cavity . The rats were then injected intraperitoneally (IP) with 0.5 ml doses of each test fraction in saline, physiological at the selected concentration. Two rats were injected at each concentration. A saline injection was used as a control. Four hours after the injection, the animals were sacrificed using methods according to the Animal Welfare act. The pleural exudate was removed and the leukocytes were fixed for evaluation. Fifty microliters of the exudate were loaded into a corresponding labeled slide and loaded into a cytospin. After completion of the cytospin cycle the slides were sprayed with fixative cytoprep
(Fisher Scientific Co. Atlanta) and stained with hematoxylin. Five representative areas of each slide were valued and the number of leukocytes was counted with the Optimum Image Analysis Program (Bioscan Image Analysis Program). The average of all five counts was used for the total average white blood cell count for each rat. The percent inhibition, compared to saline, control injections, was calculated for each concentration of the sample.
Example 6a Comparison of the Anti-inflammatory Activity of Egg White, Egg Yolk, Hyperimmune and Table Eggs Purified fractions of egg yolk and egg white, degreased, dehydrated by spraying table eggs and the hyperimmunized eggs, as described in example 4, were analyzed for anti-inflammatory activity. The infiltrated product of less than 3,000 molecular weight of the clear egg, table, hyperimmunized egg yolk and hyperimmunized egg white were obtained after ultrafiltration as described in Example 4 and 5. Two animals for Each sample was injected intraperitoneally with either saline, 1, 5, 10 or 20 mg of the test fractions and evaluated as described in example 6. Figure 11 shows that the fractions of the egg yolk, highly purified , molecular weight of less than 3,000 of the hyperimmunized chickens demonstrated. 65% inhibition of migration of leukocytes in a dose of 1 mg / rat. These fractions of the egg yolk, hyperimmune also showed a higher level of activity (65% inhibition in a dose of 1 mg / rat) than fractions obtained from the egg white, hyperimmune (38%) in the same dose. The control egg white showed lower anti-inflammatory activity (25% inhibition at a dose of 1 mg / rat). Non-hyperimmunized egg fractions also contain anti-inflammatory activity since all chickens were vaccinated and subjected to natural immunization from the presence of microorganisms and allergens in the environment. However, surprisingly, the level of the anti-inflammatory composition was increased in both the yolk and the clear by the hyperimmunization process. Since the components in the egg are generally divided into compartments in either the lipid-rich or the clear egg yolk, it is surprising that the anti-inflammatory activity was found in the fraction of less than 3,000 molecular weight of both the egg yolk and egg white. This example shows that the anti-inflammatory composition is less than 3,000 daltons, it was found in the egg yolk and the egg white and it was found in effective amounts and much higher in the hyperimmune egg fractions than in the table eggs, of control. In fact, the highly pure anti-inflammatory composition is found in hyperimmunized eggs in an amount which is effective in the treatment of inflammation.
Example 6b Effect of DEAE Fractions, Negatively Loaded on Leukocyte Migration Following the anti-inflammatory bioassay procedure, DEAE peak 2, negatively charged, eluted with NH4OAc and the 3K and 10K ultrafiltered products of the egg yolk hyperimmune were analyzed by activity. Each sample was analyzed in the indicated dose. Figure 12 shows that the fraction of DEAE, negatively charged, the 3K and 10K ultrafiltered product of the eggs of the hyperimmunized chickens shows a high level of anti-inflammatory activity when compared to the control of the saline solution.
Example 6c Comparison with Anti-Inflammatory Drugs The anti-inflammatory drugs of indomethacin and aspirin were compared with the 3K ultrafiltered egg product by the inhibition of leukocyte migration in vivo. In this analysis, intraperitoneal injections were given in the doses of: 1000 μg, 100 μg, 10 μg, 1 μg, for indomethacin, aspirin and the anti-inflammatory composition of egg, 3K of the hyperimmune egg. Figure 13 shows that the 3K ultrafiltered product and aspirin showed similar levels of anti-inflammatory activity. The level of inhibition of leukocyte migration by indomethacin seems to be reduced at lower doses
(1-100 μg / rat). This example shows that the anti-inflammatory fractions of the egg have a comparable effect in the analysis of leukocyte migration for the known anti-inflammatory drugs.
Example 7 Purification of Anti-Inflammatory Factor by Reverse Phase High Pressure Liquid Chromatography (HPLC) DEAE fractions of less than 3K
10K, substantially pure from the egg yolk and the DEAE fraction, from 30K of the egg white were separated on a reversed phase symmetry column
Waters C8 (3.9 x 250 mm). A linear gradient of methanol 10% - 80% and H20 was used as the mobile phase in a CLAP Waters Model 2010 with an autosampler
717 plus and a photo-diode array detector 996.
The lyophilized samples were diluted in 100 to 200 ml of doubly distilled, sterile water. The column was eluted at a flow rate of 2 ml per minute and the data from the diode array, effluent were stored at all wavelengths from 200 nm to 350 nm. Figure 14 shows a MAX graphic chromatogram of the separation of the 3K infiltrated product. This chromatogram shows the two highest peaks of molecular weight less than 3K which result in a highly purified anti-inflammatory egg composition. Figure 15 shows the separation of the composition of a 10K infiltrated product which was separated in a DEAE ion exchange column as summarized in example 5. Two peaks similar to those observed with the fraction of the ultrafiltration product were obtained. 3K. These results indicate that both fractions, of less than 3K and of DEAE which also showed an anti-inflammatory activity (Example 7), can be used to obtain a highly negatively charged component. purified. Composition analyzes of the egg yolk fraction of 3k showed the presence of an initial series of elution peaks in one to two minutes and three elution peaks of 10 to 20 minutes. This example shows that the anti-inflammatory composition is of molecular weight less than 3,000 and can be separated by the method in a relatively pure peak.
Preparation of CLAP to obtain large quantities of the anti-inflammatory factor after ultrafiltration, the preferred step is to separate the composition, of molecular weight less than 3,000, highly purified in a preparative CLAP column. Accordingly, a 5.08 X 20 cm column was packed with a Zorbax C8 packing material and 4 grams of the ultrafiltered product of molecular weight less than 3,000 were purified during each run. Using a mobile phase of H20 / 80/20 methanol with 0.05% TFA, at a flow rate of 90 ml per minute, the infiltrated product was separated into a highly purified, harvested and lyophilized composition. Then the collected peak was analyzed again on a C8 analytical column. With the described method, large quantities of the ultrapure peak were obtained by preparative CLAP and analyzed by the bioassay activity, the elemental composition and the chemical structure. Mass spectroscopy, elemental analysis, infrared spectroscopy and nuclear magnetic resonance, NMR, were used to determine the structure of the factor.
Example 8 Physical Characteristics of the Anti-inflammatory Egg Composition This example illustrates the heat stability, the changes in acid and base, the proteinase treatment.
Four milligrams of the ultrafiltered product of molecular weight less than 3,000 were diluted in sterile water and held at 100 ° C for 30 minutes. Samples of the ultrafiltered product of molecular weight less than 3K were subjected to acidic HCl, pH 2.0, pH neutral 7.4 or NaOH base pH 9.0, for 30 minutes before neutralization. The 3K fractions were also treated with a 1 unit of the pronase enzyme for one hour. All sterile samples were filtered, lyophilized, weighed and analyzed for biological activity. Comparisons of the bioassay activity of the treated fractions (high temperature, changes in pH and digestion with pronase) with the untreated control animals injected with saline show surprising stability for these treatments. Stability at high temperatures, changes in pH, and treatment with pronase in addition to small size
(molecular weight less than 3,000) indicate that the anti-inflammatory composition is not protein. It was determined that the anti-inflammatory, highly pure composition has the following characteristics. It has a molecular weight of less than 3,000 daltons. This characteristic is inferred from the isolation and purification of the composition, where the isolation and purification process uses an ultrafiltration membrane that does not allow the passage of molecular species greater than 3,000 daltons through. The composition is determined to be non-proteinaceous and non-spheroidal because it is small in size and is not degraded by the enzymes which degrade the proteins. On the other hand, the composition is orally active and is not degraded by digestive enzymes. The stable, small form of the composition (as it differs from the proteins which are much larger) facilitates the absorption of the digestive system. The composition is resistant to acid and bases, returning to an active form even after treatment with pH 9.0 conditions. In addition, the composition is thermostable at 100 ° C for at least 30 minutes and has a negative charge at a neutral pH. The anti-inflammatory composition of egg can be isolated from the whole egg, from the egg yolk and from the egg white. The anti-inflammatory composition, isolated from the egg yolk, shows an anti-inflammatory activation, higher than the purified anti-inflammatory composition of the egg white. Supranormal levels of the anti-inflammatory composition of the hyperimmune whole egg, the hyperimmune egg yolk and the hyperimmune egg white can be isolated.
It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.
Having described the invention as above, the content of the following claims is claimed as property.
Claims (40)
1. An anti-inflammatory composition, in a highly purified form, wherein the composition is characterized by: (a) it is present in the egg white and the egg yolk of an egg produced by an egg-producing animal; (b) is thermostable at 100 ° C for at least 60 minutes; (c) has a negative ionic charge at a neutral pH; (d) it is resistant to acid and base under conditions of pH 4.0 - pH 9.0; (e) is orally active; and (f) has a molecular weight of less than 3,000 daltons.
2. A method of preventing, eliminating, or reducing inflation in a subject, characterized in that it comprises administering to a subject an effective amount of the anti-inflammatory composition of claim 1.
3. A method according to claim 2, characterized in that the effective amount of the anti-inflammatory composition ranges from 5 micrograms to 300 milligrams.
4. A method of inhibiting the migration of leukocytes in a subject, characterized in that the method comprises administering to the subject the composition of claim 1, in a dose sufficient to inhibit the migration of leukocytes. «
5. A method for highly purifying an anti-inflammatory composition of an egg of an egg-producing animal, characterized in that it comprises: (a) isolating a water soluble fraction from the whole egg, the egg yolk or the egg white; (b) separating an infiltrated product of less than 3,000 daltons from the water soluble fraction; (c) fractionating the infiltrated product of less than 3,000 daltons to recover a biologically active fraction.
6. The method according to claim 5, characterized in that the fractionation step further comprises high pressure, reverse phase liquid chromatography of the infiltrated product of less than 3,000 daltons.
7. The method according to claim 5, characterized in that it also comprises the degreasing of the whole egg or the egg yolk before step (a).
8. The method according to claim 5, characterized in that it also comprises the lyophilization of the infiltrated product of < 3,000 daltons from step (b).
9. The method according to claim 5, characterized in that the separation step further comprises the ultrafiltration of the water-soluble fraction through a filter with an intercept of molecular weight of 3,000 daltons.
10. The method according to claim 5, characterized in that the egg producing animal is maintained in a hyperimmunized state.
11. The method according to claim 10, characterized in that the hyperimmunized state is induced by an antigenic, genetic or bioengineered vaccine.
12. The method according to claim 11, characterized in that the genetic vaccine comprises at least one antigen coding DNA construct selected from the group consisting essentially of pure DNA fragments, plasmid DNA, viral DNA, DNA expression libraries, DNA-RNA antigens, DNA-protein conjugates and DNA-liposome conjugates and combinations thereof.
13. The method according to claim 11, characterized in that the antigenic vaccine comprises at least one antigen selected from the group consisting of bacterial, viral, protosoar, fungal and cellular antigens.
14. An anti-inflammatory composition, in a highly purified form, produced by a process characterized in that it comprises: (a) isolating a water-soluble fraction from the whole egg, the egg yolk or the egg white; (b) separating an infiltrated product of less than 3,000 daltons from the water soluble fraction; (c) fractionating the infiltrated product of less than 3,000 daltons to recover a biologically active fraction.
15. The anti-inflammatory composition according to claim 14, characterized in that the egg producing animal is in a hyperimmunized state.
16. The anti-inflammatory composition according to claim 14, characterized in that the hyperimmunized state is induced by an antigenic, genetic or bioengineered vaccine.
17. The . anti-inflammatory composition according to claim 16, characterized in that the genetic vaccine comprises at least one antigen coding DNA construct selected from the group consisting essentially of pure DNA fragments, plasmid DNA, viral DNA, expression collections of DNA, DNA-RNA antigens, DNA-protein conjugates and DNA-liposome conjugates and combinations thereof.
18. The anti-inflammatory composition according to claim 16, characterized in that the antigenic vaccine comprises at least one antigen selected from the group consisting of bacterial, viral, protosoar, fungal and cellular antigens and combinations thereof.
19. A method of preventing, eliminating or reducing inflammation "in a subject characterized in that it comprises administering to a subject an effective amount of the anti-inflammatory composition of claim 14.
20. A method according to claim 19, characterized in that the effective amount of the anti-inflammatory composition ranges from 5 micrograms to 300 milligrams.
21. A method of inhibiting the migration of leukocytes in a subject, characterized in that the method comprises administering the anti-inflammatory egg composition of claim 14, to the subject, in an amount sufficient to inhibit the migration of leukocytes.
22. An egg or an anti-inflammatory egg fraction, characterized in that it is prepared by the hyperimmunization of an egg producing animal.
23. The egg or anti-inflammatory egg fraction according to claim 22, characterized in that the egg-producing animal is hyperimmunized with an antigenic, genetic or bio-designed vaccine.
24. The egg or the anti-inflammatory egg fraction in accordance with the claim 23, characterized in that the genetic vaccine comprises at least one DNA construct encoding antigens selected from the group consisting essentially of pure DNA fragments, plasmid DNA, viral DNA, bacterial DNA, DNA expression collections, DNA-antigens. RNA, DNA-protein conjugates and DNA-liposome conjugates and combinations thereof.
25. The egg or anti-inflammatory egg fraction according to claim 23, characterized in that the antigenic vaccine comprises at least one antigen selected from the group consisting of bacterial, viral, protosoar, fungal and cellular antigens.
26. The egg or antiinflammatory egg fraction according to claim 22, characterized in that it comprises at least one anti-inflammatory composition, the anti-inflammatory composition is present at supranormal levels, the levels are effective for the treatment of inflammation in a subject.
27. The egg or anti-inflammatory egg product according to claim 26, wherein the anti-inflammatory composition is characterized in that: (a) it is present in the egg white and the egg yolk of the anti-inflammatory egg; (b) is thermostable at 100 ° C for at least 30 minutes; (c) has a negative ionic charge at a neutral pH; (d) it is resistant to acid and base under conditions of pH 4.0 - pH 9.0; (e) is orally active; and (f) has a molecular weight of less than 3,000 daltons.
28. The egg or anti-inflammatory egg fraction according to claim 26, wherein the anti-inflammatory composition is highly purified from the egg or the anti-inflammatory egg product by a process comprising: (a) isolating a fraction soluble in water of the whole egg, egg yolk or egg white; (b) separating an infiltrated product of less than 3,000 daltons from the water soluble fraction; (c) fractionating the infiltrated product of less than 3,000 Daltons to recover a biologically active fraction.
29. A method of preventing, eliminating or reducing inflammation in a subject having a weight, the method is characterized in that it comprises administering to a subject an effective amount of the egg or anti-inflammatory egg product according to claim 22.
30. The method according to claim 29, characterized in that the effective amount of the egg or product of the anti-inflammatory egg varies from 1 to 40 grams per kilogram of the weight of the subject.
31. A method of inhibiting the migration of leukocytes in a subject, characterized in that the method comprises administering an effective amount of the egg or anti-inflammatory egg product according to claim 22 to a subject.
32. An anti-inflammatory composition, in a substantially purified form, produced by a process characterized in that it comprises: (a) isolating a water-soluble fraction from eggs produced by an egg-producing animal; (b) separating an infiltrated product of less than 30,000 daltons from the water soluble fraction to recover a biologically active infiltrated product.
33. The anti-inflammatory composition according to claim 32, characterized in that it also comprises holding the filtered product of < 30,000 daltons to ion exchange chromatography after step (d).
34. The anti-inflammatory composition according to claim 33, characterized in that the separation step further comprises the ultrafiltration of the water-soluble fraction through a filter with molecular weight intercept of 30,000 daltons.
35. The anti-inflammatory composition according to claim 32, characterized in that it further comprises removing the endotoxins from the infiltrated product of < 30,000 daltons
36. The anti-inflammatory composition according to claim 35, characterized in that the egg-producing animal is maintained in a hyperimmunized state.
37. The anti-inflammatory composition according to claim 36, characterized in that the egg-producing animal is hyperimmunized with an antigenic, genetic or bio-designed vaccine.
38. The anti-inflammatory composition according to claim 37, characterized in that the antigenic vaccine comprises at least one antigen selected from the group consisting of bacterial, viral, protrosoar, fungal and cellular antigens.
39. The anti-inflammatory composition according to claim 37, characterized in that the genetic vaccine comprises at least one DNA construct encoding antigens selected from the group consisting of pure DNA fragments, plasmid DNA, viral DNA, bacterial DNA, collections of DNA expression, DNA-RNA antigens, DNA-protein conjugates and DNA-lipo conjugates = omas and combinations thereof.
40. The anti-inflammatory composition according to claim 32, characterized in that it further comprises the degreasing of the eggs before step (a).
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US014097 | 1996-03-26 | ||
US814187 | 1997-03-10 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA98007754A true MXPA98007754A (en) | 1999-09-01 |
Family
ID=
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