KR100934411B1 - Enamel Substrate Protein Compositions for Modulating Immune Responses - Google Patents

Abstract

The present invention relates to the use of an active enamel matrix material formulation, such as amellogenin, for the preparation of a pharmaceutical composition for modulating an immune response. The composition can be used to prevent and / or treat a mammalian condition or disease that is characteristic of a mammal that exhibits an imbalance in its natural immune response to internal and / or external stimuli. That is, at least a portion of the mammalian immune system is non-discriminatory to stimulate hypersensitivity to or fail to respond to the immunogen. Such conditions are typically systemic or local, such as systemic and / or post-traumatic systemic inflammation or autoimmune disease.

Active enamel substrate, hypersensitivity, immunogen, amegenin, autoimmune disease

Description

Enamel matrix protein compositions for modulating immune response

The present invention relates to the use of active enamel matrix material preparations, such as amelogenin, processed amellogenin products or metabolites thereof, for the preparation of pharmaceutical compositions for modulating an immune response. The composition can be used to prevent and / or treat a mammalian condition or disease characterized by an imbalance in the natural immune response to internal and / or external stimuli. The condition may typically be systemic or local, such as systemic inflammation or autoimmune disease following systemic and / or trauma.

The normal function of the immune system is to protect the body by attacking and destroying foreign microorganisms such as bacteria and viruses. Under normal conditions these foreign microorganisms will be recognized as intruders and will be harmless.

It is the lymphatic system that is involved in the human immune response; Lymphatic system refers to the collection of lymphocytes, their precursors and derivatives and all supporting cells. Under abnormal conditions or when the immune system is imbalanced, the lymphatic system becomes unable to respond in response to foreign microorganisms, and misrecognizes its cells and tissues as foreign substances to attack or improperly activate them in a sensitive manner.

Immune responses to antigenic agents, either foreign or autoantigens, are generally characterized by antibody production by B lymphocytes and destruction of cells representing these antigens by T lymphocytes or natural killer (NK) cells. However, defects in B or T lymphocytes can lead to immunodeficiency diseases or impaired immune response function. Immunodeficiency or defects can be inherent, such as caused by mutations in a gene, or acquired through viral infection or as a result of the aging process. Thus, the defect that occurs may be fatal or nonfatal, depending on the stage of stem cell or lymphocyte differentiation in which the defect occurs.

In addition to antigens, lymphocytes require lymphokines to be fully activated. For example, T-helper cells do not proliferate unless they receive a signal from macrophages, and T-cytotoxin cells and B-cells rely on signals from T-helper cells and possibly macrophages to develop fully. . A suitable immune response requires a combination of several cell types, which means that modulating the expression or activity of lymphokines has a beneficial effect on a bad immune response.

After shock, burns, extensive surgical or traumatic injury, the action of the immune system can be excessively stimulated with systemic, non-discriminatory, excessive systemic inflammation, while other actions are markedly paralyzed. When the immunological host response is not controlled, the observed imbalance of cellular activation results in a continuous clinical condition. This condition may be an acute phase reaction, systemic inflammation, anergy, sepsis, infection or organ damage and may eventually result in multiple organ damage. Multiple organ damage or dysfunction is the most common cause of death in the intensive care unit. At present, therapies involving the prevention or amelioration of uncontrolled immune responses have not been able to significantly alter the outcome.

Another example of an imbalance situation is when the immune system attacks your body. These situations are different in terms of the origin and mechanism of the antigen causing the attack and the expression of the attack. These reactions are often referred to as allergy, delayed type of hypersensitivity, allograft and autoimmunity, respectively.

Anergi is another type of imbalance, in which the human body cannot respond to injected allergens or antigens. Anergi is seen as the result of intracellular signals after interaction between the T-cell receptor (TCR) and the peptide-providing major histocompatibility complex (MHC) in the absence of "co-stimulatory" signals. Such costimulatory signals are usually provided on the cell surface of antigen-presenting cells (APCs).

The immune system regulates its action locally and systemically. Delayed types of hypersensitivity, for example, can occur locally in characteristic skin reactions, or as systemic reactions characterized by fever, discomfort and pain in the joint area and a decrease in the number of circulating lymphocytes as large amounts of antigen enter the bloodstream. Inflammation is generally defined as a local response to cellular damage, while systemic inflammation or multiple organ damage is defined as systemic inflammation.

After trauma, monocytes and macrophages immediately hypersensitivity to excessive release of proinflammatory cytokines. This excessive release leads to the development of systemic inflammation, which in turn causes paralysis of the cell in most patients. After 3 to 5 days, the condition is overcome by newly supplemented monocytes / macrophages that are immature and do not have a full spectrum of activity. In addition, the patient may undergo an anti-inflammatory phase (complementary anti-inflammatory response syndrome) and optionally display a mixed reaction (mixed antagonistic response syndrome) with pro- and anti-inflammatory components.

Post-traumatic impact on cell mediated immune regulation is mainly caused by simultaneous attack on monocyte / macrophage bonds and T cells, resulting in degradation of complete cell interactions. Traumatic stress not only affects the ability of the appropriate and specific performance of each cell type, but also affects the control and regulatory surveillance that monocyte / macrophage bonds and T cells retain each other in multiple control loops. Loss of control action occurs simultaneously with the moment of injury.

Inflammatory cytokines known to cause the early systemic superinflammatory described above are, for example, tumor necrosis factor-a (TNF-alpha), interleukin-1 (IL-1), IL-6 and interferon gamma, which are TNF-alpha Synergistically with Clinical trials aimed at downregulation of such mediators using, for example, antibodies against endotoxin, TNF-alpha, antagonists of IL-1 or platelet activating factors have generally been disappointing. These substances have not been found to even have modulatory effects, and they have increased mortality.

In addition, under stressed conditions, monocyte / macrophage bonds are readily stimulated to produce and release prostaglandin E ₂ (PGE ₂ ), which may be the most potent endogenous immunosuppressant. PGE ₂ is an inhibitor of T cell mitosis, IL-2 receptor expression and IgM antibody synthesis by B cells. Through an intracellular increase in cAMP levels, PGE ₂ negatively controls monocyte / macrophage bond synthesis of TNF _α and IL-1. PGE ₂ can inhibit the synthesis of TH ₁ cytokines (IL-2, IFN _γ ) as well as the synthesis of IL-4 by TH ₂ cells. Thus, PGE ₂ secretion suggests a favored balance of TH ₂ type responses resulting in a change in B cell production from IgM to IgGl and IgE. Information on balance will therefore influence the development of a TH ₁ or TH ₂ -dominant response.

Traditional reagents and methods used to modulate the immune response of a patient often result in unwanted side effects. For example, immunosuppressive agents such as cyclosporin A, azathioprine, and prednisone are used to suppress the immune system in patients with autoimmune disease or undergoing transplantation. However, these reagents inhibit the patient's overall immune response, thus impairing the patient's ability to initiate an immune response against an infectious agent not originally involved in the disease. Due to these harmful side effects and the medical importance of immunomodulation, the study of reagents and methods for regulating certain parts of the immune system has been the subject of many years of research.

Enamel matrix proteins present in enamel substrates are often best known as precursors to enamels. Prior to cementitious formation, enamel matrix proteins are deposited on the root surface at the apical end of the growing root. The deposited enamel substrate is an initiator for forming cementitious. Again, the formation of cementitious itself is associated with the development of periodontal ligaments and alveolar bone. As has been found by the inventors prior to the present invention, enamel matrix proteins can promote periodontal regeneration through mimicking natural adhesion development in teeth (Gestrelius S, Lyngstadaas SP, Hammarstroem L. Emdogain-periodontal regeneration based on biomimicry.Clin Oral Invest 4: 120-125 (2000)).

Enamel substrates consist of a number of proteins, such as amelagenin, enamelin, tuft protein, protease and albumin. Amellogenin, a major component of the enamel substrate, is a collection of hydrophobic proteins derived from a single gene by selective splicing and controlled post secretory processing. They are highly conserved through vertebrate differentiation and show high overall sequence homology levels (> 80%) among all the higher vertebrates investigated. In fact, the sequences of porcine and human amelogenin gene transcripts differ only by 4% of their bases. Thus, an enamel matrix protein of swine origin can be recognized as "self" when found in the human body and can promote dental regeneration in the human body without causing an allergic or other undesirable reaction.

Enamel matrix proteins and enamel matrix derivatives (EMDs) are used for hard tissue formation (ie, enamel formation, US Pat. No. 4,672,032 (Slavkin)), binding between hard tissues (EP-B-0 337 967 and EP-B-0 263 086). And (WO 99/43344) have been described in the patent literature for a long time.

Summary of the Invention

The present invention is based on the surprising finding that active enamel substances have the ability to modulate the immune mechanisms of mammals. Immunregulation herein, which is defined as increasing or enhancing the degree of immune response, is by a variety of specific and non-specific means.

Thus, a preferred embodiment of the present invention is the use of an active enamel material for the preparation of a pharmaceutical composition for the prevention and / or treatment of a mammalian condition characterized by a heterogeneous immune response of a mammal against an immunogen such as an external or internal stimulating factor. It includes.

"Immune response imbalance" can occur after shock, burns, extensive surgical procedures and / or traumatic injuries. The term describes a situation / condition in which the action of one or more immune systems may be excessively stimulated systemically, non-discriminatory, excessive systemic inflammation, while other actions may be insufficiently stimulated simultaneously or independently. The term “immune imbalance” refers to an imbalanced state of cell activation that has been observed to result in uncontrolled situations / conditions and subsequent clinical conditions. This condition may be an acute phase reaction, systemic inflammation, anergy, shock, sepsis, infection and / or organ damage and finally may result in multiple organ damage.

Another example of an "immune imbalance" is a situation / condition in which a person's inactive immune system attacks his own body. These situations differ in terms of the origin of the antigen causing the attack and the mechanism and indication of the attack. These reactions are often referred to as allergy, delayed type of hypersensitivity, allograft and autoimmune reactions.

The term “immune imbalance” may describe both local and systemic situations as described above. For example, inflammation is generally defined as a local response to cellular damage, while systemic inflammation or multiple organ damage is an example of systemic inflammation.

In the present invention, the term "immunogen" is used to describe a factor that can elicit an immune response. This may be, for example, toxins, fungi, parasites, viruses, bacteria, pollen, dust, contaminating particles, cytokines, hormones, stress, surgery, burns, transplanted organs or blood, tissue fragments, shock, trauma or non-self antigens or autoantigens. Can be.

Immunological hypersensitivity is an immune response that results in tissue damage or impaired body action. They are classified into five types (I-V) according to the mechanism of tissue damage, and they are involved in a number of important human diseases and disorders such as hypersensitivity, asthma, food allergies, myasthenia gravis and discoid lupus erythematosus.

In one embodiment of the invention, the active enamel material is used to prepare a pharmaceutical composition for preventing and / or treating a condition in a mammal in which at least part of the immune system is hypersensitive to the immunogen. In a preferred embodiment, at least part of the immune system of the mammal is thus non- discriminatively stimulated.

Benefits from the present invention include those in which the imbalance of the mammalian immune response is local and / or systemic. In this regard, systemic is defined as being related to or common to the whole body, ie, generally affecting the whole body as opposed to local. Such conditions include, for example, rheumatoid arthritis, skin diseases, allergies, sclerosis, atherosclerosis, multiple sclerosis, asthma, psoriasis, lupus, systemic disc lupus erythematosus, diabetes, myasthenia gravis, chronic fatigue syndrome, fibromyalgia, chronic fatigue syndrome, Crohn's disease , Hashimoto's thyroiditis, Graves' disease, Addison's disease, Guillian Barre syndrome and scleroderma.

In the context of the present invention, the term “autoimmune disease” refers to at least 80 different serious chronic diseases. Autoimmune diseases are the third major disease group in the United States after cancer and heart disease. 75-90% of all autoimmune diseases occur in women, often at childbirth age. In addition to organs in the body, autoimmune diseases can also affect nerves, muscles, blood, connective tissue, gastrointestinal tract and all systemic systems.

The causes of autoimmune diseases are not fully known or known, but hormones, genetics and environmental factors are presumed to play a major role in their cause and invention. Most pathways for these diseases are individual and very difficult to predict. Autoimmune diseases do not represent a generalized impairment of resistance mechanisms, but they reflect the appearance of specific responses to specific autoantigens.

Other conditions contemplated by the present invention are characterized by systemic inflammation and / or infection, which may be associated with trauma, burns, surgery, dialysis, shock and / or bacterial infections or viral infections. In a preferred embodiment of the present invention, the systemic condition is extracorporeal membrane oxygenation (ECMO), shock, systemic inflammatory response syndrome (SIRS), sepsis, multiple organ damage, rejection of grafts such as organs and / or prostheses, heart and lung side tubes Surgery, chronic inflammation, asthma and / or stress-related diseases of the lungs, bacterial infections, necrosis, soft tissue infections, and wound infections.

Some serious trauma, such as major surgery, systemic inflammation, burns, or shock, can cause significant damage to the patient's immunological responsiveness and injuries to serious systemic infections that can result in severe tissue destruction and organ damage as a result of its clinical consequences. The individual's sensitivity is increased.

In one embodiment of the present invention, an active enamel material is prepared for the preparation of a pharmaceutical composition for preventing and / or treating a condition in a mammal wherein at least a portion of the immune system attacks the molecules, cells, or tissues of the organ that produces them. Used for Active enamel materials are used for the preparation of pharmaceutical compositions for the prophylaxis and / or treatment of mammalian conditions characterized by systemic inflammation and / or infection.

In systemic, non-discriminatory, excessive systemic inflammation, part of the immune system is stimulated, while other actions in the complex of cell-mediated immunity (CMI) are markedly paralyzed. Post-traumatic immunodeficiency results in a complex grade as a result of cellular activation, which is not yet well known. One known fact is that traumatic stress leads to the separation of complete monocyte-T cell interactions, which is associated with significant changes in monocyte regulation and actual degradation of T cell action. In a preferred embodiment of the invention, the active enamel material is used to prepare a pharmaceutical composition for restoring the balance of monocyte-T cell interactions in a patient, ie for restoring healthy monocyte control and T cell function.

In this regard, inflammation is characterized by capillary dilatation, leukocyte infiltration, flushing, fever, pain, edema, and / or loss of function, and is usually associated with cell damage that acts as a mechanism to initiate removal of toxic substances and damaged tissue. It is defined as the local response to it.

Active enamel materials are used in the present invention to prepare pharmaceutical compositions for modulating the concept of T-helper cell activation in mammals. The active enamel material included in the pharmaceutical composition can modulate the level of expression, release and / or action of intracellular and / or intercellular signaling substances. The active enamel material included in the pharmaceutical composition may alternatively regulate the expression, release and / or level of action of one or more cytokines involved in the mammalian immune mechanism, or may be involved in the immune mechanism of the same mammal. Cell signaling cascades can be regulated.

Many common and clinically important disease states, such as rheumatoid arthritis, asthma, tuberculosis, leprosy, schistosomiasis, chronic hepatitis, thyroiditis and multiple sclerosis, are examples of chronic inflammation and its consequences. Chronic inflammation may be due to the failure to remove the acute inflammatory stimulant from the autoimmune response to autoantigens, or it may be caused by sustained weak intensity natural chronic stimulants. It is characterized by simultaneous inflammation and treatment by the gradual and activation of macrophages, lymphocytes and other cells caused by the co-action of cytokines and growth factors. Preferred embodiments of the invention include the use of active enamel materials to prepare pharmaceutical compositions for treating and / or preventing chronic inflammation.

Chronic inflammation is best defined in terms of process, and simultaneous inflammation and tissue treatment attempts by treatment occur simultaneously. Chronic inflammation is often simply defined over time, but the presence of lesions over six weeks is traditionally considered chronic, and this definition is at its sole discretion. At the microscopic level, chronic inflammation is sometimes defined in terms of cellular response patterns, but this is variable and not entirely reliable.

Characteristic characteristics of this process are best understood by comparison with an acute inflammatory response. In contrast to acute inflammation in which the main response causes recovery, including removal of the irritant, followed by tissue regeneration or treatment, chronic inflammation is characterized by simultaneous, rather than continuous, inflammation and treatment. Treatment is always a hallmark of chronic inflammation because it involves stimulants that cause destruction of tissue structure. Treatment is typically achieved by internal growth of granulated tissue, including macrophages, fibroblasts and new blood vessels.

Another important distinction between acute and chronic inflammation relates to the relative balance between exudation and cell swelling as well as cell types predominant in the inflammatory response. For chronic inflammation, the exudative response is less pronounced and inflammatory cell buildup typically increases, which can be accompanied by local cell proliferation. In contrast to acute inflammation, which is usually characterized by the increase in the number of neutrophil leukocytes, the invading cells that predominate in all forms of chronic inflammation are macrophages. Depending on the nature of the irritant, different mediators of inflammation mediators and growth factors (collectively called cytokines) are produced locally, leading to different morphological patterns of chronic inflammation.

The systemic effects of inflammation are more pronounced in chronic inflammatory diseases and can have a significant impact on clinical outcome. These systemic effects are primarily mediated by cytokines. Most famous systemic effects of acute inflammation are fever and leukocytosis, while chronic inflammation is usually associated with fatigue, drowsiness, weight loss and weakness.

If the condition and / or disease related to the invention is characterized as an infection, the condition is capable of releasing endotoxin peptidoglycan (PepG), which is a special cell wall component of gram-positive and gram-negative microorganisms. for example Staphylococcus aureus (Staphylococci), and Streptococcus (streptococci); And 2) G-: by an infection with bacteria selected from, but not limited to, coliforms and other microorganisms that release lipopolysaccharide (LPS) from foreign cell envelope during infection. May be induced. Immunological conditions are caused by endotoxins and / or exotoxins produced by the infectious microorganism.

In the present invention, the cellular response in human blood induced by endotoxin (PepG) obtained from Staphylococcus aureus and endotoxin (LPS) obtained from Escherichia coli is ameliorated as shown in Example 1. Administration can be made effectively by administering genine. These results clearly demonstrate the potent regulatory effect of active enamel substances on immune mechanisms in mammals.

In another experiment carried out by the inventor and appended with citation evidence as Example 2 in the present application, the ability to treat enamel matrix derivatives in vivo is investigated in vivo using pigs as a model for sepsis.

EMD, which is also delivered by big bolus injection, is in Experiment 2, which has been shown to be stable for systemic administration of high doses. In addition, systemic injection of enamel matrix derivatives has been found to counteract the effects of endotoxin (LPS) in pigs. In general, initiation of sepsis shock in treated pigs is delayed compared to pigs with Siamese surgery. This delay is dose dependent and 1 mg / kg (body weight) causes a short delay (about 30 minutes), while a 5 mg / kg dose prevents the onset of sepsis shock and associated onset of multiple organ damage. Or at least significantly delay (> 5 hours).

One possible mechanism that could explain the beneficial effects of enamel substrate derivatives on LPS induced shock is to inactivate the toxic effects by binding the enamel substrate derivatives to LPS. Alternatively, a similarly conceivable possibility is that enamel matrix derivatives compete with LPS for the same receptors on, for example, white blood cells, macrophages, bone blast cells and / or mast cells. Accordingly, preferred embodiments of the present invention relate to the use of active enamel materials for the manufacture of pharmaceutical compositions, wherein the active enamel materials are directly bound to endotoxins and / or exotoxins and / or endotoxins and / or Or by binding to cellular receptors for exotoxins, thereby inactivating the toxic effects of endotoxins and / or exotoxins produced by the microorganisms.

The third most likely mechanism is that the enamel substrate derivative directly modulates the immune response by regulating the expression cascade of cytokines and signal molecules that play a major role in LPS-induced sepsis shock. Based on previous in vitro experiments not described in this application, it would be reasonable to view at least some of the effects of the enamel substrate derivatives on the latter mechanism. In addition, the blood assays conducted also suggest that enamel matrix derivatives have a direct effect on cytokine expression in white blood cells. This effect appears to have a specificity that shows a strong effect on TNF and IL-6 but no effect on IL-1β. After all, it is also possible that all of the mechanisms presented above are a combination of these effects which involve and yield the beneficial effect of the enamel matrix derivative in the model.

Delayed onset of sepsis shock as reported in Example 2 has significant clinical significance. In one foreseeable embodiment of the invention, said delay is directly related to the amount of "free" LPS in the blood and the enamel matrix derivative is delivered as an additive to antibiotics in infections such as severe bacteremia and meningitis to effect the toxin. Delay and / or decrease. Such treatment will be critical for the patient's survival during the critical time following initiation of antibiotic treatment.

In another, equally preferred embodiment, the effect of the enamel matrix derivative is caused by direct regulation of the inflammatory response, so the enamel matrix derivative can be used in various clinical situations where shockable complications are present. This includes all major surgeries, transplants, traumas, ECMOs, acute dialysis, serious infections, severe allergies and many others.

One theoretical explanation of the mechanism by which the active enamel material described herein, which is not intended to limit the scope of the present invention, can modulate the immune system of mammals is described in the following sections: ICAM, HLA antigen, interleukin 1 receptor, interleukin 2 receptor, interleukin 3 receptor, Interleukin 4 receptor, interleukin 5 receptor, interleukin 6 receptor, interleukin 7 receptor, interleukin 8 receptor, interleukin 9 receptor, interleukin 10 receptor, interleukin 11 receptor, interleukin 12 receptor, interleukin 13 receptor, interleukin 14 receptor, interleukin 15 receptor, interleukin 16 Cell surface, selected from the group consisting of receptor, interleukin 17 receptor, interleukin 18 receptor, IFNα receptor, IFNβ receptor, IFNΔ receptor, IFNγ receptor, IFNΩ receptor PDGF receptor, TGF receptor, TNF receptor, EGF receptor, CD44 variant and integrin receptor family Receptor and / or cell surface spheres With a interaction.

Cell surface receptors are known mediators of cell delivery when activated by each signal molecule, ligand. Cell surface receptors react with ligands with high or low affinity to convert extracellular signals into one or more intracellular signals that alter the behavior of the target cell. Active enamel materials can modulate the immune system by altering the behavior of target cells in a similar manner. Thus, in one embodiment of the present invention, the active enamel material causes the receptor specific intracellular signal cascade and alters the expression of one or more genes by activating cell surface receptors resulting in alteration of said cell behavior. .

In another embodiment of the invention, the active enamel material is an enzyme called a catalytic receptor, capable of activating cell surface receptors that act directly, for example, transmembrane with cytoplasmic domains that act as tyrosine kinases. A) protein.

In another preferred embodiment of the invention, the active enamel material activates G-protein-bound surface receptors that indirectly activate or inactivate separate plasma membrane binding enzymes or ion channels. Interactions between receptors and enzymes and / or ion channels typically result in third proteins such as G proteins (GTP-binding regulatory proteins) that activate intracellular mediators such as, but not limited to, cyclic AMPs, InsP3, and Ca ²⁺ . Is mediated by.

Cytokines such as interleukin, IFNγ and TNFα play an important role in the regulation of immune responses. Special attention has been paid to cytokine gene regulation as T cells differentiate into TH ₁ and TH ₂ subsets. The effects of cytokines can be mediated, for example, through specific receptors that initiate signal transduction pathways, which in themselves can lead to the release of cytokines in a feedback mechanism.

As illustrated in Example 1, amelogenin has a modulating effect on cytokine release and regulation of inflammatory surface receptors corresponding to bacterial cell wall products. Accordingly, embodiments of the present invention provide an active enamel material for preparing a pharmaceutical composition for modulating an immune response by mediating signals through cytokine receptors such as IL-2R, IL-4R, IL-10R, IFNγR and TNFαR. Discloses the use of. Alternatively, since the release of cytokines is regulated by their expression levels, active enamel substances can modulate the immune response by initiating up and / or down regulation of the expression level of one or more cytokines in a mammal, It modulates the release and / or action of one or more cytokines.

In a particularly preferred embodiment of the invention, the active enamel material mediates the regulation of the immune response via TNFαR. TNFα receptors are expressed on all somatic types except red blood cells. 55 kDa (also referred to as TNF-R1; CD120a: also referred to as TNF receptor superfamily member 1A (TNF-RSF1A)) and 75 kDa (TNF-R2; also referred to as CD120b, also referred to as TNF receptor superfamily member 1B (TNF-RSF1B) Two receptors of) have been described. TNFαR is related to the low affinity receptor of NGF and human cell surface antigen CD40 and is strongly expressed on stimulated T-cells and B-lymphocytes. The third receptor subtype is expressed in normal human liver. It binds to TNFα but not only to TNFβ.

Different effects of the two receptor subtypes have been found. Involvement of the p55 receptor specifically leads to the introduction of cellular adhesion molecules ICAM-1, E-selectin, V-CAM-1 and CD44, whereas involvement of p55 and p75 receptors induces expression of α-2 integrins do. Apart from membrane-bound receptors, several soluble proteins that bind to TNF have been described. About 30 kDa of these proteins, called T-BP-1 and T-BP-2 (tumor necrotic factor binding protein), are derived from the TNF-binding domain of the membrane receptor. They act as physiological modulators of TNF activity by inhibiting TNF from binding to its receptor.

The biological activity of IL-2 is mediated by membrane receptors expressed only on exclusively activated T-cells and not on resting T-cells. IL-5 and IL-6 regulate the expression of the IL-2 receptor. In another preferred embodiment of the invention, the active enamel material comprises mediating the regulation of the immune response via IL-2R.

Three different types of IL-2 receptors are distinguished as being different and independently expressed. High affinity IL-2 receptors make up about 10% of all IL-2 receptors expressed by cells. This receptor is a membrane receptor complex consisting of two subunits IL-2R-alpha (TAC antigen = T-cell activating antigen; p55) and IL-2R-beta (p75; newly designated CD122). p75 is constitutively expressed on resting T-lymphocytes, NK-cells, and many other cell types, whereas expression of p55 has been commonly observed only after cell activation. p75 is involved in the introduction of IL-2 mediated signals. In addition, IL-2 receptors are associated with a number of other proteins (p22, p40, p100) that are believed to be involved in mediating structural changes, receptor-mediated corrosion, and other signal transduction processes. One of the proteins identified is the 95 kDa cell adhesion molecule ICAM-1 that focuses IL-2 receptors in the cell-to-cell contact region and thus mediates paracrine activity during IL-2 mediated stimulation of T-cells. can do. Another protein associated with p75 is a tyrosine-specific protein kinase called Ick and other kinases may be associated with IL-2 receptors. Two such kinases called fyn and lyn have been identified. In addition, the IL-2 receptor signal can also be mediated by vav. A third 64 kDa subunit of the IL-2 receptor, also expressed as gamma, has recently been disclosed. This subunit is required for the production of highly substituted and moderately affinity IL-2 receptors but does not bind to IL-2 itself. The gamma subunit of the IL-2 receptor has recently been found to be a component of the receptor for IL-4 and IL-7. It is also a component of the IL-13 receptor.

Activated lymphocytes subsequently secrete 42 kDa fragments of TAC antigen. This fragment circulates between serum and plasma and acts as a soluble IL-2 receptor (sIL-2R). The concentration of soluble receptors varies markedly with different pathogenic situations.

In another preferred embodiment of the invention, the active enamel material activates IFNγR. Many binding proteins with molecular weights ranging from 70 to 160 kDa are described as IFNγR. They are expressed on all types of human cells except mature red blood cells. Receptors expressed in monocytes and other hematopoietic cells have a molecular weight of 140 kDa. 54 kDa protein is observed in other cell types.

The biological activity of IL-4 is mediated by the IL-4 receptor (IL-4R). In a preferred embodiment of the invention, the active enamel material activates IL-4R. The extracellular domain of the IL-4 receptor is associated with the beta chains of the receptors of Epo, IL-6 and IL-2 receptors. This was named CD124. Two types of receptors have been described, one of which is secreted. Secreted receptors contain only extracellular IL-4 binding domains and can block IL-4 activity. IL-4 binding proteins (IL-4-BP) that bind IL-4 with the same affinity as the IL-4 receptor have been shown to be soluble IL-4 receptor mutations. Soluble receptors act as physiological modulators of cytokine activity by inhibiting receptor binding or by acting as transport proteins. Little is known about the mechanism of IL-4 mediated intracellular signal transduction. IL-4 affects intracellular calcium levels and also the metabolism of inositol phospholipids and protein kinase C.

In humans, IL10 is activated by CD8 ⁺ peripheral blood T-cells, B-cell lymphoma by Th ₀ , Th ₁ -and Th ₂ -like CD4 ⁺ T cell clones after antigen-specific and polyclonal activation. And by LPS-activated monocytes and mast cells. IL-4 and IL-10 inhibit the synthesis of IL10 by monocytes. In a preferred embodiment of the invention, the active enamel material activates the IL-10 receptor (IL-10R). Rat IL-10 receptor was cloned. This receptor is about 110 kDa protein that specifically binds to mouse IL-10. This receptor is structurally related to the receptor for IFN.

The effect of enamel matrix derivatives on peripheral lymphocytes shows that porcine amelogenin derivative Emdogain® slightly increases CD25 / CD4 positive lymphocyte proliferation in vitro and induces simultaneous reduction of CD19 positive B-lymphocytes (Petinaki, E , S. Nikolopoulos and E. Castanas. 1998. Low Stimulation of peripheral lymphocytes, following in vitro application of Emdogain. J Clin Periodontol. 25: 715-20). However, the authors have not demonstrated any significant effect on immunoglobulin and / or cytokine (IL-2 and IL-6) production.

Interestingly, we found that various cell cultures of fibroblasts (periodontal ligaments, fish fins or embryos derived from the skin of birds, skin) were stimulated with EMDOGAIN® (manufactured by BIORA AB, Sweden) and with unstimulated cultures. In comparison, it was found to produce about two times the transforming growth factor (TGF-β1). This finding supports the notion that active enamel material has a potent regulatory effect on immune response imbalance.

Enamel substrates are precursors of enamel and can be obtained from related natural sources, ie mammals in which teeth are developing. Suitable sources are developing teeth obtained from slaughtered animals such as calves, pigs or sheep. Another source is, for example, fish fins.

Enamel substrates can be prepared from developing teeth as described in the prior art (EP-B-0 337 967 and EP-B-0 263 086). The enamel substrate can be scraped and the enamel substrate derivative (EMD) can be extracted using an aqueous solution such as a buffer, dilute acid or base or water / solvent mixture, followed by size exclusion extraction, desalting or other purification steps, or otherwise Is prepared by lyophilization. The enzyme may be deactivated by heat treatment or solvent treatment, in which case the derivative may be stored in liquid form without lyophilization.

As other sources of enamel matrix derivatives or proteins, cultured eukaryotic cells and / or prokaryotic cells that can be modified by use of generally available synthetic routes well known to those skilled in the art, or optionally by DNA techniques Can be used. The enamel matrix protein may be of recombinant origin and may be otherwise genetically modified (see, eg, Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).

In the context of the present invention, enamel matrix derivatives, or enamel matrix protein derivatives (EMDs) may be prepared by enzymatic or chemical degradation of naturally or naturally-length proteins by conjugation (splicing) or processing, or By synthesis of a polypeptide in vitro or in vivo (recombinant DNA method or culture of diploid cells, which are plant or animal cells), one or several enamel substrate proteins or derivatives of an enamel substrate comprising part of such proteins. Enamel substrate protein derivatives also include enamel substrate related polypeptides or proteins. These polypeptides or proteins can be linked to suitable biodegradable carrier molecules such as polyamine acids or polysaccharides or combinations thereof. The term enamel matrix derivatives also includes synthetic analogue materials.

Proteins are biological polymers composed of amino acid residues joined together by peptide bonds. Proteins that are linear polymers of amino acids are also called polypeptides. Typically, a protein has 50-800 amino acid residues and thus has a molecular weight in the range of about 6,000 to about several hundred thousand daltons or more. Small proteins are called peptides or oligopeptides.

Enamel substrate proteins are proteins that are normally present in enamel substrates, ie precursors of enamel (Ten Cate: Oral Histology, 1994; Robinson: Eur. J. Oral Science, Jan. 1998, 106 Suppl. 1: 282-91), or such It is a protein that can be obtained by breaking down proteins. In general, such proteins have a molecular weight of 120,000 Daltons or less and include amelogenin, non-amelogenin, proline-rich non-amelogenin and tuftelin.

Examples of proteins that can be used in accordance with the present invention include amellogenin, proline-rich non-amelogenin, tuftelin, tuft protein, serum protein, saliva protein, ameloblastine, enamel second protein (sheathlin) and Derivatives thereof, and mixtures thereof. Formulations containing active enamel materials for use in accordance with the present invention may contain two or more of the aforementioned proteinaceous materials. In addition, other proteins for use according to the invention can be found in the commercially available product EMDOGAIN® (manufactured by BIORA AB, Sweden).

EMDOGAIN® (manufactured by BIORA AB, S-205 12 Malmo, Sweden) is a 30 mg enamel matrix protein and 1 ml carrier solution (propylene glycol alginate) heated at about 80 ° C. for 3 hours to inactivate the remaining protease. Which are mixed before application unless the protein and carrier are tested separately. The weight ratio is 80/8/12 between the major protein peaks at 20, 14 and 5 kDa, respectively.

In general, the main protein of the enamel substrate is known as amelogenin. These constitute about 90% w / w of matrix proteins. The remaining 10% w / w comprises proline-rich non-amelogenin, tuftelin, tuft protein, serum protein and one or more saliva proteins; However, other proteins can be, for example, amelin (ameloblastine, enamel super protein) found to be associated with enamel substrates. In addition, various proteins can be synthesized and / or processed in several different sizes (ie, different molecular weights). Thus, the major protein in the enamel substrate, amellogenin, was found to exist in several different sizes that together form the supermolecular weight aggregates. These are prominent hydrophobic substances that form aggregates under physiological conditions. These may be carriers for or to deliver other proteins or peptides.

The amelogenin of the tooth enamel formed is a tissue specific protein, rich in proline, leucine, histidine and glutamyl residues and synthesized by enamel blast cells (ameloblast) cells of the inner enamel epithelium. These proteins become mature enamels including masses of extracellular matrix that are mineralized by the hydroxyapatite phase. Examination of the amino acid sequence of amelogenin from a number of mammals indicates a high degree of evolutionary sequence conservation, suggesting specific functions. It has now been found that a plurality of amelogenin components found in substrates are produced by a series of proteolytic treatments after secretion and alternatively by the expression of conjugated mRNA produced from the amellogenin genes that take up on the sex chromosome. lost. Physicochemical studies of recombinant amelogenin have shown that they undergo self-assembly in vitro to produce ultra-molecular weight 'nanosphere' structures, and recent discoveries in vivo have revealed the organization of the first inorganic crystallites. It presents a functional role for nanospheres of ultrastructured tissues of inorganic crystalline secretory enamel substrates that contribute to formation.

Recent studies on the expression of amelogenin alternatingly conjugate mouse amellogenin RNA to produce seven distinct mRNAs, which encode an amegenin protein of 194 to 44 amino acid residues in length.

Other protein materials may also be suitably used according to the present invention. Examples are proteins such as proline-rich proteins and polyproline. Other examples of materials that can be considered suitable for use in accordance with the present invention include aggregates, enamel substrate derivatives and / or enamel substrate proteins as well as metabolites of enamel substrates, enamel substrate derivatives and enamel substrate proteins. The metabolite may range in size from the size of the protein to the size of the short peptide.

As noted above, proteins, polypeptides or peptides that may be used in accordance with the present invention typically have a maximum of 120 kDa, such as at most 100 kDa, 90 kDa, 80 kDa, 70 kDa or 60 kDa, as determined by SDS PAGE electrophoresis. Has a molecular weight.

Proteins that can be used according to the invention can usually be in the form of a preparation, wherein the protein content of the active enamel material in the preparation is from about 0.005% w / w to 100% w / w, such as about 0.5-99% w / w. , About 1-95% w / w, about 10-95% w / w, about 10-90% w / w, about 15-90% w / w, about 20-90% w / w, about 30-90 % w / w, about 40-85% w / w, about 50-80% w / w, about 60-70% w / w, about 70-90% w / w, or about 80-90% w / w Range.

Preparations of active enamel materials for use according to the invention may also contain mixtures of active enamel materials with different molecular weights.

Enamel matrix proteins can be roughly divided into high and low molecular weight portions, and well-defined fractions of enamel matrix proteins have been found to possess important properties related to the treatment of periodontal defects (ie, periodontal wounds). This fraction contains an acetic acid extractable protein, commonly referred to as amelogenin, and also constitutes the low molecular weight portion of the enamel substrate (see EP-B-0 337 967 and EP-B-0 263 086).

In the context of the present invention, the active protein is not limited to the low molecular weight portion of the enamel substrate. At present, preferred proteins include enamel matrix proteins such as amelogenin, tuftelin, etc. having a molecular weight of about 60,000 Daltons or less (measured in vitro using SDS-PAGE), while proteins having a molecular weight of 60,000 Daltons or more It has promising properties as a substance for promoting connective tissue growth.

Thus, the active enamel material for use in accordance with the present invention has a molecular weight of about 40,000 Da or less, such as about 5,000 Da and about 25,000 Da.

By separating the protein by precipitation, ion exchange chromatography, conservative electrophoresis, gel permeation chromatography, reverse phase chromatography or affinity chromatography, amelogenin having a different molecular weight can be purified.

The combined molecular weight of amelogenin can vary from predominant 20 kDa compounds to aggregates of amelagenin with different molecular weights ranging from 40 to 5 kDa and to predominant 5 kDa compounds. Other enamel matrix proteins, such as tuftelin or proteolytic enzymes commonly found in enamel substrates, can be added and delivered by amellogenin aggregates.

In a particularly preferred embodiment of the invention, the active enamel material used to prepare a pharmaceutical composition for the prophylaxis and / or treatment of a mammal's condition characterized by an imbalance of the mammalian immune response to an immunogen is shown in FIG. Included in the eluate represented by the third and / or fourth peaks of the HPLC analysis of the processed amelogenin as shown in 2, such as 2-4.5 kDa, 3-5.5 kDa, 4-6.5 kDa, 5- And soluble amelogenin based on peptides having an amino acid length of about 2-13 kDa, including soluble amelogenin based on peptides having a 7.5 kDa or 5-13 kDa amino acid length.

In a preferred embodiment of the invention, the processed amellogenin product is a 5 kDa polypeptide.

According to the invention, the active enamel material is combined with other active drug substances such as antimicrobial, anti-inflammatory, antiviral, antifungal substances or with local chemotherapy, inducers of apoptosis, growth factors such as TGFβ, PDGF, IGF, FGF , EGF, keratinocyte growth factor or peptide analogs thereof. Enzymes inherent or added to an enamel substrate or formulation thereof can be used with enamel substrates, enamel substrate derivatives and / or enamel substrate proteins, in particular proteases.

When administered to an individual (animal or human), the active enamel material and / or formulation thereof is preferably formulated into a pharmaceutical composition containing the active enamel material and optionally one or more pharmaceutically acceptable excipients.

Compositions comprising the active enamel material to be administered may be modified for administration by any suitable route, such as by systemic administration to a patient via a hose, syringe, spray or drain device. In addition, the compositions may be modified for administration in connection with surgery, such as systemic administration by infusion into blood, lymph, ascites or spinal fluid, or by inhalation.

In the following, examples of suitable compositions containing active enamel materials are described.

For administration to an individual (animal or human), the substance is preferably formulated into a pharmaceutical composition containing the substance and optionally one or more pharmaceutically acceptable excipients.

The composition may be in the form of a liquid, semisolid or solid composition, but sterile saline, Ringer's solution, glucose solution, phosphate buffered saline, blood, plasma or water, powder, microcapsules, microspheres, nanoparticles, sprays, aerosols, inhalation devices It is not limited to dissolved infusion liquids such as solutions, dispersants, suspensions, emulsions, mixtures.

The composition can be formulated according to conventional pharmaceutical practice. See, eg, "Remington: The sceince and practice of pharmacy" 20 ^th ed. Mack Publishing, Easton PA, 2000 ISBN 0-912734-04-3 and "Encyclopaedia of Pharmaceutical Technology", edited by Swarbrick, J. & JC Boylan, Marcel Dekker, Inc., New York, 1988 ISBN 0-8247-2800- See 9.

Pharmaceutical compositions comprising the active substance act as a drug delivery system. In the context of the present invention, the term "drug delivery system" means a pharmaceutical composition (pharmaceutical formulation or dosage form) which, when administered, provides the active substance in the human or animal body. The term "drug delivery system" thus includes common pharmaceutical compositions such as liquids, powders and sprays.

The choice of pharmaceutically acceptable excipients and their optimal concentrations in the compositions for use according to the invention is generally unpredictable and should be determined on the basis of their experimental measurements. Whether a pharmaceutically acceptable excipient is suitable for use in a pharmaceutical composition generally depends on the type of dosage form selected. However, those skilled in the art of pharmaceutical compositions are entitled to "Remington: The sceince and practice of pharmacy" 20 ^th ed. Mack Publishing, Easton PA, 2000 ISBN 0-912734- 04-3 ".

Pharmaceutically acceptable excipients are substances that are substantially harmless to the individual to which the composition is to be administered. Such excipients generally meet the requirements by natural medicines. Formal pharmacopoeia, such as the British Pharmacopoeia, the US Pharmacopoeia, and the European Pharmacopoeia, set the criteria for well-known pharmaceutically acceptable excipients.

The following outlines the relevant pharmaceutical compositions for use in accordance with the present invention. This overview is based on specific routes of administration. However, where pharmaceutically acceptable excipients may be used in different dosage forms, the application of certain pharmaceutically acceptable excipients is not limited to particular dosage forms or specific excipient actions.

Parenteral Composition:

For systemic application, the compositions according to the invention may contain conventional non-toxic pharmaceutically acceptable carriers and excipients according to the invention, including microspheres and liposomes.

Compositions for use according to the invention include all kinds of solid, semisolid and liquid compositions. Particularly relevant compositions are, for example, solutions, suspensions, emulsions.

Pharmaceutically acceptable excipients may include solvents, buffers, preservatives, chelating agents, antioxidants, stabilizers, emulsifiers, suspending agents, diluents. Examples of different materials are shown below.

In the most preferred embodiment, the lyophilized powder of Emdogain® (Bilmo, Biomo AB, Malmo, Sweden) is dissolved in phosphate buffered saline (PBS) at a final concentration of 30 mg / ml.

Other, equally preferred embodiments have a final concentration of 0.01-50 mg / ml, such as 0.01-10 mg / ml, 0.1-10 mg / ml, 0.01-5 mg / ml, 0.1-5 mg / ml, 0.01-1 dissolved in phosphate buffered saline (PBs) at mg / ml, 0.1-1 mg / ml or 0.01-0.05 mg / ml, about 0.01 mg / ml, 0.02 mg / ml, 0.03 mg / ml, 0.04 mg / ml, 0.05 mg / ml, 0.06 mg / ml, 0.07 mg / ml, 0.08 mg / ml, 0.09 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, Emdogain® (manufactured by BIORA AB, containing concentrations of 1 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml or 50 mg / ml Lyophilized powder, Malmö, Sweden.

As described in Example 2, the dosage weight of the enamel matrix derivative administered to a mammal in need of administration is of course adjusted according to the individual weight of the mammal to be treated and the desired effect of the EMD, and most preferably enamel Depending on whether the substrate derivative is administered at a high or low dose, about 0.01-100 mgEMD / kg body weight range, such as about 0.01-50 mg EMD / kg, 0.05-10 mgEMD / kg, 0.01-1 mg EMD / kg, 0.1-50 mg EMD / kg, 0.1-25 mg EMD / kg, 0.1-15 mg EMD / kg or 0.1-10 mg EMD / kg or 0.1-1 mg EMD / kg body weight.

Typical low doses are at most 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.15, 0.17, 0.2, 0.5, 1, 1.2, 1.5, 1.6, 1.8, 2, 2.5, Typical high doses include at most 10, 15, 18, 20, 25, 50, 75 or 50 mg EMD / kg body weight, whereas 5, 6, 7, 8, 9 or 9.9 mg EMD / kg body weight. .

The dosage described above may be at least once per week, such as 1, 2, 3, 4, 5, 6, 7 or more per week for at least 1 to 30 days, or alternatively for at least 10 to 90 days or longer. It is administered 1 week.

Examples of various materials are as follows:

Examples of the solvent are water, alcohol, blood, plasma, spinal fluid, ascites fluid and lymph fluid.

Examples of buffers include citric acid, acetic acid, tartaric acid, lactic acid, hydrogen phosphate, bicarbonate, phosphate, diethylamine, and the like, but are not limited to these.

Examples of chelating agents are, but are not limited to, sodium EDTA and citric acid.

Examples of antioxidants include, but are not limited to, butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, cysteine, and mixtures thereof.

Examples of powder components are alginate, collagen, lactose, powders that can form gels (absorb liquid / wound exudates) when administered to wounds, but are not limited to these.

Examples of diluents and disintegrants are, but are not limited to, lactose, saccharose, mdex, calcium phosphate, calcium carbonate, calcium sulfate, mannitol, starch and microcrystalline cellulose.

Examples of binders include, but are not limited to, saccharose, sorbitol, gum acacia, sodium alginate, gel latin, starch, cellulose, sodium carboxymethylcellulose, methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone and polyethylene glycol. .

List of references:

Figure 1:

Whole blood samples treated with LPS, PepG and matrix protein compositions were incubated with 10 μl FITC or PE conjugated monoclonal antibodies. Antigen expression was analyzed by flow cytometry using 10 μl anti-CD14, anti-CD44, anti-ICAM-1 as well as isotope negative controls anti-IgG ₁ and anti-IgG ₂ . All samples were analyzed on FACScan using CellQuest software.

Figure 2:

According to Gestrellius et al. 1997, amelogenin was extracted from the enamels of immature pigs. After making into a solution in 30% acetonitrile, the samples were separated using a size-exclusion column on Jasco HPLC. Four distinct peaks represent complex, full length protein and soluble, processed amelogenin peptides.

Figure 3:

Enamel matrix derivatives resulted in decreased production of the matched pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) by increased release of anti-inflammatory cytokine IL-10.

Experiment part

Example 1

The purpose of this study was to study the potent regulatory effects of amelogenin on natural immune mechanisms in human whole blood. Of particular importance is the cytokine release and regulation of inflammatory surface receptors corresponding to bacterial cell wall products.

Materials and reagents.

Peripheral venous blood was taken from healthy staff and collected in Becutainer (Becton Dickinson Vacutainer Systems Eur, Medex, France), containing 0.129 M CitNa. Lyophilized Emdogain® (B Malmo, Sweden) powder was dissolved in phosphate buffered saline (PBS) at a final concentration of 30 μg / ml. Escherichia coli O26: B6 lipopolysaccharide (LPS; manufactured by Difco Laboratories, Detroit, Mich.) Was suspended in pyrogen-free sterile saline and 10 ng / ml LPS was added directly to blood samples in microcentrifuge tubes. . Lipoteichoic cid (LTA) was purchased from Sigma Chemicals, St. Louis, Missouri. Peptidoglycan (PepG) was isolated from Staphylococcus aureus as described previously (Foster, SJ 1992 J Bacteriol. 174: 464-70). Analysis of autolysines of Bacillus subtilis 168 during trophic growth and differentiation was performed using regenerated polyacrylamide gel electrophoresis, which was added directly to blood samples at a concentration of 10 μg / ml. It became.

Whole blood experiment.

Peripheral venous blood was taken from healthy staff and collected in Becutainer (Becton Dickinson Vacutainer Systems Eur, Medex, France), containing 0.129 M CitNa. Whole blood samples were divided into equal portions and placed in 1.5 ml microcentrifuge tubes (Solenson Biosciences, Salt Late City, Utah). Prior to stimulation with LPS, PepG or LTA, blood samples were preincubated for 2 hours at 37 ° C. with Emdogain® dissolved at final concentrations of 0, 45, 150, 300, 450, 600 or 750 g / ml. Blood samples were stimulated for 4 or 6 hours with 10 ng / ml LPS, 10 g / ml PepG or 100 g / ml LTA. The blood was cooled on ice and centrifuged (1 minute at 14,000 g). Blood was then cooled on ice, centrifuged (1 min at 14,000 g) and then the plasma was pipetted for cytokine protein detection. For control, Eastern skin 0.9% NaCl was added instead of bacterial product. Basal cytokine levels were examined immediately after bleeding. Preliminary studies have revealed that PepG and LTA show a 6 hour peak value of TNF-a after stimulation as opposed to 4 hours by LPS stimulation. Therefore, 4 hours of LPS stimulation and 6 hours of PepG and LTA stimulation were selected.

ELISA technique.

Plasma concentrations of TNF-α, IL-6 and IL-10 were determined using manufacturer's instructions using a commercially available solid sandwich enzyme-linked immunosorbent assay (ELISA) kit (Pelikin Compact, CLB Labs, Amsterdam, The Netherlands). Thus decided. Detection limits of TNF-α, IL-6 and IL-10 were 3, 0.4 and 3 pg / ml, respectively. Plates were read at 450 nm with an ELISA reader (Thermo max microplate reader, Molecular Devices, Menlo Park, Calif.).

Antibody Labeling and Flow Cytometry.

After stimulation, whole blood samples were immediately divided into 100 μl equivalents and placed in Falcon polystyrene tubes (Becton Dickinson, Lincoln Park, NJ) with 10 μl FITC or PE conjugated monoclonal antibody for 30 minutes in the dark at room temperature. Incubated. Prior to washing and fixation, erythrocyte cells were lysed with FACS lysate (Becton Dickinson, San Jose, Calif.). Samples were washed three times using CellWash (Becton Dickinson, Inc.) prior to fixation using formaldehyde 1% (CellFIX, Beckton Dickinson, Erembodegem, Belgium). Antigen expression is 10 μl anti-CD14 (18D11, manufactured by Ostec, Oslo, Norway), anti-CD44 (made by Diatec AS, Oslo, Norway), and anti-ICAM-1 (manufactured by Diactec AS, Oslo, Norway) But was also analyzed by flow cytometry using isotopic negative control anti-IgG ₁ (1B9) and anti-IgG ₂ (5A7) (both manufactured by Diatec AS). All samples were analyzed on FACScan (Becton Dickinson) using CellQuest software. The results are shown in FIG.

In each experiment, monocyte populations were identified as CD14 positive cells with characteristic positions on the scatter diagram. Fluorescence intensity (FI) was measured in the monocyte population after appropriate gating according to these features, and the average FI (geometric mean) was recorded for each measurement.

Static analysis.

Data are expressed as mean ± standard deviation of the mean (SEM). These data were followed by a one-way analysis of variation (ANOVA) followed by a Turkish test. P <0.05 was considered significant.

As shown in FIG. 3, enamel matrix derivatives resulted in decreased production of pre-inflammatory cytokine tumor necrosis factor alpha (TNF-α), comparable to increased release of anti-inflammatory cytokine IL-10. These results indicate that enamel matrix derivatives have potent immunomodulatory properties. It also suggests that enamel matrix derivatives may have therapeutic efficacy in sepsis associated with multiple organ damage. In addition, enamel matrix derivatives may have therapeutic efficacy in other diseases associated with acute or chronic inflammation, such as inflammatory bowel disease, rejection of transplant organs or orthopedic grafts, rheumatoid arthritis, arteriosclerosis and some diseases.

Example 2

In this study using a pig model of sepsis, the therapeutic efficacy of enamel matrix derivatives was investigated in vivo. This model is based on the continuous fusion of E. coli LPS, resulting in an acute inflammatory response. After a while (typically 30 minutes to 1 hour after LPS injection), the animal entered sepsis shock and showed significant pathophysiological characteristics of some sepsis. This model is a powerful tool to link in vitro findings with possible clinical trials.

Thirty minutes before the onset of sepsis (= injection of LPS, 1.7 μg / kg body weight / hour), the pigs were given an enamel substrate derivative (maximum dose of 5 mg / kg body weight, ie a calculated serum concentration of about 100 mg / l) intravenously. Administration was by bolus injection. It has been reported that the half-life in the blood of enamel matrix derivatives is 240 minutes. Thus, intravenous infusion equipment was used to deliver 50 mg of enamel substrate derivative in 100 ml Ringer's solution (pH = 6) per hour to maintain therapeutic serum levels of EMD. For control, three "Sham" pigs were treated identically except for injection injection of serum albumin ("Placebo") instead of administration of EMD.

Hematopoietic parameters were continuously registered and recorded every hour from 5 hours after the injection of bacterial toxin (LPS) to the end of the experiment. Records of blood samples and clinical variables were associated with sepsis from the start of the experiment (time 0), immediately after initiation of the enamel matrix derivative or placebo injection (time 0-a) and from the hepatic and portal vein, the abdominal artery, and the pulmonary artery. Measured hourly after this initiation and measured for blood homeostasis parameters, organ action markers (serum asperate aminotransferase (ASAT), serum alumina aminotransferase (ALAT) and serum alkaline phosphatase activity and kidney function markers) The markers gamma-glutamyltransferase (γ-GT) and creatine, inflammatory mediators (TNF-α, IL-1β and IL-6) were analyzed for organ function markers every hour during the experiment at predetermined time points. Analyzes were performed on blood samples taken from pigs Blood samples were analyzed using standard hospital procedures of hematopoietic laboratories at Norwegian National Hospital. TNF-α / TNFSF2 Quantikine P ELISA Kit (# PTA00), Porcine IL-6 Quantikine P Immunoassay Kit (# P6000) and Porcine IL-1β / IL-1F2 Quantikine P ELISA Kit (# PTA00) (both are R & D Systmes Inc. (manufactured by Minneapolis, Minn.) Was applied to analyze in the same blood sample, using the standard techniques suggested by the manufacturer without omission or modification.

After completion of the experiment (after animals were killed or 5 hours after continuous injection of enamel matrix derivative or placebo and LPS), animals were euthanized and necropsied. Internal circulation was visually examined and tissue sampled for histological evaluation according to the study plan.

result

Pig 1

Physical data: magnetic; 23.5 kg total body weight; Healthy

Surgery: no complications                 

Treatment: placebo (25 mg serum albumin in 50 ml PBS bolus injection, 10 mg SA in 100 ml Ringer's solution per hour infusion, total SA dose = 75 mg), LPS (40 μg / hour in 4.5 ml PBS, 5 h) Continuous injection; total LPS dose = 200 μg).

Hemodynamic Observation: This animal did not respond to placebo injections. Experiments showed a strong initial response to LPS injection after being treated with sustained sepsis shock conditions. The onset of shock appeared one hour before the start of the LPS injection.

Anatomical and visual appearance of circulatory organs in lethality: All circulatory organs showed normal structure. Pathological signs were found in the liver showing signs of congestion and hemostasis.

Hematology: The animals initially displayed normal hematological values. Most of the values were within the normal range during the experiment. An exception was observed 4 and 5 hours after LPS injection, with an increase in B-neutrophils and a decrease in B lymphocytes.

Biochemistry: All enzymatic activity and creatinine remained within normal values during the experiment. A slow but constant decrease in creatinine was observed, but the value never decreased below normal. Pigs initially had a slight increase in ALP, but normalized before administration of LPS. ASAT values were on the high side during the experiment, but did not show a significant change from LPS injection.

Cytokines: These pigs showed a spike in TNF values after LPS administration. This value reached above the detection limit (1500 μg / l) 1 hour before the start of LPS injection and remained at this level during the experiment. IL-6 also spiked after initiation of LPS injection. IL-6 levels increased consistently throughout this experiment, reaching 3500 μg / l. IL-1β shows a slow increase caused by LPS injection, reaching about 700 μg / l after 5 hours.

Explanation: Hematopoietic parameters indicated hepatic and lung damage at the end of the experiment. Low diuresis and reduced diuresis showed kidney disability. The first signs of organ disability were observed 2 hours after the start of the LPS injection. Convulsions were observed 30 minutes after administration of LPS. Hematology and biochemical values indicated that the life support system was functioning normally, and the observed effects were caused by toxin injection and not by surgical trauma. Inflammatory conditions indicated that the pig had septic shock. Cytokine values showed no signs of recovery. The pig is very unlikely to survive during this experiment.

Pig 2

Physical data: magnetic; 21.5 kg total body weight; Healthy

Surgery: no complications

Treatment: Enamel Substrate Derivatives (21.5 mg Enamel Substrate Derivatives in 50 ml PBS cyclic injections, 10 mg Enamel Substrate Derivatives in 100 ml Ringer's solution per hour infusion, Total Enamel Substrate Dose = 71.5 mg), LPS (in 4.5 ml PBS) 37 μg / hour, continuous injection for 5 hours; total LPS dose = 185 μg).                 

Hemodynamic Observation: This animal did not respond to bolus injection of EMD. About 1 hour after initiation of LPS injection a very rapid and strong initial response to LPS injection followed by onset of sepsis shock. At this stage, early signs or multiple organ damage were observed. However, hemodynamic values stabilized and organ function recovered.

Anatomical and visual appearance of circulatory organs in lethality: All circulatory organs showed normal structure. No gross signs of pathology were found in the organs investigated.

Hematology: The animals initially displayed normal hematological values. Most of the values were within the normal range during the experiment.

Biochemistry: All enzymatic activity and creatinine remained within normal values during the experiment. Pigs initially had a slight increase in ALP, but remained unchanged by enamel matrix derivative or LPS injection. ASAT values slightly increased towards the end of the experiment but did not show significant changes from enamel substrate derivative or LPS injection.

Cytokines: Injection of enamel substrate derivatives did not cause any increase in the circulating cytokines analyzed. However, the pig showed a spike in TNF values after administration of LPS. This value reached or exceeded the detection limit (1500 μg / l) before 1 hour after initiating LPS injection and remained at this level for about 4 hours. After 4 hours, TNF values decreased and showed signs of normalization. IL-6 also spiked after initiation of LPS injection. IL-6 levels increased constantly during this experiment, reaching 4800 μg / l levels after 4 hours. After 5 hours the IL-6 value seemed to decrease, but due to the (planned) end of the experiment, this trend was not confirmed. IL-1β showed a slow increase induced by LPS injection and reached about 1000 μg / l after 5 hours.

Comment: As observed by hemodynamic variables and by the recovery of renal function (diuresis) and the reduction of ascites, this animal appeared to have recovered from the onset of obvious sepsis shock and tracheal insufficiency. Convulsions were observed 1 hour after LPS administration. These findings are uncommon and lead to a strong initial response to endotoxin (LPS). Hematology and biochemistry values indicated that the life support system was functioning normally, and the observed effects were caused by toxin injection and not by surgical trauma. Inflammatory conditions indicated that the pig had septic shock. However, signs of slow recovery were seen in IL-6 and TNF values. IL-1β values did not appear to be affected by administration of enamel substrate derivatives. Given the continuous reduction and time of inflammatory cytokine levels, this pig is likely to survive during this experiment.

Pig 3

Physical data: magnetic; 25.0 kg total body weight; Healthy

Surgery: no complications

Treatment: placebo (125 mg SA in 100 ml PBS bolus injection, 50 mg SA in 100 ml Ringer's solution per hour infusion, total SA dose = 375 mg), LPS (43 μg / hour in 4.5 ml PBS for 5 hours) Continuous injection; total LPS dose = 215 μg).

Hemodynamic Observation: This animal did not respond to placebo injections. Experiments showed a low or moderate initial response to LPS injection after treatment in sustained sepsis shock conditions. The onset of shock was 1 hour after the start of the LPS injection.

Anatomical and visual appearance of circulatory organs in lethality: All circulatory organs showed normal structure. Pathological signs were found only in the liver that showed signs of congestion (edema and red phase) and hemostasis.

Hematology: This animal initially had normal hematological values. Most of the values were within the normal range during the experiment. An exception was observed 4 and 5 hours after LPS injection, with an increase in B-neutrophils and a decrease in B lymphocytes.

Biochemistry: All enzymatic activity and creatinine remained within normal values during the experiment. A slow but constant decrease in creatinine was observed, and 2 hours after LPS injection, creatinine levels decreased below normal and remained low for the remainder of the experiment. This pig initially had a slight increase in ALP, but this value was stable and unaffected by LPS injection. Inflammatory conditions indicate that the pig has septic shock. There was no sign of recovery in cytokine values. During this experiment, the pig will be very unlikely to survive.

Cytokines: These pigs showed a spike in TNF values after LPS administration. This value reached the detection limit (1500 μg / l) or more before 1 hour after initiating LPS injection and remained at this level during the experiment. IL-6 also spiked after initiation of LPS injection. IL-6 levels increased consistently throughout this experiment, reaching 5000 μg / l levels after 3 hours of LPS injection and ending at 3900 μg / l. IL-1β produced a slow increase induced by LPS injection and finished at about 700 μg / l after 5 hours.

Explanation: Hematopoietic parameters indicated hepatic and lung damage at the end of the experiment. Low diuresis and reduced diuresis showed kidney disability. The first signs of organ disability were observed 3 hours after the start of the LPS injection. Convulsions were observed 30 minutes after administration of LPS. Hematology and biochemical values indicated that the life support system was functioning normally, and the observed effects were caused by toxin injection and not by surgical trauma. Inflammatory conditions indicated that the pig had septic shock. Cytokine values showed no signs of recovery. The pig is very unlikely to survive during this experiment.

Pig 4

Physical data: magnetic; 24.5 kg total body weight; Healthy

Surgery: no complications

Treatment: Enamel Substrate Derivatives (125 mg Enamel Substrate Derivatives in 100 ml PBS cyclic injections, 50 mg Enamel Substrate Derivatives in 100 ml Ringer's solution per hour infusion, Total Enamel Substrate Dose = 375 mg), LPS (in 4.5 ml PBS) 42 μg / hour, 5 hours continuous injection; total LPS dose = 210 μg).

Hemodynamic Observation: This animal did not respond to bolus injection of EMD. A strong initial response to LPS injection was shown followed by a mild sepsis shock state 3 hours after initiation of LPS injection. However, after this time, hemodynamic values stabilized and all organ function was restored.

 Anatomical and visual appearance of circulatory organs in lethality: All circulatory organs showed normal structure. No gross signs of pathology were found in the organs investigated.

Hematology: This animal initially had normal hematological values. Most of the values were within the normal range during the experiment.

Biochemistry: All enzymatic activity and creatinine remained within normal values during the experiment. The pig initially had a slight increase in ALP but remained unchanged by enamel matrix derivative or LPS injection. ASAT values slightly increased towards the end of the experiment but did not show significant changes from enamel substrate derivative or LPS injection.

Cytokines: Injection of enamel substrate derivatives did not cause any increase in the circulating cytokines analyzed. However, the pig showed a spike in TNF values after administration of LPS. This value reached the detection limit (1500 μg / l) or more before 1 hour after initiating LPS injection and remained at this level during the experiment. IL-6 also spiked after initiation of LPS injection. IL-6 levels increased constantly during this experiment and increased above the detection level (> 5000 μg / l) after 3 hours. After 4 hours the IL-6 value seemed to decrease and this trend was also observed at 5 hours. IL-1β showed a slow increase induced by LPS injection and finished at about 700 μg / l after 5 hours.

Commentary: As observed by clinical and hemodynamic variables, this animal appeared to be protected from obvious sepsis shock and organ failure. No organ failure was observed, but diuresis was low throughout the experiment. The production of ascites was very small and no cramps were observed. These findings are uncommon and lead to a strong initial response to endotoxin (LPS). Hematology and biochemistry values indicated that the life support system was functioning normally, and the observed effects were caused by toxin injection and not by surgical trauma. Inflammatory conditions indicated that the pig had septic shock. However, signs of slow recovery were seen in IL-6 and TNF values. IL-1β values did not appear to be affected by administration of enamel substrate derivatives. Given the continuous reduction and time of inflammatory cytokine levels, this pig is likely to survive during this experiment.

Pig 5

Physical data: magnetic; 25.0 kg total body weight; Healthy

Surgery: no complications

Treatment: Enamel Substrate Derivatives (125 mg Enamel Substrate Derivatives in 100 ml PBS cyclic injections, 50 mg Enamel Substrate Derivatives in 100 ml Ringer's solution per hour infusion, Total Enamel Substrate Dose = 375 mg), LPS (in 4.5 ml PBS) 43 μg / hour, 5 hours continuous injection; total LPS dose = 215 μg).

Hemodynamic Observation: This animal showed an initial increase in pulmonary blood pressure in response to the initiation of a bolus injection of EMD. This effect is very temporary (moisture) and not dose dependent, indicating that the enamel matrix derivative solution has a simple vasoconstrictive effect on pulmonary capillaries. The observed effect normalized before the injection injection was complete. No other effect was observed from the injection of enamel matrix derivatives. This animal did not respond significantly to LPS injection. No signs of sepsis shock were found and organ circulation was stable during the experiment. The LPS flow and the position of the LPS catheter were adjusted several times during the experiment to ensure that the LPS was delivered according to the experimental design.

Anatomical and visual appearance of circulatory organs in lethality: All circulatory organs showed normal structure. No gross signs of pathology were found in the organs investigated.

Hematology: This animal initially showed normal hematologic values, except that the number of leukocytes increased significantly (twice the normal value) from the onset of surgery (0-sample). The relative values of the different white blood cells seemed normal but remained stable during the experiment. All other values remained within the normal range during the experiment.

Biochemistry: All enzymatic activity and creatinine remained within normal during the experiment except for ALP which increased from the start of the experiment but remained stable during the observation period. ALP levels remained unchanged by enamel substrate derivative or LPS injection. The ASAT value slightly increased towards the end of the experiment. However, this increase was so small that it barely rose above normal limits.

Cytokines: Injection of enamel substrate derivatives did not cause any increase in the circulating cytokines analyzed. However, the pig showed a marked increase in TNF values even before initiation of the experiment (> 1500 μg / l). This value was beyond the detection range until 2 hours after the start of LPS injection. However, a sharp drop in TNF values was observed and TNF levels were nearly normal before the end of this experiment. IL-6 spiked immediately after initiation of LPS injection. IL-6 levels increased consistently throughout this experiment, reaching 2500 μg / l. IL-6 values then decreased and reached normal values 5 hours after LPS injection. IL-1β showed a slow but constant increase induced by LPS injection and finished at about 900 μg / l after 5 hours.

Commentary: As observed by clinical and hemodynamic variables, this animal appeared to be protected from obvious sepsis shock and sluggish organs. No organ sluggishness was observed, but diuresis was good and stable throughout the experiment. The production of ascites was very small and no cramps were observed. These findings are uncommon and lead to a strong initial response to endotoxin (LPS). Hematology and biochemistry values indicated that the life support system was functioning normally, and the observed effects were caused by toxin injection and not by surgical trauma. Inflammatory conditions indicated that the pig had septic shock. However, clear signs of rapid recovery were seen in IL-6 and TNF values. IL-1β values did not appear to be affected by administration of enamel substrate derivatives. There is no doubt that this animal will survive the experiment.

Pig 6

Physical data: magnetic; 23.0 kg total body weight; Healthy

Surgery: no complications                 

Treatment: Enamel Substrate Derivatives (115 mg Enamel Substrate Derivatives in 100 ml PBS cyclic injections, 50 mg Enamel Substrate Derivatives in 100 ml Ringer's solution per hour infusion, Total Enamel Substrate Dosage = 365 mg), LPS (in 4.5 ml PBS) 39 μg / hour, continuous injection for 5 hours; total LPS dose = 195 μg).

Hemodynamic Observation: This animal showed an initial increase in pulmonary blood pressure in response to the initiation of a bolus injection of EMD. This reaction is immediate and transient (moisture) and not dose dependent, indicating that the enamel matrix derivative solution has a simple vasoconstrictive effect on pulmonary capillaries. The observed effect normalized before the injection injection was complete.

A strong initial response to LPS injection was observed (a rapid and transient increase in heart rate and arterial flow) followed by a normal high specific gravity that persisted during the experiment. No signs of sepsis shock were found and no signs of tracheal insufficiency.

Anatomical and visual appearance of circulatory organs in lethality: All circulatory organs showed normal structure. No gross signs of pathology were found in the organs investigated.

Hematology: This animal initially had normal hematological values. All other values remained within the normal range during the experiment.

Biochemistry: All enzymatic activity and creatinine remained within normal during the experiment except for ALP which increased from the start of the experiment but remained stable during the observation period. ALP and ASAT levels remained unchanged by enamel substrate derivative or LPS injection.                 

Cytokines: Injection of enamel substrate derivatives did not cause any increase in the circulating cytokines analyzed. However, the pig showed a spike in TNF values after administration of LPS. This value was above the detection limit (1500 μg / l) before 1 hour after initiating LPS injection and this level was maintained for about 3 hours. After 3 hours, TNF values dropped sharply and normalized at the end of the observation period. IL-6 spiked immediately after initiation of LPS injection. IL-6 levels reached 2700 μg / l 2 hours after LPS injection. However, at the end of the experiment, IL-6 values were reduced to nearly normal values (350 μg / l). IL-1β showed a slow but constant increase induced by LPS injection and reached about 1200 μg / l after 4 hours.

Comment: As observed by hemodynamic and clinical variables, this animal appeared to be protected from severe sepsis shock and tracheal insufficiency. No organ failure was observed and diuresis was high and stable throughout the experiment. Ascites production was very low and spasm was observed about 4 hours after LPS injection. These findings are uncommon and lead to a strong initial response to endotoxin (LPS). Hematology and biochemistry values indicated that the life support system was functioning normally, and the observed effects were caused by toxin injection and not by surgical trauma. The inflammatory state indicated that the pig was suffering from a strong sepsis shock (a sharp and strong increase in TNF and IL-6). However, clear signs of rapid recovery were seen in IL-6 and TNF values. IL-1β values did not appear to be affected by administration of enamel substrate derivatives. There is no doubt that this animal will survive the experiment.                 

summary

Control (Pigs 1 and 3)

Controls all reacted normally. That is, they all entered sepsis shock state about 30 minutes after the start of LPS injection. Both pigs survived 5 hours after LPS injection, but they showed clear clinical signs of severe uncontrolled sepsis shock, indicating increased body temperature, multiple organ insufficiency, kidney failure, hypertension and decreased heart rate. Blood analysis also showed clear signs of sepsis shock. The inflammatory state corresponded to acute toxin-induced sepsis shock, indicating strong expression of TNF-α and interleukin-6. It was determined that near the end of the experiment the condition of these pigs was so severe that it would be difficult to continue to survive.

Low Dose Enamel Substrate Derivatives (Pig 2)

Pigs administered low doses of enamel matrix derivatives also entered sepsis shock. However, the onset of an acute inflammatory response to LPS was delayed by about 30 to 60 minutes compared to Siamese animals. After initiation of sepsis, the pigs stabilized and then slowly recovered from shock. This animal showed slightly better clinical variables and no signs of kidney failure (measured by diuresis). Pigs were euthanized as planned 5 hours after the start of LPS injection. The injection test and subsequent injection of low dose enamel substrate derivatives did not cause any clinical signs of any adverse reactions in the pig and did not result in a controlled variable response to injection of enamel substrate derivatives. This pig showed a tendency to decrease in TNF and IL-6 values. In addition to these observations, clinical observations suggest that the animals are recovering from shock.

High-dose enamel matrix derivatives (pig 4, pig 5 and pig 6)

None of the three pigs administered the high dose of enamel substrate derivative experienced severe sepsis shock from LPS injection. Two animals (pig 4 and pig 6) experienced an initial transient endotoxin response, but this condition was stabilized immediately and the animals recovered immediately and remained in a “controlled state” during the experiment. The third animal (pig 5) did not respond to LPS injection at all. None of these animals showed signs of multiple organ failure or kidney failure. Only one of the pigs (Swine 6) experienced typical cramps induced by LPS injection. The onset of convulsions was significantly delayed (4 hours) compared to the control. Five hours after the start of the LPS injection, the pigs were euthanized as planned.

Injection of the "high dose" enamel substrate derivative following the injection test did not cause any profound clinical signs or adverse reactions in these pigs. In two of the pigs (pig 5 and pig 6), an initial response to bolus injection was observed. This response is found to be a rapid but transient increase in pulmonary arterial blood pressure, which means that a central bolus injection (approximately 1.25 mg / ml) of a high-laboratory enamel matrix derivative solution causes natural contraction of the pulmonary artery and / or capillary. do. This effect is immediate and not dose related. The observed effect is normalized in part and the pigs are stable, indicating normal hematopoietic parameters and clinical condition prior to completion of the bolus injection.

These pigs tended to decrease in TNF and Il-6 values after the initial LPS response. Indeed, TNF and Il-6 values were nearly normalized at the end of the observation period in two of the three animals (pig 5 and pig 6). In addition to these observations, clinical observations suggest that these animals are protected from LPS-induced shock and consequent multiple organ failure. Enamel matrix analog injections administered to these pigs protected the animals from severe injury from toxin injection, thus keeping the animals from sepsis shock. All three animals survived the experiment.

Claims (102)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete A pharmaceutical composition of amellogenin for systemic administration for preventing or treating systemic inflammatory response syndrome (SIRS) in a mammal. delete delete delete delete The pharmaceutical composition of claim 69, wherein the systemic inflammatory response syndrome (SIRS) results from trauma, burns, surgery, dialysis, shock or bacterial or viral infection. 70. The system of claim 69, wherein the systemic inflammatory response syndrome (SIRS) is extracorporeal membrane oxygenation (ECMO), shock, sepsis, multiple organ damage, rejection of grafts such as trachea or prosthesis, heart and pulmonary side surgery, chronic inflammation, asthma Or a pharmaceutical composition resulting from a disease selected from the group consisting of stress-related diseases of the lungs, bacterial infections, necrosis, soft tissue infections and wound infections. 75. The pharmaceutical composition of claim 74, wherein the systemic inflammatory response syndrome (SIRS) is caused by contamination by bacteria selected from the group consisting of gram negative microbes and gram positive microbes. 77. The pharmaceutical composition of claim 76, wherein systemic inflammatory response syndrome (SIRS) is caused by endotoxin or exotoxin produced by the microorganism. 78. The pharmaceutical composition of claim 77, wherein the systemic inflammatory response syndrome (SIRS) is caused by PepGido (PeptidoGlycan) from Staphylococcus aureus . 78. The pharmaceutical composition of claim 77, wherein said systemic inflammatory response syndrome (SIRS) is caused by lipopolysaccharide (LPS) from Escherichia coli . delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete 70. The method of claim 69, wherein the content of amellogenin is 0.005% w / w to 100% w / w, 0.5-99% w / w, 1-95% w / w, 10-95% w / w, 10 -90% w / w, 15-90% w / w, 20-90% w / w, 30-90% w / w, 40-85% w / w, 50-80% w / w, 60-70 Pharmaceutical compositions in the range of% w / w, 70-90% w / w, or 80-90% w / w. The pharmaceutical composition of claim 69, wherein the pharmaceutical composition also comprises a pharmaceutically acceptable excipient. 102. The pharmaceutical composition of claim 101, wherein said pharmaceutically acceptable excipient is phosphate buffered saline (PBS).

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