Methods of reducing extravasation of inflammatory cells

BACKGROUND

[0002] 1. Field of the Invention

[0003] The present disclosure relates to methods of reducing penetration of inflammatory cells into tissue, in particular to methods of reducing extravasation of macrophage cells from blood vessels, methods of preventing, regulating and suppressing inflammatory response, and methods of treating inflammatory states.

[0004] 2. Description of Related Art

[0005] The inflammatory response evolved to protect organisms against injury and infection. Following an injury or infection, a complex cascade of events leads to the delivery of blood-borne leukocytes to sites of injury to kill potential pathogens and promote tissue repair. However, the powerful inflammatory response has the capacity to cause damage to normal tissue, and dysregulation of the innate or acquired immune response is involved in different pathologies. It has long been known that Multiple Sclerosis is an inflammatory disease of the brain, but it is only in recent years that it has been suggested that inflammation may significantly contribute to diseases such as stroke, traumatic brain injury, HIV-related dementia, Alzheimer's disease and prion disease. The recognition of an inflammatory component in the pathology of these and other diseases has come from the development of new techniques and reagents for the study of inflammation biology.

[0006] The inflammatory response is a part of innate immunity. Inflammation occurs when tissues are injured by viruses, bacteria, trauma, chemicals, heat, cold, or any other harmful stimuli. Chemicals, such as bradykinin, histamine, and serotonin, are released by specialized cells and attract tissue macrophages and white blood cells to localize in an area to engulf (phagocytize) and destroy foreign substances. A byproduct of this activity is the formation of pus—a combination of white blood cells, bacteria, and foreign debris. The chemical mediators released during the inflammatory response give rise to the typical findings associated with inflammation.

[0007] Inflammation constitutes the body's response to injury and is characterized by a series of events that includes the inflammatory reaction per se, a sensory response perceived as pain, and a repair process. The inflammatory reaction is characterized by successive phases: (1) a silent phase, where cells, including resident cells in the damaged tissue, release the first inflammatory mediators, (2) a vascular phase where vasodilation and increased vascular permeability occur, and (3) a cellular phase, which is characterized by the infiltration of leukocytes to the site of injury. The repair process includes tissue cell division, neovascularization and reinnervation of repaired tissues. In many diseases such as arthritis, inflammatory bowel disease, and asthma, the inflammatory process is not appropriately regulated. As a result, significant tissue dysfunction (leading to the generation of the symptoms that typify these diseases), and tissue re-structuring occur (e.g., fibrosis) that can further impair tissue function.

[0008] The very first event of the inflammatory reaction, the “silent phase” is based upon the reaction of cells and resident cells of the damaged tissue. These resident cells release mediators, such as nitric oxide (NO), histamine, kinins, cytokines, or prostaglandins. The release of these vasomotor mediators from resident cells leads to the second phase of the inflammatory reaction: the vascular phase. Vascular tone and permeability are regulated by an endothelial-dependent mechanism involving the release of nitric oxide. Certain agonist signal to sensory afferents, including the release of neuropeptides which are known to act on vascular beds to induce vasodilation and increased permeability. Increased vasodilatation and permeability provoke plasma leakage from the blood to the inflamed tissues, and facilitate the passage of leukocytes from the blood flow to the tissues.

[0009] The third phase of the inflammatory process is the cellular phase, which is characterized by the arrival of leukocytes circulating in the blood. In order to be recruited to the site of inflammation, circulating leukocytes roll onto the venular endothelial surfaces, adhere to the endothelium, and then transmigrate across the endothelial barrier. The process by which cells leave the blood stream and penetrate tissue parenchyma is known as extravasation. The events of rolling, adhesion, and transmigration (extravasation) are regulated by several cell adhesion molecules, known as CAMs and receptor molecules expressed by the endothelium and the leukocytes.

[0010] The inflammatory response to tissue damage may be of great value. By isolating the damaged area, mobilizing effector cells and molecules to the site, and—in the late stages—promoting healing, inflammation may protect the body. However, inflammation is more often associated with pain, injection and diseased states. Often the inflammatory response is out of proportion to stimulus which activated the response. The inflammatory process inevitably causes tissue damage and is accompanied by simultaneous attempts at healing and repair. Tissue destruction is caused by both the caustic agents and by the inflammatory process itself. The result can be more damage to the body than the agent itself would have produced. For example, all the many types of allergies and many of the autoimmune diseases are examples of inflammation in response to what should have been a harmless, or at least a noninfectious, agent. Some examples of chronic inflammatory diseases include Asthma, Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), Systemic Lupus Erythematosus (SLE), psoriasis, and Chronic Obstructive Pulmonary Disease (COPD). In many of these cases, the problem is made worse by the formation of antibodies against self antigens or persistent antigens from smoldering infections. Additionally, any disease with an inflammatory component may be treated by a better understanding of the immune system and the disease-fighting responses to toxins, injury, viruses and bacteria. Recently, it has been shown that inflammation has been link to cardiovascular diseases.

[0011] Traditional therapeutics for the treatment of inflammation include anti- inflammatory agents and steroids. Inappropriate inflammation can be treated with steroids like the glucocorticoid cortisol, nonsteroidal anti-inflammatory drugs (NSAIDs) like aspirin and ibuprofen, and a number of proteins produced by recombinant DNA technology.

[0012] The NSAIDs achieve their effects by blocking the activity of cyclooxygenase. The body produces several different forms of cyclooxygenase (COX), including COX-1, which is involved in pain, clotting, and protecting the stomach; COX-2, which is involved in the pain produced by inflammation. Most of the NSAIDs inhibit both COX-1 and COX-2. However, some newer drugs, the so-called COX-2 inhibitors, such as rofecoxib (Vioxx®) and celecoxib (Celebrex®) are much more active against COX-2 than COX-1.

[0013] Recombinant DNA and monoclonal antibody technology have produced some new therapies that are being enlisted in the battle against damaging inflammation. Examples of these therapeutics include: (1) an IL-1 antagonist that binds and inactivates the IL-1 receptor; (2) etanercept (Embrel®), which a soluble version of the TNF-α receptor, which binds TNF-α preventing it from carrying out its many inflammatory actions; (3) recombinant protein C, which helps the body dissolve the tiny clots that are triggered during inflammation; (4) infliximab (Remicade®), which is a monoclonal antibody that binds to TNF-α, particularly promising against some inflammatory diseases such as rheumatoid arthritis; and (5) the antibody natalizumab (Antegren®; Biogen Inc., Cambridge, Mass.), which functions by blocking the adhesion of immune cells to blood vessels and can inhibit movement of immune cells from the blood into the brain. Several of these therapies carry a severe risk of allowing infections to develop. In fact, the more powerful the anti-inflammatory agents (e.g., glucocorticoids), the greater the risk of infection.

[0014] Reducing inflammation and regulating the inflammatory response also is beneficial in the prevention of various cancers and other cell conditions. Chronic inflammation is a recognized cause of cancer. For example, liver cancer is often the sequel to years of inflammation caused by infection hepatitis B or C infection. Lung cancer often is the end stage of years of chronic inflammation caused by inhaled irritants, such as tobacco smoke. Cervical cancer can follow chronic infection and inflammation caused by papilloma viruses and chlamydia. Similarly, bladder, colon, pancreas, stomach, and other cancers may be the final stage of years of inflammation.

[0015] The strong link between chronic inflammation and cancer should not be surprising considering that the reactive oxygen species (ROS) liberated during inflammation are powerful DNA-damaging agents. Additionally, increased mitosis in response to inflammation puts more cells at risk of mutations as they replicate their DNA during S phase. Furthermore, apoptosis, the programmed death of damaged cells, is suppressed in inflamed tissue. So precancerous cells with genetic mutations, which should have committed suicide, continue and ultimately develop into cancerous cells. Therefore, the regulation of the inflammatory response including reducing inflammation in tissues is beneficial to the prevention of various cancers.

[0016] The penetration of inflammatory cells into and through the parenchyma of tissues is intimately involved in the pathophysiology of numerous disease, injury and inflammatory states. The ability to prevent penetration of inflammatory cells into tissue has beneficial therapeutic effects. It is known that reduction of tissue penetrating inflammatory cells has been accomplished to a degree with steroid, anti-integrin and other anti-inflammatory compounds. The access of cells through the blood vessel wall and into tissues is, in part, dependent upon the extracellular matrix, receptor molecules, CAMs, the cell membranes of the vascular endothelium and the migrating cell. Interfering with one or more of these signals or receptors disrupts the recruitment of inflammatory cells to the tissue. This disruption thus leads to reduced inflammation in the tissue.

SUMMARY OF THE INVENTION

[0017] It has been found that the use of enzymes capable of cleaving chondroitin sulfate proteoglycans are effective in altering the extravasation of cells associated with inflammation, such as macrophages from blood vessels into tissue.

[0018] One embodiment of the present invention is a method for modifying access of cells to extravascular spaces and regions comprising administering to a patient an enzyme that cleaves chondroitin sulfate proteoglycans. Another embodiment of the present invention is a method of reducing penetration of cells associated with inflammation into tissue of a patient. In several methods of the invention, an enzyme selected from the group consisting of chondroitinase ABCType I, chondroitinase ABCType II, chondroitinase AC, chondroitinase B, Hyaluronidase 1, Hyaluronidase 2, Hyaluronidase 3, Hyaluronidase 4, and hyaluronoglucosaminidase, fragments thereof, and combinations thereof are used. In several embodiments of the invention enzymes directed against integrins containing chondroitin sulfate proteoglycan expressed on the cell surface of cells are used.

[0019] Another embodiment of the present invention is a method for inhibiting extravasation of cells associated with inflammation from blood vessels comprising administering to a patient an enzyme that cleaves chondroitin sulfate proteoglycans. In several embodiments of the invention an enzyme prevents cells selected from the group selected from the group consisting of white blood cells, leukocytes, neutrophils, eosinophils, basophils, lymphocytes, B-cells, T-cells, monocytes, and macrophages cells from leaving the blood stream.

[0020] Another embodiment of the invention is a method of treating inflammation in a patient comprising administering to the patient an enzyme that cleaves chondroitin sulfate proteoglycans. In several embodiments of the present invention, inflammation is associated with disease or injury, such as chronic inflammatory diseases and central nervous system disease.

[0021] Another embodiment of the invention is a method of preventing inflammation in a patient comprising administering to the patient an enzyme that cleaves chondroitin sulfate proteoglycans.

[0022] Another embodiment of the present invention is a method of treating inflammation of a patient comprising extracting cells associated with inflammation from a patient, subjecting the cells to an enzyme that cleaves chondroitin sulfate proteoglycans ex vivo to modify the cells, and administering the modified blood cells into the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] For a fuller understanding of the nature and advantages of the present invention, reference should be had to the following detailed description taken in connection with the accompanying drawings, in which:

[0024]
FIG. 1 is schematic drawing of the four major families of cell adhesion molecules.

[0025]
FIG. 2 is a schematic drawing of the sequence of cell-cell interactions leading to tight binding of leukocytes to activated endothelial cell and subsequent extravasation.

DETAILED DESCRIPTION

[0026] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0027] It must also be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to an “enzyme” is a reference to one or more enzymes and equivalents thereof known to those skilled in the art, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0028] It has been found that the administration of certain enzymes to the blood stream disrupts the mechanism by which cells move from the blood stream to the tissues. When cells, primarily macrophages and other immunological cells or cells associated with inflammation, are signaled to enter a tissue, extravasation ensues. The cells circulating in the blood stream migrate to the blood vessel walls where they encounter specific receptors, known as cell adhesion molecules. Through specific receptor based adhesion to the vessel wall, the cells roll along the wall until they encounter an endothelial cell junction where they can squeeze out of the vessel and enter the extravascular space. Some of these adhesion molecules or receptors contain carbohydrate chains made up of chondroitin sulfates. Digestion of these chains with enzymes interrupts extravasation. Several methods are provided for the regulation, prevention, treatment and suppression of the inflammatory response comprising administering enzymes that digest chondroitin sulfates. Also provided are methods of treating and preventing infection and diseases associated with inflammatory response.

[0029] The process of “extravasation” is known as the transmigration of cells, such as leukocytes, from a blood vessel into the extravascular space, and may further include migration into surrounding tissue. As used herein the term “leukocyte” is used to refer to the class of cells associated with inflammation, which may also be defined as any of the various blood cells that have a nucleus and cytoplasm. Also known as white blood cells, leukocytes include neutrophils, eosinophils, basophils, lymphocytes, such as B-cells, T-cells, monocytes and macrophages. Four types of leukocytes are particularly important in immune defense, including neutrophils, which release several antibacterial proteins; monocytes, which are the precursors of macrophages that engulf and destroy foreign particles, and T and B lymphocytes, which are the antigen-recognizing cells of the immune cells.

[0030] The term “therapeutic agent” used in connection with the inflammatory response therefore means an agent that functions as a blocker, suppressor, regulator or modulator of the inflammatory response. For example, several therapeutic enzymes described herein function as blockers, suppressors, regulators or modulators of the inflammatory response.

[0031] Inflammation is a defense reaction caused by tissue damage or injury, characterized by redness, heat, swelling, and pain. The inflammatory response involves the dilation of capillaries to increase blood flow; microvascular structural changes and escape of plasma proteins from the bloodstream; and leukocyte transmigration through endothelium and accumulation at the site of injury. As explained above, the ability to control the inflammatory response is beneficial in reducing inflammation associated with disease or infection and is also beneficial in treating a number of diseases with an inflammatory component.

[0032] Several types of leukocytes participate in the defense against infection caused by foreign invaders (e.g., bacteria and viruses) and tissue damage due to trauma or inflammation. To fight infection and clear away damaged tissue, these cells must move rapidly from the blood stream, where they circulate as unattached, relatively quiescent cells, into the underlying tissue at sites of infection, inflammation or damage.

[0033] Extravasation requires the successive formation and breakage of cell-cell contacts between leukocytes in the blood and endothelial cells lining the vessels. This successive formation and breakage is also known as the “leukocyte adhesion cascade”, which is described as a sequence of adhesion and activation events that ends with extravasation of the leukocyte, whereby the cell exerts its effects on the inflamed tissue site. Some sources have described the cascade as having at least five steps of adhesion termed capture, rolling, slow rolling, firm adhesion, and transmigration. For example, R. O. Hynes and A. Lander, 1992, Cell 68:3030, herein incorporated by reference, describes the sequence in five steps. See also, Jung U, Norman K E, Scharffetter-Kochanek K, Beaudet A L, Ley K, Transit Time of Leukocytes Rolling through venules Controls Cytokine-induced Inflammatory Cell Recruitment In Vivo. Journal of Cinical Investigation. 1998; 102:1526-1533, herein incorporated by reference. Each of these five steps appears to be necessary for effective leukocyte recruitment, therefore blocking any of the five can severely reduce leukocyte accumulation in the tissue. These steps are not phases of inflammation, but represent the sequence of events from the perspective of each leukocyte. At any given moment, the events of capture, rolling, slow rolling, firm adhesion and transmigration occur in parallel, involving different leukocytes in the same microvessels.

[0034] Cells in tissue can adhere directly to one another (cell-cell adhesion) through specialized integral membrane proteins called cell-adhesion molecules (CAMs). There are four principal classes of CAMs: cadherins (or mucins), the immunoglobulin (Ig) superfamily, selectins, and integrins. See for example, “Molecular Cell Biology”, 5th Edition, Lodish, et al. Chpt 6, “Integrating Cells into Tissue”, herein incorporated by reference in its entirety, which describes the general categories of CAMs. FIG. 1 is a schematic drawing of these four major categories of CAMs. CAMs mediate through their extracellular domains adhesive interactions between cells of the same type or between cells of a different type. The homophilic interactions are coordinated by the cadherins and the Ig-superfamily. While the heterophilic interactions occur with integrins and selectins.

[0035] The structures of the various CAMs vary according to function. The selectins, shown as dimers in FIG. 1, contain a carbohydrate-binding lectin domain that recognizes specialized sugar structures on glycoproteins (shown here) and glycolipids on adjacent cells.

[0036] Selectins mediate leukocyte-vascular cell interactions. For example, P-selectin is localized to the blood-facing surface of endothelial cells. Selectins contain a Ca2+-dependent lectin domain, which is located at the distal end of the extracellular region of the molecule and recognizes oligosaccharides in glycoproteins or glycolipids. For example, the primary ligand for P- and E-selectins is an oligosaccharide called the sialyl Lewis-x antigen, a part of longer oligosaccharides present in abundance on leukocyte glycoproteins and glycolipids. P-selectin exposed on the surface of activated endothelial cells mediates the weak adhesion of passing leukocytes. Because of the force of the blood flow and the rapid “on” and “off” rates of P-selectin binding to its ligands, these trapped leukocytes are slowed but not stopped and literally roll along the surface of the endothelium. Among the signals that promote activation of the endothelium are chemokines, a group of small secreted proteins produced by a wide variety of cells, including endothelial cells and leukocytes.

[0037] All known selectin ligands are transmembrane glycoproteins which present oligosaccharide structures to the selectins. Transient bond formations between the selectins and their ligands mediate the early steps of the adhesion cascade. All three selectins can recognize glycoproteins and/or glycolipids containing the tetrasaccharide sialyl-Lewisx (sialyl-CD15). This tetrasaccharide is found on all circulating myeloid cells and is composed of sialic acid, galactose, fucose, and N-acetyl-galactosamine.

[0038] PSGL-1 (P-selectin Glycoprotien Ligand) has been characterized as a ligand for P-selectin. PSGL-1 is a glycoprotein expressed on blood cells and contains the sialyl-Lewisx tetrasaccharide. Another P-selectin ligand is CD24, which appears to be important for tumor cell binding to P-selectin. For L-selectin, four possible ligands have been identified: GlyCAM-1 (Glycosylation-Dependent Cell Adhesion Molecule), CD34, and MAdCAM-1 (Mucosal Addressin Cell Adhesion Molecule), and PSGL-1. Evidence suggests that both sulfate and sialic acid in an α(2,3) linkage are essential to L-selectin ligand activity. Although specific ligands for E-selectin are not yet known, candidate molecules include fucosylated, sialyated oligosaccharides found as components of glycoprotein and glycolipid molecules.

[0039] The P-, E-, and L-selectins have long chains of short consensus repeats, such as SRC, also found in complement regulating proteins. These long chains reach out from the cell surface and at the end of the SRC chain there is a EGF domain and finally the C-type lectin domain. The S-type selectins also known as gallectins, bind to gallactose. The collectins have a collagen like tail, such as mannose binding protein.

[0040] Other CAMs include selectin ligands. The sugars that the selectins bind to are mainly sialylated and fucosylated tetrasaccharide antigens called Lewis sugars. Examples include Lex (LPE), 3-sulpho Lex (E) and 3-sialo Lex (L), and sulphated tyrosines (of PSGL) (P). The N-linked sugars are usually bi- or teraantennary. The conformation and linkage of the sugars are very important for the overall shape (e.g., a 1-3 and a 1-4 linkage can cause the sugars to look completely different).

[0041] Integrins are a large family of heterodimeric transmembrane glycoproteins that attach cells to extracellular matrix proteins. Integrins contain large (α) and small (β) subunits of sizes 120-170 kDa and 90-100 kDa, respectively. For tight adhesion to occur between activated endothelial cells and leukocytes, β2-containing integrins on the surfaces of leukocytes also must be activated by chemokines or other local activation signals such as platelet-activating factor (PAF). Activated integrins on leukocytes then bind to each of two distinct IgCAMs on the surface of endothelial cells: ICAM-2, which is expressed constitutively, and ICAM-1. ICAM-1, whose synthesis along with that of E-selectin and P-selectin is induced by activation, does not usually contribute substantially to leukocyte endothelial cell adhesion immediately after activated, but rather participates at later times in cases of chronic inflammation. The resulting tight adhesion mediated by the Ca2+ independent integrins-ICAM interactions leads to the cessation of rolling and to the spreading of leukocytes on the surface of the endothelium. The adhered cells then move between adjacent endothelial cells and into the underlying tissue. Integrins contain binding sites for divalent cations Mg2+ and Ca2+, which are necessary for their adhesive function. Mammalian integrins form several subfamilies sharing common β subunits that associate with different a subunits. Integrin ligands include NCAM and Fibronectin. These ligands have Ig domains and can be anchored to membrane by GPI anchor or TM helix. Other CAMs include clustered determinants, known as the CD molecules, which mostly have Ig domains. Other CAMs include pMHC and TCR; proteoglycans, having long unbranched glycans; and growth factors (FGF, EGF and NGF), which bind to haparan sulphate to be recognized by receptor.

[0042] The activated lymphocyte has many glycoproteins and cell adhesion molecules on its surface, eg TCR, CD43 (hyperglycosylated spacer) and ICAM. These molecules are important for the recognition of foreign antigens, activation of APCs and toxic effects on other cells. The lymphocytes interact with the APC via specific interactions (TCR) and non-specific interactions (CD2—LFA3 and ICAM-1—LFA1).

[0043] Upon infection of tissue, cytokines are secreted that make the endothelial cells express E- and P-selectins on their surface. These will interact with glycoproteins on the surface on the leukocytes in the blood stream, making the cells slow down by a rolling movement. The target of P-selectin is PSGL on the surface of the leukocyte, having a heavily O- and N-linked glycoprotein tyrosine-sulphate content. The target of E-selectin is unknown. The leukocyte also has a selectin on its surface (L). The targets of this are CD34, MAdCAM-1 (both with IgSF domains and lots of N- and O-linked glycosylation) and GlyCAM-1 (soluble inhibitor of the mucin family). When the leukocyte is stopped it will exrtavasate aided by chemoattractants (such as RANTES) and integrin adhesion to fibronectin of epithelia.

[0044] Lymphocytes are capable of a remarkable level of recirculation, continuously moving through the blood and lymph to various lymphoid organs. About 40-50% of the lymphocytes will be recruited to the spleen, about 38-48% will be recruited to various peripheral lymph nodes and the remainder will be recruited to various mucosal-associated lymphoid tissues (MALT) or SALT (skin associated lymphoid tissues). In order for recirculating lymphocytes to enter various lymphoid organs or inflammatory-tissue spaces, the lymphocytes must adhere to a pass between the endothelial cells lining the walls of blood vessels by extravasation. Vascular endothelial cells possess cell adhesion molecules on their surface. Some of these are expressed constitutively and others are inducible (expressed primarily in response to certain cytokines produced during an inflammatory response).

[0045] Selectins are membrane glycoproteins with a distal lectin-like domain which enables the protein to bind to specific carbohydrate moieties. L-selectin is expressed on all leukocytes while E- and P-selectin are expressed on vascular endothelial cells. Cadherins (or mucins) are serine- and threonine-rich proteins that are heavily glycosylated. They present sialyated carbohydrate ligands to selectins. For instance, L selectin on leukocytes recognizes sialylated carbohydrates on two mucin-like molecules (CD34 and GlyCAM-1) expressed on certain endothelial cells of lymph nodes. PSGL-1 on neutrophils interacts with E and P-selectin on inflamed endothelium. Other CAMs include the category known as Integrins, which generally have a αβ-heterodimers structure. Integrins are integral membrane proteins expressed by leukocytes which facilitate adherence to the vascular endothelium. Different integrins, including LFA-1, are expressed by different populations of leukocytes, allowing these cells to bind to different CAMs belonging to the Ig family expressed along the vascular endothelium. LAD (leukocyte adhesion deficiency) is an autosomal recessive disease characterized by recurrent bacterial infections and impaired wound healing. Abnormal synthesis of the B chain in integrins characterize the LAD disorder. As a result of the abnormal synthesis, leukocytes cannot extravasate. The Ig superfamily of CAMs are proteins which contain at least one Ig-domain such as ICAM-1,2,3 and VCAM on vascular endothelial cells. The protein MAdCAM has both Ig and mucine-like domains expressed by mucosal endothelial cells. It directs lymphocytes into the mucosa. It binds to integrins via its immunoglobulin-like domain and to selectins via its mucin-like domain.

[0046] During an inflammatory response cytokines and other chemicals act on local blood vessels (dilation and increased permeability) to show an increased expression of CAMs. When this happens the vascular endothelium is said to be inflamed or activated. Neutrophils are among the first cells to bind and extravasate. This process is similar for monocytes but better studied for neutrophils. Extrasvastion of neutrophils includes: rolling chemoattractant activating stimulus arrest and adhesion and transendothelial migration rolling. Initially low affinity selectin-carbohydrate interaction is observed during inflammation. E and P selectins expressed in higher levels on vascular endothelial cells bind to mucin-like cell adhesion molecules on the neutrophil membrane. As the neutrophil rolls, it is activated by various chemoattractants that are localized on the endothelial cell surface or secreted locally by cells involved in the inflammatory process. Chemoattractive cytokines (or chemokines) include IL-8, macrophage inflammatory protein (MIP), PAF, and C5a. Adhesion binding to chemokines triggers G protein activated signal transduction pathway, which induces a conformational change in integrins, increasing their affinity for Ig superfamily CAMs on the endothelium. Extravasation directed migration of the neutrophil through interendothelial junctions has not been well characterized.

[0047] Antigen specific T cells are known to disappear from the circulation in less than 48 hours. Recruitment of T cells occurs especially at locations called High Endothelial Venules (HEVs), which are plump, cuboidal cells present in all secondary lymphoid sites except the spleen. Appearance or expression of HEVs dependent upon antigen stimulation. These HEVs express on their surface special CAMs. Antigen stimulation results in an increased expression of these CAMs thereby facilitating extravasation. Germ-free animals do not have HEVs. Recirculating lymphocytes, monocytes, and granulocytes bear adhesion-molecule receptors for: CAMs of the selectin family, the mucin family (GlyCAM-1 and CD34), and the Ig superfamily (ICAM-1-3, VCAM-1, and MAdCAM-1). When distributed in a tissue-specific way these molecules are termed: vascular adressins. The corresponding receptors on endothelial cells have come to be called homing receptors. The homing receptors recruit different subsets of lymphocytes, which migrate differentially into different tissues based on the expression of the homing receptors and also due to chemokine secretion patterns and differential responses. For example, MIP (macrophage inhibitory protein) preferentially attracts naive T cells. Monocyte chemoattractant protein (MCP) and RANTES preferentially attract memory T cells. Naive lymphocytes initial attachment to HEVs are generally mediated by binding of homing receptor L-selectin to vascular adressins such as GlycCAM-1 and CD34 on HEVs. If the lymphocytes encounter antigens within the node, they become activated and enlarge into lymphoblasts and are retained for at least 48 hours. The lymphoblasts experience rapid proliferation and differentiation to effector and memory cells which then leave the node. Effector and Memory Lymphocytes tend to home to regions of infection. Memory lymphocytes are attracted to the type of tissue where they originally encountered the antigen. These cells express high levels of certain homing receptors, this allows tissue-specific homing behavior.

[0048] Table 1 below summarizes the circulating cellular and endothelial interactions of several embodiments of the present invention.

[0049] The leukocyte cascade sequence is illustrated at FIG. 2. In the absence of inflammation or infection, leukocytes and endothelial cells lining blood vessels are in a resting state. Inflammatory signals released in areas of inflammation or infection activate resting

1EndothelialcomponentCell componentGlycosylationL-SelectinCounter receptors onCounter receptors O-linked(cd62L)leukocytesglycosylationPSGL-1 (cd162)MadCAM-1CD34GlyCAM-1P SelectinPSGL-1Counter receptors O-linkedCD-24glycosylationE SelectinPSGL-1 (hypothesized)Counter receptors O-linkedESL-1 (hypothesized)glycosylationBeta IntegrinsLFA-1, MAC-1Beta integrins glycosylatedICAM-1,2(Intercellular)PECAM-1(Platelet, endoth)VCAM-1(Vascular)NeurothelinNeurothelin glycosylated(cd147)CD151Glycosylated(Transmembrane4 superfamilyprotein:PETA-3, SFA-1)BST-1Glycosylated(Bone marrowstromal cellantigen-1)CD18, 29, 49VLA-4LPAM-2I-selectinCD34: FLT-3,CD34-GlycosylatedFLT-3Ligand

[0050] endothelial cells to move vesicle-sequestered selectins to the cell surface. The exposed selectins mediate loose binding of leukocytes by interacting with carbohydrate ligands on leukocytes. Activation of the endothelium also causes synthesis of platelet-activating factor (PAF) and ICAM-1, both expressed on the cell surface. PAF and other usually secreted activators, including chemokines, then induce changes in the shapes of the leukocytes and activation of leukocyte integrins such as αLβ2, which is expressed by T lymphocytes. The subsequent tight binding between activated integrins on leukocytes and CAMs on the endothelium (e.g., ICAM-2 and ICAM-1) results in firm adhesion and subsequent movement (extravasation) into the underlying tissue.

[0051] The selective adhesion of leukocytes to the endothelium near sites of infection or inflammation thus depends on the sequential appearance and activation of several different CAMs on the surfaces of interacting cells. It has been found that several CAMs and associated circulating leukocytes express carbohydrate chains comprising chondroitin sulfate proteoglycans (CSPG).

[0052] CSPGs are a family of proteoglycans composed of a core protein and covalently linked sulfated glycosaminoglycans. Each proteoglycan is determined by the glycosaminoglycan side chains. For CSPGs these side chains are made up of approximately 40 to 100 sulfated disaccharides composed of chondroitin 4, 6 and dermatan sulfates. The protein component of the CSPG is ribosomally synthesized and the glycosylation occurs in the endoplasmic reticulum and Golgi apparatus. The sugar chains are then sulfated at the 4 or 6 positions by several glycosaminoglycan sulfotransferases.

[0053] Disruption of the glycosylation interaction between CAMs and associated receptors in the extravasation cascade through targeting of carbohydrate chains. A disruption of cell-cell adhesion thus disrupts extravasation of leukocytes into tissue cells. It has been found that the use of enzymes directed against the CSPG carbohydrate chains allows for regulation and modification of extravasation processes. The enzyme may be directed against the CSPG carbohydrate chains of ligands expressed on circulating leukocytes. Thus, the leukocytes are not able to adhere to the integrins, mucins, or selectins localized on the blood-facing surface of endothelial cells. As described herein, the ligands for endothelial cell surface CAMs include the ligands for adhesion to the selectin family, the mucin family and the integrin family. Any ligand expressed on leukocytes containing CSPG carbohydrate chains may be targeted in the several treatments and methods of the present invention.

[0054] As used herein, a compound directed against CSPG is a molecule that can degrade CSPG chains, cleave CSPG, bind to CSPG, or inhibit other compounds or cells from adhering to CSPG. A non-limiting example of compounds directed against CSPG includes the chondroitinase enzymes. The administration of compounds directed against CSPG may be used to control extravasation of leukocytes, thus modification of the immune response is possible.

[0055] Thus the use of enzymes directed against CSPG may be used to prevent, treat and alleviate symptoms of inflammation and inflammatory states. The enzyme may also prevent, treat and alleviate symptoms of chronic inflammatory diseases and central nervous system disorders. The enzyme may be used to treat inflammation associated with pain, injection and diseased states. The enzyme may be used to prevent tissue damage that is associated with inflammatory processes. Several conditions may benefit from controlled immune response. The many types of allergies and many of the autoimmune diseases are examples of inflammation in response to what should have been a harmless, or at least noninfectious, agent. Some examples of chronic inflammatory diseases include Asthma, Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), Systemic Lupus Erythematosus (SLE), and Chronic Obstructive Pulmonary Disease (COPD). The use of enzymes directed against CSPG may also be used to regulate the inflammatory state associated with one or more disease selected from the group consisting of central nervous system disorders, central nervous system diseases, spinal cord injury, and cardiovascular diseases.

[0056] Inflammatory diseases, autoimmune diseases, and diseases with an inflammatory component may also include Multiple Sclerosis, Meningitis, Encephalitis, Rheumatoid arthritis, Osteo arthritis, Lupus, Wegener's granulomatosis, Inflammatory bowel disease: Crohn's colitis, ulcerative colitis, Asthma, Chlamydia infections, Syphilis, Thyroiditis, Temporal arteritis, Polymyalgia rheumatica, Ankylosing spondylitis, Psoriasis, Vasculitiditis such as: temporal arteritis, Takayasu arteritis, syphilitic aortitis, infectious aneurisms, atherosclerotic aneurisms, inflammatory abdominal aortic aneurysms, polyarteritis nodosa, Kawasaki disease, Churg-Strauss, hypersensitivity vasculitis, Buerger's disease, mesenteric inflammatory veno-occlusive disease, phlebitis, thrombophlebitis, Churg-Strauss, primary angiitis of the CNS, drug induced vasculitis, any secondary arteritis or venulitis, Gout, Pseudogout, Sarcoidosis, Sjogren's Syndrome, Myelitis, Salpingitis of any etiology, Uveitis, Pelvic Inflammatory Disease, Glomerulonephritis of any etiology, Goodpasture's syndrome, Pericarditis, Myocarditis, Endocarditis, and Pancreatitis.

[0057] Suitable enzymes that are directed against CSPGs include chondroitinase ABC type I, chondroitinase ABC Type II, chondroitinase AC and Chondroitinase B or mammalian enzymes with chondroitinase-like activity such as hyaluronidasel, hyaluronidase 2, hyaluronidase 3, hyaluronidase 4, cathepsins, and ADAMTs, fragments thereof or mixtures thereof.

[0058] Chondroitinases are enzymes of bacterial origin that act on chondroitin sulfate, a component of the proteoglycans that are expressed in various CAMs and receptors associated with inflammatory response. Examples of chondroitinase enzymes are chondroitinase ABC, which may be produced by the bacterium Proteus vulgaris (P. vulgaris), and chondroitinase AC, which may be produced by A. aurescens. Chondroitinases ABC and AC function by degrading polysaccharide side chains in protein-polysaccharide complexes, without degrading the protein core.

[0059] It has now been found that the CSPG-degrading enzymes such as chondroitinase ABCTypeI, chondroitinase ABCTypeII, chondroitinase AC, chondroitinase B or mammalian enzymes with chondroitinase-like activity such as Hyal 1, Hyal 2, Hyal 3, and Hyal 4 are useful in controlling and/or inhibiting the effects of chondroitin sulfates and in developing therapeutics for the regulation, treatment and prevention of inflammation associated with various disease states.

[0060] Yarnagata et al. (J. Biol. Chem. 243:1523-1535, 1968) describe the purification of the chondroitinase ABC from extracts of P. vulgaris. This enzyme selectively degrades the glycosaminoglycans chondroitin-4-sulfate, dermatan sulfate, and chondroitin-6-sulfate (also referred to respectively as chondroitin sulfates A, B, and C which are side chains of proteoglycans) at pH 8 at higher rates than it degrades chondroitin or hyaluronic acid. P. vulgaris chondroitinase I migrates with an apparent molecular mass of about 110 kDa when resolved by SDS-PAGE. The products of the degradation are high molecular weight unsaturated oligosaccharides and an unsaturated disaccharide. However, chondroitinase ABC does not act on keratosulfate, heparin or heparitin sulfate.

[0061] Another chondroitinase, chondroitinase II, has also been isolated and purified from P. vulgaris, Chondroitinase II is a polypeptide of 990 amino acids with an apparent molecular mass by SDS-PAGE of about 112 kDa. Its molecular mass as determined by electrospray and laser desorption mass spectrometry is 111,772.+0.27 and 111,725.±−0.20 daltons, respectively. Chondroitinase II has an isoelectric point of 8.4-8.45. Its enzymatic activity is distinct from, but complementary to, that of chondroitinase I. Chondroitinase I endolytically cleaves proteoglycans to produce end-product disaccharides, as well as at least two other products which are thought to be tetrasaccharides, Chondroitinase II digests at least one of these tetrasaccharide products of chondroitinase I digestion of proteoglycan.

[0062] Chondroitinase AC and chondroitinase B are chondroitin lyase enzymes, which may be derived from various sources. Any chondroitinase AC or B may be used in the present method embodiments including, but not limited to chondroitinase AC (derived from Flavobacterium heparinum; T. Yamagata, H. Saito, O. Habuchi, S. Suzuki, J. Biol. Chem., 243, 1523 (1968)); chondroitinase AC II (derived for Arthobacter aurescens; K. Hiyama, S. Okada, J. Biol. Chem., 250, 1824 (1975), K. Hiyama, S. Okada, J. Biochem. (Tokyo), 80, 1201 (1976)); chondroitinase AC III (derived from Flavobacterium sp. Hp102; H. Miyazono, H. Kikuchi, K. Yoshida, K. Morikawa, K. Tokuyasu, Seikagaku, 61, 1023 (1989)); chondroitinase B (derived from Flavobacterium heparinum; Y. M. Michelaaci, C. P. Dietrich, Biochem. Biophys. Res. Commun., 56, 973 (1974), V. M. Michelaaci, C. P. Dietrich, Biochem. J., 151, 121 (1975), K. Maeyama, A. Tawada, A. Ueno, K. Yoshida, Seikagaku, 57, 1189 (1985)); and chondroitinase B (derived from Flavobacterium sp. Hp102; H. Miyazono, H. Kikuchi, K. Yoshida, K. Morikawa, K. Tokuyasu, Seikagaku, 61, 1023 (1989)). Suitable chondroitinase AC and chondroitinase B are commercially available from Seikagaku America, Falmouth, Mass., USA. Additionally, the enzymes may be produced by the methods disclosed in U.S. Pat. No. 6,093,563 by Bennett et al., the disclosure of which is incorporated herein. Chondroitinase I and chondroitinase II are exo- and endo-lyases respectively which cleave both chondroitin and dermatan sulfates (Hamei et al 1997). Mammalian enzymes with chondroitinase-like activity have been identified. For example, certain hyaluronidases such as Hyal 1, Hyal 2, Hyal 3, and Hyal 4 also degrade CSPGs and can be used in the present invention.

[0063] Hyaluronidase is an enzyme that catalyzes the breakdown of hyaluronic acid in the body, thereby increasing tissue permeability to fluids. It is also known as a spreading factor. The hyaluronidase (endo-N-acetylhexosaminidase) enzyme acts on not only hyaluronan but also chondroitin, chondroitin 4-sulfate and chondroitin 6-sulfate. Besides the hydrolytic reaction, hyaluronidase is also known to catalyze the reverse reaction, transglycosylation.

[0064] Hyaluronidase may be purified from mammalian testicular and spleen tissue (common sources are bovine and ovine testes), venom from bees and snakes, and many bacteria. The enzyme has a molecular weight of 55,000 daltons. The function of this enzyme is to catalyze the depolymerization of mucopolysaccharides, hyaluronic acid, and the chondroitin sulfates A and C. This enzyme is commonly used in medical practice to encourage intradermal drug absorption in patients and in increasing the efficiency of local anesthetics.

[0065] An enzyme that is directed against both hyaluronic acid and chondoritinase sulfate is also suitable in the several method embodiments of the present invention. Treponema denticola, Treponema vincentii and Treponema socranskii produce an enzyme that hydrolyses hyaluronic acid and chondroitin sulfate. The secreted enzyme is specifically inhibited by gold sodium thiomalate and anti-bee-venom antibodies. The affinity-purified extracellular enzyme of T. denticola contains a single molecular species with a molecular mass of 59 kDa. Since it hydrolyses both HA and CS, this enzyme is termed a hyaluronoglucosaminidase (HGase).

[0066] Another embodiment of the present invention is a method of treating inflammation in a patient comprising extracting circulating cells from a patient, subjecting the cells to an enzyme that cleaves the expressed chondroitin sulfate proteoglycan ex vivo to modify the cells, and administering the modified blood cells into the patient. Therefore, the use of enzymes directed against CSPG described herein may also be directed to ex vivo treatments.

[0067] Extraction of cells may be accomplished by a variety of methods including, but not limited to, intravenous blood withdrawal, transfusion, dialysis, bypass, organ transplant and other similar methods that result in removal of cells from the body. Administration of the cells may be accomplished by the same methods used to extract the cells, including, but not limited to, intravenous blood withdrawal, transfusion, dialysis, bypass, organ transplant and the like.

[0068] A circulating leukocyte with ligands expressed on its surface containing carbohydrate chains may be extracted from a patient and modified ex vivo by the presently described enzymes. Extraction may be accomplished by blood draw, transfusion, dialysis, bypass, or organ transplant. As described, the enzymes would modify the carbohydrate chains, specifically any compound directed against CSPG would be suitable to modify the extracted leukocytes. Once modified, the leukocytes may be reintroduced into a patient's blood stream. Modified leukocytes will be incapable of adhering to endothelial expressed selecting, mucins, and integrins. Timing of such extraction and reintroduction into the bloodstream may be optimized by observing the inflammatory response and the appearance of leukocytes in the blood stream, once said cells are signaled to specific sites of injury or infection. As a result, extravasation of leukocytes into tissue may be regulated, prevented, reduced, or controlled. Such regulation may be used in methods and treatments as directed to control and treat inflammatory response and diseases with an inflammatory component.

[0069] Enzyme activity can be stabilized by the addition of excipients or by lyophilization. Stabilizers include carbohydrates, amino acids, fatty acids, and surfactants and are known to those skilled in the art. Examples include carbohydrate such as sucrose, lactose, mannitol, and dextran, proteins such as albumin and protamine, amino acids such as arginine, glycine, and threonine, surfactants such as TWEEN® and PLURONIC®, salts such as calcium chloride and sodium phosphate, and lipids such as fatty acids, phospholipids, and bile salts. The stabilizers are generally added to the protein in a ration of 1:10 to 4:1, carbohydrate to protein, amino acids to protein, protein stabilizer to protein, and salts to protein; 1:1000 to 1:20, surfactant to protein; and 1:20 to 4:1, lipids to protein. Other stabilizers include high concentrations of ammonium sulfate, sodium acetate or sodium sulfate, based on comparative studies with heparinase activity. The stabilizing agents, such as the ammonium sulfate or other similar salt, are added to the enzyme in a ratio of 0.1 to 4.0 mg ammonium sulfate/IU enzyme.

[0070] Enzymes may be administered intravenously, through local or systemic injection. Systemic administration is preferable for greater control of application. Other administrations include subcutaneous, transdermal, transmucosal, and enteric coating formulations. The enzymes, singularly or in combination, can be mixed with an appropriate pharmaceutical carrier prior to administration. Examples of generally used pharmaceutical carriers and additives are conventional diluents, binders, lubricants, coloring agents, disintegrating agents, buffer agents, isotonizing agents, preservants, anesthetics and the like. Specifically pharmaceutical carrier that may be used are dextran, sucrose, lactose, maltose, xylose, trehalose, mannitol, xylitol, sorbitol, inositol, serum albumin, gelatin, creatinine, polyethylene glycol, non-ionic surfactants (e.g. polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hardened castor oil, sucrose fatty acid esters, polyoxyethylene polyoxypropylene glycol) and similar compounds.

[0071] A pharmaceutical carrier may also be used. Suitable carriers include polyethylene glycol and/or sucrose, polyoxyethylene sorbitan fatty acid esters, polyethylene sorbitan monooleate, and the like, and combinations thereof.

[0072] The dose will also vary depending on the manner of administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician. Dosage levels of the therapeutic CSPG enzymes for human subjects range between about 0.001 mg per kg and about 100 mg per kg per patient per treatment.

[0073] The treatment regimens of the present invention may be carried out by a means of administering chondroitinase ABCTypeI, chondroitinase ABCTypeII, chondroitinase AC and chondroitinase B or mammalian enzymes with chondroitinase-like activity such as Hyal1, Hyal2, Hyal3, and Hyal4 in vivo or ex vivo. In vivo administration may include systemic intravenous administration to the area of inflammation. The mode of administration, the timing of administration and the dosage are carried out such that the inflammation is prevented or reduced by modifying extravasation of cells. The treatments of the present disclosure deliver an effective amount of chondroitinase ABCTypeI, chondroitinase ABCTypeII, chondroitinase AC and chondroitinase B or mammalian enzymes with chondroitinase-like activity such as Hyal 1, Hyal 2, Hyal 3, and Hyal 4, or fragments or combinations thereof to the injured site. The term “effective amount” means an amount sufficient to degrade the CSPGs or an amount sufficient to modify extravasation of cells. The effective amount of chondroitinase can be administered in a single dose or a plurality of dosages.

[0074] Although it is to be understood that the dosage may be administered at any time, in one embodiment, the dosage is administered within hours of the signaling of inflammatory response, or as soon as is feasible. In another embodiment, the dosage is administered to a mammal in one, two or a plurality of dosages; such dosages would be dependant on the severity of the injury and the amount of CSPGs present. Where a plurality of dosages is administered, they may be delivered on a daily, weekly, or bi-weekly basis. The delivery of the dosages may be by means of catheter or syringe. Alternatively, the treatment can be administered during surgery to allow direct application to the site of inflammation or injury. Subject to the judgment of the physician, a typical therapeutic treatment includes a series of doses, which are usually administered concurrent with the monitoring of clinical endpoints.

[0075] The therapeutic CSPG enzymatic agents may be administered by methods known in the art, such as by bolus injection, intravenous delivery, continuous infusion, or sustained release formulation, such as implants and the like.

[0076] Formulations suitable for injection are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Such formulations include antibacterial preservatives, buffers, solubilizers, antioxidants, and other pharmaceutical adjuncts. Such formulations must be sterile and non-pyrongenic, and generally will include purified therapeutic porcine B7-1 agents in conjunction with a pharmaceutically effective carrier, such as saline, buffered (e.g., phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions, and the like. The formulations may contain pharmaceutically acceptable auxiliary substances as required, such as, tonicity adjusting agents, wetting agents, bactericidal agents, preservatives, stabilizers, and the like.

EXAMPLES

Example 1

Toxicity Studies

[0077] Female Long Evans rats from Charles River Laboratories, weighing approximately 210 grams were housed in the Acorda Animal Care Facility for 5 days prior to injection to ensure health and weight stability. Rats were anesthetized with isoflurane and injected i.v. via tail veins with chondroitinase ABCI (Seikagaku; Cat number 100332, lot number E02201). Animals were injected with either 0, 0.2, 0.775 or 7.775 mg/kg with solutions containing 0, 0.2, 0.775 and 7.775 mg/ml, respectively in Hank's balanced salt solution.

[0078] Additionally, toxicity studies were conducted for Intrathecal (IT) catheter administration. Intrathecal catheters were placed in 16 normal, un-injured female rats at about the T13/L1 vertebral junction for delivery of chondroitinase. Catheters were fed rostrally to rest at the T9/T10 level to simulate previous chondroitinase studies. Twenty-four hours after intrathecal catheter placement animals were dosed with 0, 0.06, 0.6 or 6.0 Units of Acorda chondroitinase ABCI (100 Units/milligram) in 20 microliters of artificial cerebrospinal fluid over a 20 minute period.

[0079] Animals were observed for 24 hours or 7 days and their weights and temperatures were followed. Animals were monitored for pain and distress and body weights were acquired daily. No overt reactions were observed during or immediately after chondroitinase ABCI dosing. No swelling, inflammation, bruising or necrosis was noted at the injection site for the IV experiment. No changes in body temperature were observed in animals treated via the IT route. No alterations in feeding, grooming or vocalizations were noted. Animals were assessed for motor behavior in an open pool. No abnormalities were noted by the animal care staff or behavioral specialists. Animals displayed no signs of joint tenderness or swelling. There were no significant differences in weight change between the treatment groups. The results demonstrate that chondroitinase ABCI treatment is not associated with acute toxicity using IV doses. Additionally, Chondroitinase appears safe in single intrathecal (I.T.) doses.

[0080] What has been described and illustrated herein are embodiments of the invention along with some of their variations. The terms, descriptions and figures used herein are set forth by way of illustration only and are not meant as limitations. Those skilled in the art will recognize that many variations are possible within the spirit and scope of the invention, which is intended to be defined by the following claims and their equivalents in which all terms are meant in their broadest reasonable sense unless otherwise indicated.

Methods of reducing extravasation of inflammatory cells

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