Disease-targeted biomarker panels based on the multifactor ontology of glycocalyx disruption

Abstract

The present disclosure provides biomarkers, as well as related compositions, kits, and methods, useful as companion diagnostics for detecting glycocalyx-based diseases amenable to treatment with compounds designed to improve the state of the glycocalyx and / or reduce inflammation and / or oxidative damage.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 907,389, filed Sep. 27, 2019, which is incorporated by reference in its entirety. [Background technology]

[0002] Epithelium is one of the four basic types of animal tissue, along with connective tissue, muscle tissue, and nervous tissue. Epithelial tissue lines cavities and surfaces of structures throughout the body. Many glands are composed of epithelial cells. The functions of epithelial cells include secretion, selective absorption, protection, transcellular transport, and sensory detection. The cells of epithelial tissue are tightly packed and form a continuous sheet. Epithelial cells form glands and constitute the major layer in mucous membranes. Epithelial cells at mucosal surfaces are constantly engaged in the important function of forming a protective apical barrier that prevents cell damage and infection while allowing the exchange of molecules with the extracellular environment. Loss of barrier function is the cause of many mucosal pathologies, such as dry eye, severe asthma, and inflammatory bowel disease. Epithelial tissue lines the mouth, alveoli, and kidney tubules. The inner lining of blood and lymphatic vessels is a specialized form of epithelium called the endothelium.

[0003] The primary functions of epithelial tissue are (1) to protect underlying tissues from radiation, desiccation, toxins, invasion by pathogens, and physical trauma; (2) to regulate and exchange chemicals between underlying tissues and body cavities; (3) to secrete hormones into the vascular system and / or secrete sweat, mucus, enzymes, and other products delivered by the ducts; and (4) to provide sensation.

[0004] Most epithelial cells, including some bacteria, have a fluffy coat on the outer surface of their cell membrane, called the glycocalyx, which is a glycoprotein-polysaccharide covering that surrounds the cell membrane. The existence of the glycocalyx was discovered about 40 years ago, when it was described as a thin layer at the surface of the endothelium (1966. Fed Proc 25:1773-1783). However, the importance of this structure was not recognized, in part because it is destroyed during conventional tissue fixation and is not visible in most light microscopic examinations. The glycocalyx is a protective lining at the surface of the endothelium found in all healthy blood vessels, and it is composed of proteoglycans, a complex network of proteins (glycoproteins) and disaccharides (glycosaminoglycans) that serve as supporting scaffold molecules. In general, the carbohydrate moieties of glycolipids found on the surface of cell membranes help contribute to cell-cell recognition, communication, and intracellular adhesion. This complex network (derived from plasma and vascular wall) forms a dynamic layer between the flowing blood and the endothelium, constantly changing in thickness depending on the shear or blood flow pressure. Thus, the shear generated by the blood flow regulates the equilibrium between the biosynthesis and shedding of the various glycocalyx components. The core protein group of this layer is the syndecans and glypicans indiscriminately bound to different glycosaminoglycans, including heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan (or hyaluronic acid). In the vascular system, heparan sulfate accounts for almost 50-90% of the total amount of proteoglycans, followed by chondroitin sulfate, in a typical ratio of 4:1, respectively (2007.Pflugers Arch;454:345-359).

[0005] The glycocalyx can also be found at the apical end of microvilli in the digestive tract, particularly in the small intestine. It creates a 0.3 micrometer thick meshwork and is composed of acidic mucopolysaccharides and glycoproteins that protrude from the apical cell membrane of epithelial absorptive cells. It provides an additional surface for adsorption and contains enzymes secreted by the absorptive cells that are essential for the final steps of protein and sugar digestion.

[0006] Each cell is surrounded by a glycocalyx. The glycocalyx layer of connective cells in tissues forms a glycocalyx layer on the surface of tissues, creating a barrier. Once destroyed, the underlying cells become vulnerable to destruction and immune attack by macrophages and other organisms. The glycocalyx of endothelial cells, such as the endometrium, the lining of the lungs, the microvilli of the kidneys, and the pancreas, forms a seal of cells.

[0007] Furthermore, at the cellular level, the glycocalyx supports the structural and functional integrity of glycoproteins and other biomolecules that pass through it. Biomolecules that form channels, receptors, and other functional components of cell membranes structurally and functionally coexist with and through the glycocalyx. Disruption of the glycocalyx results in the disruption of the structure and function of those biomolecules, thereby disrupting the structure and function of cells, and the tissues and organs that are composed of those cells.

[0008] Other systemic functions affected by the state of the glycocalyx include protection (it buffers cell membranes and protects them from chemical damage), immunity to infection (it allows the immune system to recognize and selectively attack foreign bodies), defense against cancer (changes in the glycocalyx of cancerous cells allow the immune system to recognize and destroy them), transplant compatibility (it forms the basis of compatibility for blood transfusions, tissue transplants, and organ transplants), cell adhesion (it binds cells together so tissues do not fall apart), inflammation regulation (the glycocalyx coating on the endothelial walls in blood vessels prevents white blood cells from rolling / binding in healthy conditions), fertilization (it allows sperm to recognize and bind to the egg), and embryonic development (it guides embryonic cells to their destination). [Prior art documents] [Patent documents]

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[0010] [Non-Patent Document 1] 1966.Fed Proc 25:1773-1783 [Non-Patent Document 2] 2007.Pflugers Arch;454:345-359 [Non-Patent Document 3] Fundamental Immunology, edited by WE Paul, Raven Press, NY (1993) [Non-Patent Document 4] Huston et al. (1988) Proc. Nat. Acad. Sci. USA, 85:5879-5883 [Non-Patent Document 5] Reiter et al. (1995) Protein Eng. 8:1323-1331 [Non-Patent Document 6] Kohler and Milstein.(1975)Nature, 256:495-497 [Non-Patent Document 7] Hongo et al. (1995) Hybridoma, 14(3):253-260 [Non-Patent Document 8] Harlow et al. (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) [Non-Patent Document 9] Hammerling et al. (1981): Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY) [Non-Patent Document 10] Clackson et al. (1991) Nature 352:624-628 [Non-Patent Document 11] Marks et al. (1992) J. Mol. Biol. 222:581-597 [Non-Patent Document 12] Sidhu et al. (2004) J.Mol.Biol.338(2):299-310 [Non-Patent Document 13] Lee et al. (2004) J.Mol.Biol.340(5):1073-1093 [Non-Patent Document 14] Jakobovits et al. (1993) Nature 362:255-258 [Non-Patent Document 15] Bruggemann et al. (1993) Year in Immunol. 7:33 [Non-Patent Document 16] Marks et al. (1992) Bio / Technology 10:779-783 [Non-Patent Document 17] Lonberg et al. (1994) Nature 368:856-859 [Non-Patent Document 18] Morrison(1994)Nature 368:812-813

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[0011] Various embodiments contemplated herein may include, but are not necessarily limited to, one or more of the following. [Means for solving the problem]

[0012] Embodiment 1: Hyaluronan Synthetase-1 (HAS-1), Hepa La 1. A kit comprising a means for measuring at least two biomarkers selected from the group consisting of: syndecan-SO4 (HS), syndecan-1 (SDC-1), plasminogen activator inhibitor (PAI-1), gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A), wherein at least one of the at least two biomarkers is selected from the group consisting of gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0013] Embodiment 2: The kit of embodiment 1, wherein the at least two biomarkers are selected from the group consisting of gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0014] Embodiment 3: The kit of embodiment 2, wherein the kit comprises means for measuring at least three biomarkers, the at least three biomarkers comprising gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0015] Embodiment 4: A kit according to any one of embodiments 1 to 3, wherein the means for measuring the biomarker comprises a binding partner that specifically binds to the biomarker.

[0016] Embodiment 5: The kit of embodiment 4, wherein each binding partner in the kit is labeled with a different detectable label.

[0017] Embodiment 6: The kit of embodiment 4 or embodiment 5, wherein the binding partner comprises a detectably labeled antibody.

[0018] Embodiment 7: A method of treating a subject having a disease characterized by disruption of the glycocalyx, comprising administering to a subject a compound of Formulae I-XI: [ka] [ka] [ka] The method includes administering to a subject, or having a subject receive, one or more compositions comprising: The subject is hyaluronan Synthetase-1 (HAS-1), Hepa La The subject has previously been identified as having a disease characterized by disruption of the glycocalyx by measuring at least two biomarkers selected from the group consisting of glycosyltransferase (HS), syndecan-1 (SDC-1), plasminogen activator inhibitor (PAI-1), gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein-A (PAPP-A).

[0019] Embodiment 8: The method of embodiment 7, wherein at least one of the at least two biomarkers measured is selected from the group consisting of gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0020] Embodiment 9: The method of embodiment 8, wherein the at least two biomarkers measured are selected from the group consisting of gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0021] Embodiment 10: The method of embodiment 9, wherein at least three biomarkers are measured, and the at least three biomarkers include gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0022] Embodiment 11: The method of any one of embodiments 7 to 10, further comprising measuring or causing the measurement of biomarkers.

[0023] Embodiment 12: A method for detecting biomarkers of glycocalyx integrity in a subject, comprising measuring, or causing to be measured, the levels of at least two biomarkers in a biological sample obtained from the subject, wherein the at least two biomarkers are hyaluronan, Synthetase-1 (HAS-1), Hepa La and wherein at least one of the at least two biomarkers measured is selected from the group consisting of gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0024] Embodiment 13: The method of embodiment 12, wherein the at least two biomarkers measured are selected from the group consisting of gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0025] Embodiment 14: The method of embodiment 13, wherein at least three biomarkers are measured, and the at least three biomarkers include gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0026] Embodiment 15: The method of any one of embodiments 12 to 14, wherein at least two biomarkers are measured.

[0027] Embodiment 16: The method of embodiment 15, wherein the biological sample is selected from the group consisting of blood, plasma, urine, saliva, tears, and cerebrospinal fluid.

[0028] Embodiment 17: The method of embodiment 15 or embodiment 16, wherein the biomarker is measured using an immunoassay or mass spectrometry.

[0029] Embodiment 18: The method of any one of embodiments 12 to 17, wherein an elevated level of one of the at least two biomarkers compared to a predetermined normal level indicates that the subject has a disease characterized by disruption of the glycocalyx.

[0030] Embodiment 19: The method of any one of embodiments 12 to 18, wherein the subject has one or more symptoms consistent with at least two possible diseases or two possible stages of a disease, comprising measuring, or causing measurements to be made of, at least two of said biomarkers to obtain a biomarker signature, and identifying the subject as a candidate for treatment of one of the at least two possible diseases or stages of a disease based on the biomarker signature.

[0031] Embodiment 20: The method of embodiment 19, comprising measuring, or causing the measuring of, at least three of said biomarkers to obtain a biomarker signature.

[0032] Embodiment 21: The method of embodiment 19, further comprising treating the subject for said one of at least two possible diseases or disease stages.

[0033] Embodiment 22: A method of treating a subject having one or more symptoms consistent with at least two possible diseases or two possible stages of a disease, comprising administering to the subject a hyaluronan Synthetase-1 (HAS-1), Hepa La and a subject who has previously been identified as having one of two possible diseases or disease stages by measuring at least two biomarkers selected from the group consisting of HS, syndecan-1 (SDC-1), plasminogen activator inhibitor (PAI-1), gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A) to determine a biological signature indicative of one of the at least two possible diseases, wherein the method comprises treating the subject for said one of the at least two possible diseases or disease stages.

[0034] Embodiment 23: Formulas I-XI: [ka] [ka] [ka] 23. The method of embodiment 22, comprising administering to the subject or having the subject administer one or more compositions characterized by one or more of:

[0035] Embodiment 24: The method of any one of embodiments 12 to 17, wherein the subject comprises a non-human subject.

[0036] Embodiment 25: The method of embodiment 24, comprising administering a candidate drug to a subject.

[0037] Embodiment 26: The method of embodiment 25, wherein the candidate drug is administered to the subject prior to measuring the level of the biomarker.

[0038] Embodiment 27: The method of any one of embodiments 24 to 26, wherein the subject comprises an animal model of a disease characterized by disruption of the glycocalyx, endothelial inflammation, or oxidative damage to the endothelium.

[0039] Embodiment 28: The method of embodiment 27, wherein the condition comprises arteritis and / or plaque.

[0040] Embodiment 29: The method of any one of embodiments 24 to 28, wherein the subject is produced from the mammal by one or more treatments selected from the group consisting of administering a xenobiotic to the mammal, administering a pathogen to the mammal, and feeding the mammal a diet with at least 21% (w / w) fat.

[0041] Embodiment 30: The method of embodiment 29, wherein the subject is produced by administering polychlorinated biphenyls (PCBs) to the mammal, administering the bacteria to the mammal, and feeding the mammal a diet of at least 50% fat.

[0042] Embodiment 31: The method of embodiment 30, wherein the subject is a mouse subject produced by administering 3,3',4,4'-tetrachlorobiphenyl (PCB-77) to the mouse, administering Porphyromonas gingivalis to the mouse, and feeding the mammal a diet of at least 60% fat.

[0043] Embodiment 32: PCB-77 is administered at a dose of at least 150 μmol / kg and Porphyromonas gingivalis is administered at a dose of at least 3×10 11 32. The method of embodiment 31, wherein the bacterium is administered in a dose of 10 bacteria / mouse.

[0044] Embodiment 33: The method of any one of embodiments 24 to 32, wherein the candidate drug has been demonstrated to have an activity selected from the group consisting of anti-inflammatory activity, antioxidant activity, activity in reducing breakdown of the glycocalyx, and any combination thereof.

[0045] Embodiment 34: The method of embodiment 33, wherein the candidate drug has been demonstrated to have anti-inflammatory activity, antioxidant activity, and activity in reducing the breakdown of the glycocalyx, and the candidate drug comprises a combination of FTX compounds.

[0046] Embodiment 35: Hyaluronan Synthetase-1 (HAS-1), Hepa La 6. The kit according to any one of embodiments 1 to 5, comprising means for measuring at least four biomarkers selected from the group consisting of hyaluronan SO4 (HS), syndecan-1 (SDC-1), plasminogen activator inhibitor (PAI-1), gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A). Synthetase-1 (HAS-1), Hepa La 35. The method of any one of embodiments 7 to 34, wherein at least four biomarkers selected from the group consisting of HS, syndecan-1 (SDC-1), plasminogen activator inhibitor (PAI-1), gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A) are measured.

[0047] Embodiment 36: Hyaluronan as a biomarker Synthetase-1 (HAS-1), Hepa La 6. The kit according to any one of the preceding embodiments, comprising means for measuring hyaluronan, serotonin-SO4 (HS), syndecan-1 (SDC-1), plasminogen activator inhibitor (PAI-1), gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A), or the biomarkers measured are hyaluronan, serotonin-dependent protein B (SEQ ID NO: 1), serotonin-dependent protein C (SEQ ID NO: 2), and serotonin-dependent protein D (SEQ ID NO: 3). Synthetase-1(HAS-1), Hepa La 35. The method of any one of embodiments 7 to 34, comprising administering to the patient a therapeutically effective amount of a medicament for the treatment of a patient having a pulmonary embolism comprising isoform SEQ ID NO4 (HS), syndecan-1 (SDC-1), plasminogen activator inhibitor (PAI-1), gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

[0048] Embodiment 37: Hyaluronan as a biomarker Synthetase-1 (HAS-1), Hepa La 6. The kit according to any one of the preceding claims, comprising a means for measuring hyaluronan, serotonin-SO4 (HS), syndecan-1 (SDC-1), plasminogen activator inhibitor (PAI-1), gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A), or the biomarkers measured are hyaluronan, serotonin-dependent protein B (SEQ ID NO: 1), serotonin-dependent protein C (SEQ ID NO: 2), serotonin-dependent protein D (SDC-1), serotonin-dependent protein D (SDC-1), serotonin-dependent protein D (SDC-1), serotonin-dependent protein E (SDC-1), serotonin-dependent protein F (SDC-1), serotonin-dependent protein F (SDC-1), serotonin-dependent protein F (SDC-1), serotonin-dependent protein D ... Synthetase-1 (HAS-1), Hepa La 35. The method of any one of embodiments 7 to 34, comprising administering to the patient a therapeutically effective amount of a medicament for the treatment of a patient having a pulmonary embolism comprising administering to the patient a therapeutically effective amount of a medicament for the treatment of a patient having a pulmonary embolism comprising administering to the patient a therapeutically effective amount of a medicament for the treatment of a patient having a pulmonary embolism comprising administering to the patient a therapeutically effective amount of a medicament for the treatment of a patient having a pulmonary embolism.

[0049] Embodiment 38: A kit according to any one of embodiments 1 to 5, comprising a means for measuring the biomarker gamma fibrinogen (GF), or a method according to any one of embodiments 7 to 34, wherein at least one biomarker measured comprises gamma fibrinogen (GF).

[0050] Embodiment 39: The kit or method of embodiment 38, wherein the kit further comprises a means for measuring the biomarker Growth Differentiation Factor 15 (GDF-15), or wherein at least one biomarker measured comprises Growth Differentiation Factor 15 (GDF-15).

[0051] Embodiment 40: A kit according to any one of embodiments 1 to 5, comprising means for measuring the biomarker Growth Differentiation Factor 15 (GDF-15), or a method according to any one of embodiments 7 to 34, wherein at least one biomarker measured comprises Growth Differentiation Factor 15 (GDF-15).

[0052] Embodiment 41: The kit or method of embodiment 38 or embodiment 40, wherein the kit further comprises a means for measuring the biomarker Pregnancy Associated Plasma Protein A (PAPP-A) or wherein at least one biomarker measured comprises Pregnancy Associated Plasma Protein A (PAPP-A).

[0053] Embodiment 42: A kit according to any one of embodiments 1 to 5, comprising a means for measuring the biomarker Pregnancy Associated Plasma Protein A (PAPP-A), or a method according to any one of embodiments 7 to 34, wherein at least one biomarker measured comprises Pregnancy Associated Plasma Protein A (PAPP-A).

[0054] Embodiment 43: The kit comprises a biomarker, hyaluronan Synthetase-1 (HAS-1), or at least one biomarker measured is hyaluronan. Synthetase-1 43. The kit or method of any one of embodiments 38 to 42, which is a method comprising (HAS-1).

[0055] Embodiment 44: The kit comprises a biomarker, La The method further comprises a means for measuring hepatic sulphate (HS), or at least one biomarker measured is hepatic sulphate. La 44. The kit or method according to any one of embodiments 38 to 43, wherein the kit or method comprises the compound SO4 (HS).

[0056] Embodiment 45: A kit or method according to any one of embodiments 38 to 44, wherein the kit further comprises a means for measuring the biomarker syndecan-1 (SDC-1) or wherein at least one biomarker measured comprises syndecan-1 (SDC-1).

[0057] Embodiment 46: A kit or method according to any one of embodiments 38 to 45, wherein the kit further comprises a means for measuring the biomarker Plasminogen Activator Inhibitor (PAI-1) or wherein at least one biomarker measured comprises Plasminogen Activator Inhibitor (PAI-1).

[0058] Embodiment 47: The method of any one of embodiments 7 to 11 and 23, wherein a composition comprising formula I is administered.

[0059] Embodiment 48: The method according to any one of embodiments 7 to 11 and 23, wherein a composition comprising formula II is administered.

[0060] Embodiment 49: The method of any one of embodiments 7 to 11 and 23, wherein a composition comprising formula III is administered.

[0061] Embodiment 50: The method of any one of embodiments 7 to 11 and 23, wherein a composition characterized by formula IV is administered.

[0062] Embodiment 51: The method of any one of embodiments 7 to 11 and 23, wherein a composition characterized by formula V is administered.

[0063] Embodiment 52: The method of any one of embodiments 7 to 11 and 23, wherein a composition having the formula VI is administered.

[0064] Embodiment 53: The method of any one of embodiments 7 to 11 and 23, wherein a composition having formula VII is administered.

[0065] Embodiment 54: The method of any one of embodiments 7 to 11 and 23, wherein a composition comprising formula VIII is administered.

[0066] Embodiment 55: The method of any one of embodiments 7 to 11 and 23, wherein a composition having formula IX is administered.

[0067] Embodiment 56: The method of any one of embodiments 7 to 11 and 23, wherein a composition characterized by formula X is administered.

[0068] Embodiment 57: The method of any one of embodiments 7 to 11 and 23, wherein a composition having formula XI is administered.

[0069] Embodiment 58: The method of any one of embodiments 47 to 57, wherein a second composition is co-administered with the composition, and the second composition is different from the composition, and the second composition is characterized by one of formulas I to XI.

[0070] Embodiment 59: The method of embodiment 58, wherein a third composition is co-administered with the composition and the second composition, wherein the third composition is different from the composition and the second composition, and wherein the third composition is characterized by one of formulas I-XI.

[0071] Embodiment 60: The method of embodiment 59, wherein (a) the composition is characterized by Formula I, the second composition is characterized by Formula II, and the third composition is characterized by Formula III; (b) the composition is characterized by Formula I, the second composition is characterized by Formula VI, and the third composition is characterized by Formula VII; (c) the composition is characterized by Formula I, the second composition is characterized by Formula IV, and the third composition is characterized by Formula V; and (d) the composition is characterized by Formula II, the second composition is characterized by Formula VI, and the third composition is characterized by Formula VII.

[0072] Embodiment 61: The method of any one of embodiments 47 to 60, comprising administering to the subject or having the subject administer a therapeutic agent selected from the group consisting of antihistamines, anti-infective agents, anti-tumor agents, autonomic agents, blood products, hematopoietic agents, coagulants, thrombosis agents, cardiovascular agents, cell therapy, central nervous system agents, contraceptives, dental agents, diagnostic agents, bactericides, electrolyte agents, caloric agents, water balance agents, enzymes, airway agents, ophthalmic preparations, otic preparations, nasal preparations, laryngeal preparations, gold compounds, heavy metal antagonists, hormones or synthetic substitutes therefor, uterotonics, radioactive FTX compounds, serum, toxoids, vaccines, skin and / or mucosal agents, smooth muscle relaxants, vitamins, and combinations thereof.

[0073] Embodiment 62: The method according to any one of embodiments 7 to 23, the kit or method according to any one of embodiments 38 to 46, or the method according to any one of embodiments 47 to 60, wherein the subject has a disease characterized by disrupted glycocalyx, endothelial inflammation, oxidative damage to the endothelium, or any combination thereof.

[0074] Embodiment 63: The method of embodiment 62, wherein the subject has cardiovascular disease (CVD).

[0075] Embodiment 64: The method of embodiment 63, wherein the subject has a form of cardiovascular disease (CVD) selected from the group consisting of coronary heart disease, myocardial infarction, stroke, hypertension, atrial fibrillation, congestive heart failure, congenital heart disease, peripheral arterial disease, venous thrombosis, deep vein thrombosis, pulmonary embolism, and any combination thereof.

[0076] Embodiment 65: The method of embodiment 62, wherein the subject has a condition selected from the group consisting of cancer, diabetes, arthritis, Alzheimer's disease, or any combination thereof.

[0077] Embodiment 66: The method of any one of embodiments 62 to 65, wherein the subject is known to have or is at risk for said condition or disease.

[0078] Embodiment 67: The method of any one of embodiments 62 to 66, comprising improving glycocalyx integrity, reducing endothelial inflammation or oxidative damage to the endothelium, or any combination thereof.

[0079] Embodiment 68: The method of any one of embodiments 62 to 67, comprising measuring glycocalyx disruption, endothelial inflammation, oxidative damage to the endothelium, or any combination thereof, after administration of the composition.

[0080] Embodiment 69: The method of any one of embodiments 62 to 68, wherein the glycocalyx or endothelium is in an area of ​​the body selected from the group consisting of glands, mouth, lungs, kidneys, eyes, blood vessels, and the endometrium or gastrointestinal lining.

[0081] Embodiment 70: The method of any one of embodiments 62 to 69, wherein the composition is administered to the subject in an oral formulation.

[0082] Embodiment 71: The method of any one of embodiments 62 to 70, wherein the composition is administered at a dose ranging from 0.05 mg / kg to 200.0 mg / kg.

[0083] Embodiment 72: The method of embodiment 71, wherein the dose is in the range of 0.1 mg / kg to 100 mg / kg. [Brief description of the drawings]

[0084] [Figure 1A] 1 is a chart of the experimental protocol. [Figure 1B] 1 is a chart of the experimental protocol. [Figure 2A] 1 is a photomicrograph of sections from Group 3 from Example 1. [Figure 2B] 1 is a photomicrograph of sections from Group 4 from Example 1. [Diagram 3] 1 is a graph of total plasminogen activator inhibitor-1 levels from Example 1. [Figure 4] 1 is a graph of heparan sulfate levels from Example 1. [Diagram 5] 1 is a graph of hyaluronan synthase 1 levels from Example 1. [Figure 6] 1 is a graph of syndecan-1 levels from Example 1. [Figure 7] 1 is a graph of thrombin and antithrombin III levels from Example 1. [Figure 8] 1 is a graph of antithrombin levels from Example 1. [Figure 9] 1 is a graph of mean disease score by treatment group and sacrifice time from Example 1. [Figure 10] FIG. 1 shows a photograph of a plaque at 10x, a photograph of a plaque at 40x, and a photograph of a normal artery wall. [Figure 11] Depiction of three enzymes associated with the glycocalyx that regulate blood flow. [Figure 12] 12A-H are photographs of therapeutic and preventative histopathology of arterial blood vessels for combinations of compounds designated B, F, I, and C (the compounds of each combination are identified in FIG. 14): preventative (A) and therapeutic (B) results for combination B ("compound B"); preventative (C) and therapeutic (D) results for combination F ("compound F"); preventative (E) and therapeutic (F) results for combination I ("compound I"); and preventative (G) and therapeutic (H) results for combination C ("compound C"). [Figure 13] FIG. 1 shows sample biomarker "signatures" defined by patterns of absolute or relative biomarker levels for three different cardiovascular diseases when clinical data is analyzed using a combination of seven biomarkers. [Figure 14] FIG. 1 is a chart showing the effect of three different drug combinations assessed using the biomarkers hyaluronan synthase-2 ("hyaluronan"), heparan sulfate, and plasminogen activator inhibitor ("PAI-1"). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] In some embodiments, the present disclosure is generally directed to methods and compositions for repairing the glycocalyx. Disruption of the glycocalyx is a contributing factor in many diseases, particularly cardiovascular disease. The compositions of the present disclosure maintain the integrity of the glycocalyx in many different membranes.

[0086] definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0087] As used herein, "vascular disease" refers to any disease that affects the circulatory system of arteries, veins, capillaries, and lymphatic vessels in the body. Vascular diseases can include, but are not limited to, peripheral arterial disease, aneurysms, renal artery disease, Raynaud's disease, Buerger's disease, peripheral venous disease, varicose veins, blood clots (thromboembolism), blood clotting disorders, and lymphedema.

[0088] A "thrombus" is a solid mass composed of platelets, fibrin and blood components.

[0089] An "embolus" is a piece of blood clot that breaks off and is carried into the bloodstream.

[0090] A "thromboembolism" is a free-floating embolus that lodges in a blood vessel and blocks blood flow.

[0091] As used herein, "thromboembolism" refers to the obstruction of a blood vessel by a blood clot, which can occur in a family of vascular diseases that include coronary heart disease (CHD), acute myocardial infarction (MI), stroke, hypertension, atrial fibrillation, congestive heart failure (CHF), congenital heart disease, peripheral arterial disease (PAD), chronic venous insufficiency (CVI), deep vein thrombosis (DVT), and pulmonary embolism (PE).

[0092] "Cardiovascular disease" (CVD) includes a family of diseases that affect both the arteries and veins and the heart; diseases of the arteries include coronary heart disease (CHD), myocardial infarction (MI), stroke, hypertension, atrial fibrillation, congestive heart failure (CHF), congenital heart disease, and peripheral arterial disease (PAD); diseases of the veins include venous thrombosis, deep vein thrombosis (DVT), and pulmonary embolism (PE).

[0093] "Coronary heart disease" (CHD) results from the effects of atherosclerotic plaque formation in the coronary arteries. Reduced blood supply to the heart muscle reduces the efficiency of the heart and can lead to heart failure. One of the first major symptoms of this condition is angina (chest pain caused by reduced blood flow to the heart muscle).

[0094] A "myocardial infarction" (MI), commonly known as a heart attack, is the irreversible death of the heart muscle due to prolonged interruption of the blood supply (ischemia). The heart requires a constant supply of oxygen and nutrients, and if one of the arteries or branches is suddenly blocked, the heart becomes starved of oxygen, a condition called "cardiac ischemia." If cardiac ischemia continues for too long, the starved heart tissue dies, which is called a heart attack (myocardial infarction), literally "death of the heart muscle."

[0095] A "stroke" occurs when brain cells die due to lack of blood supply, which can be classified as ischemic or hemorrhagic; an ischemic stroke involves a reduction in blood supply to an area of ​​the brain, resulting in brain cell death and thereby impaired brain function, and a hemorrhagic stroke results from a ruptured blood vessel or abnormal vasculature, causing the accumulation of blood in an area of ​​the brain. The majority of strokes (80%) are ischemic in nature.

[0096] "Hypertension" or "high blood pressure" is defined as a condition in which the blood pressure flowing through the blood vessels remains high for a long period of time, regardless of the body's needs. Increased blood pressure makes the heart work harder and makes the heart and arteries more susceptible to damage. Hypertension further increases the risk of events such as heart attack, heart failure, and atherosclerosis.

[0097] Cardiac arrhythmia is a heart rhythm problem that occurs when the heartbeat is not well coordinated due to inappropriate electrical impulses. This can cause the heart to beat too fast (tachycardia) or too slowly (bradycardia). Arrhythmias are generally harmless and momentary, but frequent rhythm disturbances increase the risk of stroke and congestive heart failure. Atrial fibrillation is the most common sustained arrhythmia.

[0098] "Congestive heart failure" (CHF) is a condition in which the heart is unable to pump blood to various parts of the body. This may be due to narrowed arteries, myocardial infarction, valvular heart disease, high blood pressure, cardiomyopathy, or congenital abnormalities.

[0099] "Peripheral Arterial Disease" (PAD) is a vascular disorder in which thickening of the arteries causes reduced blood flow to the limbs, resulting in intermittent leg pain while walking. This disease is an indicator of atherosclerosis. It leads to pain (which does not heal) and gangrene.

[0100] "Deep vein thrombosis" (DVT) is a blood clot that forms in the deep veins, usually in the legs or arms, and can block venous return. DVT can cause leg pain or swelling, but may not cause symptoms. DVT is usually not life-threatening, but can become life-threatening if the clot breaks off and lodges in the lungs. This is known as a "pulmonary embolism" (PE).

[0101] As used herein, the term "healthy" refers to the state of an organ or individual that is free of disease (e.g., vascular disease), in good health, and has no specific known physiologically-based risk of developing a disease (e.g., vascular disease).

[0102] As used herein, the term "compound" refers to "a substance containing atoms or ions of two or more different elements in definite proportions combined in molecules by chemical bonds."

[0103] As used herein, the term "composition" refers to a substance that includes a compound, often in combination with other compounds or elements.

[0104] As used herein, "disrupting" or "disruption of" the glycocalyx refers to any process or disease state that affects the glycocalyx so that it does not function normally. Disruption can be caused by inflammation or oxidation in the body. Disruption can cause the glycocalyx to thin and lose its component proteoglycans. For example, the dimensions or percentages of the glycocalyx associated with blood vessels are as follows: Vascular diameter (nm) Glycocalyx thickness (nm) Glycocalyx % Venules 20,900 638 3.05 Arteriole 18,000 551 3.06 Capillaries 8,200 348 4.24 Thus, disruption refers to abnormal shedding of the glycocalyx, resulting in loss of integrity and thickness, specifically, glycocalyx thickness of less than 3.0% of the diameter of a venule or arteriole, and less than 4.2% of the diameter of a capillary.

[0105] An agent is said to have "activity in reducing the breakdown of glycocalyx" if the agent reduces the breakdown of glycocalyx as determined by any means described herein or known in the art.

[0106] As used herein, "inflammation" refers to a defensive response of tissues to injury or destruction to exclude or block any harmful agents and damaged tissue and initiate tissue repair. Inflammation can cause pain, heat, redness, swelling, and loss of function. Inflammatory mediators (cytokines and chemotactic factors) can cause the shedding of the glycocalyx. Inflammation can also cause white blood cells to degranulate, releasing enzymes that can degrade the glycocalyx.

[0107] As used herein, "anti-inflammatory" refers to a molecule, compound, or composition that inhibits any inflammatory process or symptoms thereof, such as those described herein or otherwise known in the art. An anti-inflammatory is said to have "anti-inflammatory activity."

[0108] As used herein, "oxidative damage," "oxidative stress," or "oxidation" refers to an imbalance of reactive oxygen species (ROS) and the body's ability to detoxify reactive intermediates and repair damage caused by ROS. Inflammation can cause the release of ROS. The presence of ROS can cause significant damage to cellular structures, including the glycocalyx. At the molecular level, "oxidation" refers to the loss of electrons during a reaction by a molecule, atom, or ion.

[0109] As used herein, the term "symptom" refers to a mental or physical symptom that is considered to be indicative of a disease or condition.

[0110] As used herein, the term "symptom-targeting drug" refers to a drug that improves the symptoms of a disease or condition, which may or may not address the underlying pathology.

[0111] For example, when used in connection with treating a disease or condition, the term "treat" refers to the alleviation and / or elimination of one or more symptoms of the disease or condition, and / or the delay in the progression of the disease or condition and / or the reduction in the incidence or severity of one or more symptoms, and / or the prevention of the disease or condition. The term treat encompasses therapeutic treatment, including delaying the onset or preventing the onset of a disease or condition, and prophylactic treatment.

[0112] An amount of a therapeutic compound is said to be "s" if that amount is effective to effect an improvement, including, but not limited to, improved survival or more rapid recovery, or improvement or elimination of symptoms and other indicators (such as biomarkers) as selected as appropriate measures by one of skill in the art.

[0113] As used herein, "antioxidant" refers to a molecule that can inhibit the oxidation of other molecules and neutralize or scavenge ROS. Antioxidants are said to have "antioxidant activity."

[0114] As used herein, the term "assay" refers to a procedure for determining the amount of a particular component of a mixture or sample. "Assay" is used interchangeably herein with the term "test."

[0115] As used herein, the term "biomarker" refers to a substance such as, but not limited to, a protein, a DNA sequence, an RNA sequence, or other biological substance or material that, when detected, indicates a particular healthy or unhealthy state of an individual with respect to disease (e.g., vascular disease).

[0116] As used herein, the term "sample" typically refers to a biological sample from an individual, which may be, but is not limited to, blood, plasma, urine, saliva, tears, or cerebrospinal fluid (CSF).

[0117] As used herein, the term "biomarker panel" generally refers to a combination of reagents useful for detecting multiple biomarkers. A biomarker panel is typically provided in a kit for detecting two or more biomarkers. In the art, the term "biomarker panel" may be used to refer to a combination of biomarkers themselves (as opposed to reagents for detecting the biomarkers). The meaning of this term as used herein will be readily apparent to one of skill in the art from the context in which the term is used.

[0118] A "detectable label" includes any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful labels include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, etc., e.g., Molecular Probes, Eugene, Oregon, USA), chemiluminescent compounds, e.g., acridinium (e.g., acridinium-9-carboxamide), phenanthridinium, dioxetane, luminol, etc., radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), catalysts such as enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase and others commonly used in ELISA), and colorimetric labels such as colloidal gold (e.g., gold particles in the size range of 40-80 nm in diameter that scatter green light with high efficiency) or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0119] As used herein, "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are typically classified as either kappa or lambda. Heavy chains are typically classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0120] A typical full-length (intact) immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The variable light chain (VLC) is L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.

[0121] Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments that can be produced, inter alia, by digestion with various peptidases. Thus, for example, pepsin digests antibodies under the disulfide bonds in the hinge region, itself splitting them into V fragments separated by disulfide bonds. H -C H F(ab)', a dimer of Fab, a light chain linked to 1 2 F(ab') 2 can be reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby forming (Fab') 2The dimer is converted to a Fab' monomer, which is essentially a Fab with part of the hinge region (see Fundamental Immunology, ed. W. E. Paul, Raven Press, NY (1993) for a more detailed description of other antibody fragments). Although various antibody fragments have been defined in terms of the digestion of intact antibodies, one of skill in the art will appreciate that such Fab' fragments can be synthesized de novo, either chemically or by utilizing recombinant DNA methodology. Thus, as used herein, the term antibody also includes whole antibodies, antibody fragments produced by the modification of whole antibodies, or synthesized de novo using recombinant DNA methodology. In certain embodiments, an antibody comprises a single chain antibody (an antibody that exists as a single polypeptide chain), e.g., a single chain Fv antibody (scFv) in which a variable heavy chain and a variable light chain are linked together (directly or via a peptide linker) to form a continuous polypeptide. In certain embodiments, a single chain Fv antibody comprises VFvs linked directly or by a peptide-encoding linker. H and V L A covalently linked V that can be expressed from a nucleic acid comprising a sequence encoding H -V L It is a heterodimer (see, e.g., Huston et al., (1988) Proc. Nat. Acad. Sci. USA, 85:5879-5883). H and V L are connected together as a single polypeptide chain, whereas V H and V LThe domains are non-covalently associated. The first functional antibody molecules expressed on the surface of filamentous phages were single chain Fv's (scFv), although alternative expression strategies have been successful. For example, Fab molecules can be displayed on phages when one of the chains (heavy or light) is fused, for example, to the g3 capsid protein, and the complementary chain is transported to the periplasm as a soluble molecule. The two chains can be encoded on the same or different replicons. The key is that the two antibody chains of each Fab molecule assemble post-translationally, and the dimer is incorporated into the phage particle via binding of one of the chains, for example to g3p (see, for example, U.S. Pat. No. 5,733,743). Those skilled in the art are aware of scFv antibodies and many other structures that convert the naturally aggregated but chemically separated light and heavy polypeptide chains from an antibody V region into molecules that fold into a three-dimensional structure substantially similar to that of an antigen-binding site (see, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778). Thus, in certain embodiments, anti-Fc receptor antibodies include, but are not limited to, any displayed on phage or yeast (e.g., scFv, Fv, Fab, and disulfide-linked Fv (see, e.g., Reiter et al. (1995) Protein Eng. 8:1323-1331)).

[0122] Antibodies also include "single domain" antibodies (sdAbs), also known as nanobodies. Single domain antibodies consist of a single monomeric variable antibody domain. Like common "whole antibodies", they can selectively bind to a specific antigen. With a molecular weight of only 12-15 kDa, single domain antibodies are much smaller than common antibodies (150-160 kDa), which are composed of two heavy protein chains and two light chains, and are also smaller than Fab fragments (about 50 kDa, one light chain and half a heavy chain) and single chain variable fragments (about 25 kDa, two variable domains, one from a light chain and one from a heavy chain). Some species, such as camelids, naturally produce single domain antibodies.

[0123] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual members of the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation, etc.) that may be present in minor amounts. Monoclonal antibodies are typically highly specific and directed against a single epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The term "monoclonal" indicates the character of an antibody as being obtained from or being one of a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced by hybridoma techniques (see, e.g., Kohler and Milstein. (1975) Nature, 256:495-497; Hongo et al., (1995) Hybridoma, 14(3):253-260; Harlow et al., (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.); Hammerling et al., (1981): Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (see, e.g., Clackson et al., (1991) Nature, 352:624-628; Marks et al., (1992) J. Mol. Biol. 222:581-597; Sidhu et al., (2004) J. Mol. Biol. 338(2):299-310; Lee et al., (2004) J. Mol. Biol.340(5):1073-1093), and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin loci, or genes encoding human immunoglobulin sequences (see, e.g., PCT Patent Publication Nos. WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; and WO 1991 / 10741; U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Jakobovits et al. (1993) Nature 362:255-258; Bruggemann et al. (1993) Year in Immunol. 7:33; Marks et al. (1992) Bio / Technology 10:779-783; Lonberg et al. (1994) Nature 368:856-859; Morrison (1994) Nature 368:812-813; Fishwild et al. (1996) Nature Biotechnol. 14:845-851; Neuberger (1996) Nature Biotechnol. 14:826; Lonberg and Fluszar (1995) Intern. Rev. Immunol. 13:65-93, etc.).

[0124] As used herein, the phrase "causing a measurement to be made" refers to any action that results in a measurement being made. For example, when a physician orders a test for a biomarker to be performed on a sample from a given subject, the physician causes a measurement of that biomarker to be made.

[0125] As used herein, "biomarker signature" refers to a pattern of absolute or relative biomarker levels for two or more biomarkers that is characteristic of a particular disease or condition, or a stage of a disease or condition.

[0126] As used herein with respect to a disease or condition, the term "stage" refers to the level of biological severity of the disease or condition. Many diseases have clearly defined staging criteria.

[0127] As used herein, the term "xenobiotic" refers to a substance, typically a chemical, that is foreign to the body, and whose chronic exposure to the body produces a deleterious effect (to the body) that manifests as one or more chronic diseases, defined herein as a "xenodisease."

[0128] A "pathogen" is a microorganism (eg, a bacterium or virus) that can cause infectious disease.

[0129] As used herein, the term "differential diagnosis" refers to determining which of two or more diseases with similar symptoms is most likely to be the cause of a subject's symptoms based on an analysis of clinical data.

[0130] The term "prognosis" is used herein to refer to the likely course of a disease or condition.

[0131] Glycocalyx and Disease The glycocalyx is a critical structure for maintaining the integrity of the vascular wall and the proper functioning of many organs. Disruption of the glycocalyx can result from contact with fluid flow stress or low shear stress, especially in arterial bends and branches, physical damage or injury, infection or exposure to xenobiotics, oxidation and inflammation, and loss of protective enzymes and proteins. Unhindered blood flow, especially in straight sections of arterial vessels where shear stress is high, is typically characterized by a thick glycocalyx layer and the absence of plaque. Thin glycocalyx promotes plaque accumulation, especially in vessel bends where low shear blood flow vortices are present. Plaques are essentially patches that cover small gaps to maintain osmotic balance of the membrane. Small gaps in the membrane can leak electrolytes both inside the cell (Na+Cl-, Ca+, HCO3) and outside (K+, PO4-, Mg+), resulting in a family of cardiovascular diseases. Destruction can also be caused by debris trapped in stagnant blood flow, which leads to oxidation and inflammation.

[0132] Any disruption or loss of thickness of the glycocalyx can result in many different conditions, including chronic vascular disease (2010. Cardiovascular Research. Vol. 87, No. 2, pp. 300-310). For example, chronic stagnant blood flow, common at arterial bifurcations, can lead to shedding of the glycocalyx and plaque formation. In the heart, disruption of the glycocalyx in coronary arteries can result in insufficient blood flow (coronary perfusion); at the arteriole level, damaged glycocalyx can slow blood flow, reduce nitric oxide (NO) production, and constrict blood vessels; at the capillary level, disruption of the glycocalyx can reduce blood flow to tissues or muscles. In addition, the glycocalyx harbors a variety of enzymes that regulate proper blood flow, including superoxide dismutase (SOD), an enzyme that neutralizes reactive oxygen species, antithrombin (AT-III), a natural anticoagulant (anticoagulant), and lipoprotein lipase (LPL), an enzyme that releases triglycerides from chylomicrons and very low density lipoproteins (VLDL) for energy. See FIG. 11.

[0133] In the event of cardiac ischemia / reperfusion injury (myocardial injury due to obstruction of blood flow followed by restoration of blood supply), disruption of the glycocalyx results in coronary artery stenosis, insufficient blood flow, and edema. However, pretreatment of the heart with antithrombin reduces glycocalyx shedding and restores coronary artery function (2009. Cardiovascular Research. Vol. 83, No. 2, pp. 388-396).

[0134] Other more common consequences of glycocalyx disruption include osmotic gradient shifts, leakage between cells (such as blood vessels, kidney, and lung cells), macrophage infiltration and inflammation, and tissue dysfunction. Ultimately, glycocalyx dysfunction can lead to obstruction of flow in the vasculature, kidney, pancreas, and other organs and tissues.

[0135] cardiovascular disease Cardiovascular disease (CVD) is the leading cause of death worldwide and remains a major subject of pathological research due to its complexity and manifesting clinical sequelae. Although members of the CVD family are quite different in clinical presentation, they share a common characteristic of being fundamentally related to atherosclerosis and vascular damage, especially to the endothelial glycocalyx. When the vasculature is damaged, a thromboembolic cascade ensues. Thromboembolism as a process leading to the formation of a thrombus (blood clot), when this thrombus dislodges from its original site, forms an embolism and travels downstream in the vascular tree as a thromboembolism, blocking blood flow and potentially becoming fatal.

[0136] Blood pressure generated by the pumping action of the heart fluctuates and blood flow is particularly slow at arterial bifurcations and bends, especially in the coronary arteries. A high-fat diet increases blood viscosity, further slowing blood flow, which reduces shear forces and results in the shedding or destruction of the endothelial glycocalyx. Glycocalyx thickness ranges from 2-3 μm in small arteries to 4.5 μm in carotid arteries (2007. J Vasc Res 44:87-98), and shedding or damage of this layer reduces its function as a protective shield, leading to leakage of nutrients (extravasation) and tissue edema, loss of nutrient blood flow, and increased coagulability due to aggregation (adhesion) of platelets and leukocytes.

[0137] The endothelial glycocalyx provides a "nest" for protective enzymes, including the anticoagulant antithrombin (AT-III), antioxidants (SOD), and antihypertensive lipoprotein lipase (LpL). Thus, loss of endothelial glycocalyx results in the accumulation of fibrin, inhibition of fibrinolysis, and promotion of plaque formation. Further inflammation predisposes plaques to rupture, and ruptured plaques lead to clot formation, which can be exacerbated by seed clots formed by Roleaux cells, resulting in significant thrombosis. Detached clots can lodge on rigid blood vessels narrowed by plaque, especially in individuals who already have atherosclerosis, causing stroke (blockage of arteries to the brain), heart attack (blockage of arteries to the heart), or PAD (blockage of arteries to the arms or legs).

[0138] Thus, protection and / or restoration of endothelial glycocalyx presents a promising therapeutic target both in acute critical care situations and in the treatment of chronic vascular diseases. Drugs that can specifically increase the synthesis of glycocalyx components, regenerate them, or selectively block their enzymatic degradation have not been widely available (Cardiovascular Research, Vol. 87, No. 2, pp. 300-310, 2010). However, compounds aimed at restoring and maintaining glycocalyx are described in U.S. Pat. No. 9,867,842 (issued Jan. 16, 2019 by Tunan, Inc.), which is incorporated herein by reference for this description.

[0139] Under inflammatory conditions, the integrity of the endothelial glycocalyx is compromised to various degrees, especially during systemic inflammatory responses, but the glycocalyx can recover to its original thickness after appropriate treatment of the inflammatory condition (2008. Circulation Research, Vol. 102, No. 7, pp. 770-776). Thus, therapeutic strategies may directly aim to preserve, support, or reconstitute glycocalyx structure or structure, indirectly by downregulating inflammatory processes, or directly by inhibiting glycocalyx degradation with antioxidants (2006. American Journal of Physiology: Heart and Circulatory Physiology, Vol. 290, No. 6, pp. H2247-H2256). An example of an anti-inflammatory drug is etanercept (Enbrel), which inhibits TNF-α and reduces shedding of glycocalyx components, coagulation activation, and functional vascular function in humans (2009. Atherosclerosis, Vol. 202, No. 1, pp. 296-303).

[0140] Another approach to improve the status of the glycocalyx is antithrombin therapy, since thrombin is known to cleave syndecan components of the glycocalyx (2009. Circulation Research, Vol. 104, No. 11, pp. 1313-1317). Indeed, antithrombin therapy protects the glycocalyx from TNF-α and ischemia / reperfusion-induced shedding in the heart (2009. Basic Research in Cardiology, Vol. 104, No. 1, pp. 78-89; 2010. Shock, Vol. 34, No. 2, pp. 133-139), which may result in reduced leukocyte adhesion, reduced vascular permeability, reduced coronary leakage, and reduced interstitial edema after ischemia in the heart (2009. Basic Research in Cardiology, Vol. 104, No. 1, pp. 78-89).

[0141] Biomarkers for Cardiovascular Disease Biomarkers have been identified that may be useful in identifying individuals at risk for vascular disease. For example, biomarkers of inflammation may indicate the presence of atherosclerosis or plaque (e.g., C-reactive protein, IL-18, IL-6). Biomarkers of lipid accumulation may indicate the presence of plaque (e.g., lipoprotein-associated phospholipase A2). Biomarkers of thrombosis may indicate the presence of plaque instability or progression of carotid artery disease (e.g., tissue plasminogen activator (t-PA), fibrinogen, plasminogen activator inhibitor-1 (PAI-1)). However, such biomarkers are not currently used by physicians as diagnostic tools.

[0142] U.S. Patent Application Publication No. 2007 / 0269836 by McPherson et al. discloses methods and compositions for diagnosing venous thromboembolic disease, pulmonary embolism, and / or deep vein thrombosis, and for risk stratification in such conditions. Assays can be performed from test samples obtained from subjects to diagnose the subject, including markers, such as thrombin-antithrombin complex (TAT), antithrombin III (ATIII), and PAI-1, used individually or in combination.

[0143] U.S. Patent No. 8,759,095 to Vink et al. discloses diagnostic and therapeutic tools for diseases that alter vascular function. In particular, disruption of the endothelial glycocalyx can be diagnosed in a sample from a subject by detecting heparan sulfate (HS) (heparan sulfate in the sample), hyaluronidase (HAD), and syndecan-1.

[0144] U.S. Patent Application No. 2013 / 0273096 by Daniels discloses a method of treating disorders affecting the endothelial glycocalyx. Characteristics of the endothelial glycocalyx can be determined by detecting markers, such as heparan sulfate (HS), hyaluronidase (HAD), and syndecan-1, in a sample from a subject.

[0145] Biomarkers with particular utility in the methods described herein Biomarkers useful in the methods described herein include those described in PCT Publication No. WO2016 / 123163 (filed by Tunan on January 27, 2016), as well as newly described biomarkers for this purpose. The biomarkers described herein may be used individually or in any combination, depending on the particular type of condition or disease to be detected. Exemplary combinations of biomarkers have been developed, as described below and in the Examples.

[0146] Biomarkers described in PCT Publication No. WO2016 / 123163 In certain embodiments, the disclosure of U.S. Patent Application No. 16 / 060,840 is directed to a "panel" of biomarkers used to detect diseases characterized by disruption of the glycocalyx, endothelial inflammation, and / or oxidative damage to the endothelium (e.g., vascular diseases), and in particular, biomarkers indicative of abnormal biochemical elements involved in the blood clotting cascade, as well as abnormal levels of enzymes and structural components on the vascular surface (e.g., due to oxidative damage to the blood vessels; PCT Publication No. WO2016 / 123163 is incorporated by reference herein for this discussion).

[0147] Most commonly, a biomarker panel includes a set of chemical, immunochemical and / or enzymatic assays or tests that can be used together to monitor the levels of a set of biomarkers. A biomarker panel can be used to determine the presence of a disease or the propensity of an individual to develop a disease. A biomarker panel can also be used to mark the progression of a disease. Assessment of different stages or components of vascular disease is important for intervention or reversal of the effects of the disease. For each of the biomarkers discussed herein, a baseline level of the biomarker is known or can be established to reflect the level of a healthy individual. A healthy individual should have a lower level of the biomarker than an individual suffering from a disease characterized by breakdown of the glycocalyx, endothelial inflammation, and / or oxidative damage to the endothelium. If the biomarker level is above the baseline level in a sample from a subject, the subject can be determined to have a disease characterized by breakdown of the glycocalyx, endothelial inflammation, and / or oxidative damage to the endothelium (e.g., vascular disease) or to be at risk for developing such a condition. Other levels can determine the stage or progression of the disease.

[0148] 4-marker panel: soluble fibrin, thrombin-antithrombin complex, antithrombin III, and plasminogen activator inhibitor In some embodiments, the biomarker panel may include a four-marker test for endothelial glycocalyx health that detects soluble fibrin (SF), thrombin-antithrombin complex (TAT), antithrombin III (ATIII), and plasminogen activator inhibitor (PAI-1), which are intended to assess clotting or clotting risk in a subject.

[0149] Soluble fibrin (SF) is composed of fibrin monomers and fibrinogen derivatives present in the circulating blood of patients with thrombosis. Its detection and quantification is useful to obtain information about the state and extent of intravascular coagulation in early thrombosis. The level of SF increases during clotting, which is associated with the production of blood factor VIII. Thus, factor VIII circulates in the plasma bound to von Willebrand factor (vWf). Thrombin cleaves and activates factor VIII, releasing vWf. vWf is then free to bind to the surface of ruptured endothelial cells, where it activates platelet aggregation. The released FVIIIa acts as a cofactor for factor IXa to generate factor Xa. In the presence of Ca2+ and phospholipids, FX is activated to FXa by FIXa. FVIIIa is a cofactor for FIXa, so it greatly stimulates the reaction. Healthy individuals should have lower levels of SF than those in disease states. If a level with a biomarker above the baseline level is detected, the individual can be determined to have vascular disease or to be at risk for developing vascular disease, particularly thrombosis. Other levels can determine the stage or progression of vascular disease.

[0150] Another blood component reflecting blood clotting is the formation of thrombin-antithrombin complex (TAT). TAT complex is a parameter of coagulation and fibrinolysis. Elevated concentrations are associated with vascular diseases. Antithrombin deficiency promotes arterial and / or venous clot formation and is associated with a high risk of thromboembolic disorders. TAT can be conveniently detected using commercially available microtiter plates. These microtiter plates are pre-coated with an antibody specific for thrombin. Calibrators or samples are added to appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for ATIII. Avidin conjugated to horseradish peroxidase (HRP) is then added to each microplate well and incubated. HRP TMB substrate solution is then added to each well. Only those wells containing TAT, biotin-conjugated antibody and enzyme-conjugated avidin show a color change. The enzyme-substrate reaction is stopped by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm±10 nm. The concentration of TAT in the sample is then determined by comparing the OD of the sample to the standard curve.

[0151] Antithrombin III (AT III) is a vitamin K-independent protease enzyme that functions as a natural anticoagulant and inhibits clotting. AT III deficiency results in an increased risk of developing life-threatening blood clots that block blood flow. For example, deep vein thrombosis (DVT) occurs when a clot, or thrombus, develops in one of the deep veins, most commonly found in the legs. The level of AT III drops when blood clots, which is determined by commercially available test kits. One such example of a test kit is LS-F13067, which is a 96-well enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of bovine antithrombin-III in plasma and serum samples. It is based on the principle of a sandwich assay and can be used to detect levels of antithrombin-III as low as 78 picograms per milliliter. Another example is the AssayMax Mouse AT III ELISA kit (LSBio, Seattle WA 98121), which is designed to detect mouse AT III in plasma, serum and cell culture supernatant. This assay utilizes a quantitative sandwich enzyme immunoassay technique that measures AT III in 4 hours. Thus, microtiter plates pre-coated with polyclonal antibodies specific for mouse AT III are commercially available. Mouse AT III in standards and samples is sandwiched between immobilized antibodies specific for mouse AT III and biotinylated polyclonal antibodies that are recognized by streptavidin-peroxidase conjugate. Then, all unbound material is washed away and peroxidase enzyme substrate is added. Color development is stopped and color intensity is measured. A baseline level of AT III can be established to reflect the level of a healthy individual. A healthy individual should have a lower level of AT III than an individual in a disease state. If a level with the biomarker panel above the baseline level is detected, the individual can be determined to have or be at risk for developing vascular disease. Other levels can determine the stage or progression of vascular disease.

[0152] Plasminogen activator inhibitor-1 (PAI-1), also known as endothelial plasminogen activator inhibitor or serpin E1, is a protein that is a central regulator of the blood fibrinolytic system and its production precedes thrombosis. In other words, increased levels of PAI-1 increase the risk of thrombosis, whereas decreased levels cause recurrent bleeding. PAI-1 is the main inhibitor of plasminogen activators and is therefore a key component of the coagulation system that downregulates fibrinolysis. Decreased levels of PAI-1 result in increased fibrinolysis and an associated bleeding diathesis. Another PAI, plasminogen activator inhibitor-2 (PAI-2), is secreted by the placenta and is present in significant amounts only during pregnancy. Test kits for PAI-1 are commercially available (a kit for human PAI-1 is available from Sigma-Aldrich) and free, latent or complexed PAI-1 present in plasma reacts with a capture antibody that has been coated and dried onto a microtiter plate. Any unbound PAI-1 is washed away and an anti-PAI-1 primary antibody is added. Excess primary antibody is washed away and the bound antibody, which is proportional to the total PAI-1 present in the sample, is then reacted with an HRP-labeled secondary antibody. After an additional washing step, TMB is then used for color development at 450 nm. The amount of color development is directly proportional to the concentration of total PAI-1 in the sample.

[0153] 3-marker panel: syndecan-1, heparan sulfate, and hyaluronidase In some embodiments, the biomarker panel can include a three-marker test for endothelial glycocalyx health that detects syndecan-1 (SDC1), heparan sulfate (HS), and hyaluronidase (HAD), which are intended to assess the integrity of the glycocalyx.

[0154] Syndecans are transmembrane domain proteins with three to five heparan sulfate and chondroitin sulfate chains that have a variety of important ligands, including fibroblast growth factor, vascular endothelial growth factor, transforming growth factor-beta, fibronectin, and antithrombin-1. Syndecan-1 (SDC1) is a cell surface heparan sulfate proteoglycan that is a key component of the protective endothelial glycocalyx that covers the luminal surface of blood vessels. An important role of SDC1 is mechanosensing in the endothelium and regulating endothelial integrity and function. Shedding of syndecan-1 and heparan sulfate into the circulation is associated with inflammatory diseases and atherosclerosis. Test kits for syndecans, such as those precoated with monoclonal antibodies specific for SDC1, are commercially available. Samples are added to appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for SDC1. Avidin conjugated to HRP is then added to each microplate well and incubated. TMB substrate solution is then added to each well. Only those wells containing SDC1, biotin-conjugated antibody and enzyme-conjugated avidin show a color change. The enzyme-substrate reaction is stopped by the addition of sulfuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450 nm±10 nm. The concentration of SDC1 in the sample is then determined by comparing the OD of the sample to the standard curve.

[0155] Heparan sulfate (HS) is a strongly negatively charged polysaccharide with 1→4 linked sulfated glucosamine and uronic acid repeating disaccharide units. HS is present on cell surfaces and in the extracellular matrix and binds to proteins involved in anticoagulation, angiogenesis, microbial infection, and monocyte adhesion. HS are glycoproteins with the common feature of containing one or more covalently linked chains, including syndecans and glycosylphosphatidylinositol-anchored proteoglycans (glypicans), secreted extracellular matrix HSPGs (agrin, perlecan, type XVIII collagen), and the secretory vesicle proteoglycan, serglycin. HS are involved in the pathogenesis of atherosclerosis by their ability to trap plasma lipoproteins in the arterial wall and their effects on cell migration, adhesion, and proliferation. Intact HS chains are antiatherogenic. ELISA test kits for heparan sulfate are commercially available. This test involves pretreatment of serum with proteinase (actinase E) to digest serum proteins. One volume of dissolved actinase E (20 mg / mL in actinase E dissolution buffer) can be added per 10 volumes of serum and then mixed. Proteins can be digested for 16-20 hours at 55°C in a water bath. After digestion, the mixture can be boiled for 5 minutes to stop the digestion. After boiling, the mixture can be brought to room temperature (15-25°C) and then centrifuged at 3,000 rpm for 10 minutes. After centrifugation, the supernatant can be taken and mixed thoroughly. The supernatant can then be subjected to a heparan sulfate ELISA kit. The HS value can be calculated on the pretreated samples according to the procedure of the heparan sulfate ELISA kit. To determine the HS concentration in serum, the calculated HS value must be multiplied by the dilution factor as follows: HS concentration = calculated HS value x dilution factor x 1.1.

[0156] Hyaluronic acid (HA, also called hyaluronan or hyaluronate) is a large, negatively charged, non-sulfated, linear glycosaminoglycan (a class of negatively charged polysaccharides) of the repeating disaccharide structure D-glucuronic acid (UDP-GlcA) and N-acetylglucosamine (UDP-GlcNac), which is the major component of the endothelial glycocalyx. HA is widely distributed throughout connective, epithelial, and neural tissues. HA is the simplest glycosaminoglycan that confers compressive strength, lubrication, and hydration. Disruption of HA is atherogenic. Removal of HA-rich glycocalyx by hyaluronidase is associated with increased vascular permeability, resulting in atherogenic injury. Increased plasma HA and hyaluronidase (HAD) levels are found to be associated with endothelial glycocalyx damage, the presence of microvascular disease, and carotid intima-media thickening. In an exemplary assay, a coated well immunoenzymatic assay for quantitative measurement of hyaluronidase (HAD) utilizes a polyclonal anti-HAD antibody and a HAD-HRP conjugate. The assay sample and buffer are incubated with the HAD-HRP conjugate in the pre-coated plate for 1 hour. After the incubation period, the wells are decanted and washed 5 times. The wells are then incubated with a substrate for the HRP enzyme. The product of the enzyme-substrate reaction forms a blue complex. Finally, a stop solution is added to stop the reaction, after which the solution turns yellow. The color intensity is measured spectrophotometrically at 450 nm in a microplate reader. The HAD from the sample and the HAD-HRP conjugate compete for anti-HAD antibody binding sites, so the color intensity is inversely proportional to the HAD concentration. Because the number of sites is limited, the more sites occupied by HAD from the sample, the fewer sites are left to bind to the HAD-HRP conjugate. Standards of known HAD concentration are run simultaneously with the samples being assayed and a standard curve relating color intensity (OD) to the concentration of HAD is plotted. The concentration of HAD in each sample is interpolated from this standard curve.

[0157] 3-4-marker panel: hyaluronan synthase-1, heparan sulfate, plasminogen activator inhibitor, and optionally syndecan-1 Biomarker panels can also include any combination of the above biomarkers. That is, they are not limited to being used in combination with only three marker tests and a total of four tests. For example, another preferred combination can include a panel of hyaluronan synthase-1 (HAS-1), heparan sulfate (HS), and plasminogen activator inhibitor (PAI-1) as blood tests that define vascular leakage and clot development to correlate with plaque formation. As shown in the following example, these three biomarkers are highly correlated with plaque formation. In certain embodiments, syndecan-1 (SDC-1) is added to these biomarkers to form a four-marker panel.

[0158] Additional biomarkers In addition to the above, three other biomarkers associated with cell destruction are useful in the methods described herein and, in some embodiments, can be combined with any of the above biomarkers and panels to complement the diagnosis of a broader range of chronic diseases: gamma (γ') fibrinogen (GF), growth differentiation factor-15 (GDF-15), and pregnancy associated plasma protein-A (PAPP-A).

[0159] Gamma fibrinogen Plasma fibrinogen is a clotting factor and an acute-phase inflammatory marker involved in the pathophysiology of cardiovascular disease (CVD) (2005. JAMA. 294:1799-1809). Fibrinogen is a key component of the hemostatic system and plays a role in both primary and secondary responses. Fibrinogen is composed of three pairs of non-identical polypeptide chains. "Gamma (γ')" fibrinogen (GF) refers to the gamma chain. Thrombin-catalyzed cleavage of fibrinopeptides (Fp) A and B converts fibrinogen to fibrin, which spontaneously polymerizes and forms two-stranded profibrils that assemble into branched fibrin fibers, forming a fibrin clot (2008. Cardiovasc Hematol Agents Med Chem 6:181-189). GF is a biomarker of early clotting. GF has been demonstrated to be significantly associated with coronary artery disease and myocardial infarction in the Stockholm Coronary Artery Risk Factor Study and the Framingham Heart Study (2007. J Thromb Haemost 5:766-73), and to be significantly associated with stroke as seen in the Erasmus Stroke Study and others (2012. Thrombosis Research 129:807-809). GF is increased during inflammation and is regulated differently than total fibrinogen under pathological conditions as demonstrated in the Periodontitis and Vascular Events Study (2011. Thromb Haemost 105:605-9).

[0160] Elevated human levels of GF have been reported in various studies.

[0161] Study of 133 patients diagnosed with coronary artery disease (CAD): 0.299 g / L in controls vs. 0.413 g / L in disease (1996. J Biol Chem 271(38):23121-23125).

[0162] Epidemiology study of myocardial infarction (MI) in the Stockholm Coronary Risk Factor Cohort: 0.28 g / L higher in disease than in controls (2007. J Thromb Haemost 5:766-73).

[0163] Study of patients with a history of CVD and periodontal disease: greatly elevated compared to controls, 0.622 g / L (2010 Clin Chem 2010;56:781-8).

[0164] Study validating GF as an independent predictor of CAD: 405.70 mg / dL in controls vs. 433.36 in hypertensive participants (2017. Rev Esp Cardiol. 70:34-41).

[0165] Studies showing that GF is positively associated with peripheral artery disease (PAD), heart failure (HF), and mortality from CVD death: lowest quartile 8.0-24.34 mg / dl; highest quartile ≥ 35.19 mg / dl (2015. Arterioscler Thromb Vasc Biol. 35(12):2700-2706).

[0166] A study of 3,042 participants in the Framingham Heart Study Offspring Cohort: 0.258 mg / ml in individuals without high-risk CVD vs. 0.278 mg / ml in individuals with high-risk CVD (2011. Arterioscler Thromb Vasc Biol. Oct;31(10):2345-2352).

[0167] Physician's Health Study of 14,916 subjects: Level of 343 mg / dL, 2-fold increased risk of myocardial infarction (2013. University Heart Journal 9:40-46).

[0168] Studies reporting GF were significantly higher in patients with ischemic stroke, 0.37 g / L compared to 0.32 g / L in controls (2011. Thromb Haemost, 105:430-4).

[0169] Growth differentiation factor-15 Growth differentiation factor-15 (GDF-15) is a protein belonging to the transforming growth factor beta superfamily, which functions in regulating inflammatory pathways, apoptosis, and cell repair and proliferation associated with cardiovascular and neoplastic injury (2000.Molecular and Cellular Biology.20(10):3742-51). GDF-15 functions as a prognostic protein in patients with different diseases such as heart disease and cancer, is expressed at low concentrations in most organs, and is upregulated due to organ damage such as liver, kidney, heart, and lung (2005.Shock.23(6):543-8). GDF-15 is a stress-responsive cytokine that increases during tissue injury and inflammatory conditions and is associated with cardiometabolic risk. Increased levels of GDF-15 are associated with cardiovascular diseases such as hypertrophy, heart failure, atherosclerosis, endothelial dysfunction, obesity, insulin resistance, diabetes, and chronic kidney disease in diabetes. GDF-15 is an inflammatory marker associated with increased cardiovascular and non-cardiovascular mortality and plays a pivotal role in the development and progression of cardiovascular diseases, such as heart failure, coronary artery disease, atrial fibrillation, diabetes, cancer, and cognitive impairment (2013. Clinical Chemistry 59:1550-1552, 2014. Circulation 130:1847-1858). Increased levels of GDF-15 are associated with the progression and prognosis of disease states.

[0170] Elevated human levels of GDF-15 have been reported in various studies.

[0171] One study stratified blood GDF-15 levels into three categories: normal (<1200 pg / mL), moderately elevated (1200-1800 pg / mL), and severely elevated (>1800 pg / mL) (2010. Aging Cell, 9:1057-1064).

[0172] Elevated GDF-15 levels above 1800 ng / L have been reported to be associated with a higher risk of death within one year (2008. BMC Public Health, 8:148).

[0173] Another study showed that GDF-15 concentrations above 1800 ng / L correlated with a higher risk of all-cause and cardiovascular mortality compared with those below 1200 ng / L (2012. Clinical Chemistry 58:172-182).

[0174] Elevated GDF-15 levels have been reported to be associated with reduced endothelium-dependent vasodilation in resistance vessels below 948 ng / L (1st quartile) to above 1390 ng / L (4th quartile) (2009. Eur Heart J. 30:2346-2353).

[0175] Another study, considering a median concentration of 1253 ng / L at baseline, found that the hazard ratios (HRs) for the highest compared to the lowest quartile for CV death were 2.63; for sudden death, 3.06; for heart failure (HF) death, 4.3; for cancer death, 2.5; for HF hospitalization, 5.8 (3.2 to 10); for MI, 1.4; and for stroke, 1.8 (2017. Clinical Chemistry 63:1 140-151) (2017).

[0176] Pregnancy-associated plasma protein-A Pregnancy-associated plasma protein-A (PAPP-A) levels are an independent predictor of acute cardiovascular events. PAPP-A is associated with thin-cap plaque leakage and a greater burden of coronary thin-cap fibrous atheroma (TCFA). PAPPP-A is elevated in patients with acute coronary syndromes and those with risk factors such as obesity, hypertension, and / or diabetes compared to healthy subjects (2015. Biomark Med. 9:731-741). PAPP-A is highly expressed in vulnerable atherosclerotic plaques (2016. Medicine (Baltimore) 95:e2563; 2004. Circulation 109:1724-1728; 2005. Clin Chem 52:1096-1103).

[0177] Elevated human levels of PAPP-A have been reported in various studies.

[0178] PAPP-A levels are an independent predictor of acute cardiovascular event occurrence, are associated with thin-cap plaque leakage and greater burden of coronary thin-cap fibrous atheroma (TCFA), and are elevated in patients with acute coronary syndromes and those with risk factors such as obesity, hypertension, and / or diabetes compared to healthy subjects (2015. Biomark Med. 9:731-741), and are highly expressed in vulnerable atherosclerotic plaques (2016. Medicine (Baltimore) 95:e2563). Patients with ≥3 VH-TCFAs have been reported to have higher PAPP-A levels than patients with 1–3 VH-TCFAs or no VH-TCFAs (13.3±11.8 vs. 7.8±4.7 vs. 7.4±4.7 mIU / L, respectively, P<0.001) (2016. Medicine (Baltimore). 95(3):e2563).

[0179] PAPP-A levels in acute coronary syndrome: PAPP-A levels have been reported to be significantly elevated in patients with acute myocardial infarction (AMI) and unstable angina (UA) with mean levels of 64.26 and 36.23 ng / ml, respectively, whereas the mean PAPP-A level in controls was 10.68±1.04 ng / ml (2015. Indian J Clin Biochem. 30(2):150-154).

[0180] PAPP-A has been reportedly correlated with cardiovascular events in diabetic hemodialysis patients, with a median PAPP-A concentration of 17 mIU / patient in the fourth PAPP-A quartile (≤20.9 mIU / L) having an adjusted 2.6-fold increased risk for sudden death and a 2.8-fold increased risk for stroke compared with patients in the first quartile (≤13.4 mIU / L) (2014. Atherosclerosis. 236:263-269).

[0181] 3-marker panel: gamma fibrinogen, growth differentiation factor-15, and pregnancy-associated plasma protein-A In some embodiments, a biomarker panel useful in any of the methods described herein includes gamma fibrinogen (GF), growth differentiation factor-15 (GDF-15), and pregnancy associated plasma protein-A (PAPP-A).

[0182] 7-marker panel: hyaluronan synthase-1, heparan sulfate, plasminogen activator inhibitor, syndecan-1, gamma fibrinogen, growth differentiation factor-15, and pregnancy-associated plasma protein-A In certain embodiments, a biomarker panel useful in any of the methods described herein includes hyaluronan synthase-1 (HAS-1), heparan sulfate (HS), plasminogen activator inhibitor (PAI-1), syndecan-1 (SDC-1), gamma fibrinogen (GF), growth differentiation factor-15 (GDF-15), and pregnancy associated plasma protein-A (PAPP-A).

[0183] This 7-biomarker combination is useful for determining a biomarker signature indicative of a disease type and / or a disease stage. Thus, the 7-marker combination can be used in making a differential diagnosis between at least two diseases and / or two stages of a disease, allowing treatments to be targeted to specific diseases and / or disease stages.

[0184] This 7-biomarker combination is also useful for associating disease signatures with specific diseases. For example, the panel can be used to detect the levels of the seven biomarkers in various diseases or disease stages, and this raw data can be analyzed to define a disease signature for a given disease or disease stage. It can be determined that for a particular disease, a combination of 2, 3, 4, 5, or 6 biomarkers provides a reliable biomarker signature for the disease or disease stage and / or allows for the discrimination of two or more possible diseases or disease stages, facilitating differential diagnosis and thereby facilitating treatment targeted to the specific disease or disease stage identified in the subject. Thus, the 7-biomarker panel can be used to determine that only a subset of these markers is necessary to identify or distinguish a given disease and / or disease stage from another. In this way, the 7-biomarker panel can be used in studies aimed at identifying useful biomarker panels composed of a subset of the seven biomarkers.

[0185] Biomarker Panels and Uses Thereof General The biomarker panels described herein can be used alone or in combination. Biomarker panels can be used individually, or combinations of biomarker panels can be used to ensure reliability, for example, if a strong correlation has been established for any of the biomarkers in that particular panel.

[0186] The biomarker panel can be used in the following manner to detect the presence of, or a propensity to develop, a disease characterized by breakdown of the glycocalyx, endothelial inflammation, and / or oxidative damage to the endothelium (e.g., vascular disease), or a propensity to develop such a condition. In an exemplary embodiment, a sample is obtained or obtained (preferably by a medical professional) from a subject and tested using a biomarker panel (either a 4-panel test for coagulation, a 3-panel test for glycocalyx integrity, both of the above, or any combination of the above biomarkers). If any or all of the biomarkers are detected, it can be determined whether the subject has or is at risk for a disease characterized by breakdown of the glycocalyx, endothelial inflammation, and / or oxidative damage to the endothelium (e.g., vascular disease) by comparing the biomarker levels to known baseline levels for healthy individuals. In other words, if levels of two biomarkers above the baseline levels are detected, it can be determined that the individual has or is at risk for a disease characterized by breakdown of the glycocalyx, endothelial inflammation, and / or oxidative damage to the endothelium (e.g., vascular disease).

[0187] In some embodiments, the biomarker panel can be used to determine the stage of a disease (e.g., vascular disease) characterized by glycocalyx disruption, endothelial inflammation, and / or oxidative damage to the endothelium in an individual (e.g., to monitor the progression of the individual's vascular disease). The stage of a disease (e.g., vascular disease) characterized by glycocalyx disruption, endothelial inflammation, and / or oxidative damage to the endothelium can be determined by comparing the results to known stage levels. Based on the results of the vascular disease stage, medication appropriate for that particular stage can be prohibited to the individual.

[0188] In some embodiments, a biomarker panel can be used to determine a prognosis for a disease characterized by disruption of the glycocalyx, endothelial inflammation, and / or oxidative damage to the endothelium in an individual (e.g., vascular disease). For example, a poor prognosis can lead physicians and patients to choose a more aggressive form of treatment than would normally be used at the time of diagnosis and treatment.

[0189] In certain embodiments, the biomarker panels can also be used in methods for monitoring the effectiveness of drugs or other treatments for diseases characterized by glycocalyx disruption, endothelial inflammation, and / or oxidative damage to the endothelium (e.g., cardiovascular diseases or other diseases involving inflammation, vascular disruption, plaque, or blood clots). Such monitoring can be performed during drug development (e.g., in animal models or human subjects as described herein) or during actual patient treatment. Exemplary treatments that may be monitored include, but are not limited to, anti-inflammatory drugs (such as nonsteroidal anti-inflammatory drugs (NSAIDS), steroids, or immunoselective anti-inflammatory derivatives (ImSAIDs)), anticoagulants (such as alteplase, ardeparin, dalteparin, danaparoid, enoxaparin, fondaparinux, lepirudin, urokinase, or warfarin), antioxidants (such as glutathione, alpha lipoic acid, CoQ10, resveratrol, carotenoids, astaxanthin vitamin C, or vitamin E), supplements, and any other suitable therapeutic agents.

[0190] In some embodiments, the biomarkers included in the panels of the present disclosure measure factors produced early in the clot formation process. Thus, each of these biomarkers is useful alone and together in the panel in predicting the onset of biological processes (e.g., oxidation and immunogenic and / or inflammatory processes) that lead to the formation of clots. The biomarker panels described herein may be useful in conjunction with the "lipid panel" (measurement of cholesterol and triglycerides) devised by the American Heart Association, but since lipid panels cannot reliably predict cardiovascular disease, it is contemplated that one or more of the biomarker panels of the present disclosure may be substituted for this lipid panel for routine diagnosis.

[0191] As one of skill in the art will readily appreciate, absolute levels of biomarkers may vary depending on the biological sample (e.g., blood or blood fraction) tested and the particular assay used (as assays vary with respect to sensitivity and dynamic range). It is within the level of skill in the art to select and design assays suitable for discriminating between healthy individuals and those suffering from diseases characterized by disruption of the glycocalyx, inflammation, and / or oxidative damage.

[0192] Sampling and processing The assay methods described herein are generally performed on biological samples derived from animals, in some embodiments mammals, and in certain embodiments, humans.

[0193] The methods described herein can be performed using any sample relevant to the particular glycocalyx at issue (e.g., blood or blood fractions for atherosclerosis). Exemplary samples include, for example, blood, plasma, urine, saliva, tears, and cerebrospinal fluid, or any fraction thereof (e.g., fluid or tissue fractions, cells, or proteins).

[0194] The sample may be pretreated, if necessary, by dilution in an appropriate buffer solution or concentrated, if desired. Any of a number of standard aqueous buffer solutions utilizing any of a variety of buffers, such as phosphate, Tris, etc., at physiological pH, and / or protease inhibitors may be used.

[0195] Biomarker assays The biomarkers described herein may be detected and quantified by any of a number of methods well known to those of skill in the art, including analytical biochemical methods such as electrophoresis, capillary electrophoresis, electrochemiluminescence, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, mass spectrometry, or various immunological methods such as, but not limited to, Western blot, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioreceptor assay, proteomic methods (such as mass spectrometry), or quantitative immunostaining methods.

[0196] The sample reacts with various reagents in the panel based on the presence of the above biomarkers. In some embodiments, the sample applied to the panel is sent to a laboratory for analysis. Any method of detection and quantification may be used. In some embodiments, the panel contains one or more reagents (such as antibodies) conjugated to a detectable label. Those skilled in the art can easily determine the appropriate detection and quantification method for a given detectable label. For example, the assays described herein can provide colorimetric results and can be read with a colorimeter.

[0197] In certain embodiments, biomarkers are detected and / or quantified in biological samples using any of a number of well-known immunoassays (see, e.g., U.S. Patent Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a general review of immunoassays, see also Methods in Cell Biology Vol. 37: Antibodies in Cell Biology, Asai, ed., Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th ed., Stites & Terr, eds. (1991).

[0198] Traditional immunoassays often utilize a "capture agent" to specifically bind to the analyte, often to immobilize the analyte on a solid phase. In a preferred embodiment, the capture agent is an antibody.

[0199] Immunoassays also typically utilize a labeled detection agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeled detection agent may itself be one of the moieties that make up the antibody / analyte complex. Alternatively, the labeled detection agent may be a third moiety, such as another antibody, that specifically binds to the capture agent / analyte complex. Other polypeptides capable of specifically binding to immunoglobulin constant regions, such as polypeptide A or polypeptide G, may also constitute the labeled detection agent. These polypeptides are normal components of the cell wall of streptococci. They show strong non-immunogenic reactivity with immunoglobulin constant regions from various species (see generally Kronval et al., (1973) J. Immunol., 111:1401-1406, and Akerstrom (1985) J. Immunol., 135:2589-2542).

[0200] Immunoassays for detecting target biomarkers can be either competitive or non-competitive. Non-competitive immunoassays are assays in which the amount of captured analyte is measured directly. In competitive assays, the amount of analyte in a sample is measured indirectly by measuring the amount of added (exogenous) labeled analyte displaced (or competed away) from the capture agent by the analyte present in the sample. In a competitive assay, a known amount of labeled biomarker is added to the sample, and the sample is then contacted with the capture agent. The amount of labeled biomarker bound to the antibody is inversely proportional to the concentration of biomarker present in the sample.

[0201] Biomarkers can also be measured by any available proteomics method, such as mass spectrometry (MS), which measures the mass-to-charge ratio (m / z) of gas-phase ions. A mass spectrometer consists of an ion source that converts analyte molecules into gas-phase ions, a mass analyzer that separates the ionized analytes based on their m / z ratio, and a detector that records the number of ions at each m / z value. MS is particularly suitable for the analysis of GAGs to accurately determine the molecular weight of oligosaccharides and their distribution within a mixture. Two approaches for MS proteomics are total protein analysis ("top-down") and analysis of enzymatically or chemically produced peptides ("bottom-up"). Either or both can be used to measure any of the biomarkers described herein.

[0202] antibody Antibodies useful in the immunoassay methods described herein include polyclonal and monoclonal antibodies. Polyclonal antibodies are raised by injecting (e.g., subcutaneously or intramuscularly) an immunogen into a suitable non-human mammal (e.g., a mouse or rabbit). In general, the immunogen should induce the production of high titers of antibodies with relatively high affinity to the target antigen.

[0203] If desired, the antigen can be conjugated to a carrier protein by conjugation techniques well known in the art. Commonly used carriers include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The conjugate is then used to immunize the animal.

[0204] The antibodies are then obtained from blood samples taken from the animals. Techniques used to produce polyclonal antibodies have been extensively described in the literature (see, for example, Methods of Enzymology, "Production of Antisera With Small Doses of Immunogen: Multiple Intradermal Injections", edited by Langone et al. (Acad. Press, 1981)). The polyclonal antibodies produced by the animals can be further purified, for example, by binding to and elution from a matrix to which the target antigen is bound. Those skilled in the art will be aware of the various techniques common in the immunological field for purifying and / or concentrating polyclonal and monoclonal antibodies. See, for example, Coligan et al. (1991) Unit 9, Current Protocols in Immunology, Wiley Interscience.

[0205] For many applications, monoclonal antibodies (mAbs) are preferred. The general method used for the production of hybridomas secreting mAbs is well known (Kohler and Milstein (1975) Nature, 256:495). Briefly, as described by Kohler and Milstein, the technique involves isolating lymphocytes from local draining lymph nodes of five separate cancer patients with either melanoma, teratocarcinoma, or cervical, glioma, or lung cancer (samples were obtained from surgical specimens), pooling the cells, and fusing the cells with SHFP-1. The hybridomas were screened for the production of antibodies that bound to the cancer cell lines. Confirmation of specificity between mAbs can be performed using routine screening techniques (such as enzyme-linked immunosorbent assay, or "ELISA") to determine the basic reactivity pattern of the mAbs of interest.

[0206] As used herein, the term "antibody" encompasses antigen-binding antibody fragments, e.g., single chain antibodies (scFv or otherwise), which can be produced / selected using phage display technology. The ability to express antibody fragments on the surface of viruses that infect bacteria (bacteriophages or phages) allows the production of single binding antibody fragments, e.g., in the range of 10 to 150 uL. 10 This allows the isolation of antibody fragments from a library of more than 10 non-binding clones. To express antibody fragments on the surface of phages (phage display), the antibody fragment gene is inserted into a gene encoding a phage surface protein (e.g., pIII) and the antibody fragment-pIII fusion protein is displayed on the phage surface (McCafferty et al., (1990) Nature, 348:552-554; Hoogenboom et al., (1991) Nucleic Acids Res. 19:4133-4137).

[0207] Since the antibody fragments on the surface of the phage are functional, the antigen-binding antibody fragments carried by the phage can be separated from non-binding phage by antigen affinity chromatography (McCafferty et al., (1990) Nature, 348:552-554). Depending on the affinity of the antibody fragments, enrichment factors of 20-fold to 1,000,000-fold can be obtained for a single round of affinity selection. However, by infecting bacteria with the eluted phage, more phage can be propagated and subjected to another round of selection. Thus, a 1000-fold enrichment in one round can become 1,000,000-fold in two rounds of selection (McCafferty et al., (1990) Nature, 348:552-554). Thus, even if the enrichment is low (Marks et al., (1991) J. Mol. Biol. 222:581-597), multiple rounds of affinity selection can result in the isolation of rare phages. Selection of a phage antibody library against an antigen results in enrichment, so that the majority of clones bind to the antigen after only 3-4 rounds of selection. Therefore, only a relatively small number of clones (a few hundred) need to be analyzed for binding to the antigen.

[0208] As one of skill in the art will readily appreciate, antibodies may be prepared by any of a number of commercial services (eg, Berkeley Antibody Laboratory, Bethyl Laboratories, Anawa, Eurogenetec, etc.).

[0209] solid phase For embodiments of the biomarker assay that utilize a solid phase as a support for the capture agent, the solid phase can be any suitable porous material with sufficient porosity to allow access by the reagents and suitable surface affinity for binding the capture agent. Microporous structures are generally preferred, but materials with a gel structure in the hydrated state can be used as well. Useful solid supports include natural polymeric carbohydrates and their synthetically modified, cross-linked, or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gum, cellulose esters, mixed cellulose esters, and cellulose ethers, especially with nitrates and carboxylic acids, nitrogen-containing natural polymers, such as proteins and derivatives, including cross-linked or modified gelatins, natural hydrocarbon polymers, such as latex and rubber, synthetic polymers that can be prepared in a suitably porous structure, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and partially hydrolyzed derivatives thereof, polyacrylamides, polymethacrylates, copolymers of the polycondensates of the above. and terpolymers, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides, porous inorganic materials, such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, alkali and alkaline earth metal silicates, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolites, silica gel, or glass (these materials can be used as filters with the above polymeric materials); and mixtures or copolymers of the above classes, such as graft copolymers obtained by initiating the polymerization of synthetic polymers on existing natural polymers. All of these materials can be used in suitable shapes, such as films, sheets, or plates, or they can be coated, adhered, or laminated to a suitable inert carrier, such as paper, glass, plastic film, cloth, etc.

[0210] Nitrocellulose's porous structure has excellent absorption and adsorption qualities for a wide variety of reagents, including monoclonal antibodies. Nylon also has similar characteristics and is also suitable.

[0211] Porous solid phases useful in the assays described herein may be in the form of sheets having a thickness of about 0.01 to 0.5 mm, for example about 0.1 mm. The pore size may vary within wide limits, preferably from about 0.025 to about 15 microns, especially from about 0.15 to about 15 microns.

[0212] Preferred solid phase materials for flow-through assay devices include filter paper, e.g., porous fiberglass materials or other fibrous matrix materials. The thickness of such materials is not critical and is a matter of choice based primarily on the characteristics of the sample or analyte being assayed, such as the flowability of the biological sample.

[0213] Alternatively, the solid phase can consist of microparticles. Microparticles useful in the assays described herein can be selected by one of skill in the art from any suitable type of particle material, including those composed of polystyrene, polymethylacrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar materials.

[0214] The microparticles may be suspended in a mixture of soluble reagents and the biological sample, or may be retained and immobilized by a support material, in which case the microparticles on or in the support material cannot substantially migrate to other locations within the support material.

[0215] The methods of the present disclosure may be adapted for use in systems utilizing microparticle technology, including automated and semi-automated systems in which the solid phase comprises a microparticle, including those described in pending U.S. Application No. 425,651 and U.S. Patent No. 5,089,424, which correspond to published EPO applications EP0425633 and EP0424634, respectively, and U.S. Patent No. 5,006,309.

[0216] In certain embodiments, the solid phase comprises one or more electrodes. The capture agent can be attached directly or indirectly to the electrode. In one embodiment, for example, the capture agent can be attached to a magnetic or paramagnetic microparticle, which is then placed near the electrode surface using a magnet. Systems in which one or more electrodes serve as the solid phase are useful when detection is based on electrochemical interactions. Exemplary systems of this type are described, for example, in U.S. Patent No. 6,887,714 (issued May 3, 2005). Basic methods for electrochemical detection are further described below.

[0217] The capture agent can be attached to the solid phase by adsorption onto a porous material and held by hydrophobic forces, or the surface of the solid phase can be activated by a chemical process that causes the covalent attachment of the capture agent to the support.

[0218] To change or enhance the inherent charge of the solid phase, a charged substance may be directly coated on the solid phase material or on the microparticles, which are then retained by the solid phase material. Ion trapping procedures for immobilizing immobilizable reaction complexes with negatively charged polymers, as described in US Application No. 150,278, corresponding to EP Publication No. 0326100, and US Application No. 375,029 (EP Publication No. 0406473), can be used to affect rapid solution-phase immunochemical reactions. In these procedures, the immobilizable immune complexes are separated from the rest of the reaction mixture by ionic interactions between the negatively charged polyanion / immune complexes and the previously treated, positively charged porous matrix, and are detected by using any of several signal generating systems, including chemiluminescence systems, as described, for example, in US Application No. 921,979, corresponding to EPO Publication No. 0273,115.

[0219] When the solid phase is silicon or glass, the surface must generally be activated prior to attachment of a specific binding partner. Activated silane compounds such as triethoxyaminopropylsilane (available from Sigma Chemical Co., St. Louis, Mo.), triethoxyvinylsilane (Aldrich Chemical Co., Milwaukee, Wis.), and (3-mercapto-propyl)-trimethoxysilane (Sigma Chemical Co., St. Louis, Mo.) can be used to introduce reactive groups such as amino-, vinyl, and thiol, respectively. Such activated surfaces can be used to directly link capture agents (in the case of amino or thiol) or they can be further reacted with linkers such as glutaraldehyde, bis(succinimidyl)suberate, SPPD9 succinimidyl 3-[2-pyridyldithio]propionate), SMCC (succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate), SIAB (succinimidyl[4-iodoacetyl]aminobenzoate), and SMPB (succinimidyl 4-[1-maleimidophenyl]butyrate) to separate the capture agent from the surface. The vinyl groups can be oxidized to provide a means for covalent attachment. The vinyl groups can also be used as anchors for polymerization of various polymers such as polyacrylic acid, which can provide multiple attachment points for a particular capture agent. The amino groups can be reacted with oxidized dextrans of various molecular weights to provide hydrophilic linkers of different sizes and capacities. Examples of oxidizable dextrans include dextran T-40 (molecular weight 40,000 daltons), dextran T-110 (molecular weight 110,000 daltons), dextran T-500 (molecular weight 500,000 daltons), dextran T-2M (molecular weight 2,000,000 daltons) (all of which are available from Pharmacia, Piscataway, NJ), or Ficoll (molecular weight 70,000 daltons; available from Sigma Chemical Co., St. Louis, Mo.).Additionally, polyelectrolyte interactions can be used to immobilize specific capture agents on solid phases using techniques and chemistries described in U.S. application Ser. No. 150,278, filed Jan. 29, 1988, and U.S. application Ser. No. 375,029, filed Jul. 7, 1989, each of which is incorporated herein by reference.

[0220] Other considerations influencing the choice of solid phase include its ability to minimize non-specific binding of labeled entities and its compatibility with the labeling system being utilized, for example, solid phases used with fluorescent labels should have sufficiently low background fluorescence to permit signal detection.

[0221] After attachment of the specific capture agent, the surface of the solid support can be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding.

[0222] Labeling System As discussed above, many immunoassays utilize a labeled detection agent.

[0223] The label may be attached to the detection agent before, during, or after contact with the biological sample. So-called "direct labels" are detectable labels that are directly attached to or incorporated into the detection agent prior to use in the assay. Direct labels may be attached to or incorporated into the detection agent by any of a number of means well known to those of skill in the art.

[0224] In contrast, so-called "indirect labels" typically bind to a detection agent at some point during the assay. In many cases, indirect labels bind to a moiety that is attached to or incorporated into the detection agent prior to use. Thus, for example, an antibody used as a detection agent ("detection antibody") can be biotinylated prior to use in an assay. During the assay, an avidin-conjugated fluorophore can bind to a detection agent bearing biotin to provide a label that is easily detected.

[0225] In another example of indirect labeling, polypeptides capable of specifically binding to immunoglobulin constant regions, such as polypeptide A or polypeptide G, can also be used as labels for detection antibodies. Thus, such polypeptides can be labeled and added to the assay mixture, where they bind to the detection antibody.

[0226] Some labels useful in the assays described herein may require the use of an indicator reagent to generate a detectable signal. In an ELISA, for example, an enzyme label (e.g., beta-galactosidase) requires the addition of a substrate (e.g., X-gal) to generate a detectable signal.

[0227] In some embodiments, the biomarker panel can use a support structure such as a flat microwell plate (such as an ELISA plate) with multiple wells to hold the samples. Various enzymes or antibodies can be applied to the wells as needed for each test, such as those listed above. To prevent contamination or unwanted diffusion of the sample, a housing can surround the biomarker panel, which can be made of plastic or another suitable material.

[0228] kit The biomarker panels described herein can be included in a kit. The kit includes one or more reagents useful for performing one or more assays described herein. The kit can include a biomarker panel (a 4-panel or a 3-panel or any combination of the above biomarkers), instructions for use, materials for taking and applying samples to the panel (such as but not limited to swabs, syringes, or vials), and an explanation of biomarker levels and their meanings (such as normal values). The kit can include various antibodies as needed to detect the biomarkers.

[0229] In some embodiments, the kit comprises a package having one or more containers holding the reagents as one or more separate compositions, or optionally, where compatibility of the reagents allows, as a mixture. The kit can also include other materials that may be desirable from a user's perspective, such as buffers, diluents, standards, and / or any other materials useful for processing the sample, washing, or performing any other steps of the assay.

[0230] Kits useful for the assays described herein preferably include instructions for carrying out one or more of the assays. Instructions included in the kits of the invention may be attached to packaging materials or may be included as a package insert. The instructions are typically, but are not limited to, written or printed. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" may include the address of an internet site that provides the instructions.

[0231] Use of biomarker signatures in differential diagnosis and treatment The biomarkers described herein have been found to have different absolute and / or relative levels that correlate with different disease types. Figure 13 demonstrates that the above 7-biomarker panel provides different biomarker signatures or "fingerprints" for each of the three types of cardiovascular disease: coronary heart disease (CHD), hypertension (HTN), and heart failure (HF). Blood marker levels were obtained from published clinical data. Individual markers were associated to some degree with any of the three diseases, but as a panel, they became more specific and accurate.

[0232] A biomarker signature can be composed of individual biomarker levels and / or parameters derived therefrom (e.g., ratios of the level of one biomarker to the level of another). Biomarker signatures can be derived from the use of algorithms, pattern recognition, dot matrices, and the like.

[0233] Arterial plaque animal model Knockout ApoE mice (apoE * Although the 3-Leiden, apoE- / -) is a standard model of atherosclerosis, it does not represent the clinical setting: (1) there are no studies yet showing plaques in this model that spontaneously break down (2005. Circ Res. 96:667-674), and (2) although the fatal human plaque is a fibrous lesion without a necrotic core (1996. Circulation. 93:1354-1363), a suitable animal model of this was not available (2001. J Pathol. 195:257-263; 2001. Arterioscler Thromb Vasc Biol. 21:1470-1476).

[0234] Using an accidental rat model, hyperglycemia (2011. Anesth Analg. 112(6):1289-1295), Glycocalyx disruption (syndecan-1 and heparan sulfate shedding) has been evaluated in hemorrhagic shock (2013. J Trauma Acute Care Surg. 75(5):759-66), inflammation and ischemia-reperfusion injury (2004. Am J Physiol Heart Circ Physiol. 286(5):H1672-80), and coagulation function after hemorrhagic shock (2013. J Trauma Acute Care Surg. 75(5):759-66). However, there were no animal models that represent glycocalyx disruption associated with cardiovascular disease (CVD).

[0235] To develop the biomarkers described herein and to provide for the evaluation of FTX drugs discussed above, we developed the first mouse model mimicking the CVD and thromboembolic disease cascade, called the Tunanac Arterial Plaque (TAP) Mouse™ model (2017. J Clin Exp Cardiolog Suppl 8:1). To generate this model, mice were fed a high fat diet and challenged with xenobiotics and pathogens. This process is illustrated in Example 1, where mice were fed a 60% fat (by mass) diet and treated with the bacteria Porphyromonas gingivalis and polychlorinated biphenyls, resulting in mice that developed well-formed subendothelial plaques (Figures 10A-10C).

[0236] In various embodiments, the animal can be any typical laboratory animal (eg, a mammal, such as a mouse, rat, dog, cat, non-human primate, etc.).

[0237] In some embodiments, the high fat diet is at least 21%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% fat by weight. In various embodiments, the percentage of fat in the diet is within a range bounded by any of these values, such as 30%-70%, 35%-69%, 40%-68%, 45%-67%, 50%-65%, 55%-65% fat by weight.

[0238] The xenobiotics used are typically those that cause disruption of the glycocalyx, endothelial inflammation, oxidative damage to the endothelium, or any combination thereof. Exemplary xenobiotics include, but are not limited to, polychlorinated biphenyls, heavy metals (e.g., lead, arsenic, cadmium, mercury, nickel), phytoestrogens (e.g., resveratrol, caffeine), dioxins (e.g., chlorinated waste), phthalates (e.g., plasticizers), flame retardants (e.g., halocarbons), phenols, polycyclic aromatic hydrocarbons, pesticides / insecticides / herbicides, microorganisms, drugs (e.g., aromatic sulfonic acids, alkylbenzene sulfonates, polychlorinated, and those containing diazo bonds), smoke, and other particulate matter, etc. While not limited to any particular mechanism of action, most xenobiotics, in this context, are believed to disrupt the electronic bonds of molecules, e.g., take up electrons. Thus, molecules that lose electrons are oxidized, and the oxidized molecules can become antigenic and / or induce inflammation and proinflammatory cytokines.

[0239] The pathogen may be any pathogen that can infect the animal used in the model. Examples of pathogens that can be used in typical experimental animals (e.g., mammals such as mice, rats, dogs, cats, non-human primates, etc.) include, but are not limited to, intestinal microorganisms (Chlamydia sp., H. pylori, Enterobacter sp., Cytomegalovirus), dental microorganisms (Porphyromonas gingivalis, Prevotella sp., Tannerella sp., Aggregatibacter sp.), etc.

[0240] Treatment regimens may vary depending on the particular high-fat diet, xenobiotics, and pathogens used, and the appropriate regimen in a particular case can be determined by one of skill in the art given the guidance provided herein (see, e.g., Example 1).

[0241] Glycocalyx repair and maintenance compositions The present disclosure provides a composition for treating multiple diseases characterized by the breakdown of the glycocalyx. The composition includes at least one glycocalyx repair and maintenance compound. The composition preferably treats glycocalyx breakdown, inflammation, and oxidative damage. The composition can also treat any one of these problems individually. The glycocalyx repair and maintenance compound can be any suitable compound capable of performing one or more of these functions in the body.

[0242] For example, glycocalyx repair and maintenance compounds can be peptides and homologs of glycopeptides in the glycocalyx that act to stimulate glycoprotein synthesis. During glycoprotein synthesis, the peptide portion of the molecule is synthesized first, and then the sugar portion is incorporated. Attachment of the peptide portion to the surface appears to be through an association between a region of repeated amino acids and a component of the glycocalyx.

[0243] In some embodiments, the glycocalyx repair and maintenance compound may be any of the compounds FTX-214, FTX-218, FTX-219, FTX216-4, FTX224-2, FTX226-4, FTX229, FTX230, or FTX231 (described below), either alone or in combination. While each compound may be effective in its own right for the indications and repair of the glycocalyx described below (and thus they may be used individually in the methods herein), they may be used synergistically in combination to repair and maintain the glycocalyx and reverse inflammation and oxidative damage that may contribute to glycocalyx damage and destruction. In an exemplary embodiment, the glycocalyx repair and maintenance compound may be used in combination with the compounds FTX-214, FTX-218, and FTX-219.

[0244] FTX-214 FTX-214 (indole acetamide) is an antioxidant that increases antioxidant capacity by enhancing the antioxidant enzymes GSH, SOD, and CAT, thereby preventing the accumulation of reactive oxygen species that damage the glycocalyx. FTX-214 is shown in Formula I. FTX-214 is shown as a salt in combination with trifluoroacetic acid in Formula IA, which is the form of FTX-214 used in Example 2. Other salts of FTX-214 are contemplated and are described below under "Derivatives of FTX Compounds." As one of skill in the art will appreciate, compounds may be formed as salts to increase water solubility, thereby reducing toxicity. [ka]

[0245] The IUPAC numbering and nomenclature for Formula I is shown below: [ka]

[0246] FTX-218 FTX-218 (lipoic acid choline) is anti-inflammatory, neutralizes cytokines, and promotes glycocalyx synthesis. FTX-218 is shown in Formula II. [ka]

[0247] The IUPAC numbering and nomenclature for Formula II is shown below: [ka]

[0248] FTX-219 FTX-219 (lipoate-cysteine-glutamic acid tripeptide) repairs the glycocalyx, restores constituent moieties, and enhances synthesis of the glycocalyx. FTX-219 is shown in Formula III. FTX-219 is shown as a salt in combination with trifluoroacetic acid in Formula IIIA, which is the form of FTX-219 used in Example 2. Other salts of FTX-219 are contemplated and are described below under "Derivatives of FTX Compounds." [ka]

[0249] Formula II To I The IUPAC numbering and nomenclature for these compounds is shown below. [ka]

[0250] FTX216-4 FTX216-4 (6-N-oxidoribose-phenazinol or myxin-1β xyloside) has the basic function of 6-N-oxidoribose-phenazinol, as illustrated in Formula IV. [ka]

[0251] The IUPAC numbering and nomenclature for Formula IV is shown below: [ka]

[0252] The composition of formula IV stimulates the synthesis of chondroitin sulfate, the second most common glycosaminoglycan (GAG) in the endothelial cell glycocalyx. β-D-xylosides act as primers for GAG chain initiation and compete with xylosylated core proteins for the addition of galactose to the xylose residues of the core protein. Xyloside activity varies with the aglycone (the type of aglycone is important because the primer competes with endogenous substrates and inhibits the synthesis of proteoglycans (PGs) and glycoproteins). The composition is a broad-spectrum antimicrobial agent.

[0253] FTX-224-2 FTX224-2 (dioxidoisothiocyanatoindole, also known as dioxidoisothiocyanatopyrrole and dioxidoisothiocyanatocholine) is designed to inhibit blood clotting and is shown in Formula V. FTX224-2 is shown as a salt in combination with trifluoroacetic acid in Formula VA, which is the form of FTX-224-2 used in Example 2. Other salts of FTX-224-2 are contemplated and are described below under "Derivatives of the FTX Compounds." [ka]

[0254] The IUPAC numbering and nomenclature for Formula V is shown below: [ka]

[0255] Compounds of formula V also act as anti-inflammatory, anti-proliferative and anti-angiogenic agents and prevent glutathione depletion in the liver.

[0256] FTX-226-4 FTX226-4 is piperidine ribose, as illustrated in formula VI. FTX226-4 is shown as a salt in combination with HCl in formula VIA, which is the form of FTX226-4 used in Example 2. Other salts of FTX226-4 are contemplated and are described below under "Derivatives of FTX Compounds." [ka]

[0257] The IUPAC numbering and nomenclature for Formula VI is shown below: [ka]

[0258] The compound of formula VI inhibits the production of two important proinflammatory mediators, IL6 and PGE2 (which cause pain), and enhances the bioavailability of drugs by inhibiting drug metabolism or by increasing absorption. This compound may be useful in combination treatment with other drugs by improving the therapeutic effect or reducing the required dose of other drugs when administered with disease-modifying antirheumatic drugs (DMARDs) as a therapeutic or dietary supplement. This compound is an antihypertensive agent, as it inhibits platelet aggregation and stabilizes and increases the activity of eNOS, resulting in a reduction in blood pressure.

[0259] FTX-229 FTX-229 is a nicotinylcholine, as exemplified by Formula VII. [ka]

[0260] The IUPAC numbering and nomenclature for Formula VII is shown below: [ka]

[0261] After the discovery of the nicotinic acid receptor GPR109A in adipocytes and immune cells, new direct immunomodulatory properties of nicotinic acid were identified, including its direct anti-inflammatory activity in other cells (such as hepatocytes and endothelial and vascular cells) that were previously shown to be important players in the release of inflammatory mediators from adipose tissue and in atherogenesis, and nicotinic acid prevents macrophages from invading the atherosclerotic vessel wall by activating its receptor, thereby stopping chronic inflammation. On the other hand, choline performs various functions in the mammalian body, namely in the structure of cell membranes, in preventing the liver from accumulating fat, and as a precursor molecule for the neurotransmitter acetylcholine. Choline, via its metabolic product trimethylglycine (betaine), is the main source of methyl groups involved in the S-adenosylmethionine synthesis pathway, and is used in treating hepatitis, glaucoma, atherosclerosis, and possibly neurological disorders.

[0262] FTX-230 FTX-230 is an ammonium lipoate, as illustrated in Formula VIII. [ka]

[0263] The IUPAC numbering and nomenclature for Formula VIII is shown below: [ka]

[0264] Lipoic acid is reduced to dihydrolipoic acid and functions as an antioxidant. Reduced glutathione (GSH) is the most abundant non-protein thiol in mammalian cells and is the preferred substrate for several enzymes in xenobiotic metabolism and antioxidant defense, but the direct use of GSH as a therapeutic agent is limited due to its unfavorable biochemical and pharmacokinetic properties.

[0265] FTX-231 FTX-231 (melatonin 6,β-D xyloside) is shown in formula IX. This compound can improve the condition of the glycocalyx by inducing at least glycosaminoglycan (GAG) chain synthesis, independent of the proteoglycan core protein (Thorsheim K. et al., 2016. Glycoconj J. 33:245-57). Biosynthesis of GAG chains is initiated by the addition of xylose, followed by galactose, to the primer core protein. The enzyme xylosylprotein β1,4-galactosyltransferase 7 (β4GalT7) is an essential enzyme in the biosynthesis of GAG chains, with xylose being the optimal acceptor substrate (Siegbahn, A. et al., 2914. Chem. Sci., 5:3501-3508). Thus, the xylosylated core of GlcUA-Gal-Gal-Xyl-proteins is galactosylated by β4GalT7 to produce chondroitin sulfate (CS) or heparan sulfate (HS). Alternatively, the galactosyltransferase β4GalT7 transfers D-xylose from UDP-xylose to the core protein to produce heparan sulfate, heparin, chondroitin sulfate, and dermatan sulfate, depending on the tissue. [ka]

[0266] The IUPAC numbering and nomenclature for Formula IX is shown below: [ka]

[0267] FTX-216-1 FTX-216-1 (myxin-1 beta xyloside, also known as 6-N-oxidribose-phenazinol) produces xylose for glycosaminoglycan (GAG) maintenance and repair. GAGs are linked to proteoglycan proteins that make up the glycocalyx. FTX-216 is shown in formula X. [ka]

[0268] The IUPAC numbering and nomenclature for formula X is shown below: [ka]

[0269] FTX-226 FTX-226 (trans-trans-trans piperine ribose) targets very low density lipoprotein (VLDL) and is shown in Formula XI. [ka]

[0270] The IUPAC numbering and nomenclature for Formula XI is shown below: [ka]

[0271] It should also be understood that any of the above compounds may be used individually and in any combination to achieve the desired effect, either alone or together, to prevent damage or shedding of the existing glycocalyx layer and further provide any of the functionality described above.

[0272] Synthesis scheme of FTX compounds FTX-214 [ka] [ka]

[0273] Synthesis process: 1. 6-(Benzyloxy)-1H-indole was treated with formaldehyde, acetic acid and N,N-dimethylamine to give gramine. 2. Gramine was converted to cyano using NaCN, DMF aqueous solution at 80°C. 3. Cyano to LiAlH 4 Reduction with ethyl acetate at 0° C. in ether gave the amine. 4. The amine is dissolved in dichloromethane and Ac 2 0, Et 3 N was used to convert it to N-acetyl. 5. Pd-C / H in ethyl acetate 2 Debenzylation by. 6. NaCNBH in dichloromethane 3 Reduction of the indole double bond by . 7. Aniline was converted to its TFA salt. [ka]

[0274] Synthesis process: 8. 6-(Benzyloxy)-1H-indole was treated with formaldehyde, acetic acid and N,N-dimethylamine to give gramine. 9. Gramine was converted to cyano using NaCN, DMF aqueous solution at 80°C. 10. Cyano to LiAlH 4 Reduction with ethyl acetate at 0° C. in ether gave the amine. 11. The amine is dissolved in dichloromethane and Ac 2 0, Et 3 N was used to convert it to N-acetyl. 12. Pd-C / H in ethyl acetate 2 Debenzylation by. 13. NaCNBH in dichloromethane 3 Reduction of the indole double bond by . 14. Aniline was converted to its TFA salt.

[0275] FTX-218 [ka]

[0276] Synthesis step: To a solution of 1 (1.10 g, 5.34 mmol) and A (0.475 g, 5.34 mmol) in anhydrous DCM (40 mL) was added EDCI (1.22 g, 6.41 mmol) and DMAP (65.7 mg, 0.0534 mmol) under N2 at -20°C. After addition, the mixture was warmed to 20°C until no starting material was detected by TLC. Then, all volatiles were removed under reduced pressure and it was purified by silica gel column chromatography to give 2 (1.20 g, yield: 81.1%) as a clear yellow oil. [ka]

[0277] Synthesis step: Methyl iodide (0.681 g, 4.76 mmol) was added to a solution of 2 (1.2 g, 4.33 mmol) in anhydrous DCM (20 mL). The reaction mixture was stirred at 20° C. overnight until no starting material was detected by TLC. With vigorous stirring, the mixture was slowly poured into diethyl ether (250 mL). The product was isolated by filtration as a yellow solid (1.40 g, yield: 77.3%).

[0278] FTX-219 [ka]

[0279] Synthesis process: 1. The thiol was converted to trityl protection in DMF, TrCl. 2. Lipoic acid, EDC, HOBt, Et 3 N, C.H. 2 Cl 2 The amine was coupled via 3. LiOH, THF, H 2 0, ester hydrolysis at room temperature. [ka]

[0280] Synthesis process: 1. Amino acids, BOC 2 , THF, H 2 The BOC protection was achieved via O. 2. The acid was converted to the tert.butyl ester. 3. Pd-C / H 2 , Cbz deprotection via ethyl acetate. [ka]

[0281] FTX-219 (Lipoate-Cysteine-Glutamic Acid Tripeptide) (TFA Salt)

[0282] Synthesis process: 1. EDC, HOBt, Et 3 N, C.H. 2 Cl 2 Amide formation with amines and acids via. 2.CH 2 Cl 2 Boc and trityl deprotection with TFA in gives the TFA salt.

[0283] FTX-216-4 [ka]

[0284] Synthesis process: 1. Pyrogallol was converted to a ketal in the presence of triethoxyformate, cat p-TSA, in toluene at 100°C. 2. Free hydroxyl groups are converted to phenylalanine by the addition of BnBr, K in DMF. 2 CO 3 The benzyl protection was carried out in the presence of 3. Ketal deprotection with methanol, cat p-TSA. 4. The diol was converted to a diketone. 5. Diketones coupled with 1,2-diaminobenzene give tricyclic compounds. 6. Pd-C / H in ethyl acetate 2Debenzylation with gives the free hydroxyl group. [ka]

[0285] Synthesis process: 1. D-Ribose, MeOH, cat.H 2 SO 4 The methyl glycoside was converted to the corresponding methyl glycoside in the presence of 2. Converted to acetal protection in the presence of acetone and cat p-TSA. 3. Hydroxy was converted to tosyl in Py, TsCl. [ka]

[0286] Synthesis process: 4. Ether coupling with tosyl gives the sugar-linked compound. 5. The N group was converted to an N-oxide. 6. Deprotection of methyl glycosides in aqueous HCl. [ka]

[0287] FTX-224-2 [ka]

[0288] FTX-224 (Dioxidoisothiocyanate Indole, also known as Dioxidoisothiocyanate Choline) (HCl salt)

[0289] Synthesis process: 1. Thiol alkylation with TrCl. 2. The hydroxyl was converted to a Ts group using TsCl / Py. 3. Alkylation of amines with tosyl groups. 4. The thiol group was converted to S-oxide.

[0290] FTX-226-4 [ka]

[0291] Synthesis process: 1. Hydroxy was converted to Ts group. 2. Alkylation of Ts group with hydroxyl group. 3. Deprotection of Boc, acetyl and methyl glycosides.

[0292] FTX-229 [ka]

[0293] 1.Synthetic route: [ka]

[0294] General Reagents and Conditions: a.SOCl 2 , reflux 2 hours; b. Dehydrated CH 2 Cl 2 , room temperature 2 hours, 63.5%; c.CH 3 CH 2 OH, room temperature, 2 hours, 61.5%

[0295] 2. Experimental Section: 2.1. Synthesis of compound 3: [Table 1]

[0296] A flask containing 1 (2.70 g, 22 mmol) and 2 (10 mL, 139 mmol) was filled with N 2 The mixture was heated under reflux for 2 hours. Excess SOCl 2 Remove under reduced pressure and then dehydrate 2 Cl2 (30 mL).

[0297] 2-2. Synthesis of compound 5: [Table 2]

[0298] The final reaction mixture is dehydrated and cooled. 2 Cl 2 (20 mL) was added dropwise to a solution of 4 in 100 mL of CH. The mixture was then stirred for 2 h. TLC analysis showed that the starting material had disappeared, and ammonia solution was added to the mixture until it became strongly alkaline. 2 Cl 2 (100mL x 3) and Na 2 SO 4 and then subjected to flash column chromatography (eluent: CH 2 Cl 2 / CH 3 The product was purified by hexane / TEA (100 / 6 / 1). This protocol yielded 2.972 g of product, 63.g% yield.

[0299] 2-3. Synthesis of compound 7: [Table 3]

[0300] 4.69g of 6, N 2 It was slowly poured into a mixture of 5 in 40 mL ethanol under atmosphere, which was stirred for 2 h. The mixture was filtered and the solid was recrystallized from hot ethanol to give 4.537 g of product, 61.5% yield.

[0301] FTX-230 [ka]

[0302] FTX-231 FTX-216-1 [ka]

[0303] Synthesis process: 7. Pyrogallol was converted to a ketal in the presence of triethoxyformate, cat p-TSA, in toluene at 100 °C. 8. Free hydroxyl groups are converted to phenylalanine by the addition of BnBr, K in DMF. 2 CO 3 The benzyl protection was carried out in the presence of 9. Ketal deprotection with methanol, cat p-TSA. 10. Diols were converted to diketones. 11. Coupling of diketones with 1,2-diaminobenzene gives tricyclic compounds. 12. Pd-C / H in ethyl acetate 2 to give the free hydroxyl group. [ka]

[0304] Synthesis process: 7. D-Ribose, MeOH, cat.H 2 SO 4 The methyl glycoside was converted to the corresponding methyl glycoside in the presence of 8. Converted to acetal protection in the presence of acetone and cat p-TSA. 9. Py: Hydroxyl was converted to tosyl in TsCl. [ka]

[0305] 10. Ether coupling with tosyl gives the sugar-linked compound. 11. The N group was converted to the N-oxide. 12. Deprotection of methyl glycosides in aqueous HCl.

[0306] Derivatives of FTX compounds The FTX compounds may be administered in their "natural" form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, etc., provided that the salts, esters, amides, prodrugs, or derivatives are pharmacologically suitable, i.e., effective in at least one of the methods described herein. Salts, esters, amides, prodrugs, and other derivatives of the FTX compounds are known to those skilled in the art of synthetic organic chemistry and can be prepared using standard procedures, for example, as described in March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Edition, NY Wiley-Interscience.

[0307] Pharmaceutically acceptable salts of FTX compounds include those derived from pharma- ceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, gluconic acid, isethionic acid, glycinic acid, malic acid, mucoic acid, glutamic acid, sulfamic acid, ascorbic acid; toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, trifluoroacetic acid, and benzenesulfonic acid. Salts derived from suitable bases include, but are not limited to, alkalis such as sodium and ammonium. In some embodiments, one or more of the compounds described herein may be prepared by solubility in water, for example, NaCl, NH 4 F, MgCO 3 , and Fe 2 (HPO 4 ) 3 It is used as a salt.

[0308] For example, acid addition salts are typically prepared from free bases using conventional methodologies involving reaction with a suitable acid. Generally, the base form of the drug is dissolved in a polar organic solvent, such as methanol or ethanol, and an acid is added thereto. The resulting acid can precipitate or be removed from solution by adding a less polar solvent. Suitable acids for preparing acid addition salts include both organic acids, such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, and inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Acid addition salts can be reconverted to free bases by treatment with a suitable base. Exemplary acid addition salts of FTX compounds herein are halide salts, such as those that can be prepared using hydrochloric acid or hydrobromic acid. Conversely, basic salts of the FTX compounds described herein are prepared in a similar manner using pharma- ceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, etc. Exemplary basic salts include alkali metal salts, e.g., sodium salts, and copper salts.

[0309] Acid addition salts useful in the methods described herein include physiologically compatible acid addition salts, most preferably the dihydrochloride salt. Bis-quaternary salts useful in the methods described herein include physiologically compatible bis-quaternary salts such as methiodides and dimethiodides.

[0310] The preparation of esters typically involves functionalization of hydroxyl and / or carboxyl groups and / or other reactive groups that may be present in the molecular structure of the drug.Esters are typically acyl-substituted derivatives of free alcohol groups, i.e., moieties derived from carboxylic acids of the formula RCOOH, where R is alkyl, preferably lower alkyl.Esters can be reconverted to the free acids, if desired, by using conventional hydrogenolysis or hydrolysis procedures.

[0311] Amides and prodrugs may also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from anhydrides or acid chlorides by reaction with ammonia or a lower alkyl amine. Prodrugs are typically prepared by covalent attachment of a moiety that results in a compound that is therapeutically inactive until modified by the individual's metabolic system.

[0312] When the FTX compounds (or derivatives) described herein contain chiral or prochiral centers, they may exist in different stereoisomeric forms, including (+) and (-) type enantiomers or mixtures thereof. The present disclosure includes within its scope both individual isomers and mixtures thereof. It will be understood that when a mixture of optical isomers exists, they can be separated according to classical resolution methods based on their different physicochemical properties, for example, by fractional crystallization of their acid addition salts with suitable optically active acids, or by chromatographic separation with suitable mixtures of solvents.

[0313] Pharmaceutical preparations The FTX compounds (or derivatives) described herein are typically combined with a pharma- ceutically acceptable carrier (excipient) as described in Remington's Pharmaceutical Sciences (1980) 16th ed., edited by Osol, 1980. The pharma- ceutically acceptable carrier can contain one or more physiologically acceptable compounds that act, for example, to stabilize the composition or to increase or decrease absorption of the FTX compound (or derivative). Pharmaceutically acceptable carriers suitable for use in the methods described herein are non-toxic to cells, tissues, or subjects at the dosages employed, and can include buffers (such as phosphate buffers, citrate buffers, and buffers made from other organic acids), antioxidants (e.g., ascorbic acid), low molecular weight (less than about 10 residues) peptides, polypeptides (such as serum albumin, gelatin, and immunoglobulins), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, arginine, and / or lysine), monosaccharides, disaccharides, and / or other carbohydrates (including glucose, mannose, and dextrins), chelating agents (e.g., ethylenediaminetetraacetic acid [EDTA]), sugar alcohols (such as mannitol and sorbitol), salt-forming counterions (e.g., sodium), and / or anionic surfactants (such as Tween™, Pluronics™, and PEG). In one embodiment, the pharma-ceutically acceptable carrier is an aqueous pH buffered solution.

[0314] Other pharma- ceutically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms.Various preservatives are well known and include, for example, phenol and ascorbic acid.Those skilled in the art will understand that the selection of a pharma-ceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the FTX compound (or derivative) and their particular physicochemical properties.

[0315] Suitable pharmaceutical formulations can be administered in a variety of unit dosage forms depending on the method of administration. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injections, implantable sustained release formulations, lipid complexes, etc. In another embodiment, one or more components of the solution can be provided as a "concentrate", for example, in a storage container ready for dilution (e.g., in a pre-measured volume) or in a soluble capsule ready to be added to a volume of water.

[0316] The pharmaceutical preparations described herein can be stored in any standard form, including, for example, aqueous solution or lyophilized cake.Such compositions are typically sterilized when administered to a subject.The sterilization of aqueous solution can be easily achieved by filtration through a sterile filtration membrane.When the composition is stored in lyophilized form, the composition can be filtered before or after lyophilization and reconstitution.

[0317] In certain embodiments, the FTX compound (or derivative) may also be delivered through the skin using a conventional transdermal drug delivery system, i.e., a transdermal "patch," in which the FTX compound (or derivative) is typically contained within a laminated structure that functions as a drug delivery device that is applied to the skin. In such structures, the drug composition is typically contained in a layer, or "reservoir," that is underneath an upper backing layer. It will be understood that the term "reservoir" in this context refers to the amount of "active ingredient" that is ultimately available for delivery to the surface of the skin. Thus, for example, the "reservoir" may contain the active ingredient in an adhesive on the backing layer of the patch, or in any of a variety of different matrix formulations known to those skilled in the art. The patch may contain a single reservoir, or it may contain multiple reservoirs.

[0318] In one embodiment, the reservoir comprises a polymer matrix of a pharma- ceutically acceptable contact adhesive material that serves to attach the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylene, polysiloxane, polyisobutylene, polyacrylate, polyurethane, and the like. Alternatively, the drug-containing reservoir and the skin contact adhesive are present as separate and distinct layers, and the adhesive underneath the reservoir can in this case be a polymer matrix as described above, or it can be a liquid or hydrogel reservoir, or it can take some other form. The backing layer in these laminates, which serves as the top surface of the device, preferably serves as the main structural element of the "patch" and provides the device with most of its flexibility. The material selected for the backing layer is preferably substantially impermeable to the FTX compound (or derivative) and any other materials present.

[0319] In certain embodiments, one or more active FTX compounds (or derivatives) described herein are administered alone or in combination with other therapeutic agents in an implantable (e.g., subcutaneous) matrix, referred to as a "depot formulation."

[0320] A major problem with standard drug dosing is that the typical delivery of a drug results in a rapid burst of medication upon dosing, followed by a rapid loss of the drug from the body. Most of the drug's side effects occur during the burst phase of its release into the bloodstream. Secondly, the time that the drug is present in the bloodstream at therapeutic levels is very short, and most is used and removed during the short burst.

[0321] Drugs embedded in various matrix materials for sustained release (e.g., FTX compounds or derivatives described herein) can alleviate these problems. For example, drugs embedded in polymer beads or polymer wafers have several advantages. First, most systems allow for sustained release of the drug, so that small levels of drug are sustained dosing to the body. This prevents the side effects typically associated with high burst levels of regular injection or pill-based drugs. Second, these polymers can be made to release over hours to months, so the therapeutic period of the drug is significantly increased. In many cases, by mixing different ratios of the same polymer components, polymers with different degradation rates can be made, allowing for significant flexibility depending on the drug being used. Extended drug release is not only beneficial for people who may have difficulty maintaining normal dosages, such as the elderly, but also represents an increased ease of use that anyone can appreciate. Most polymers can be made to be degraded and removed by the body over time, so they do not remain in the body after the therapeutic interval.

[0322] Another advantage of polymer-based drug delivery is that polymers can often stabilize or solubilize proteins, peptides, and other large molecules that cannot be used as medicines in other ways.Finally, many drug / polymer mixtures can be placed directly in diseased areas, allowing specific targeting of medicines where they are needed without losing the drug to the "first pass" effect.This is certainly effective in treating the brain, which is often depleted of medicines that cannot pass the blood / brain barrier.

[0323] A wide variety of approaches to design depot formulations that provide sustained release of active agents are known and suitable for use in the methods described herein. In general, the components of such formulations are biocompatible and can be biodegradable. Biocompatible polymeric materials have been widely used in therapeutic drug delivery and medical implant applications to provide localized and sustained release. See Leong et al., "Polymeric Controlled Drug Delivery," Advanced Drug Delivery Rev., 1:199-233 (1987); Langer, "New Methods of Drug Delivery," Science, 249:1527-33 (1990); Chien et al., Novel Drug Delivery Systems (1982). Such delivery systems offer the potential to enhance therapeutic efficacy and reduce overall toxicity.

[0324] Examples of classes of synthetic polymers that have been investigated as possible solid biodegradable materials include polyesters (Pitt et al., "Biodegradable Drug Delivery Systems Based on Aliphatic Polyesters: Applications to Contraceptives and Narcotic Antagonists," Controlled Release of Bioactive Materials, 19-44 (Richard Baker, ed., 1980); poly(amino acids) and pseudopoly(amino acids) (Pulapura et al., "Trends in the Development of Bioresorbable Polymers for Medical Applications," J. Biomaterials Appl., 6:1, 216-50 (1992); polyurethanes (Bruin et al., "Biodegradable Lysine Diisocyanate-based Poly(Glycolide-co-.epsilon.Caprolactone)-Urethane Network in Artificial Skin," Biomaterials, 11:4, 291-95 (1990); polyorthoesters (Heller et al., "Release of Norethindrone from Poly(Ortho and polyanhydrides (Leong et al., "Polyanhydrides for Controlled Release of Bioactive Agents", Biomaterials 7:5, 364-71 (1986).

[0325] Thus, for example, the FTX compound (or derivative) can be incorporated into a biocompatible polymer composition and formed into a desired shape outside the body. This solid implant is then typically inserted into the subject's body through an incision. Alternatively, small individual particles composed of these polymer compositions can be injected into the body, for example, using a syringe. In an exemplary embodiment, the FTX compound (or derivative) can be encapsulated in microspheres of poly(D,L-lactide) polymer suspended in a diluent of water, mannitol, carboxymethylcellulose, and polysorbate 80. The polylactide polymer is gradually metabolized into carbon dioxide and water, releasing the FTX compound (or derivative) into the system.

[0326] In yet another approach, the depot formulation can be injected by syringe as a liquid polymer composition.Liquid polymer compositions useful for biodegradable controlled release drug delivery systems are described, for example, in U.S. Patent Nos. 4,938,763; 5,702,716; 5,744,153; 5,990,194; and 5,324,519.After being injected in liquid state, or alternatively as a solution, the composition solidifies to become solid.

[0327] One type of polymer composition suitable for this application comprises a non-reactive thermoplastic polymer or copolymer dissolved in a body fluid dispersion solvent. The polymer solution is placed in the body where the polymer coagulates or precipitates and solidifies upon disappearance of the solvent or diffusion of the solvent into surrounding body tissues. See, for example, Dunn et al., U.S. Patent Nos. 5,278,201; 5,278,202; and 5,340,849 (which disclose thermoplastic drug delivery systems in which a solid, linear, biodegradable polymer or copolymer is dissolved in a solvent to form a liquid solution).

[0328] The FTX compound (or derivative) may also be adsorbed onto a membrane, such as a silastic membrane, which may be implanted, as described in International Publication No. WO 91 / 04014. Other exemplary implantable sustained release systems include, but are not limited to, Re-Gel®, SQ2Gel®, and Oligosphere® by MacroMed, ProLease® and Medisorb® by Alkermes, Paclimer® and Gliadel® wafers by Guilford pharmaceuticals, Duros implants by Alza, Acoustic biSpheres by Point Biomedical, Intelsite capsules by Scintipharma, and the like.

[0329] Treatment The glycocalyx repair and maintenance compounds described above can be used alone or in any combination to treat, repair, and maintain any membrane that has a glycocalyx, and / or reduce endothelial inflammation and / or oxidative damage to the endothelium. The membrane to be treated can be, but is not limited to, blood vessels, lungs, endometrial lining, gastrointestinal lining, epithelium, or any other lining in the body.

[0330] The integrity of the endothelial glycocalyx is determined by the equilibrium between shedding and synthesis, but under pathological conditions, the equilibrium is disrupted, resulting in the shedding of one or more of its components (e.g., heparan sulfate, syndecan-1, or hyaluronic acid) into the blood. However, the equilibrium can be restored, and the glycocalyx can be reconstituted to its native hydrodynamic thickness by self-assembly within as few as 5-7 days (2009. Circul Res 2009;104:1318-1325). It has been demonstrated that heparan sulfate (HS) is repaired on the surface of endothelial cells within 20 hours in vitro (2013. Cell Mol Bioeng 6:160-174). Example 2 below demonstrates the ability of the combination of the glycocalyx repair and maintenance compounds described above to prevent and / or heal arterial plaque in a proprietary animal model of atherosclerosis.

[0331] The present disclosure provides a method for treating the causes of multiple diseases by administering to an individual a composition comprising a glycocalyx repair and maintenance compound to repair the glycocalyx, reverse inflammation, and reverse oxidative damage. The glycocalyx repair and maintenance compound treats the underlying cause of the disease, repairs the glycocalyx, and maintains the glycocalyx. The glycocalyx repair and maintenance compound can be any of those described above. Normal blood flow shear is required for balanced shedding and synthesis of the proteoglycan components of the glycocalyx, as well as for maintaining the residence of various enzymes and signaling molecules, including the antioxidant superoxide dismutase (SOD), the anti-inflammatory antithrombin (AT-III), and proteases (thrombin, plasmin, protease-3, and elastase, which are important in blood clotting, immunity, and inflammation). Disruption of the balance of these resident enzymes leads to shedding of the glycocalyx, followed by a series of pathological events. Thus, the therapeutic approaches disclosed herein that improve the structure and function of the glycocalyx may also inhibit pathological processes associated with vascular inflammation. The compositions may restore the balance of the above enzymes.

[0332] More specifically, in certain embodiments, the disease to be treated can be any cardiovascular disease (CVD), since CVD involves the destruction of the glycocalyx, inflammation, and oxidative damage, ultimately resulting in clot formation and migration of clots to small blood vessels, resulting in impaired flow (i.e., stroke, etc.). Thus, the present disclosure provides a method of treating CVD by administering to an individual a composition comprising a glycocalyx repair and maintenance compound to repair the glycocalyx, reverse inflammation, or reverse oxidative damage (preferably achieving all three). The CVD to be treated can be, but is not limited to, coronary heart disease, myocardial infarction, stroke, hypertension, atrial fibrillation, congestive heart failure, congenital heart disease, peripheral arterial disease, venous thrombosis, deep vein thrombosis, and pulmonary embolism.

[0333] The disease treated with the glycocalyx repair and maintenance compounds can also be any disease with indications of disrupted glycocalyx, inflammation, and / or oxidative damage. For example, a disrupted glycocalyx may be manifested in damage to the body (because the glycocalyx buffers cell membranes and protects them from chemical damage), impaired immunity to infection (because the glycocalyx allows the immune system to recognize and selectively attack foreign bodies), cancer (because changes in the glycocalyx of cancerous cells allow the immune system to recognize and destroy them), transplant rejection (because the glycocalyx forms the basis for compatibility of blood transfusions, tissue transplants, and organ transplants), cell adhesion problems (because the glycocalyx binds cells together so that tissues do not fall apart), inflammatory regulation disorders (because the glycocalyx coating on the endothelial walls in blood vessels prevents white blood cells from rolling / binding in healthy conditions), fertilization problems (because the glycocalyx allows sperm to recognize and bind to the egg), problems with embryonic development (because the glycocalyx guides embryonic cells to their destination), and diabetes.

[0334] Inflammation may be involved in plasma cell leukemia, rheumatoid arthritis, multiple myeloma, Lennert's syndrome, Castleman's disease, cardiac myxoma, liver cirrhosis, chronic polyarthritis, bacterial and viral meningitis, graft-versus-host reaction, chorioamnionitis, inflammatory gastrointestinal diseases, many cancers and advanced cancers (including pancreatic cancer), encephalitis, decreased gene expression, schizophrenia, depression, bacterial, viral, fungal, and parasitic infections, exposure to microbial toxins, tissue necrosis, presence of foreign bodies, immune responses, acne vulgaris, asthma, autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, hypersensitivity, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, graft rejection, vasculitis, interstitial cystitis, atherosclerosis, allergies, myopathies, leukocyte deficiencies, endometriosis, and multiple sclerosis.

[0335] Oxidative damage may be involved in cancer, Parkinson's disease, Alzheimer's disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, sickle cell disease, lichen planus, vitiligo, autism, infections, and chronic fatigue syndrome.

[0336] It should be understood that glycocalyx repair and maintenance compounds may not only reverse the above-listed diseases, but may also prevent their occurrence and inhibit their progression.

[0337] In certain embodiments, the present disclosure generally provides a method of treating multiple causes of disease by administering a combination therapy to an individual and targeting multiple causes of the disease. The combination therapy has multiple components necessary to target each underlying cause of the disease. Many diseases (such as CVD, cancer, diabetes, or any other disease described below) have multiple mechanisms involved in their development. For example, the cause of the disease may include glycocalyx destruction, inflammation, and oxidative damage. To treat this disease, the combination therapy may include a component that can target glycocalyx destruction, a component that can target inflammation, and a component that can target oxidative damage. The multiple components may be in a single polypill. An example of a combination therapy is the combination glycocalyx repair and maintenance compounds of FTX-214, FTX-218, and FTX-219 described above. Previously, diseases were treated only with their symptoms and not their underlying causes, or a single composition was given to treat the underlying cause (similar to antibiotics). The present disclosure allows for the administration of multiple components (preferably within a single pill) that each target a different cause of a disease, making it easier for patients to take their medication and also targeting the underlying cause of their disease.

[0338] Because the glycocalyx repair and maintenance compounds can treat any one of glycocalyx destruction, inflammation, and oxidative damage, either individually or in combination, the present disclosure also includes the following methods: A method of repairing glycocalyx is provided by administering a composition including a glycocalyx repair and maintenance compound to an individual to repair the glycocalyx. A method of reversing inflammation is provided by administering a composition including a glycocalyx repair and maintenance compound to an individual to reverse inflammation and repair the glycocalyx. In other words, by reversing inflammation that may cause glycocalyx destruction and damage, the glycocalyx may be restored to normal function. A method of reversing oxidative damage is provided by administering a composition including a glycocalyx repair and maintenance compound to an individual to reverse oxidative damage and repair the glycocalyx. In other words, by reversing oxidative damage that may cause glycocalyx destruction and damage, the glycocalyx may be restored to normal function.

[0339] Route of administration and dosage The compositions of the present disclosure are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, the scheduling of administration, the patient's age, sex, weight, and other factors known to physicians. Thus, for purposes herein, a pharma- ceutically "effective amount" is determined by such considerations as are known in the art.

[0340] For example, the multiple FTX compounds (or derivatives) used in any of the combination therapies described herein can be administered by the same or different administration routes. When possible, it is generally desirable to administer these agents by the same administration route, preferably in the same formulation. However, due to differences in pharmacodynamics, pharmacokinetics, or other considerations, it may be decided to co-administer the selected compound and the additional agent in separate formulations.

[0341] In the treatment method of the present disclosure, the composition of the present disclosure can be administered in various ways. It should be noted that they can be administered as one or more active ingredients, including one or more of the compounds, alone or in combination with one or more pharma-ceutically acceptable carriers, diluents, adjuvants and vehicles. The compound can be administered topically, orally, buccal, subcutaneously, rectally, intravaginally, or parenterally, including intravenously, intraarterially, intradermally, intramuscularly, intracisterna, intraperitoneally, intratonsillarly, and intranasally, as well as intrathecally, and by injection. Implants of the compound are also useful.

[0342] The subject to be treated is a warm-blooded animal, particularly a mammal, including humans.Examples of suitable subjects include, for example, laboratory animals or pets, such as mice, rats, guinea pigs, rabbits, cats, dogs, and monkeys and other primates.In certain embodiments, the subject is a human.Pharmaceutically acceptable carriers, diluents, adjuvants and vehicles, and implant carriers generally refer to inserts, non-toxic solid or liquid fillers, diluents or encapsulating materials that do not react with the active ingredients of the present disclosure.

[0343] In various embodiments, the FTX compounds (or derivatives) described herein can be administered orally, in which case delivery can be enhanced by the use of protective excipients. This is typically accomplished by complexing the FTX compounds (or derivatives) with compositions to make them resistant to acid and enzymatic hydrolysis, or by packaging the drug in a suitable resistant carrier, such as liposomes. Means of protecting drugs for oral delivery are well known in the art (see, for example, U.S. Patent No. 5,391,377).

[0344] When the compound of the present disclosure is administered parenterally, it is generally formulated in a unit dosage injectable form (solution, suspension, emulsion).The pharmaceutical composition suitable for injection includes sterile aqueous solution or dispersion and sterile powder for reconstitution into sterile injectable solution or dispersion.The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oil.

[0345] Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil, and esters such as isopropyl myristate, may also be used as solvent systems for the compound compositions. In addition, various additives that enhance the stability, sterility, and isotonicity of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it is desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents that delay absorption, for example, aluminum monostearate and gelatin. However, according to the present disclosure, any vehicle, diluent, or additive used must be compatible with the compound.

[0346] Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present disclosure in the required amount of the appropriate solvent with various other ingredients, as desired. Sterility can be ensured by the use of sterile components and processes during formulation or by sterilization after formulation.

[0347] The composition comprising the compound of the present disclosure can be parenterally administered to the subject in the form of sustained release subcutaneous implant or targeted delivery system, such as monoclonal antibody, vector delivery, iontophoresis delivery, and / or delivery using polymer matrix, liposome, and / or microsphere.Examples of delivery systems useful for the present disclosure include U.S. Patent Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196.Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0348] In an exemplary embodiment, the compounds described herein may be administered orally, preferably in a single dosage form. The dose may be administered as a single dose or multiple doses over several days. Treatment generally has a length that varies depending on the length of the disease process and the effectiveness of the drug and the species of the subject being treated.

[0349] In therapeutic applications, one or more of the active agents described herein are administered to a subject in an amount sufficient to treat glycocalyx disruption, inflammation, and / or oxidative damage. Amounts effective for this use may depend on the disease state, the degree of improvement sought, and the general state of the subject's health. Single or multiple administrations of the active agent may be administered depending on the dosage and frequency required and tolerated by the subject.

[0350] The concentration of the active agent can vary widely and is selected primarily based on the volume of liquid, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the subject. According to standard practice, the clinician can titrate the dosage and modify the route of administration as necessary to obtain the optimal therapeutic effect. Generally, the clinician will start with a low dose and increase the dosage until the desired therapeutic effect is achieved. The starting dose for a given active agent can be estimated, for example, from in vitro and / or animal data.

[0351] In certain embodiments, the concentration of the FTX compound (or derivative) is typically selected to provide a dosage in the range of about 10 μg / kg / day to about 200 mg / kg / day, sometimes more. In various embodiments, the dosage is in the range of 25 μg / kg / day to about 175 mg / kg / day, particularly about 50 μg / kg / day to about 150 mg / kg / day, more particularly about 75 μg / kg / day to about 125 mg / kg / day, and even more particularly about 90 μg / kg / day to about 100 mg / kg / day, for example, about 0.1 to 100 mg / kg / day. It will be understood that such dosages may be varied to optimize the treatment regimen in a particular subject or group of subjects, and thus any of these values ​​may represent the upper or lower limits of a preferred dosage range (e.g., about 10 μg / kg / day to about 100 mg / kg / day) according to the present invention.

[0352] When used in combination, the compounds may be used in the same or different dosages. Thus, the compounds of two drug combinations may be used in a 1:1 ratio (by weight) or, for example, 1:2, 1:3, 1:4, 1:5, or other ratios. The compounds of three drug combinations may be used in a 1:1:1 ratio (by weight) or any number of possible ratios, including, but not limited to, for combinations containing additional compounds, for example, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:2:2, 1:2:3, 1:2:4, 1:2:5, 1:3:3, 1:3:4, 1:3:5, 1:4:4, 1:4:5, 1:5:5, etc. In some embodiments, the effect of the individual compounds is additive. In some embodiments, the effect of the individual compounds is synergistic. In some embodiments, the use of a combination of compounds allows for the use of a lower dose of one or more of the compounds in the combination. Determination of a suitable dose of a compound for treating a particular condition in a particular subject is within the level of skill in the art in light of the guidance provided herein.

[0353] In exemplary embodiments, the dose for any of the compounds may be from 5 mg to 750 mg (per average 70 kg human body weight). In a preferred combination, the dose may be from 50 mg FTX-214, 50 mg FTX-218, and 50 mg FTX-219 (effective dose) up to 750 mg FTX-214, 750 mg FTX-218, and 750 mg FTX-219 (maximum tolerated dose). The 50 mg dose was shown to prevent or reverse the breakdown of the glycocalyx as evidenced by the reversal of plaque formation, shown in Figures 12A-12H (B; FTX-226-4 + FTX-229 + FTX-214; F: FTX-224-2 + FTX-216 + FTX-214; I: FTX-216 + FTX-214 + FTX-218; and K: FTX-214 + FTX-218 + FTX-219).

[0354] Coadministration with other FTX compounds Compositions comprising the glycocalyx repair and maintenance compounds described herein can be administered in combination with one or more other therapeutic agents to treat certain diseases and conditions. Therapeutic agents include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDS), such as, but not limited to, acetaminophen, salicylates (e.g., aspirin, diflunisal, salsalate), acetic acid derivatives (e.g., indomethacin, ketorolac, sulindac, etodolac, diclofenac, nabumetone), propionic acid derivatives (e.g., ibuprofen, naproxen, flurbiprofen, ketoprofen, oxaprozin, fenoprofen, loxoprofen), fenamic acid derivatives (e.g., meclofenamic acid, mefenamic acid, flufenamic acid, tolfenamic acid), oxicams (e.g., enolic acid, These may include aryl alkanoic acid derivatives (e.g., piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam), arylalkanoic acid derivatives (e.g., tolmetin); or selective COX-2 inhibitors (e.g., celecoxib, rofecoxib, valdecoxib, parecoxib, lumiracoxib, etoricoxib, firocoxib), and narcotic analgesics (e.g., morphine, codeine, oxycodone, and other opiates). The therapeutic agents may also generally be from one or more of the following classes: antihistamines, anti-infectives, anti-tumor agents, autonomic agents, blood products, hematopoietic agents, coagulants, thrombosis agents, cardiovascular agents, cellular therapies, central nervous system agents, contraceptives, dental agents, diagnostic agents, bactericides, electrolytes, caloric agents, and water balance agents, enzymes, airway agents, ophthalmic, otic, nasal, and pharyngological preparations, gold compounds, heavy metal antagonists, hormones and their synthetic substitutes, uterotonics, radioactive FTX compounds, serum, toxoids, vaccines, skin and mucosal agents, smooth muscle relaxants, and vitamins. These therapeutic agents may be administered simultaneously with, before, or after the glycocalyx repair and maintenance compounds, they may be in separate or the same dosage forms, and they may have different or the same release profiles.

[0355] The compositions disclosed herein offer the advantage over conventional therapies of treating the underlying cause of a disease, not just the symptoms. In some cases, however, it may be advantageous to do both. Thus, the present disclosure provides methods that entail co-administering a composition comprising a glycocalyx repair and maintenance compound with a drug targeted to one or more symptoms. There are many symptom-targeted drugs currently marketed for cardiovascular disease, including cholesterol-lowering drugs such as statins and fibrates for CHD; diuretics, ACE inhibitors, ARBs, calcium inhibitors, and beta-blockers for hypertension; and anticoagulants for stroke, such as anticoagulants (e.g., heparin, rivaroxaban, low molecular weight heparin, dabigatran etexilate methanesulfonate, bivalirudin, coumadin, abciximab, eprifibatide, tirofiban), antiplatelet agents (e.g., clopidogrel bisulfate, prasugrel, ticagrelor, cilostazol, aspirin, terutroban, dipyridamole), and fibrinolytic agents (e.g., tissue plasminogen activator (tPA), streptokinase).

[0356] Receptor function Receptors are embedded in and pass through the glycocalyx: transmembrane glycoprotein receptors account for a significant number of membrane-bound receptors, and disruption of the glycocalyx can render these membrane-bound receptors dysfunctional and structurally disrupted. Glycocalyx repair and maintenance compounds repair the glycocalyx and restore structural integrity and function to these receptors. Thus, the present disclosure provides methods for restoring the structural and functional integrity of receptors in the glycocalyx by administering glycocalyx repair and maintenance compounds to an individual, and for restoring the structural integrity and function of receptors embedded in and passing through the glycocalyx.

[0357] Receptors in the glycocalyx can be for a variety of antigens and antibodies, both polyclonal and monoclonal. By restoring receptor integrity, the overall systemic action of the ligand (e.g., antigen or antibody) on the receptor can be restored to a healthy state and effectively increased. The activity can be metabolic, immunological, or any other activity that the receptor controls. The response of antibodies can be enhanced by administration of glycocalyx repair and maintenance compounds, because the receptors they bind to are restored.

[0358] Thus, the present disclosure provides methods of repairing the glycocalyx and receptors therein and enhancing drug response by administering to an individual suffering from a disease a composition comprising a glycocalyx repair and maintenance compound, co-administering an antibody, repairing the glycocalyx, repairing receptors in the glycocalyx, and enhancing response to the antibody. The present disclosure also provides compositions for treating a disease comprising a glycocalyx repair and maintenance compound and an antibody. The components of the combination may be in the same dosage form or different dosage forms and may be administered with different or the same release profiles.

[0359] The disease treated by co-administration of a glycocalyx repair and maintenance compound with an antibody can be any disease or condition that antibodies can be used to treat, such as, but not limited to, an autoimmune disease, cancer, a metabolic disorder, or an infectious disease. The receptor to be repaired can be any receptor that the antibody specifically binds to or otherwise interacts with.

[0360] The antibody may generally be any suitable monoclonal or polyclonal antibody, such as, but not limited to, 3F8, 8H9, abagovomab, abciximab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anifrolumab, anrukinzumab, apolizumab, arcitumomab, acelizumab, atinumab, atotumab, tet ... Lizumab, atorolumumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, beziotoxumab, biciromab, bimagrumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, Catumaxomab, cBR96-doxorubicin immunoconjugate, CC49, cedelizumab, certolizumab pegol, cetuximab, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab (cnatumumab), concizumab, CR6261, crenezumab, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dinutuximab, zirridabumab, dorlimomab alitox, droditumab, durigotumab , dupilumab, durvalumab, ducidizumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elotuzumab, ersilimomab, emibetuzumab, enabatuzumab, enfortumab vedotin, enlimomab pegol, enokizumab, enoticumab, encituximab, epitumomab situxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab,Farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, fizitumumab, framvotumab, fretikumab, fontolizumab, foralumab, forvirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomilikimab, guselkumab, ibalizumab, ibritumomab tiuxetan, icurus Kumab, Igovomab, IMAB362, Imciromab, Imgatuzumab, Inclacumab, Indatuximab-ravtansine, Infliximab, Inolimomab, Inotuzumab-ozogamicin, Intetumumab, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Remaresomab, Lerdelimumab, Lexatumumab, Ribivirumab, Rifastuzumab-vedotin, Ligelizumab, Lintuzumab, Lirilumab, Roderucizumab, Lorvotuzumab, Lorvotuzumab Mab-mertansine, lucatumumab, lurizumab pegol, rumiliximab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narutumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotu uzumab, nivolumab, nofetumomab merpentan, obiltoxaximab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratuzumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pasivaximab, palivizumab, panitumumab, pancomab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pembrolizumab,Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placlumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO140, Quilizumab, Racotumomab, Ladolezumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Lobatumumab, Lomodumab, Lomoso uzumab, rontalizumab, rovelizumab, ruplizumab, samilizumab, sarilumab, satumomab pendetide, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-CD19A, SGN-CD33A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sul Somab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, talexuzumab, tefibazumab, terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, TGN1412, ticilimumab, tigatuzumab, tildrakizumab, TNX-650, tocilizumab, toralizumab, tositumomab, tobetumab, tralokinumab, trastuzumab, TRBS 07, tregalizumab, tremelimumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vanticutumab, bapaliximab, valilumab, batelizumab, vedolizumab, veltuzumab, beparimomab, besencumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, dillalimumab, or zolimimab aritox.

[0361] One particular receptor whose integrity can be restored by glycocalyx repair and maintenance compounds is the LDL receptor, which mediates LDL endocytosis in the liver, the main pathway for LDL clearance from the circulation. It is desirable to reduce LDL levels in individuals with high cholesterol and atherosclerosis as well as other cardiovascular diseases. Proprotein convertase subtilisin / kexin type 9 (PCSK9) plays a key role in cholesterol metabolism by controlling the levels of LDL particles circulating in the bloodstream. PCSK9 increases plasma LDL cholesterol by promoting the degradation of LDL receptors. Monoclonal antibody (MAb) anti-PCSK9 (U.S. Pat. No. 8,062,640 to Sleeman et al., U.S. Pat. Nos. 8,030,457, 8,168,762 to Jackson et al., U.S. Patent Application Publication Nos. 2011 / 0027287, 2012 / 0020975, 2012 / 0027765, 2012 / 0213797, and 2012 / 0251544 to Champion et al., WO 2011 / 027257 describe various MAb anti-PCSK9) are used to bind to PCSK9, or portions thereof, to block its mechanism of action. However, when there is disruption of the glycocalyx and subsequent disruption of receptors therein, including the LDL receptor, the effectiveness of MAb anti-PCSK9 is diminished. By administering glycocalyx repair and maintenance compounds to repair the glycocalyx, the LDL receptors may be repaired as well, increasing receptor binding and thereby increasing the efficacy of MAb anti-PCSK9 and lowering LDL levels in patients suffering from cardiovascular disease.

[0362] Thus, the present disclosure provides a method of repairing the glycocalyx and receptors therein and enhancing drug response by administering a composition comprising a glycocalyx repair and maintenance compound and MAb anti-PCSK9 to an individual suffering from cardiovascular disease, repairing the glycocalyx, repairing LDL receptors in the glycocalyx, and enhancing the response of MAb anti-PCSK9. The MAb anti-PCSK9 can be any of those described above, as well as bococizumab (Pfizer RN316, described in U.S. Patent No. 8,080,243 to Liang et al.). The present disclosure also provides a composition for treating cardiovascular disease comprising a glycocalyx repair and maintenance compound and MAb anti-PCSK9. The components of the combination may be in the same dosage form or in different dosage forms and may be administered with different or the same release profiles.

[0363] Assessment of treatment effectiveness Any means of assessing the effectiveness of treatment with a glycocalyx repair and maintenance composition (or other therapeutic intervention) may be used in conjunction with the methods and compounds described herein. Traditional methods of monitoring glycocalyx destruction include (1) direct microscopy, which entails optical measurement of the distance between the endothelium and red blood cells, (2) indirect methods, in which two different sizes of dextran sulfate are injected into the bloodstream (dextran-40 and dextran-70) and the relative distribution of these dextrans is measured (theoretically, this difference reflects the volume of the glycocalyx), and (3) imaging with a digital camera placed under the tongue to measure red blood cells as they move through the perfused border zone (PBR; uninterrupted blood flow indicates a healthy glycocalyx).

[0364] Example 2 illustrates one method of evaluating the efficacy of a treatment for repairing the glycocalyx and / or reducing endothelial inflammation and / or oxidative damage to the endothelium using a proprietary animal model of atherosclerosis (also described more generally above). Such evaluations may be performed in the context of drug development using non-human animals.

[0365] These methods are cumbersome or cannot be performed on human patients. However, the biomarkers described herein provide a convenient means of assessing the effectiveness of treatment in a subject. To this end, a sample relevant to the particular glycocalyx in question (e.g., blood or blood fractions for atherosclerosis) is taken and assayed for one, two, three, four, five, six, or seven or more biomarkers (e.g., including but not limited to those described above). As above, a decrease in a previously elevated biomarker, such as those described herein, indicates a positive therapeutic effect. EXAMPLES

[0366] Arterial plaque animal models and biomarkers correlated with plaque formation or vascular inflammation A novel model of atherosclerosis in mice was developed using a high-fat diet and administration of polychlorinated biphenyl (3,3',4,4'-tetrachlorobiphenyl; PCB-77), which promotes both obesity and atherosclerosis, and further oral administration of the caries-causing bacterium, Porphyromonas gingivalis 381 (ATCC 33277).The aim of this study was to investigate the relevance of biomarkers in a mouse model of atherosclerosis.

[0367] Materials and Methods mouse For the pilot study, 48 10-week-old male C57 / Bl6 mice were obtained from Jackson Laboratories. Three mice were maintained on a normal diet from 6 weeks of age and served as controls, while the remaining 45 were maintained on a 60% fat diet (D12451, DIO series diet, Opensource Diets). For the bacterial dosing study, 16 10-week-old male C57 / Bl6 mice were divided into four treatment groups and maintained on a normal diet.

[0368] treatment 3,3',4,4'-Tetrachlorobiphenyl (PCB-77) was obtained from Neosyn Laboratories. 100 mg of dry chemical was suspended in 15.22 ml of corn oil to deliver 150 μmol / kg in 0.2 ml per mouse.

[0369] Porphyromonas gingivalis 381 (ATCC33277) was obtained from ATCC. Bacteria were stored frozen prior to use. Bacteria were grown in 40 ml of supplemented tryptic soy broth in multiple sterile tubes at 37°C under anaerobic conditions. Cultures were centrifuged, medium removed, and samples combined. 100 μl of bacterial sample was mixed with 100 μl of medium and added to a 96-well plate. Bacterial concentration was determined by measuring optical density at 600 nm using a microplate reader. Based on bacterial concentration, samples were diluted to the appropriate concentration with 2% carboxymethylcellulose in sterile phosphate-buffered saline (PBS).

[0370] Oral gavage A 20-gauge curved feeding needle was used to administer 0.2 ml of treatment into the stomach of each mouse. Light isoflurane gas anesthesia was utilized to facilitate needle introduction into the esophagus and to reduce the risk of injury to the animal due to movement during gavage.

[0371] The oral gavage schedule was carried out as described in Figure 1A. 3 × 10 per mouse 11 We were unable to grow enough bacteria to produce the proposed dosage of bacteria, which was much higher than the dosages used in the scientific literature, so we increased the dosage to 5 x 10 9 Because three mice died overnight after the first bacterial gavage, the second bacterial gavage was postponed to day 6 and the dosage was reduced to 1.5 × 10 9 was lowered to.

[0372] For dosing studies, mice received oral gavage of bacteria on days 1 and 6, mimicking the oral gavage schedule utilized in the pilot study. Figure 1B describes the bacterial dosages.

[0373] Slaughter and collection According to the experimental design, mice were sacrificed on days 10, 15, or 20 (3 each from groups 1-5). Animals were anesthetized by intraperitoneal injection of 90 mg / kg ketamine and 8 mg / kg xylazine, and isoflurane gas anesthesia. Blood was collected by retro-orbital bleeding or from the heart and mixed with 50 mg / ml heparin to prevent coagulation. The chest was opened to expose the heart, saline was injected into the left ventricle, and the right atrium was opened to drain the blood and saline. The heart was perfused with at least 5 ml of saline until no blood was observed draining from the atrium. Hearts were carefully dissected and frozen for histological sectioning. Plasma was collected from blood samples by centrifugation at 1000 rpm for 15 min and collecting the supernatant. Samples were stored at -80°C until analysis.

[0374] histology Hearts were prepared as frozen sections: they were mounted in blocks and 10 μm thick sections were cut through the aortic valve to obtain 30 sections per mouse. Oil Red O staining was used to visualize the lipid content of the plaque. Multiple 10 μm sections at the level of the aortic sinuses were analyzed for the presence of Oil Red O lipid staining, plaque size, amount of fibrous tissue, and inflammation. The percentage of the lumen occupied by the first three features was calculated using Image Pro Plus. The mean percentage of each feature was used to score the three features on the following scale: for fibrous tissue, lipid staining, and plaque size: 0=<2%, 1=≧2%, 2=≧4%, 3=≧6%, 4=≧10%. The level of inflammation in each section was scored on the following scale: 0=no inflammatory cells observed, 1=few macrophages without giant cells, 2=presence of foam cells, 3=foam cells with cholesterol, 4=presence of foam cells, giant cells, and cholesterol. Inflammation scores were averaged across all sections and then converted to a global score: for inflammation: 0=<0.2, 1=≧0.2, 2=≧0.4, 3=≧0.6, 4=≧1.

[0375] ELISA Six test kits were used to analyze collected plasma samples: thrombin-antithrombin complex ELISA (Kamiya Biomedical, Thousand Oaks, California), antithrombin III ELISA (ABCam), total plasminogen activator inhibitor-1 ELISA (Molecular-Innovive), syndecan-1 ELISA (USCN, Houston, Texas), heparan sulfate ELISA, and hyaluronan synthase 1 ELISA (antibodies-online). All tests were performed on plasma, diluted where necessary to fall within the standard curve, and performed according to the manufacturer's instructions.

[0376] result pathology No inflammation or plaques were found in groups 1, 2, 5, and 6, but inflammatory cells were observed throughout group 3 as indicated by overall lipid staining (Figure 2A), and well-defined atherosclerotic plaques were observed in group 4 (Figure 2B).

[0377] Correlation of biomarkers with plaque formation or vascular inflammation: Blood was collected from the animals at various intervals and analyzed for biomarkers. Of the different markers evaluated, three showed significant levels in animals from groups 3 and 4 (especially on days 15 and 20) and showed high correlation with inflammation or plaque formation (analyzed statistically by independent T-tests). These biomarkers included plasminogen (PAI-1), heparan sulfate, and hyaluronan synthase, with syndecan-1 being a weakly predictive biomarker.

[0378] A. Highly correlated biomarkers 1. Plasminogen activator inhibitor-1 (PAI-1): FIG. 3 shows that PAI-1 was significantly elevated at sacrifice on day 20 compared to controls (Group 6: normal diet).

[0379] 2. Heparan sulfate (HS) FIG. 4 shows that HS levels at sacrifice on day 20 were significantly higher than in the control group.

[0380] 3. Hyaluronan synthase 1 (HAS-1) FIG. 5 shows that hyaluronan synthase at sacrifice on day 20 was significantly higher than the same time point in the control group.

[0381] B. Marginal biomarker: Syndecan-1 FIG. 6 shows that syndecan-1 on day 10 was significantly higher than at the same time point in the control group.

[0382] C. Poor prognosis of inflammation or plaque formation 1. Thrombin-antithrombin (TAT) As shown in Figure 7, no significant correlation with the TAT complex was observed.

[0383] 2. Antithrombin III As shown in FIG. 8, there were no significant differences in antithrombin III levels between treatment groups at any time point, or between time points within groups, as measured by unpaired T-test.

[0384] Further conclusions FIG. 9 graphs the mean scores for each group and time point, showing that PCB77 treatment was the most significant risk factor for inflammation and plaque development. EXAMPLES

[0385] Efficacy of a glycocalyx repair and maintenance composition demonstrated in an animal model of arterial plaque The goal of this study was to examine biomarker associations with the mouse model of atherosclerosis described in Example 1 in mice treated with strategies to protect and repair the endothelial glycocalyx.

[0386] Materials and Methods Mice, treatments, and gavage Eighty-four 10-week-old male C57 / Bl6 mice were obtained from Jackson Laboratories. Thirty-two mice were fed a normal diet from 6 weeks of age and served as controls, while the remaining mice were fed a 60% fat diet (D12451, DIO series diet, Opensource Diets). 3,3',4,4'-Tetrachlorobiphenyl (PCB-77) was obtained from Neosyn Laboratories. Dry chemical was suspended in 15.22 ml corn oil and delivered by gavage at 200 μmol / kg in 0.2 ml per mouse. Otherwise, treatment and gavage were as described in Example 1.

[0387] Slaughter and collection According to the experimental design, mice were sacrificed on days 4, 11, or 18 (3 from each group). Animals were anesthetized by intraperitoneal injection of 90 mg / kg ketamine and 8 mg / kg xylazine, and isoflurane gas anesthesia. Blood was collected by retro-orbital bleeding or from the heart and mixed with 50 mg / ml heparin to prevent coagulation. The chest was opened to expose the heart, saline was injected into the left ventricle, and the right atrium was opened to allow blood and saline to drain. The heart was perfused with at least 5 ml of saline until no blood was observed draining from the atrium. Hearts were carefully dissected and frozen for histological sectioning. Plasma was collected from blood samples by centrifugation at 1000 rpm for 15 minutes and collecting the supernatant. Samples were stored at -80°C until analysis.

[0388] ELISA Collected plasma samples were analyzed using four test kits: heparan sulfate ELISA and hyaluronan synthase 1 (HAS-1) ELISA (Antibodies-Online), total plasminogen activator inhibitor-1 (PAI-1) ELISA (Molecular-Innovive), and syndecan-1 (SDC1) ELISA (USCN, Houston, Texas). All tests were performed on plasma, diluted where necessary to fall within the standard curve, and performed according to the manufacturer's instructions.

[0389] result Hyaluronan synthase 1 (HAS-1) Figure 5 shows the results for hyaluronan synthase 1 (HAS-1). It was observed that the highest HAS-1 levels occurred in mice on a high fat diet treated with PCB on days 1 and 3 and sacrificed on day 4. A decrease in HAS-1 levels was observed in mice treated with compounds designed to repair and restore the endothelial glycocalyx.

[0390] Heparan sulfate (HS) Figure 4 shows the results for heparan sulfate. An increase in HS was observed in mice treated with PCB-77 and P. gingivalis.

[0391] Total plasminogen activator inhibitor-1 (PAI-1) Figure 3 shows the results for PAI-1. It was observed that elevated PAI-1 levels occurred in all mice treated with an insult designed to induce an atherosclerotic response.

[0392] Syndecan-1 (SDC1) Figure 6 shows the results for SDC1. Elevations in SDC-1 were observed in mice treated with PCB-77 and P. gingivalis, but a high degree of variability was seen in the results for this assay.

[0393] histology The positive control group (high-fat diet, PCBs, and P. gingivalis treatment) revealed the presence of pathology consistent with plaque (Figure 10, 10x and 40x magnification), and fibrous material loosely attached to the surface of the arterial wall was observed in this sample. In contrast, the negative control group (normal diet, no PCBs, no P. gingivalis treatment) showed typical features of a normal arterial wall (10).

[0394] conclusion Three biomarkers that were found to be highly correlated with plaque production are hyaluronan synthase (HAS-1), heparan sulfate (HS), and plasminogen activator inhibitor-1 (PAI-1). The biochemical changes that define cardiovascular disease (CVD) have been difficult to quantify. In this regard, simplified predictive biomarkers have now been developed, making it possible to monitor the onset of cardiovascular disease and its progression using simple blood tests. These biomarkers have been developed to provide reliable predictors of cardiovascular events.

[0395] Results show significantly smaller endothelial glycocalyx dimensions and amounts of two of its major components, heparan sulfate and hyaluronan, in the atherogenic sinus region of the carotid bifurcation compared to the common carotid region. Disturbed endothelial glycocalyx content in prelesion regions within the arterial vascular tree leads to localized loss of endothelial cell (EC) barrier properties. A possible role for endothelial glycocalyx in controlling vessel wall permeability was revealed, as suggested by increased local intima-to-media ratios at sites of reduced endothelial glycocalyx dimensions in atherogenic risk regions. These early changes in local intima-to-media ratios, without evidence of blood cell or monocyte accumulation within the expanded intimal layer, indicate a minimal inflammatory response at this very early stage.

[0396] In conclusion, predisposed arterial vascular regions have lower amounts of glycan structures such as heparin sulfate and hyaluronan present within their luminal surface endothelial glycocalyx, resulting in locally reduced permeability barrier properties. In this study, we reveal the endothelial cell glycocalyx as a complex 3-D matrix susceptible to atherogenic risk factors, resulting in locally predisposed vulnerable arterial sites due to preexisting differences in local architecture.

[0397] As shown in Figures 12A-12H and 14, a reasonable number of the compounds tested showed effects on biomarkers in mouse models of atherosclerosis, with changes in markers of hyaluronan synthase 1 (HAS-1) and total plasminogen activator inhibitor-1 (PAI-1) in therapeutic models, and heparan sulfate (HS) and total plasminogen activator inhibitor-1 (PAI-1) in preventative protocols.

Claims

1. 1. A kit for diagnosing or monitoring a disease characterized by disruption of the glycocalyx, endothelial inflammation, and / or oxidative damage to the endothelium, comprising: A kit comprising a biomarker panel for measuring at least three biomarkers, the at least three biomarkers being gamma fibrinogen (GF), growth differentiation factor 15 (GDF-15), and pregnancy associated plasma protein A (PAPP-A).

2. The kit of claim 1, wherein the biomarker panel for measuring the biomarkers comprises binding partners that specifically bind to the biomarkers.

3. The kit of claim 2, wherein each binding partner in the kit is labeled with a different detectable label.

4. 4. The kit of claim 2 or claim 3, wherein the binding partner comprises a detectably labeled antibody.

5. The kit of claim 1, further comprising one or more reagents for measuring one or more additional biomarkers selected from the group consisting of hyaluronan synthase-1 (HAS-1), heparan SO4 (HS), syndecan-1 (SDC-1), and plasminogen activator inhibitor (PAI-1).

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