Preparation of medium molecular weight heparin

Abstract

The invention provides synthesis of medium molecular weight heparin.

Description

Technical Field

[0001] The present invention relates to the synthesis of medium molecular weight heparin, preferably by low temperature periodate oxidation. Background Art

[0002] Heparin is a naturally occurring, highly sulfated polysaccharide, characterized by a wide range of molecular weights of the polysaccharide chains. Heparin acts on various different ligands and has different effects. Heparin is a member of the glycosaminoglycan carbohydrate family and consists of repeating disaccharide units of GlcAβ1-4GlcNAcα1-4, with polydisperse sulfation, N-acetylation, and glucuronyl epimerization. Heparin is highly heterogeneous. Heparin isolated from natural sources contains polysaccharide chains with molecular weights ranging from about 3000 Da (g / mol) to about 30,000 Da (g / mol). This is called unfractionated heparin (UFH). UFH can be enzymatically or chemically treated to deliver shorter polysaccharide chains. Heparinase I cleaves at the α-1,4 bond between non-acetylated GlcNS6S and IdoA2S. The products of chemically or enzymatically treated UFH can be affinity purified to produce fractionated heparin, in which the molecular weight of the polysaccharides in each fraction can be easily determined. Low molecular weight heparin (LMWH) contains polysaccharide chains in the range of about 4000 Da (g / mol) to about 8000 Da (g / mol).

[0003] There are few known methods for preparing medium molecular weight heparin. An example of a method for preparing medium molecular weight heparin is described in: Poletti LF, Bird KE, Marques D, Harris RB, Suda Y, Sobel M. Structural aspects of heparin responsible for interactions with von Willebrand factor. Arterioscler Thromb Vasc Biol. 1997 May; 17(5):925–31. This method requires incubation at 37 °C and produces a variety of products with molecular weights ranging from 10600 g / mol down to 1900 g / mol.

[0004] Therefore, there is a need for a reliable method for preparing medium molecular weight heparin. Summary of the Invention

[0005] In a first aspect, the present invention provides a method for synthesizing medium molecular weight heparin, the method comprising the steps of: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; and (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution.

[0006] In a second aspect, the present invention provides a kit suitable for use in the preparation of medium molecular weight heparin, wherein the kit comprises: (a) unfractionated heparin; (b) an aqueous buffer solution adjusted to about pH 5.0 to about pH 9.0; (c) an oxidizing agent; and (d) an optional inactivating agent.

[0007] In a third aspect, the present invention provides a method for preparing reduced medium molecular weight heparin (MMWH-Red), which comprises: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution; and (d) incubating the medium molecular weight heparin with a reducing agent to produce MMWH-Red.

[0008] In a fourth aspect, the present invention provides reduced medium molecular weight heparin (MMWH-Red). Preferably, MMWH-Red is prepared according to the first aspect or the third aspect of the present invention.

[0009] In a fifth aspect, the present invention provides reduced medium molecular weight heparin produced according to the following steps: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution; and (d) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin.

[0010] In a sixth aspect, the present invention provides a composition comprising reduced medium molecular weight heparin.

[0011] In a seventh aspect, the present invention provides the reduced medium molecular weight heparin according to the fourth aspect or the fifth aspect of the present invention or the composition according to the sixth aspect of the present invention for use in the treatment of endotheliopathy.

[0012] In an eighth aspect, the present invention provides the use of reduced medium molecular weight heparin for treating a disease or disorder in a patient, wherein the patient has an endotheliopathy characterized by a ratio of plasma von Willebrand factor to ADAMTS13 (VWF:ADAMTS13) of at least about 2.

[0013] In a ninth aspect, the present invention provides the use of reduced medium molecular weight heparin for treating a disease or disorder in a patient, wherein the patient has an endotheliopathy characterized by a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0014] In a tenth aspect, the present invention provides a method of treating an endotheliopathy, the method comprising administering to a subject in need of treatment a therapeutically effective amount of reduced medium molecular weight heparin. Preferably, wherein the patient's plasma VWF:ADAMTS13 ratio is at least about 2.

[0015] In an eleventh aspect, the present invention provides the use of reduced medium molecular weight heparin in the preparation of a medicament for treating an endotheliopathy in a patient. Preferably, wherein the patient's plasma VWF:ADAMTS13 ratio is at least about 2.

[0016] In a twelfth aspect, the present invention provides the use of reduced medium molecular weight heparin in the preparation of a medicament for treating an endotheliopathy in a patient. Preferably, wherein the patient's plasma VWF antigen:ADAMTS13 ratio is at least about 2.

[0017] For the avoidance of doubt, the examples relating to the various aspects of the present invention apply, mutatis mutandis, to the other aspects of the present invention. From the following discussion, other aspects and embodiments of the present invention will be apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows a comparison of MMWH prepared at 4 °C with heparin treated at 37 °C, untreated heparin, and an 11 kDa standard.

[0019] Figure 2 Shows a comparison of MMWH prepared at 4 °C using only periodate with heparin treated with periodate and NaOH at 4 °C.

[0020] Figure 3 Molecular weight distribution profile of the prepared medium molecular weight heparin.

[0021] Figure 4 Activity graphs of low molecular weight (LMW) heparin, unfractionated (UF) heparin, and medium molecular weight (MMW) heparin against factor IIa.

[0022] Figure 5Activity profiles of LMW heparin, UF heparin and MMW heparin against Factor X. Detailed implementation manners

[0023] Throughout the specification, one or more aspects of the present invention may be combined with one or more features described in the specification to define different embodiments of the present invention.

[0024] In the following discussion, many of the terms cited should be understood to have the meanings provided below, unless the context clearly indicates otherwise.

[0025] Unless the context otherwise implies, references in the singular to a noun in this document cover the plural of the noun and vice versa.

[0026] Throughout the specification, the word "comprise" (or its variants, such as "comprises" or "comprising") should be understood to imply the inclusion of the stated element or whole, or group of elements or wholes, but not the exclusion of any other element or whole, or group of elements or wholes. The term "comprise" includes within its scope the terms "consist of" or "consist essentially of".

[0027] The term "consisting of" (or its variants) should be understood to imply the inclusion of the stated element or whole, or group of elements or wholes, and the exclusion of any other element or whole, or group of elements or wholes.

[0028] The term "consisting essentially of" (or its variants) should be understood to imply the inclusion of the stated element, whole or step, or group of elements, wholes or steps, and that additional components may be present, but only those that do not materially affect the basic characteristics of the formulation, composition or compound.

[0029] When defining a number or value, the term "about" in this document is used to refer to a value that is within ±5% of the specified value.

[0030] The terms "treatment" and "therapy" define a therapeutic treatment of a patient to reduce or arrest the rate of progression of a disorder or condition, or to improve or cure the disorder or condition. Prevention of a disorder or condition resulting from treatment or therapy is also included.

[0031] As used herein, the term "patient" preferably refers to a mammal. Generally, the mammal is a human.

[0032] Von Willebrand factor (VWF) is a blood glycoprotein involved in hemostasis. VWF is a large multimeric glycoprotein present in plasma and is constitutively produced as ultra-large VWF in endothelial cells (in Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a 2050-amino acid protein.

[0033] A disaccharide is a sugar whose molecule contains two monosaccharide residues.

[0034] Low molecular weight heparin is defined herein as heparin having an average molecular weight of from about 4000 Da (g / mol) to about 8000 Da (g / mol). Medium molecular weight heparin is defined herein as heparin having an average molecular weight of from about 8000 Da (g / mol) to about 13000 Da (g / mol).

[0035] Thus, in a first aspect, the present invention provides a method for synthesizing (or preparing) medium molecular weight heparin (MMWH).

[0036] The medium molecular weight heparin prepared by the method of the first aspect of the present invention may have a mass in the range of greater than about 8000 Da (g / mol) to about 13000 Da (g / mol), preferably about 10000 Da (g / mol) to about 12000 Da (g / mol). The medium molecular weight heparin prepared by the method of the first aspect of the present invention may have a mass of about 11000 Da (g / mol).

[0037] The medium molecular weight heparin prepared by the method of the first aspect of the present invention may comprise polysaccharide chains having an average molecular weight in the range of about 8000 Da (g / mol) to about 13000 Da (g / mol), preferably about 10000 Da (g / mol) to about 12000 Da (g / mol). The medium molecular weight heparin prepared by the method of the first aspect of the present invention may comprise polysaccharide chains having an average molecular weight of about 11000 Da (g / mol).

[0038] The molecular weight of the medium molecular weight heparin prepared by the method of the first aspect of the present invention can be determined by size exclusion chromatography as described herein.

[0039] The medium molecular weight heparin prepared by the method of the first aspect may comprise at least three units of GlcNS6S-IdoA2S (or IdoA2S-GlcNS6S) disaccharide. The GlcNS6S and IdoA2S monosaccharides are linked by an α1-4 bond between GlcNS6S and IdoA2S, i.e., GlcNS6Sα1-4IdoA2S. For example, the medium molecular weight heparin prepared by the method of the first aspect may comprise at least four units, preferably at least five units, preferably at least six units, preferably at least eight units, preferably at least ten units of GlcNS6S-IdoA2S disaccharide. The medium molecular weight heparin prepared by the method of the first aspect may comprise less than or equal to 25 units of GlcNS6S-IdoA2S disaccharide, such as less than or equal to 20 units. The presence of GlcNS6S-IdoA2S disaccharide units can be inferred by antibodies, mass spectrometry or from chemical and enzymatic studies. The GlcNS6S-IdoA2S units may be arranged in a continuous sequence.

[0040] "IdoA" is α-L-iduronic acid. "IdoA2S" is IdoA modified by the addition of an O-sulfate group at the carbon atom at position 2 to form 2-O-sulfo-α-L-iduronic acid. "GlcNS" is 2-deoxy-2-sulfamido-α-D-glucopyranosyl. "GlcNS6S" is 2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate. The α1-4 bond is an α-glycosidic bond between the carbon atom at position 1 of one monosaccharide and the carbon atom at position 4 of the second monosaccharide. The β1-4 bond is a β-glycosidic bond between the carbon atom at position 1 of one monosaccharide and the carbon atom at position 4 of the second monosaccharide.

[0041] The medium molecular weight heparin prepared by the method of the first aspect may contain UA2S-GlcNS6S, UA2S-GlcNS, UA-GlcNAc, where U may be iduronic acid (IdoA) or glucuronic acid (GlcA). The medium molecular weight heparin prepared by the method of the first aspect may contain at least about 60% of UA2S-GlcNS6S, UA2S-GlcNS and UA-GlcNAc. The medium molecular weight heparin prepared by the method of the first aspect may contain at least about 45%, preferably at least about 48%, preferably at least about 49%, preferably at least about 60% of UA2S-GlcNS6S. The medium molecular weight heparin prepared by the method of the first aspect may contain up to about 60%, preferably up to about 70%, preferably up to about 85% of UA2S-GlcNS6S. The medium molecular weight heparin prepared by the method of the first aspect may contain at least about 4%, preferably at least about 5%, preferably at least about 6%, preferably at least about 10% of UA2S-GlcNS. The medium molecular weight heparin prepared by the method of the first aspect may contain up to about 15%, preferably up to about 20% of UA2S-GlcNS. The medium molecular weight heparin prepared by the method of the first aspect may contain at least 4%, preferably at least 5%, preferably at least 6%, preferably at least about 10% of UA-GlcNAc. The medium molecular weight heparin prepared by the method of the first aspect may contain up to about 15%, preferably up to about 20% of UA-GlcNAc. In some embodiments, the medium molecular weight heparin prepared by the method of the first aspect may contain at least 49.2% of UA2S-GlcNS6S, 5.4% of UA2S-GlcNS and 5.4% of UA-GlcNAc. In some embodiments, the medium molecular weight heparin prepared by the method of the first aspect may contain at least 82% of UA2S-GlcNS6S, 9% of UA2S-GlcNS and 9% of UA-GlcNAc. Compared with unfractionated heparin, the percentage composition of UA-GlcNAc contained in the medium molecular weight heparin can be enriched.

[0042] "UA" is uronic acid, which is a hexose with a negatively charged carboxylate at the 6-position. Uronic acid can independently be glucuronic acid or iduronic acid. "UA2S" is UA modified by forming 2-O-sulfo-uronic acid by adding an O-sulfate group at the 2-position carbon. "GlcA" is β-D-glucuronic acid, and "GlcNAc" is 2-deoxy-2-acetamido-α-D-glucopyranosyl.

[0043] The method of the first aspect includes the following steps: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; and (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution.

[0044] UF heparin can be obtained from bovine or porcine tissues (such as porcine intestine or bovine lung).

[0045] "Buffer" refers to a chemical substance that resists changes in pH in a solution when an acid or base is added to the solution. Generally, a buffer solution (or buffer system) contains a weak acid and its conjugate base, or a weak base and its conjugate acid.

[0046] Generally, a suitable buffer contains an acid with a pKa value falling within ±1 of the desired pH of the formulation. For example, if the desired pH of the formulation is about 7.0, a suitable buffer contains a weak acid with a pKa value of about 6.0 to about 8.0. If the acid of the buffer has more than one pKa value (i.e., each molecule of the acid is capable of donating more than one proton), at least one of the pKa values should fall within the desired pH range for the buffer to be suitable.

[0047] The weak acid and conjugate base (or weak base and conjugate acid) of the buffer are in equilibrium with each other. According to Le Chatelier's principle (if a constraint is imposed on a system in equilibrium, such as changing the concentration of a reactant, the equilibrium will shift to counteract the effect of the constraint), adding an acid or a base to the solution will shift the equilibrium position in the direction favorable to the conjugate base or the weak acid, respectively. Therefore, the concentration of free protons (and thus the pH) in the formulation is relatively constant.

[0048] Suitable buffer systems include acetate and acetic acid (pKa = 4.75), citrate and citric acid (pKa = 3.13, 4.76, and 6.40), and phosphoric acid (pKa = 2.14, 7.20, and 12.37) or mixtures thereof. Phosphate buffered saline can also be used. The pKa values cited herein are those reported in water at 25°C. Generally, the buffer contains only one of the above pairs, i.e., an acid and its conjugate base. The buffer can contain acetate and acetic acid, citrate and citric acid, or phosphate and phosphoric acid.

[0049] The pH is adjusted to ensure that the pH of the first solution is between about pH 5.0 and about pH 9.0 because dissolving unfractionated heparin can cause a change in the pH of the aqueous buffer solution.

[0050] Optionally, in step (a), the aqueous buffer solution is adjusted to a pH between about 6.0 and about 8.0, more preferably to about pH 7.0. Generally, the temperature of the aqueous buffer solution in step (a) is about -2°C to about 4°C. Preferably, the temperature of the aqueous buffer solution in step (a) is about 0°C to about 2°C.

[0051] Generally, the aqueous buffer solution is a phosphate buffer solution, a citrate buffer solution or an acetate buffer solution, that is, the buffer system is phosphate, citrate or acetate. Preferably, the aqueous buffer solution is a phosphate buffer solution, and more preferably, the aqueous buffer solution is a sodium phosphate buffer solution or a potassium phosphate buffer solution, that is, the buffer system is sodium phosphate or potassium phosphate.

[0052] The buffer system in the aqueous buffer solution may be present at a concentration of about 10 mM to about 100 mM, more preferably about 20 mM to about 90 mM, more preferably about 30 mM to about 80 mM, more preferably about 40 mM to about 70 mM, more preferably about 50 mM to about 60 mM. The buffer system in the aqueous buffer solution may be present at a concentration of about 50 mM.

[0053] The concentration of UF heparin in the aqueous buffer solution may be about 0.5 mg / mL to about 10 mg / mL, more preferably about 1 mg / mL to about 8 mg / mL, more preferably about 1.5 mg / mL to about 6 mg / mL, more preferably about 2 mg / mL to about 4 mg / mL. The concentration of UF heparin in the aqueous buffer solution may be about 1.5 mg / mL, more preferably about 1.8 mg / mL, more preferably about 2 mg / mL, more preferably about 2.5 mg / mL, more preferably about 2.7 mg / mL, more preferably about 3 mg / mL.

[0054] The oxidizing agent may be a periodate, such as sodium periodate or potassium periodate. Preferably, the oxidizing agent is sodium periodate. Alternatively or additionally, the oxidizing agent may be a perchlorate, such as sodium perchlorate. Preferably, the oxidizing agent does not contain perchlorate. Using a combination of perchlorate and periodate as the oxidizing agent results in an increased level of decomposition of the sample into substances of smaller molecular weight.

[0055] The concentration of the oxidizing agent in the aqueous buffer solution may be about 1 g / L to about 10 g / L. Preferably, the concentration of the oxidizing agent may be about 2 g / L to about 9 g / L, more preferably about 4 g / L to about 8 g / L, more preferably about 5 g / L to about 7 g / L. Preferably, the concentration of the oxidizing agent is about 5.7 g / L. Preferably, the concentration of the oxidizing agent is 5.7 g / L and the oxidizing agent is sodium periodate.

[0056] The molar ratio of UF heparin to the oxidizing agent can be from about 1:1 to about 1:200, more preferably from about 1:2 to about 1:150, more preferably from about 1:10 to about 1:100, more preferably from about 1:20 to about 1:50, more preferably from about 1:30 to about 1:40. Generally, the molar ratio of UF heparin to the oxidizing agent can be about 1:40.

[0057] Generally, the incubation temperature in step (c) is from about 0 °C to about 10 °C, more preferably from about 1 °C to about 9 °C, more preferably from about 2 °C to about 8 °C, more preferably from about 3 °C to about 7 °C, more preferably from about 4 °C to about 6 °C. Preferably, the incubation temperature in step (c) is about 4 °C. Generally, the incubation step (c) is carried out for about 1 hour to about 48 hours, more preferably from about 4 hours to about 36 hours, more preferably from about 8 hours to about 30 hours, more preferably from about 12 hours to about 24 hours, more preferably from about 15 hours to about 20 hours, more preferably from about 16 hours to about 18 hours. Optionally, step (c) can be carried out at about 20 °C to about 25 °C.

[0058] The incubation step (c) of the first aspect of the present invention can be carried out in a laboratory refrigerator set to the desired temperature. The laboratory refrigerator can be set to a temperature of from about 0 °C to about 10 °C, more preferably from about 2 °C to about 8 °C, more preferably from about 3 °C to about 7 °C, more preferably from about 4 °C to about 6 °C. Preferably, the laboratory refrigerator can be set to a temperature of about 4 °C.

[0059] The method of the first aspect can further include step (d): a step of inactivating the oxidizing agent in the medium molecular weight heparin solution. The oxidizing agent can be inactivated by adding an inactivating agent selected from the group consisting of D-mannitol, glycerol, N-acetylmethionine, sodium sulfite, and combinations thereof. A particularly preferred inactivating agent is D-mannitol.

[0060] The molar ratio of the oxidizing agent to the inactivating agent can be from about 1:1 to about 1:10, more preferably from about 1:2 to about 1:8, more preferably from about 1:3 to about 1:6, more preferably from about 1:4 to about 1:5. Generally, the molar ratio of the oxidizing agent to the inactivating agent can be about 1:2 or about 1:4.

[0061] The method of the first aspect can further include step (e): a step of dialyzing the medium molecular weight heparin solution in a dialysis solution to provide a dialyzed medium molecular weight heparin sample.

[0062] "Dialysis (dialysis or dialysing)" means a process of separating molecules in a solution based on the difference in diffusion rates through a semi-permeable membrane (such as dialysis tubing). The sample to be dialyzed and the dialysis fluid (or buffer) are placed on opposite sides of the semi-permeable membrane. Target sample molecules larger than the membrane pores (such as proteins, DNA, or polysaccharides) are retained on the sample side of the membrane. Contaminants (such as small molecules and salts) can pass through the membrane into the dialysis fluid, thereby reducing the concentration of contaminants in the sample to a low level. Changing the dialysis fluid to fresh dialysis fluid removes the contaminants that have entered the dialysis fluid from the sample. This allows more contaminants to diffuse from the sample into the dialysis fluid.

[0063] Dialysis can separate small molecules (such as salts, reducing agents, or dyes) from larger macromolecules (such as proteins, DNA, or polysaccharides). Dialysis can also be used to separate polysaccharides based on molecular weight. The semi-permeable membrane is usually made of a membrane of regenerated cellulose or cellulose ester.

[0064] Dialysis can be carried out by placing the dialysis tubing containing the sample in the dialysis fluid. The "dialysis fluid" is the fluid into which materials from the dialysis tubing enter. The dialysis fluid can be updated as often as needed to achieve optimal separation. Dialysis can be carried out over a period of about 1 day to about 14 days, preferably about 5 days to about 10 days, preferably about 7 days. The dialysis fluid can be updated about 1 to about 10 times a day, preferably about 2 to about 5 times a day, preferably about 3 times a day. Usually, the dialysis fluid is several times the volume of the sample, such as about 2 to about 500 times the volume of the sample. The dialysis fluid can be about 4 times the volume of the sample.

[0065] Usually, in step (e), the dialysis fluid is water. Optionally, the dialysis fluid may contain electrolytes, such as sodium, potassium, magnesium, calcium, chloride, bicarbonate, lactate, glucose, amino acids, or a combination thereof.

[0066] The dialysis step (e) can be carried out in tubing with a molecular weight cut-off of 2kD provided by, for example, Spectra / Those skilled in the art know the appropriate tubing molecular weight cut-off sizes for different purposes. Alternatively, the dialysis step (e) can be carried out in a dialysis device or dialyzer. Suitable dialyzers can be the Slide-A-Lyzer TM , Float-A-Lyzer, Pur-A-lyzer, D-Tube, and GeBAflex dialyzer product lines.

[0067] The method of the first aspect may further include step (f): separating medium molecular weight heparin from the dialyzed heparin sample. Medium molecular weight heparin can be separated from the dialyzed heparin sample by lyophilization, centrifugation, or filtration. Preferably, medium molecular weight heparin is separated from the dialyzed heparin sample by lyophilization.

[0068] "Freeze drying" (also known as lyophilisation or cryodesiccation) is a drying process carried out at low temperatures. Freeze drying generally involves reducing the temperature and pressure below the triple point of the substance and removing the frozen solvent (such as water ice) by sublimation. For aqueous compositions (such as those disclosed herein), freeze drying can be carried out at a temperature of from about -20 °C to about -80 °C, preferably about -40 °C and a pressure of from about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).

[0069] MMWH can be purified by any suitable method known to those skilled in the art. Thus, the method can further include step (g): the step of purifying medium molecular weight heparin. For example, MMWH can be purified by thorough dialysis using phosphate buffer (pH = 7.0) or saline, a desalting column (such as Sephadex G-25 with phosphate buffer (pH = 7.0) or saline as the mobile phase), or precipitation of MMWH.

[0070] The method can include an alkali elimination step. Alternatively, the method may not include an alkali elimination step. For example, an alkali metal salt (such as sodium hydroxide, potassium hydroxide or lithium hydroxide) can be used to carry out alkali elimination. The alkali elimination step can be carried out by adding an alkali metal salt at about room temperature to increase the pH of the medium molecular weight heparin solution to about pH 10 to about pH 14, preferably about pH 12 for about 10 minutes to about 3 hours, preferably about 30 minutes.

[0071] Preferably, the method does not include an alkali elimination step. Preferably, the method does not include the addition of an alkali metal salt, for example, an alkali metal salt such as NaOH, KOH or LiOH. Preferably, the method does not include the addition of NaOH, KOH or LiOH. Unexpectedly, the method that does not include an alkali elimination step or the addition of an alkali metal salt produces medium molecular weight heparin as defined herein, which shows very low activity against factor IIa and / or Xa compared to UF heparin and low molecular weight heparin.

[0072] MMWH can be characterized by NMR, disaccharide analysis, ristocetin-induced platelet aggregation (RIPA), factor X analysis.

[0073] The method is suitable for the preparation of milligram, gram or kilogram amounts of medium molecular weight heparin.

[0074] Advantageously, the method of the first aspect reliably provides medium molecular weight heparin with good purity and reduced degradation.

[0075] The method may include a further step (h) which includes incubating medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMMWH-Red). Preferably, the reducing agent is a mild reducing agent. The reducing agent may be sodium borohydride (NaBH4), sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3) or potassium borohydride (KBH4).

[0076] Preferably, step (h) is carried out at about 10 °C to about 30 °C, or about 15 °C to about 25 °C, or about 20 °C to about 25 °C. Preferably, step (f) is carried out at about 25 °C. Generally, step (f) is carried out for about 1 hour to about 24 hours, more preferably about 2 hours to about 16 hours, more preferably about 3 hours to about 12 hours, more preferably about 6 hours to about 10 hours.

[0077] The solvent for step (h) is generally selected from the group consisting of methanol, ethanol, water, THF or combinations thereof. Preferably, the solvent in step (h) is water.

[0078] The method for synthesizing medium molecular weight heparin may consist of or consist essentially of the following steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0 °C to about 10 °C to form a medium molecular weight heparin solution; and (d) purifying the medium molecular weight heparin.

[0079] The method for synthesizing medium molecular weight heparin may consist of or consist essentially of the following steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0 °C to about 10 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidizing agent in the medium molecular weight heparin solution; and (e) purifying the medium molecular weight heparin.

[0080] The method for synthesizing medium molecular weight heparin may consist of or consist essentially of the following steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0 °C to about 10 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidizing agent in the medium molecular weight heparin solution; and (e) dialyzing the medium molecular weight heparin solution in a dialysate to provide a dialyzed medium molecular weight heparin sample.

[0081] A method for synthesizing medium molecular weight heparin may consist of or consist essentially of the following steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution; (d) inactivating the oxidizing agent in the medium molecular weight heparin solution; (e) dialyzing the medium molecular weight heparin solution in a dialysis fluid to provide a dialyzed medium molecular weight heparin sample; and (f) isolating the medium molecular weight heparin from the dialyzed heparin sample.

[0082] In a second aspect, the present invention provides a kit suitable for use in the preparation of medium molecular weight heparin, wherein the kit comprises: (a) unfractionated heparin; (b) an aqueous buffer solution adjusted to about pH 5.0 to about pH 9.0; (c) an oxidizing agent; and (d) an optional inactivating agent.

[0083] The MMWH produced by the method of the first aspect of the present invention comprises two aldehyde groups on glucuronic acid. In some cases, the reactivity of the aldehyde groups may cause the aldehyde groups to react with amine residues on in vivo proteins via a Schiff base reaction.

[0084] Therefore, in a third aspect, the present invention provides a method for preparing reduced medium molecular weight heparin (MMWH-Red). MMWH-Red does not contain aldehyde groups or contains fewer aldehyde groups than MMWH. The method of the third aspect comprises the following steps: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution; and (d) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red).

[0085] The reduced medium molecular weight heparin prepared by the method of the third aspect of the present invention may comprise polysaccharide chains having an average molecular weight in the range of about 8000 Da (g / mol) to about 13000 Da (g / mol), preferably about 10000 Da (g / mol) to about 12000 Da (g / mol). The reduced medium molecular weight heparin prepared by the method of the third aspect of the present invention may comprise polysaccharide chains having an average molecular weight of about 11000 Da (g / mol).

[0086] The molecular weight of MMWH-Red prepared by the method of the third aspect of the present invention can be determined by size exclusion chromatography as described herein.

[0087] The MMWH-Red prepared by the method of the third aspect may contain at least three units of the GlcNS6S-IdoA2S (or IdoA2S-GlcNS6S) disaccharide. The GlcNS6S and IdoA2S monosaccharides are linked by an α1-4 bond between GlcNS6S and IdoA2S, i.e., GlcNS6Sα1-4IdoA2S. For example, the MMWH-Red prepared by the method of the third aspect may contain at least four units, preferably at least five units, preferably at least six units, preferably at least eight units, preferably at least ten units of the GlcNS6S-IdoA2S disaccharide. The MMWH-Red prepared by the method of the third aspect may contain less than or equal to 25 units of the GlcNS6S-IdoA2S disaccharide, such as less than or equal to 20 units. The presence of the GlcNS6S-IdoA2S disaccharide unit can be inferred by antibodies, mass spectrometry or from chemical and enzymatic studies. The GlcNS6S-IdoA2S units may be arranged in a continuous sequence.

[0088] The MMWH-Red prepared by the method of the first aspect may contain UA2S-GlcNS6S, UA2S-GlcNS, UA-GlcNAc, where U may be iduronic acid (IdoA) or glucuronic acid (GlcA). The medium molecular weight heparin prepared by the method of the third aspect may contain at least about 60% of UA2S-GlcNS6S, UA2S-GlcNS and UA-GlcNAc. The MMWH-Red prepared by the method of the third aspect may contain at least about 45%, preferably at least about 48%, preferably at least about 49%, preferably at least about 60% of UA2S-GlcNS6S. The MMWH-Red prepared by the method of the third aspect may contain up to about 60%, preferably up to about 70%, preferably up to about 85% of UA2S-GlcNS6S. The MMWH-Red prepared by the method of the third aspect may contain at least about 4%, preferably at least about 5%, preferably at least about 6%, preferably at least about 10% of UA2S-GlcNS. The MMWH-Red prepared by the method of the third aspect may contain up to about 15%, preferably up to about 20% of UA2S-GlcNS. The MMWH-Red prepared by the method of the third aspect may contain at least 4%, preferably at least 5%, preferably at least 6%, preferably at least about 10% of UA-GlcNAc. The MMWH-Red prepared by the method of the third aspect may contain up to about 15%, preferably up to about 20% of UA-GlcNAc. In some embodiments, the MMWH-Red prepared by the method of the third aspect may contain at least 49.2% of UA2S-GlcNS6S, 5.4% of UA2S-GlcNS and 5.4% of UA-GlcNAc. In some embodiments, the MMWH-Red prepared by the method of the third aspect may contain at least 82% of UA2S-GlcNS6S, 9% of UA2S-GlcNS and 9% of UA-GlcNAc. Compared with unfractionated heparin, the percentage composition of UA-GlcNAc contained in MMWH-Red can be enriched.

[0089] The reducing agent may be sodium borohydride (NaBH4), sodium cyanoborohydride, sodium triacetoxyborohydride or potassium borohydride (KBH4). Preferably, the reducing agent is sodium borohydride.

[0090] Typically, the reducing agent is used in an amount greater than about 0.5 molar equivalents, or greater than about 1 molar equivalent, or greater than about 2 molar equivalents, or greater than about 3 molar equivalents, or greater than about 5 molar equivalents relative to the medium molecular weight heparin. Typically, the reducing agent is used in an amount less than about 15 molar equivalents, or less than about 12 molar equivalents, or less than about 10 molar equivalents, or less than about 8 molar equivalents, or less than about 6 molar equivalents, or less than about 5 molar equivalents relative to the medium molecular weight heparin. The reducing agent can be used in an amount of from about 0.5 molar equivalents to about 15 molar equivalents, or from about 1 molar equivalents to about 10 molar equivalents, or from about 2 molar equivalents to about 6 molar equivalents.

[0091] Typically, step (d) is carried out at from about 0 °C to about 30 °C, or from about 5 °C to about 25 °C, or from about 10 °C to about 20 °C. Preferably, step (d) is carried out at about 25 °C. Typically, step (d) is carried out for about 1 hour to about 24 hours, more preferably about 2 hours to about 16 hours, more preferably about 3 hours to about 12 hours, more preferably about 6 hours to about 10 hours.

[0092] The solvent for step (d) is typically selected from the group consisting of: methanol, ethanol, water, THF, dichloroethane, or combinations thereof. Preferably, the solvent in step (d) is water.

[0093] The method of the first aspect may further comprise the step of inactivating the oxidizing agent in the MMWH-Red solution. The oxidizing agent can be inactivated by adding an inactivating agent selected from the group consisting of: D-mannitol, glycerol, N-acetylmethionine, sodium sulfite, and combinations thereof. A particularly preferred inactivating agent is D-mannitol.

[0094] The molar ratio of the oxidizing agent to the inactivating agent can be from about 1:1 to about 1:10, more preferably from about 1:2 to about 1:8, more preferably from about 1:3 to about 1:6, more preferably from about 1:4 to about 1:5. Typically, the molar ratio of the oxidizing agent to the inactivating agent can be about 1:2 or about 1:4.

[0095] MMWH-Red can be purified by any suitable method known to those skilled in the art. Thus, the method may further comprise the step of purifying the medium molecular weight heparin. For example, MMWH-Red can be purified by thorough dialysis using a phosphate buffer (pH = 7.0) or saline, a desalting column (such as Sephadex G-25 with a phosphate buffer (pH = 7.0) or saline as the mobile phase), or precipitation of MMWH-Red.

[0096] For example, the method may further comprise: prior to the reduction step, dialyzing the medium molecular weight heparin solution in a dialysis fluid to provide a dialyzed medium molecular weight heparin sample.

[0097] This method is applicable to the preparation of milligram, gram or kilogram amounts of reduced medium molecular weight heparin.

[0098] Advantageously, the method of the third aspect reliably provides reduced medium molecular weight heparin with good purity and reduced degradation.

[0099] The method for synthesizing reduced medium molecular weight heparin may consist of or consist essentially of the following steps: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution; (d) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (e) purifying the MMWH-Red.

[0100] The method for synthesizing reduced medium molecular weight heparin may consist of or consist essentially of the following steps: (a) dissolving heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution; (d) inactivating the oxidizing agent in the medium molecular weight heparin solution; (e) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (f) purifying the MMWH-Red.

[0101] The method for synthesizing reduced medium molecular weight heparin may consist of or consist essentially of the following steps: (a) dissolving heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution; (d) inactivating the oxidizing agent in the medium molecular weight heparin solution; (e) dialyzing the medium molecular weight heparin solution in a dialysate to provide a dialyzed medium molecular weight heparin sample; (f) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (g) purifying the MMWH-Red.

[0102] A method for synthesizing reduced medium molecular weight heparin may consist of or consist essentially of the following steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to a pH between about 5.0 and about 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature between about 0 °C and about 10 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidizing agent in the medium molecular weight heparin solution; (e) dialyzing the medium molecular weight heparin solution in a dialysate to provide a dialyzed medium molecular weight heparin sample; (f) separating the medium molecular weight heparin from the dialyzed heparin sample; (g) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin (MMWH-Red); and (h) purifying the MMWH-Red.

[0103] Preferably, the reducing agent is a mild reducing agent. A mild reducing agent can selectively reduce aldehydes and ketones to alcohols in the presence of esters. In other words, a mild reducing agent reduces aldehydes and ketones to alcohols at a faster rate than it reduces esters to alcohols. A mild reducing agent does not reduce carboxylic acids, nitriles, and amides under normal conditions. Preferably, the mild reducing agent can be sodium borohydride (NaBH4).

[0104] Preferably, the method does not include an alkali elimination step. Preferably, the method does not include adding alkali metal salts, such as alkali metal salts of NaOH, KOH, or LiOH. Preferably, the method does not include adding NaOH, KOH, or LiOH. Unexpectedly, the method that does not include an alkali elimination step or adding alkali metal salts produces MMWH-Red as defined herein, which shows very low activity against factor IIa and / or Xa compared to UF heparin and low molecular weight heparin.

[0105] To avoid doubt, the embodiments related to the first aspect of the present invention are applicable to the third aspect of the present invention with necessary modifications in details.

[0106] Both MMWH and MMWH-Red inhibit von Willebrand factor and are thus suitable active pharmaceutical ingredients (APIs) and drug products for the purposes of the present invention.

[0107] Advantageously, compared to MMWH, MMWH-Red can show greater stability as an API and drug product. In addition, MMWH-Red can reduce the possibility of side reactions with excipients contained in the drug product, and MMWH-Red can reduce the interaction with in vivo proteins.

[0108] In a fourth aspect, the present invention provides reduced medium molecular weight heparin (MMWH-Red). The MMWH-Red can be prepared according to the first aspect or the third aspect of the present invention.

[0109] MMWH-Red does not contain aldehydes or contains fewer aldehydes than MMWH produced according to the first aspect. The presence or absence of aldehydes can be determined using a 2,4-dinitrophenylhydrazine test. When MMWH-Red reacts with 2,4-dinitrophenylhydrazine, it may not produce an orange-yellow precipitate or may produce less orange-yellow precipitate than the corresponding amount of starting material. Alternatively, the reduction of MMWH to MMWH-Red can be monitored by infrared spectroscopy.

[0110] MMWH-Red can be characterized by NMR, disaccharide analysis, ristocetin-induced platelet aggregation (RIPA), and factor X analysis.

[0111] In a fifth aspect, the present invention provides MMWH-Red produced according to the following steps:

[0112] (a) Dissolve unfractionated (UF) heparin in an aqueous buffer solution adjusted to a pH between about 5.0 and about 9.0 to provide a first solution;

[0113] (b) Add an oxidizing agent to the first solution to provide a second solution;

[0114] (c) Incubate the second solution at a temperature between about 0 °C and about 10 °C to form a medium molecular weight heparin solution; and

[0115] (d) Incubate the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin.

[0116] For the avoidance of doubt, the examples related to the first and third aspects of the present invention apply, mutatis mutandis, to the fifth aspect of the present invention.

[0117] In a sixth aspect, the present invention provides a composition comprising MMWH-Red. Preferably, the composition is a pharmaceutical composition.

[0118] For the avoidance of doubt, the examples related to the first and third aspects of the present invention apply, mutatis mutandis, to the sixth aspect of the present invention.

[0119] As described herein, endothelial diseases can be associated with many diseases. MMWH-Red inhibits von Willebrand factor. The inventors have found that MMWH-Red can be used to treat endothelial diseases, particularly in patients with high plasma von Willebrand factor levels.

[0120] Although endotheliopathy can be caused by many diseases and / or conditions as described herein, endotheliopathy with respect to COVID-19 or SARS-CoV-2 will be mainly discussed herein. Those skilled in the art will understand that this discussion is merely for providing examples and should not be considered as a limitation to the present invention.

[0121] At the end of 2019, a novel beta-coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) was identified, which caused coronavirus disease 2019 (COVID-19). Since then, the rapid geographical spread of COVID-19 eventually led the WHO to declare it a pandemic in March 2020 (1). The clinical manifestations of SARS-CoV-2-infected individuals range from asymptomatic patients to more severe pneumonia, which can lead to acute respiratory distress syndrome (ARDS) and multi-organ failure. Most symptomatic patients experience mild to moderate disease and generally do not require hospitalization (2 - 4). However, there is a subset of patients who may develop into a more severe disease form, in which the evolution of symptoms / clinical manifestations can last up to 2 weeks, starting from the initial prodromal phase until the development of ARDS (3). Currently, it is known that the subgroup of patients who become critically ill and require ventilatory support or extracorporeal membrane oxygenation (ECMO) has a very poor prognosis, with a high mortality rate approaching 90% (5).

[0122] Since the disease was first reported, it has affected over 90 million people globally. Its pathophysiological pathways are not well understood; thus, management is supportive. There is a lack of disease-modifying therapies that can be initiated while waiting for specific antiviral drugs or vaccination. Several lines of evidence suggest that endothelial dysfunction is a key pathophysiological mechanism in COVID-19. Before the current pandemic, markers of endothelial dysfunction had been shown to be associated with disease severity and mortality in patients with sepsis (6 - 9). Recently, Varga et al. (10) demonstrated widespread endocarditis affecting the pulmonary, renal, gastrointestinal, and hepatic vessels in the autopsy of three COVID-19 patients. In one of the cases, the authors reported "congestion in most small vessels", and in another case, the patient died of intestinal ischemia, with evidence of underlying endocarditis.

[0123] Recently, two proposed hemostatic mechanisms have provided insights into a better understanding of ARDS based on the molecular pathogenesis associated with endotheliopathy, which promotes inflammation and coagulation disorders in sepsis and other critical illnesses (11-14): one is the "endothelial dual activation theory," in which endothelial pathogenesis activates inflammatory pathways and microthrombosis pathways, while the other is the novel hemostatic "dual pathway unification theory," in which hemostasis initiates thrombosis and promotes microthrombosis, leading to vascular microthrombotic disease (VMTD) (11,13,15). These two theories are consistent because the endothelium contributes to the initial hemostasis and the molecular mechanisms that trigger thrombosis. ARDS is commonly associated with sepsis of various etiologies and has occurred in severe acute respiratory syndrome (SARS) caused by SARS-CoV (16), Middle East respiratory syndrome (MERS) caused by MERS-CoV (17), and now COVID-19. Sepsis-associated ARDS is often accompanied by other organ dysfunctions, such as encephalopathy (18), liver failure (19)(20), acute kidney failure, and acute necrotizing pancreatitis (21). This multi-organ involvement suggests that ARDS may not be a primary disease but rather part of a persistent systemic pathogenic mechanism triggered by infection or other critical illnesses.

[0124] On this basis, the potential physiological alterations in multi-organ failure in sepsis and other critical illnesses have been identified as circulatory dysfunction caused by endotheliopathy-related VMTD (EA-VMTD) (14,15). Thus, infection triggers endothelial injury, leading to endotheliopathy. This then results in disseminated microthrombosis (DIMT), which can trigger, for example, local hypoxemia, systemic hypoxia, and / or ischemia, as well as COVID-19, which is currently known to be associated with endocarditis as described above (10).

[0125] A series of COVID-19 lung autopsy cases have shown that, in addition to diffuse alveolar damage, there are numerous local platelet-rich microthrombi and bleeding foci in the lungs (22). The authors believe that local thromboangiitis in the lungs is the key to the pathogenesis of COVID-19, and others also believe that microthrombosis is a key driver in the disease process (23). These microcirculatory changes have been clearly demonstrated in the lungs, kidneys, and livers using contrast-enhanced ultrasound (24,25). Similar findings have also been seen in the brain (26). Thus, there is increasing evidence that COVID-19 appears to cause endocarditis as well as diffuse and extensive microthrombosis.

[0126] Hypercoagulability and COVID-19 are now widely accepted, and studies have shown abnormal D-dimer levels, with higher levels being associated with more severe disease and an increased odds ratio of in-hospital mortality (27 - 30). Several case reports have indicated that COVID-19 pneumonia patients can present with acute pulmonary embolism in the absence of major predisposing factors for venous thromboembolism (27, 31, 32). More recently, Panigada et al. have shown that, in addition to elevated D-dimer levels, the levels of factor VIII and von Willebrand factor (VWF) are also significantly increased (33). Escher et al. (34) reported that, in relation to COVID-19, VWF increased by more than 500% and factor VIII levels increased >350%. In addition, it has been demonstrated that patients with thrombocytopenia have a more than five-fold increased risk of severe disease, and patients with the lowest platelet counts are associated with the highest mortality (33, 35, 36). Thus, both hypercoagulability and thrombocytopenia appear to be harbingers of severe disease and death.

[0127] Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a key role in hemostasis and thrombosis, mediating platelet adhesion to damaged and activated blood vessels. It is synthesized only in megakaryocytes and endothelial cells (ECs), and interestingly, SARS-CoV can directly infect both of these cell types (22, 36).

[0128] The vast majority of VWF found in plasma is derived from VWF synthesized within ECs, which is stored within Weibel-Palade bodies (WPBs). Although restricted to ECs, there are differences in VWF synthesis within different vascular beds of the body, with smaller blood vessels in the lung and brain expressing higher levels of VWF than similarly sized vessels in the liver or kidney, and higher levels being expressed in venous rather than arterial ECs (37). The major portion of VWF stored within endothelial cell WPBs consists of ultra-large VWF (ULVWF). These ultra-large VWF multimers are more adhesive in circulation than smaller VWF multimers (38). After secretion, ULVWF can spontaneously bind platelets. Inflammatory cytokines (such as interleukin-1 and tumor necrosis factor (TNF)-α) can trigger exocytosis of WPBs, releasing their contents. Thus, plasma VWF levels can be used as a marker of endothelial activation and vascular inflammation, and elevated VWF levels have been shown to be associated with ARDS and sepsis and independently associated with mortality (39, 40).

[0129] After secretion from the EC, the secreted VWF enters the circulation partially and binds to the endothelium partially, and is sensitive to shear stress. This shear stress unfolds VWF and exposes sites for platelet binding, self-association, and cleavage via the enzyme ADAMTS13. It has previously been shown that these VWF molecules can self-associate into long "strings" in the direction of flow in arteries and veins, which bind to platelets and adhere to the endothelium (41-43). The protease ADAMTS13 cleaves VWF and ULVWF, and its perfusion on these platelet-VWF strings results in their rapid removal from the circulation (41). The ULVWF multimers released from WPBs have a lower shear stress for unfolding and can thus represent the starting molecules of this self-assembly process that leads to highly adhesive strings that capture platelets. The binding of platelets to VWF occurs via the GP Ib receptor. When VWF is in the globular form, the binding site of this receptor is usually not exposed and thus cannot bind to platelets. Once VWF unfolds, secondary to shear stress, the binding site is exposed and binds to platelets with high affinity. The binding of platelets to VWF can cause conformational changes, leading to the activation of integrin GPIIbIIIa (also known as α2bβ3) and promoting platelet-platelet and platelet-VWF cross-linking. Thus, the use of standard antiplatelet drugs (aspirin or P2Y12 inhibitors) may be ineffective or only partially effective in mitigating this pathological process, as shown by the cohort study of Tremblay et al. (44).

[0130] This ability to form VWF-platelet-rich thrombi in the microvascular system is a hallmark of acquired thrombotic thrombocytopenic purpura (TTP), in which autoantibodies against ADAMTS13 are present. It has also been shown that interleukin-6 (IL-6) can inhibit the cleavage of ULVWF-platelet strings (45). In addition, the synthesis of ADAMTS13, at least in cultured cells, is significantly inhibited by multiple cytokines including IL-6 and TNF-α (46). This suggests that a cytokine storm (especially IL-6) may propagate microthrombosis. However, this also suggests that if intervention is implemented early and the release of cytokines does not surge, the disease may be more easily controlled and rapid deterioration of the patient's clinical status can be avoided.

[0131] There is now a large body of evidence indicating that in COVID-19, there is a highly significant imbalance in the VWF:ADAMTS13 ratio and the levels of high-molecular-weight VWF multimers (equivalent to ULVWF). As previously mentioned, it has been shown that VWF levels are very high, and the earliest case reports by Escher et al. (34) mentioned a surge in VWF levels. Subsequently, Goshua et al. (47) demonstrated that the plasma VWF concentration was significantly elevated in hospitalized patients with COVID-19, and the elevated levels were correlated with disease severity—the mean VWF antigen level in patients admitted to the intensive care unit (ICU) was 565 ± 199%, while the mean VWF antigen level in patients not admitted to the ICU was 278 ± 133% (p < 0.0001) by comparison. Then, Rauch et al. (48) studied the relationship between the progression of COVID-19 patients and their admission VWF. Patients with the highest VWF levels required a higher level of oxygen support, while patients with normal VWF levels did not require hospitalization or supplemental oxygen (n = 10).

[0132] Shortly after Rauch et al. published, Ladikou et al. (49) showed that the VWF antigen level increased in COVID-19 patients admitted to the ICU, and the VWF level was positively correlated with patient age. However, they reported a median VWF antigen level of 350%, and critically, they also showed that the ADAMTS13 level was significantly reduced (49.7%), indicating the absence of the VWF-cleaving protease that normally degrades large VWF multimers and reduces their activity. They speculated that the excessive release of VWF observed in COVID-19 patients led to the depletion of ADAMTS13 and contributed to a prothrombotic state. Further analysis of their data showed that the median VWF level in deceased patients (477%) was significantly higher than the median VWF level in surviving patients (335%) (p = 0.015).

[0133] Helms et al. (50) recently published a multicenter prospective cohort study in France evaluating the thrombotic risk in COVID-19 patients, showing a significant increase in VWF and factor VIII. Combining the data showing increased VWF and decreased ADAMTS13, further studies indicated that the VWF:ADAMTS13 ratio was essentially disordered. Huisman et al. (51) first showed that the mean VWF:ADAMTS13 ratio in 12 patients admitted to the ICU was 8.5 (normal value 0.5 - 2). Subsequently, Mancini et al. (52) demonstrated similar findings, with an elevated ratio of von Willebrand factor antigen (VWF:Ag) to ADAMTS13 activity being closely related to disease severity, with the worst ratio of 8.3 observed in patients requiring intensive care (intubation and mechanical ventilation), compared to a ratio of 3.42 in patients requiring low-intensity care (p < 0.001).

[0134] Recently, Philippe et al. (53) published their results in a cohort of 208 patients admitted to two centers in Paris, of which 23 patients had only mild symptoms and were treated as outpatients. They found that only VWF:Ag was graded according to clinical severity, with significantly higher levels (median 507%, IQR 428 - 596) in critically ill patients compared to non-critically ill patients (288%, 230 - 350, p < 0.0001) or COVID-19 outpatients (144%, 133 - 198, p = 0.007). In the univariate analysis model, VWF:Ag levels above 423% at admission were significantly associated with higher in-hospital mortality (OR 89.7 95% CI 25.9–567.4, p < 0.001), and remained highly significant in the multivariate analysis model adjusted for age, BMI, D-dimer, and C-reactive protein (CRP) (odds ratio, OR 25.6, 95% CI 5.6 - 198.2, p < 0.001). More importantly, they showed that von Willebrand factor high molecular weight multimers (HMWM) were significantly higher in critically ill patients (median ratio 1.18, IQR 0.86 - 1.09) compared to non-critically ill patients (0.96, 1.04 - 1.39, p < 0.001). Additionally, the HMWM level (ratio) (OR 116, 95% CI 10.2 - 1943, p < 0.001) was one of the factors most significantly associated with in-hospital mortality.

[0135] It is possible to develop a unified theory of endotheliosis and endocarditis, which leads to the release of VWF and ULVWF, resulting in the formation of microthrombi. This then leads to hypoxia, and the process can be exacerbated by a "cytokine storm" and the release of IL-6, which inhibits and reduces the function of ADAMTS13, leading to a series of disseminated microthrombosis and multiple organ dysfunction and failure. It has also been suggested that microvascular thrombosis at the pulmonary level is the origin of right ventricular dysfunction (54). This mechanism can explain many of the findings currently observed, including high D-dimer levels (high due to a high level of microthrombosis), high levels of factor VIII and VWF (released from WPB in response to endothelial injury), microthrombosis and an atypical ARDS picture (55), as well as the broad clinical picture of pulmonary, neurological, and gastrointestinal symptoms. The endotheliosis and microthrombosis that we propose can also explain why patients with pre-existing endotheliosis and microangiopathy (e.g., secondary to diabetes, hypertension, or obesity) are at increased risk of severe COVID-19 (29, 56). Similarly, there is increasing evidence that patients with low ADAMTS13 levels and high VWF levels are associated with a variety of diseases that are prone to poor prognosis after infection with SARS-CoV-2 and have diverse manifestations (57-64). Given the interaction between VWF and platelets that activates the GP2b3a receptor, the use of standard antiplatelet drugs (aspirin or P2Y12 inhibitors) may also be ineffective. Although the use of caplacizumab or anfibatide to inhibit VWF-platelet binding via the GP1b receptor would be an attractive option and has been suggested (65), these drugs are not widely used, and the clinical experience with them is extremely limited. Similarly, they have significant bleeding characteristics.

[0136] Therefore, it is necessary to treat endotheliosis itself.

[0137] Heparin is a naturally occurring highly sulfated polysaccharide, characterized by a wide range of molecular weights of the polysaccharide chains. Heparin acts on various different ligands and has different effects. Heparin is a member of the glycosaminoglycan carbohydrate family and consists of repeating disaccharide units of GlcAβ1-4GlcNAcα1-4, with polydisperse sulfation, N-acetylation, and glucuronyl epimerization. Heparin is highly heterogeneous. Heparin can be isolated from natural sources, such as porcine intestine or bovine lung. Heparin isolated from natural sources contains polysaccharide chains with a molecular weight range of approximately 3000 Da to approximately 30,000 Da. This is called unfractionated heparin (UFH). UFH can be enzymatically or chemically treated to deliver shorter polysaccharide chains. Heparinase I cleaves at the α-1,4 bond between non-acetylated GlcNS6S and IdoA2S. Alkaline elimination after periodate treatment of UFH cleaves the polysaccharide chain at the unsulfated glucuronic acid unit. The products of chemically or enzymatically treated UFH can be affinity purified to produce fractionated heparin, in which the molecular weight of the polysaccharide in each fraction can be easily determined. Low molecular weight heparin (LMWH) contains polysaccharide chains in the range of approximately 4000 Da to approximately 8000 Da.

[0138] In 1991, it was first demonstrated that intravenous injection of heparin into patients during open-heart surgery induced impaired VWF-dependent platelet function without a change in plasma VWF levels (66). This inhibitory effect of heparin on VWF-dependent platelet aggregation does not depend on the affinity of heparin for antithrombin III, but rather on the molecular weight of heparin. From subsequent in vitro experiments, it was found that heparin binds to a specific amino acid sequence (residues 569-583) within the A1 domain of VWF, where basic amino acids are regularly arranged. Heparin binding induces a conformational change in the peptide of this binding site (67). Heparin binds similarly to both activated and inactivated VWF, but does not interfere with the binding of VWF to collagen. Since the platelet GpIb-binding domain (residues 524-542) is also located in the A1 domain, it was suggested that heparin inhibits platelet binding by steric hindrance and by inducing a conformational change in the domain, thereby interfering with the binding of VWF to platelet GpIb.

[0139] The structural specificity of heparin responsible for binding to VWF is centered around key disaccharide units (GlcNS6S-IdoA2S and IdoA2S-GlcNS6S). This structural unit is typically disrupted by digestion with heparinase I but was successfully deduced by competitive binding assays using heparin fractions prepared by specific methods of heparin depolymerization that yield fragments of predictable structure (68). Additionally, studies using synthetic and structurally defined oligosaccharides demonstrated that the assembly of more than 3 units of disaccharide is crucial for binding potency. Similarly, although fractionated heparins of lower molecular weight (6100 Da (g / mol)) showed higher affinity for binding to VWF, their ability to inhibit VWF activity was lower compared to UFH. This indicates that a minimum heparin molecular weight and molecular size are important for achieving steric hindrance.

[0140] Medium molecular weight heparins with specific disaccharide units (GlcNS6S-IdoA2S, also written as IdoA2S-GlcNS6S) can be generated from unfractionated heparin. These medium molecular weight heparins can be specific for inhibiting VWF-GPIb binding, thus preventing microthrombus formation, but they have little anticoagulant effect since they have little effect on antithrombin III. Thus, when considering treating patients with prothrombotic states dependent on increased VWF levels and endothelialopathy, medium molecular weight heparins of a mass of approximately 11000 Da (g / mol) may represent an ideal therapeutic option. Additionally, the results of these early studies suggest that low molecular weight heparins are unlikely to act and target the GPIb receptor, and that while UFH may contain sugar moieties that can bind to VWF, it is not optimal. Additionally, monitoring of UFH is difficult, and other fractions of UFH (such as LMWH fractions) have anticoagulant effects that can lead to dangerous bleeding events, which are unpredictable.

[0141] Further interestingly, it has recently been shown that SARS-CoV-2 binds to heparan sulfate and in particular requires the IdoA2S-GlcNS6S sugar moiety (74,75). This suggests that an exogenous supply of these sugar moieties can inhibit binding to endogenous heparan sulfate in the lung and thus serve as a potential prophylactic treatment. In summary, dedicated medium molecular weight heparins (≈11000 Da (g / mol)) with at least 3 units of the GlcNS6S-IdoA2S disaccharide can inhibit virus adhesion and replication but can also inhibit microthrombus formation triggered by VWF release secondary to virus-induced endothelialopathy.

[0142] When heparin is exposed to specific oxidants (such as periodate ion IO 4-) When this occurs, the diol moiety of glucuronic acid breaks and forms two aldehyde groups, which, as described herein, results in MMWH. As a result, the activity of heparin, as shown by conventional anti-Xa and anti-IIa assays, is significantly reduced. It has been proposed that this oxidation of glucuronic acid alters the binding ability of heparin to antithrombin III (ATIII).

[0143] However, ristocetin-induced platelet aggregation (RIPA) assays have shown an increase in the activity of MMWH. This indicates that the von Willebrand factor interaction of heparin polysaccharide remains intact and may be enhanced.

[0144] The two aldehyde groups on glucuronic acid can be reactive in some cases. Over time, this reactivity may reduce the stability of the oxidized polysaccharide. Additionally, when injected into the body, it may interact with free amines on proteins. The latter will react with the free amines to form the well-known glycated hemoglobin (commonly referred to as A1C) in the same way as glucose reacts with hemoglobin.

[0145] One potential solution is to reduce the aldehyde functional group to an alcohol functional group. This can be easily done with a mild reducing agent such as sodium borohydride (NaBH4). This reduction will not reform the cyclic ring of the glucuronic acid monosaccharide, and the binding of MMWH-Red to ATIII will not be altered. However, reduced MMWH will still maintain the interaction with von Willebrand factor and can be used to treat endothelial diseases.

[0146] The advantages of MMWH-Red include: maintaining the von Willebrand activity of MMWH, higher stability as an active pharmaceutical ingredient (API) and drug product, minimal interaction with drug excipients, reduced or no reaction with proteins in the body, and minimizing side effects.

[0147] Accordingly, in a seventh aspect, the present invention provides the use of MMWH-Red according to the fourth or fifth aspect of the present invention or a composition according to the sixth aspect of the present invention for the treatment of endothelial diseases.

[0148] MMWH-Red can inhibit von Willebrand factor (VWF). MMWH-Red can inhibit the multimers of VWF, preferably ultra-large VWF. MMWH-Red can inhibit the binding of platelets to VWF.

[0149] Preferably, the present invention provides MMWH-Red or a composition comprising MMWH-Red for use in treating endothelial diseases. Preferably, the present invention provides MMWH-Red or a composition comprising MMWH-Red for use in treating endothelial diseases in a patient having a plasma von Willebrand factor antigen to ADAMTS13 ratio of at least about 2. Preferably, the present invention provides MMWH-Red or a composition comprising MMWH-Red for use in treating endothelial diseases in a patient having a plasma von Willebrand factor to ADAMTS13 ratio of at least about 2.

[0150] The VWF:ADAMTS13 ratio in a patient can be at least about 2, at least about 4, at least about 8, or at least about 10. The VWF:ADAMTS13 ratio in a patient can be greater than about 2, greater than about 4, or greater than about 8, or greater than about 10. The VWF:ADAMTS13 ratio in a patient can be about 2-16, about 4-12, or preferably about 6-10. Patients having a VWF to ADAMTS13 ratio greater than about 8 typically indicate severe disease and typically indicate that the patient is deteriorating towards death.

[0151] The VWF antigen:ADAMTS13 ratio in a patient can be at least about 2, at least about 4, at least about 8, or at least about 10. The VWF antigen:ADAMTS13 ratio in a patient can be greater than about 2, greater than about 4, or greater than about 8, or greater than about 10. The VWF antigen:ADAMTS13 ratio in a patient can be about 2-16, about 4-12, or preferably about 6-10. Patients having a VWF antigen to ADAMTS13 ratio greater than about 8 typically indicate severe disease and typically indicate that the patient is deteriorating towards death.

[0152] The levels of VWF and ADAMTS13 in a patient can be measured using ELISA. The ratio can be calculated as described by Huisman et al. (Involvement of ADAMTS13 and von Willebrand factor in thromboembolic events in patients infected with SARS-CoV-2. Int J Lab Hematol. 2020 Oct;42(5):e211–2). Briefly, the level of VWF antigen can be determined in international units and the level of ADAMTS13 can be determined in international units, and then the VWF antigen:ADAMTS13 ratio can be determined.

[0153] Normal levels of plasma VWF are in the range of about 50 IU / dL to about 200 IU / dL. The average level of plasma VWF in the general population is about 100 IU / dL. High levels of plasma VWF are levels of about 200 IU / dL or higher, such as about 200 IU / dL to about 400 IU / dL, about 225 IU / dL to about 375 IU / dL, about 250 IU / dL to about 350 IU / dL, about 275 IU / dL to about 300 IU / dL.

[0154] A patient may have an elevated VWF antigen level of about 150% or higher, about 175% or higher, about 200% or higher, about 300% or higher, about 350% or higher, or about 400% or higher, or about 500% or higher. A patient may have a VWF antigen level as high as about 600%, as high as about 700%, as high as about 800%, or as high as about 1000%.

[0155] Notably, plasma VWF levels may be transiently elevated due to infection, inflammation, trauma, and physical and emotional stressors. Thus, a patient may have a non-transiently elevated plasma von Willebrand factor level, such as for at least about 6 hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours. Preferably, a patient may have an elevated plasma von Willebrand factor level for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, or at least about seven days. Even more preferably, a patient may have an elevated plasma von Willebrand factor level for at least about one week, at least about two weeks, at least about three weeks, or at least about four weeks. Even still more preferably, a patient may have an elevated plasma von Willebrand factor level for at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, or at least about one year. A patient may have an elevated plasma von Willebrand factor level for up to about one week, up to about four weeks, up to about two months, up to about four months, up to about six months, or up to about one year.

[0156] MMWH-Red may have a mass of about 11000 Da (g / mol). Reduced medium molecular weight heparin may comprise at least three units of the GlcNS6S-IdoA2S (or IdoA2S-GlcNS6S) disaccharide.

[0157] Endotheliopathy can be caused by any disease. In particular, endotheliopathy can be caused by COVID-19, infection, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, sepsis, septic shock, cardiovascular disease, diabetes, trauma (especially brain or head trauma), burns, inhalation injury, drugs and drug reactions, hematological disorders, subarachnoid hemorrhage, aneurysmal disease, stroke or parenchymal brain hemorrhage. Endotheliopathy can be caused by viral infection, optionally where the viral infection is SARS-CoV-2. Endotheliopathy can be caused by cancer, especially leukemia, lymphoma, myeloma or solid organ cancer, such as colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer or skin cancer.

[0158] Infections can be bacterial, fungal, or parasitic. Infections can be bacterial. Bacterial infections can be Actinomyces israelii, Bacillus anthracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdoferi, Borrelia garinii, Borrelia afzelaii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira species, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniaeStreptococcus pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroides, Rickettsia rickettsii, Salmonella, Shigella, Spirochaetes, Staphylococcus, Streptococcus, Treponema pallidum, Vibrio cholerae, or Yersinia pestis.

[0159] The infection can be fungal. The fungal infection can be Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, Histoplasma, mucormycetes, Tinea cruris, Tinea corporis, or Tinea pedis.

[0160] The infection can be parasitic. The parasitic infection can be protozoal ocular infections, Chagas' disease, leishmaniasis, toxoplasmosis, giardiasis, malaria, microsporidiosis, or Rhinosporidiosis. Preferably, the parasitic infection is malaria.

[0161] The viral infection can be SARS-CoV-2. SARS-CoV-2 is the virus that causes the COVID-19 disease. COVID-19 can cause ARDS. Endotheliopathy can be caused by SARS-CoV-2 infection. Endotheliopathy can be caused by COVID-19. Endotheliopathy can be caused by ARDS.

[0162] Endotheliopathy can be caused by cancer. The cancer can be leukemia, lymphoma, or myeloma. Alternatively or additionally, the cancer can be solid organ cancer, such as colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer, or skin cancer.

[0163] Endotheliopathy can be caused by hematological disorders, such as thrombotic thrombocytopenic purpura, anemia, or sickle cell disease.

[0164] Dysfunctional endothelial cells can allow tumor cells circulating in the blood to enter tissues. Therefore, treating endotheliopathy can prevent the hematogenous spread of bloodborne cancers. Treatment of endotheliopathy can inhibit the hematogenous spread of cancer. Medium molecular weight heparin can inhibit the hematogenous spread of cancer.

[0165] Biomarkers of endotheliopathy can include elevated levels of von Willebrand factor (VWF), ultra-large von Willebrand factor (ULVWF), factor VIII levels, and other markers such as syndecan-1, VWF antigen, VWF activity, VWF multimers, ADAMTS13 levels, platelet count, VCAM-1, ICAM-1, P-selectin levels, the VWF:ADAMTS13 ratio, or the VWF antigen:ADAMTS13 ratio. Preferably, the biomarker of endotheliopathy is the ratio of VWF:ADAMTS13 or VWF antigen:ADAMTS13.

[0166] Compared to healthy control subjects, patients may have elevated plasma von Willebrand factor (VWF) levels. Compared to healthy controls, patients may have persistently high levels of plasma VWF. Compared to healthy control subjects, plasma VWF levels can be elevated for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, or preferably for at least about one week. Compared to healthy control subjects, plasma VWF levels can be elevated for up to about one week, up to about four weeks, up to about two months, up to about four months, up to about six months, or up to about one year.

[0167] For example, plasma VWF levels can be elevated to at least about 50 nmol / L, preferably at least about 60 nmol / L, even more preferably at least about 70 nmol / L, or even still more preferably at least about 90 nmol / L. Plasma VWF levels can be elevated to about 130 nmol / L, about 150 nmol / L, or about 200 nmol / L. Plasma VWF levels can be elevated to at least about 50 nmol / L for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about one week, at least about one month, or at least about one year. Plasma VWF levels can be elevated to at least about 60 nmol / L for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about one week, at least about one month, or at least about one year. Plasma VWF levels can be elevated to at least about 70 nmol / L for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about one week, at least about one month, or at least about one year. Plasma VWF levels can be elevated to at least about 90 nmol / L for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about one week, at least about one month, or at least about one year. Plasma VWF levels can be measured using an enzyme-linked immunosorbent assay (ELISA).

[0168] Alternatively or additionally, the patient can have a plasma von Willebrand factor level of about 200 IU / dL or higher for at least about 6 hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours. Preferably, the patient can have a plasma von Willebrand factor level of about 200 IU / dL or higher for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, or at least about seven days. Even more preferably, the patient can have a plasma von Willebrand factor level of about 200 IU / dL or higher for at least about one week, at least about two weeks, at least about three weeks, or at least about four weeks. Even still more preferably, the patient can have a plasma von Willebrand factor level of about 200 IU / dL or higher for at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, or at least about one year.

[0169] Vascular endothelial function can be evaluated in the coronary arteries and the peripheral circulation. Non-invasive tests for evaluating coronary endothelial function include Doppler echocardiography, in which blood flow is measured in response to pharmacological or physiological stimuli. Other tests include positron emission tomography and phase-contrast magnetic resonance imaging. However, the gold standard test involves invasive quantitative coronary angiography to examine diameter changes in response to intracoronary infusion of an endothelium-dependent vasodilator (such as acetylcholine). Assessment of the endothelium in the peripheral circulation includes brachial artery ultrasound and strain gauge venous impedance plethysmography.

[0170] The binding of MMWH-Red to VWF can be evaluated by a competitive binding assay. Heparin-agarose beads can be incubated with labeled VWF (such as 125 I-vWF) for a period of time to allow the labeled VWF to bind to the immobilized heparin. Then different concentrations of MMWH-Red can be added, and the amount of displaced labeled VWF can be determined. Other methods for determining the binding of MMWH-Red to VWF can include surface plasmon resonance, biolayer interferometry, isothermal titration calorimetry, fluorescence polarization binding assay, ELISA, and microscale thermophoresis.

[0171] Inhibition of platelet binding to VWF can be evaluated by ristocetin-induced agglutination of immobilized platelets. Platelets can be incubated with medium molecular weight heparin and citrate-treated plasma (a source of VWF). Then ristocetin can be added, and platelet agglutination can be determined. MMWH can completely inhibit VWF-induced platelet aggregation at a concentration of 15 μM when measured by a ristocetin-induced platelet aggregation assay. Other methods for determining the inhibition of VWF binding to platelets can include ELISA, fluorescence-activated cell sorting, dynamic light scattering, or flow chamber assays.

[0172] The mass of MMWH-Red can be in the range of greater than about 8000 Da (g / mol) to about 13000 Da (g / mol), preferably about 10000 Da (g / mol) to about 12000 Da (g / mol). MMWH-Red can have a mass of about 11000 Da (g / mol). MMWH-Red can contain polysaccharide chains with an average molecular weight in the range of about 9000 Da (g / mol) to about 13000 Da (g / mol), preferably about 10000 Da (g / mol) to about 12000 Da (g / mol). MMWH-Red can contain polysaccharide chains with an average molecular weight of about 11000 Da (g / mol). For example, the molecular weight of MMWH-Red can be determined by mass spectrometry or size exclusion chromatography.

[0173] MMWH-Red may contain at least three units of GlcNS6S-IdoA2S disaccharide, such as at least four units, at least five units, at least six units, at least eight units, or at least ten units. Medium molecular weight heparin may contain less than or equal to 25 units of GlcNS6S-IdoA2S disaccharide, such as less than or equal to 20 units. The presence of GlcNS6S-IdoA2S disaccharide units can be deduced by antibodies, mass spectrometry or from chemical and enzymatic studies. GlcNS6S-IdoA2S units may be arranged in a consecutive sequence.

[0174] MMWH-Red may contain at least three units of IdoA2S-GlcNS6S disaccharide, such as at least four units, at least five units, at least six units, at least eight units, or at least ten units. Reduced medium molecular weight heparin may contain less than or equal to 25 units of IdoA2S-GlcNS6S disaccharide, such as less than or equal to 20 units. The presence of IdoA2S-GlcNS6S disaccharide units can be deduced by antibodies, mass spectrometry or from chemical and enzymatic studies. IdoA2S-GlcNS6S units may be arranged in a consecutive sequence. The number of IdoA2S-GlcNS6S units can be customized to provide the desired anti-VWF activity and / or standard anticoagulant activity.

[0175] MMWH-Red may contain UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc. MMWH-Red may contain at least about 60% of UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc. MMWH-Red may contain at least about 45%, preferably at least about 48%, preferably at least about 49%, preferably at least about 60% of UA2S-GlcNS6S. MMWH-Red may contain up to about 60%, preferably up to about 70%, preferably up to about 85% of UA2S-GlcNS6S. MMWH-Red may contain at least about 4%, preferably at least about 5%, preferably at least about 6%, preferably at least about 10% of UA2S-GlcNS. MMWH-Red may contain up to about 15%, preferably up to about 20% of UA2S-GlcNS. MMWH-Red may contain at least 4%, preferably at least 5%, preferably at least 6%, preferably at least about 10% of UA-GlcNAc. Reduced medium molecular weight heparin may contain up to about 15%, preferably up to about 20% of UA-GlcNAc. In some embodiments, MMWH-Red may contain at least 49.2% of UA2S-GlcNS6S, 5.4% of UA2S-GlcN, and 5.4% of UA-GlcNAc. In some embodiments, MMWH-Red may contain at least 82% of UA2S-GlcNS6S, 9% of UA2S-GlcNS, and 9% of UA-GlcNAc. The percentage composition of UA-GlcNAc contained in MMWH-Red may be enriched compared to unfractionated heparin.

[0176] MMWH-Red treatment of endotheliopathy may inhibit the hematogenous spread of cancer. Human tumor cells can bind to VWF under shear flow conditions, and this ability has been demonstrated in both melanoma and colon cancer cells. Immobilized platelets bound to VWF have been shown to mediate tethering, rolling, and firm adhesion of different cancer cell lines under flowing shear stress. VWF plays a key role in enabling tumor cells to firmly adhere to immobilized platelets. Existing data suggest that VWF plays an important role in tethering cancer cells. In addition, VWF-platelet binding that occurs as part of the normal thrombotic pathway can further serve to allow tumor cells to coalesce into VWF-platelets to form heteroaggregates of VWF+platelets+cancer cells, thus contributing to the hematogenous (blood-borne) spread of tumor cells. Once initial binding to VWF and platelets occurs, the process can be caused at least in part by the ability of cancer cells to translocate to the vessel wall and thereby spread to other organs. In addition, it is known that a variety of cancers cause endotheliopathy and thereby release UL-VWF. Through this mechanism, tumors trigger the release of UL-VWF, which then allows for the tethering of platelets and tumor cells and the hematogenous and metastatic spread of cancer. This cancer-induced endotheliopathy also results in an overall increased risk of thrombosis in patients with potential malignancies. Therefore, any treatment aimed at treating endotheliopathy and inhibiting the binding of platelets and / or tumor cells to VWF has the dual purpose of reducing the risk of malignancy-related thrombosis and reducing the risk of hematogenous metastatic spread.

[0177] MMWH-Red can be administered by a method of administration selected from the group consisting of: parenteral, subcutaneous, intravenous, intramuscular, intrathecal, intracutaneous, intraarterial, intraarticular, topical, transdermal, subcutaneous, depot formulations such as depot injection, intraosseous, or inhalation. Preferably, the method of administration of medium molecular weight heparin is subcutaneous, intravenous, or intramuscular. The method of administration can be inhalation, optionally via a nebulizer.

[0178] Previous studies have regarded UFH as an aerosol in various conditions. Small-scale human studies have shown that nebulized UFH can limit pulmonary fibrin deposition, slow the progression of acute lung injury, and accelerate recovery (69). Early trials in patients with acute lung injury and related conditions found that nebulized UFH can reduce pulmonary dead space, coagulation activation, microvascular thrombosis, improve lung injury, and increase the time without ventilatory support (70-73). In a pre-pandemic double-blind randomized study conducted in 256 critically ill ventilated patients, nebulized UFH limited the progression of lung injury (including acute respiratory distress syndrome) and accelerated the discharge of survivors. Therefore, MMWH-Red can be administered by inhalation via a nebulizer.

[0179] Heparin doses are usually measured in "Howell units". One unit of heparin ("Howell unit") is an amount approximately equivalent to 0.002 mg of pure heparin, which is the amount required to keep 1 ml of cat blood fluid at 0 °C for 24 hours. MMWH-Red can be administered as a bolus dose of approximately 5000 units, followed by an optional delivery via an infusion pump at a rate of approximately 1200 to approximately 1600 units per hour. MMWH-Red can be administered at a dose of approximately 18 units / kg to approximately 5000 units / kg. Preferably, MMWH-Red can be administered at a dose of approximately 100 units / kg to approximately 800 units / kg. Alternatively, MMWH-Red can be administered at a dose of approximately 18 units / kg to approximately 75 units / kg. MMWH-Red can be administered at a dose of approximately 5000 units, approximately 4000 units, approximately 3000 units, approximately 2000 units, approximately 1000 units, or approximately 500 units every 12 hours. MMWH-Red can be administered at a dose of approximately 5000 units every 12 hours.

[0180] MMWH-Red can be administered at a dose of approximately 3 units to approximately 5000 units, such as approximately 6 units to approximately 4000 units, approximately 12 units to approximately 3000 units, approximately 25 units to approximately 2000 units, approximately 50 units to approximately 1000 units, approximately 100 units to approximately 500 units, or approximately 125 units to approximately 250 units. Reduced medium molecular weight heparin can be administered at a dose of approximately 18 units / kg to approximately 5000 units / kg, such as approximately 100 units / kg to approximately 4000 units / kg, or approximately 200 units / kg to approximately 800 units / kg. Reduced medium molecular weight heparin can be administered at a dose of approximately 18 units / kg to approximately 75 units / kg. The dose can be given as a single dose or as a continuous dose. The dose can be given over a period of time. The period of time can be approximately 1 month to approximately 12 months, such as approximately 2 months to approximately 11 months, approximately 3 months to approximately 10 months, approximately 4 months to approximately 9 months, approximately 5 months to approximately 8 months, approximately 6 months to approximately 7 months. The period of time can be approximately 1 day to 7 days, approximately 2 days to approximately 6 days, approximately 3 days to approximately 5 days, approximately 4 days to approximately 5 days. The dose can be administered over approximately 1 hour to approximately 24 hours, approximately 2 hours to approximately 12 hours, approximately 3 hours to approximately 6 hours. The dose can be administered during the duration of potential endothelial disease and elevated VWF levels.

[0181] MMWH-Red can be administered at a dose of from about 0.01 mg / kg to about 10 mg / kg, such as a dose of from about 0.05 mg / kg to about 9 mg / kg, from about 0.5 mg / kg to about 8 mg / kg, from about 1 mg / kg to about 7 mg / kg, from about 1.5 mg / kg to about 6 mg / kg, or from about 2 mg / kg to about 5 mg / kg. The dose can be given as a single dose or as a continuous dose. The dose can be administered over a period of time. The period of time can be from about 1 month to about 12 months, such as from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months to about 9 months, from about 5 months to about 8 months, from about 6 months to about 7 months. The period of time can be from about 1 day to about 7 days, from about 2 days to about 6 days, from about 3 days to about 5 days, from about 4 days to about 5 days. The dose can be administered over a period of from about 1 hour to about 24 hours, from about 2 hours to about 12 hours, from about 3 hours to about 6 hours. The dose can be administered during the duration of potential endotheliopathy and elevated VWF levels.

[0182] MMWH-Red can be administered at a dose starting from about 0.01 mg / kg, from about 0.1 mg / kg, from about 1 mg / kg, from about 5 mg / kg, from about 10 mg / kg, from about 20 mg / kg, from about 30 mg / kg, from about 50 mg / kg, from about 70 mg / kg, from about 80 mg / kg, or from about 100 mg / kg. MMWH-Red can be administered at a dose of about 500 mg / kg or less, about 300 mg / kg or less, about 200 mg / kg or less, or about 100 mg / kg or less. MMWH-Red can be administered at a dose of from about 0.01 mg / kg to about 10 mg / kg, preferably from about 0.2 mg / kg to about 10 mg / kg, from about 0.2 mg / kg to about 1.6 mg / kg. MMWH-Red can be administered as a single dose or as a continuous dose. The amount of the MMWH-Red dose can depend on the VWF antigen:ADAMTS13 ratio or the overall VWF level. One skilled in the art will be able to select an appropriate amount for a patient based on the VWF antigen:ADAMTS13 ratio or the overall VWF level.

[0183] MMWH-Red can be administered at a dose of about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 8 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 2 mg / kg, about 1 mg / kg to about 1.5 mg / kg. This dose can be given as a single dose or as a continuous dose. This dose can be administered over a period of time. The period of time can be about 1 month to about 12 months, such as about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, about 6 months to about 7 months. The period of time can be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, about 4 days to about 5 days. This dose can be administered within about 1 hour to about 24 hours, about 2 hours to about 12 hours, about 3 hours to about 6 hours. This dose can be administered during the duration of potential endotheliopathy and elevated VWF levels.

[0184] MMWH-Red can be administered at a dose of about 0.1 mg to about 5000 mg, about 0.5 mg to about 2000 mg, about 1 mg to about 1000 mg, about 5 mg to about 900 mg, about 10 mg to about 800 mg, about 20 mg to about 700 mg, about 30 mg to about 600 mg, about 50 mg to about 500 mg, about 75 mg to about 400 mg, about 100 mg to about 300 mg, about 125 mg to about 250 mg, about 150 mg to about 200 mg. This dose can be given as a single dose or as a continuous dose. This dose can be administered over a period of time. The period of time can be about 1 month to about 12 months, such as about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, about 6 months to about 7 months. The period of time can be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, about 4 days to about 5 days. This dose can be administered within about 1 hour to about 24 hours, about 2 hours to about 12 hours, about 3 hours to about 6 hours. This dose can be administered during the duration of potential endotheliopathy and elevated VWF levels.

[0185] MMWH-Red can be administered at a dose of, for example, about 1 international unit (IU), about 2 IU, about 5 IU, about 10 IU, about 15 IU, about 20 IU, about 25 IU, about 50 IU, about 75 IU, about 100 IU, about 200 IU, about 300 IU, about 400 IU, about 500 IU, about 1000 IU, about 1500 IU, about 2000 IU, about 2500 IU, about 5000 IU, about 10000 IU, about 20000 IU or about 25000 IU. This dose can be given as a single dose or as a continuous dose. This dose can be administered over a period of time. The period of time can be about 1 month to about 12 months, such as about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, about 6 months to about 7 months. The period of time can be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, about 4 days to about 5 days. This dose can be administered within about 1 hour to about 24 hours, about 2 hours to about 12 hours, about 3 hours to about 6 hours. This dose can be administered during the duration of potential endotheliopathy and elevated VWF levels.

[0186] MMWH-Red can be administered at a dose of about 1 IU to about 50000 IU, about 2 IU to about 25000 IU, about 5 IU to about 20000 IU, about 10 IU to about 10000 IU, about 15 IU to about 5000 IU, about 20 IU to about 2500 IU, about 25 IU to about 2000 IU, about 50 IU to about 1500 IU, about 75 IU to about 1000 IU, about 100 IU to about 500 IU, about 200 IU to about 400 IU, about 250 IU to about 300 IU. This dose can be given as a single dose or as a continuous dose. This dose can be administered over a period of time. The period of time can be about 1 month to about 12 months, such as about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, about 6 months to about 7 months. The period of time can be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, about 4 days to about 5 days. This dose can be administered within about 1 hour to about 24 hours, about 2 hours to about 12 hours, about 3 hours to about 6 hours. This dose can be administered during the duration of potential endotheliopathy and elevated VWF levels.

[0187] MMWH-Red can be administered in proportion to the VWF antigen:ADAMTS13 ratio. For example, a higher dose of MMWH can be administered to a patient with a high VWF antigen:ADAMTS13 ratio compared to a patient with a lower VWF antigen:ADAMTS13 ratio.

[0188] MMWH-Red can be included in a pharmaceutical formulation. The pharmaceutical formulation can include excipients. The excipients can be selected from the group consisting of solvents, co-solvents, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavoring agents, lubricants, suspending agents, tonicity modifiers, surfactants, solubilizers, suspending aids, dispersants, humectants, thickening agents, coloring agents, wetting agents, defoaming agents, viscosity modifiers, sweetening agents, and combinations thereof. The pharmaceutical formulation can include additional active agents. The additional active agents can include medium molecular weight heparin or low molecular weight heparin.

[0189] The pharmaceutical formulation can include additional active agents. The additional active agents include a composition of substances having physiological effects. The additional active agents can include low molecular weight heparin or medium molecular weight heparin or reduced medium molecular weight heparin with different disaccharide compositions. The additional active agents can be selected from the group consisting of farnesoid X receptor (FXR) agonists, peroxisome proliferator-activated receptor (PPAR) agonists, aramchol, caspase inhibitors, galectin-3 inhibitors, mitogen-activated protein kinase 5 (MAPK5) inhibitors, fibroblast growth factor 19 (FGF19) agonists, FGF21 agonists, leukotriene D4 (LTD4) receptor antagonists, niacin analogs, apical sodium bile acid cotransporter (ASBT) inhibitors, apoptosis signal-regulating kinase 1 (ASK1) inhibitors, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor antagonists, chemokine receptor inhibitors, thiazolidinediones, GLP-1 analogs, biguanides, HIV replication inhibitors, metformin, opiates, anesthetics, HMG-CoA reductase inhibitors, non-steroidal anti-inflammatory drugs (NSAIDs), or any combination thereof.

[0190] MMWH-Red can include chemical modifications. The chemical modifications can be selected from the group consisting of N-acetylation, N-deacetylation, N-sulfation, O-sulfation, 2-O-desulfation, and complete desulfation.

[0191] In an eighth aspect, the present invention provides the use of MMWH-Red for treating a disease or disorder in a patient, wherein the patient has an endotheliopathy characterized by a plasma von Willebrand factor to ADAMTS13 (VWF:ADAMTS13) ratio of at least about 2.

[0192] In a ninth aspect, the present invention provides the use of MMWH-Red for treating a disease or disorder in a patient suffering from an endotheliopathy characterized by a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0193] Preferably, the disease or disorder is selected from the group consisting of: COVID-19, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, sepsis, septic shock, cardiovascular disease, diabetes, trauma (especially brain or head trauma), burns, inhalation injury, drugs and drug reactions, hematological disorders, subarachnoid hemorrhage, aneurysmal disease, stroke, or brain parenchyma, or a combination thereof. The endotheliopathy can be caused by a viral infection, optionally wherein the viral infection is SARS-CoV-2. The endotheliopathy can be caused by cancer, particularly leukemia, lymphoma, myeloma or solid organ cancer, such as colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer or skin cancer. Preferably, the endotheliopathy is caused by sepsis or septic shock. Preferably, the endotheliopathy is caused by sepsis.

[0194] In some embodiments, the present invention provides the use of MMWH-Red for treating COVID-19 in a patient suffering from an endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0195] In some embodiments, the present invention provides the use of MMWH-Red for treating viral infection in a patient suffering from an endotheliopathy characterized by a VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2. The viral infection can be SARS-CoV-2.

[0196] In some embodiments, the present invention provides the use of MMWH-Red for treating acute respiratory distress syndrome (ARDS) in a patient suffering from an endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0197] In some embodiments, the present invention provides the use of MMWH-Red for treating cancer in a patient having an endotheliopathy characterized by a VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2. The cancer can be leukemia, lymphoma, myeloma, or solid organ cancer such as colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer, or skin cancer.

[0198] In some embodiments, the present invention provides the use of MMWH-Red for treating a bacterial infection in a patient having an endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0199] In some embodiments, the present invention provides the use of MMWH-Red for treating sepsis in a patient having an endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0200] In some embodiments, the present invention provides the use of MMWH-Red for treating septic shock in a patient having an endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0201] In some embodiments, the present invention provides the use of MMWH-Red for treating cardiovascular disease in a patient having an endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0202] In some embodiments, the present invention provides the use of MMWH-Red for treating diabetes in a patient having an endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0203] In some embodiments, the present invention provides the use of MMWH-Red for treating trauma in a patient suffering from endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2. Preferably, the trauma is a brain trauma or a head trauma.

[0204] In some embodiments, the present invention provides the use of MMWH-Red for treating burns in a patient suffering from endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0205] In some embodiments, the present invention provides the use of MMWH-Red for treating inhalation injury in a patient suffering from endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0206] In some embodiments, the present invention provides the use of MMWH-Red for treating drug reactions in a patient suffering from endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0207] In some embodiments, the present invention provides the use of MMWH-Red for treating hematological disorders in a patient suffering from endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0208] In some embodiments, the present invention provides the use of MMWH-Red for treating subarachnoid hemorrhage in a patient suffering from endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0209] In some embodiments, the present invention provides the use of MMWH-Red for treating aneurysmal diseases in a patient suffering from endotheliopathy characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a ratio of von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) in plasma of at least about 2.

[0210] The use of MMWH-Red for treating endotheliopathy as defined in the seventh, eighth or ninth aspect of the present invention is particularly advantageous since said heparin can inhibit microthrombosis triggered by the release of VWF secondary to endotheliopathy caused by any disease or disorder. When the cause of the endotheliopathy is SARS-CoV-2, the MMWH-Red can additionally inhibit viral adhesion and replication.

[0211] In a tenth aspect, the present invention provides a method for treating endotheliopathy, the method comprising administering to a subject in need of treatment a therapeutically effective amount of MMWH-Red. Preferably, wherein the plasma VWF:ADAMTS13 ratio of the patient is at least about 2 or the ratio of von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) in plasma is at least about 2.

[0212] In an eleventh aspect, the present invention provides the use of MMWH-Red in the preparation of a medicament for treating endotheliopathy in a patient. Preferably, wherein the plasma VWF:ADAMTS13 ratio of the patient is at least about 2 or the ratio of von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) in plasma is at least about 2.

[0213] For the avoidance of doubt, the embodiments associated with the various aspects of the present invention apply, mutatis mutandis, to the other aspects of the present invention. From the discussion herein, other aspects and embodiments of the present invention will be apparent.

[0214] Sepsis is the body's extreme response to infection. It is characterized by a systemic inflammatory state in which, due to a dysregulated immune response, the immune system reacts to the infection by attacking the body's own tissues and organs. As a result, cells in the body are destroyed by apoptosis or necrosis, and intracellular substances eventually enter the bloodstream.

[0215] Histones are intracellular basic proteins found in the cell nucleus. DNA wraps itself around histones to form chromatin, which then tightly coils itself to facilitate the packaging of long DNA molecules into the cell nucleus. Although chromatin (and its constituent histones) is known for its role in packaging DNA into the nucleus, chromatin (and its constituent histones) has also been shown to play a role in innate immunity. During infection, neutrophils release granule proteins and chromatin, which together form extracellular fibers that bind Gram-positive and Gram-negative bacteria. These neutrophil extracellular traps (NETs) detoxify virulence factors and kill bacteria.

[0216] Circulating histones have been identified as mediators of injury in animal models of sepsis and patients. These histones may release intracellular substances due to apoptosis or necrosis that occur during sepsis, or enter the bloodstream via NET release in response to bacterial infection. Thus, histones can spread the immune system dysregulation observed during sepsis. For example, histones are damage-associated molecular patterns (DAMPs) sensed by toll-like receptors (TLRs). Activation of TLRs on innate immune cells by histones leads to the release of pro-inflammatory cytokines, thereby exacerbating the already dysregulated immune response. In particular, TLR receptors on endothelial cells can be activated by circulating histones, which results in the induction of an immune response in endothelial cells. Consequently, endothelial inflammation, endocarditis, and endotheliopathy are observed.

[0217] Thus, MMWH or MMWH-Red as defined herein can bind free histones while inhibiting VWF, thereby treating sepsis. In one embodiment, the present invention provides MMWH or MMWH-Red for treating sepsis, systemic inflammatory response syndrome (SIRS), severe sepsis, or septic shock in a subject. In some embodiments, the plasma VWF:ADAMTS13 ratio of the subject is at least about 2.

[0218] MMWH or MMWH-Red as defined herein can be a complement cascade regulator.

[0219] MMWH or MMWH-Red as defined herein can be an immune regulator.

[0220] To avoid doubt, embodiments related to various aspects of the present invention apply, with necessary modifications in details, to other aspects of the present invention. From the discussion herein, other aspects and embodiments of the present invention will be apparent.

[0221] Unless expressly excluded or otherwise limited, each document cited herein, including any cross-referenced or related patent or application, is incorporated herein by reference in its entirety.

[0222] It should be understood that various modifications can be made to the illustrated embodiments without departing from the spirit and scope of the invention as defined by the appended claims.

[0223] Example

[0224] The present invention will now be demonstrated by reference to the following non - limiting examples.

[0225] Unless otherwise stated, room temperature and pressure are 20 °C (293.15 K, 68 °F) and 1 atm (14.696 psi, 101.325 kPa), respectively.

[0226] Experimental method

[0227] Size - exclusion chromatography (SEC)

[0228] SEC was carried out on a GEC Superdex75 (10 / 100) column on an Agilent 1200 HPLC system with a variable - wavelength UV monitor or a GEC AKTA system with a variable - wavelength UV monitor. The mobile phase was an aqueous solution of 0.15 M NaCl, which passed through the column at 0.4 mL / min. The optical density was measured at 232 nm. SEC was carried out with reference to an 11 kD standard.

[0229] Freeze - drying

[0230] Freeze - drying (also known as lyophilization) is a drying process carried out at low temperature. Freeze - drying generally involves reducing the temperature and pressure below the triple point of the substance and removing the frozen solvent (such as water ice) by sublimation. For aqueous compositions (such as those disclosed herein), freeze - drying can be carried out at a temperature of about - 20 °C to - 80 °C, preferably about - 40 °C and a pressure of about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).

[0231] Preparation method of MMW heparin

[0232] 1. Development example

[0233] 1.1 Optimization of periodate concentration (periodate method)

[0234] 80 mg of a batch of unfractionated heparin was dissolved in 50 mM sodium phosphate buffer (30 mL) adjusted to pH 7.0.

[0235] Samples were prepared by adding (A) 43 mg, (B) 86 mg, (C) 173 mg, or (D) 200 mg of sodium periodate to the heparin in sodium phosphate buffer solution. The resulting solution was incubated at 4 °C for 16 to 18 hours or at 37 °C for 6 hours.

[0236] Take 200 μl aliquots before dialysis for SEC. Dialyze the remaining sample against water in tubing with a 2 kD cut-off (Spectra / Product No. 132109; 7 days: change water 3 times per day / 4 volumes). Then lyophilize the sample at -40 °C and 10 Pa. Then dissolve the sample in water and analyze by SEC on a GEC AKTA system or an Agilent 1200 system on a 75SE (10 / 300) column at 0.4 mL / min in 0.15 M NaCl.

[0237] Analyze the aliquots taken before dialysis by SEC on a GEC AKTA system or an Agilent 1200 system on a 75SE (10 / 300) column at 0.4 mL / min in 0.15 M NaCl, showing a significant amount of disaccharide-like material mixed with the reagent peak.

[0238] Perform SEC on the sample and calibrate by co-eluting with a validated 11 kDa standard. SEC analysis of the sample after dialysis gave the following results:

[0239] a. Incubation at 37 °C gave a sample with a broad peak centered at K av 0.12 and led to moderate molecular weight heparin degradation.

[0240] b. Incubation at 4 °C gave a sample with a narrower peak centered at K av 0.06 and a molecular weight of 11 kD.

[0241] Figure 1 Shows a comparison of MMWH prepared at 4 °C with heparin treated at 37 °C, untreated heparin, and 11 kDa standard.

[0242] Therefore, incubation at 4 °C favors the formation of moderate molecular weight heparin. Incubation at 37 °C mainly provides other products.

[0243] All studied sodium periodate concentrations provided the desired moderate molecular weight heparin. The most effective concentration of sodium periodate was (C) 173 mg of sodium periodate per 30 mL of heparin in sodium phosphate buffer (equivalent to 8.6 g / l.5 L used in the large-scale process example below).

[0244] 1.2 Perchlorate addition

[0245] Prepare samples according to (A) and (B) of the periodate method of (1.1) above. Add 732 mg of sodium perchlorate to samples (A) and (B) per 30 mL of sample, and incubate the resulting solution at 4 °C for 16 to 18 hours or at 37 °C for 6 hours.

[0246] Dialyze the samples against water in tubing with a 2 kD cut-off (Spectra / Product No. 132109; 7 days: change 3 times / day / 4 volumes of water). Then lyophilize the samples at -40 °C and 10 Pa. Then dissolve the samples in water and analyze by SEC on a 75SE (10 / 300) column in 0.15 M NaCl at 0.4 mL / min on a GEC AKTA system or an Agilent 1200 system. 75SE(10 / 300) column.

[0247] Perform SEC on the samples and calibrate by co-elution with a validated 11 kDa standard. SEC analysis of the samples after dialysis gave the following results:

[0248] All samples (incubated at 4 °C and 37 °C) produced a broader size range of material centered at K av 0.12. There was also a large increase in the material at the V t of the column (i.e., smaller species).

[0249] This example shows that oxidation with a mixture of periodate and perchlorate results in an increased level of breakdown into smaller species compared to oxidation with periodate alone. Thus, oxidation with periodate alone is a more effective method for preparing high-purity medium-molecular-weight heparin.

[0250] 1.3 Alkali elimination

[0251] Prepare samples according to (A) and (B) of the periodate method of (1.1) above under incubation at 4 °C and 37 °C.

[0252] Add NaOH (2 M) to the samples to adjust the pH of the samples to pH 12. Then incubate the samples at room temperature for 30 minutes. Analyze aliquots of these samples by SEC on a 75SE (10 / 300) column in 0.15 M NaCl at 0.4 mL / min on a GEC AKTA system or an Agilent 1200 system. Perform SEC on the samples and calibrate by co-elution with a validated 11 kDa standard. 75SE(10 / 300) column.

[0253] This base elimination results in further degradation, at K avThere are more distinct peaks at s 0.331, 0.35, 0.4 and 0.54, and there is a large peak at K av 0.625.

[0254] Figure 2 It is shown that MMWH prepared only with periodate at 4 °C exhibits a broad peak due to degradation compared to heparin treated with periodate at 4 °C and then with NaOH. Therefore, the preparation of high-purity medium molecular weight heparin is hindered by including an alkali elimination step because significant degradation is observed.

[0255] 2. Large-scale process example

[0256] UF heparin (heparin from porcine mucosa; Iduron catalogue number HEP001 / 100) (4 g; 2.7 mg / mL; average molecular weight 15 kDa) was dissolved in ice-cold (0 °C to 2 °C) 50 mM sodium phosphate buffer (1.5 L) adjusted to pH 7.0. Sodium periodate (8.56 g, 40 mmol) was added and the sample was incubated overnight at 4 °C. After incubation, the sodium periodate was inactivated by adding D-mannitol (30 g, 160 mmol).

[0257] Then the sample was dialyzed against water in tubing with a 2 kD cut-off (Spectra / Product number 132109; 7 days: water was changed 3 times a day / 4 volumes). Then the sample was lyophilized at -40 °C and 10 Pa to give medium molecular weight heparin in a yield of 2.1 g.

[0258] Then the medium molecular weight heparin was dissolved in water and analyzed by SEC on a 75SE (10 / 300) column in 0.15 M NaCl at 0.4 mL / min on a GEC AKTA system or an Agilent 1200 system. As shown, the average molecular weight of the medium molecular weight heparin was 11 kDa relative to an 11 kDa standard. Figure 3

[0259] As Figure 4 and Figure 5 shown, the medium molecular weight heparin produced showed very low anti-factor IIa and / or Xa activity compared to UF heparin and low molecular weight heparin. Therefore, unlike UF heparin or LMW heparin, MMW heparin does not affect factor IIa- or factor Xa-mediated coagulation.

[0260] ​Factor IIa (also known as thrombin), as a serine protease, converts soluble fibrinogen into insoluble fibrin strands and catalyzes other coagulation-related reactions. Factor Xa is the activated form of coagulation factor X. Factor X is a serine endopeptidase that plays a key role in several stages of the coagulation system.

[0261] Heparin (unfractionated heparin) and its derivatives, such as low molecular weight heparin, bind to the plasma cofactor antithrombin (AT), inactivating several coagulation factors IIa, Xa, XIa, and XIIa. This inactivation of factor Xa by heparin is termed "indirect" because it depends on the presence of AT rather than a direct interaction with factor Xa.

[0262] The activities against factor IIa and factor Xa can be measured using the Kinetichrome anti-IIa heparin kit and the Kinetichrome anti-Xa heparin kit, respectively.

[0263] Disaccharide analysis was performed on medium molecular weight heparin. The results are shown in Table 1 below.

[0264]

[0265] UF heparin derived from porcine mucosa was used for the purposes of the exemplary syntheses provided herein. For the avoidance of doubt, other sources of UFH heparin are suitable for the methods disclosed herein.

[0266] Preparation method of MMWH-Red

[0267] Heparin oxidation

[0268] The UFH heparin was oxidized as described above.

[0269] Reduction of oxidized heparin

[0270] To a solution of oxidized heparin at 10 mg / mL in deionized water (110 mL) was added 20 mg of sodium borohydride. The reaction mixture was stirred at 25 °C for 3 hours. The reduced medium molecular weight heparin was purified by thorough dialysis using phosphate buffer (pH = 7.0).

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Claims

1. A method for synthesizing medium molecular weight heparin, the method comprising the following steps: a. Dissolve unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; b. Add an oxidizing agent to the first solution to provide a second solution; and c. Incubate the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin solution.

2. The method according to claim 1, wherein the aqueous buffer solution is adjusted to about pH 7.

0.

3. The method according to claim 1 or claim 2, wherein the aqueous buffer solution is a phosphate buffer solution.

4. The method according to claim 3, wherein the phosphate buffer solution is a sodium phosphate buffer solution.

5. The method according to any one of the preceding claims, wherein the incubation step c. is carried out for about 1 hour to about 48 hours.

6. The method according to any one of the preceding claims, wherein the incubation temperature is about 4°C.

7. The method according to any one of the preceding claims, wherein the temperature of the aqueous buffer solution in step a. is about 0°C.

8. The method according to any one of the preceding claims, wherein the oxidizing agent is a periodate, preferably sodium periodate or potassium periodate.

9. The method according to any one of the preceding claims, wherein the method further comprises: d. Inactivate the oxidizing agent in the medium molecular weight heparin solution.

10. The method according to claim 9, wherein the oxidizing agent is inactivated by adding an inactivator selected from the group consisting of: D-mannitol, glycerol, N-acetylmethionine, sodium sulfite, and combinations thereof.

11. The method according to claim 9 or claim 10, wherein the oxidizing agent is inactivated by adding D-mannitol.

12. The method according to any one of claims 9 to 11, wherein the molar ratio of the oxidizing agent to the inactivator is about 1:1 to about 1:

10.

13. The method according to any one of the preceding claims, wherein the method further comprises: e. Dialyze the medium molecular weight heparin solution in a dialysis fluid to provide a dialyzed medium molecular weight heparin sample.

14. The method according to any one of the preceding claims, wherein in the dialysis step e., the dialysis fluid is water.

15. The method according to any one of the preceding claims, wherein the dialysis step e. is carried out in a tubing with a 2kD cut-off.

16. The method according to any one of claims 13 to 15, wherein the method further comprises: f. Isolate the medium molecular weight heparin from the dialyzed heparin sample.

17. The method according to claim 16, wherein the dialyzed heparin sample is lyophilized to isolate the medium molecular weight heparin.

18. The method according to any one of the preceding claims, wherein the medium molecular weight heparin has an average molecular weight greater than about 8000 Da to about 13000 Da.

19. The method according to any one of the preceding claims, wherein the medium molecular weight heparin has an average molecular weight of about 11000 Da.

20. The method according to any one of the preceding claims, wherein the medium molecular weight heparin comprises at least three units of the IdoA2S-GlcNS6S disaccharide.

21. The method according to any one of the preceding claims, wherein the method does not include an alkali elimination step.

22. The method according to any one of the preceding claims, wherein the method does not include the addition of an alkali metal salt.

23. The method according to claim 22, wherein the alkali metal salt comprises NaOH, KOH or LiOH.

24. The method according to claim 22, wherein the alkali metal salt is NaOH, KOH or LiOH.

25. A kit suitable for use in the preparation of medium molecular weight heparin, wherein the kit comprises: a. unfractionated heparin; b. an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0; c. an oxidizing agent; and d. an optional inactivating agent.

26. A method for synthesizing reduced medium molecular weight heparin (MMWH-Red), the method comprising the steps of: a. dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; b. adding an oxidizing agent to the first solution to provide a second solution; c. incubating the second solution at a temperature of about 0 °C to about 10 °C to form a medium molecular weight heparin solution; and d. incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin.

27. The method according to claim 26, wherein the reducing agent is a mild reducing agent.

28. The method according to claim 27, wherein the mild reducing agent is sodium borohydride.

29. The method according to claims 26 to 28, wherein the method does not include an alkali elimination step.

30. The method according to claims 26 to 29, wherein the method does not include the addition of an alkali metal salt.

31. The method according to claim 30, wherein the alkali metal salt comprises NaOH, KOH or LiOH.

32. The method according to claim 31, wherein the alkali metal salt is NaOH, KOH or LiOH.

33. The method according to claims 26 to 32, wherein the aqueous buffer solution is adjusted to about pH 7.

0.

34. The method according to claims 26 to 33, wherein the aqueous buffer solution is a phosphate buffer solution.

35. The method according to claim 34, wherein the phosphate buffer solution is a sodium phosphate buffer solution.

36. The method according to claims 26 to 35, wherein the incubation step c. is carried out for about 1 hour to about 48 hours.

37. The method according to claims 26 to 36, wherein the incubation temperature is about 4 °C.

38. The method according to claims 26 to 37, wherein the temperature of the aqueous buffer solution in step a. is about 0 °C.

39. The method according to claims 26 to 38, wherein the oxidizing agent is a periodate, preferably sodium periodate or potassium periodate.

40. The method according to any one of claims 26 to 40, wherein the method further comprises: Inactivate the oxidizing agent in the medium molecular weight heparin solution.

41. The method according to claim 40, wherein the oxidizing agent is inactivated by adding an inactivator selected from the group consisting of D-mannitol, glycerol, N-acetylmethionine, sodium sulfite, and combinations thereof.

42. The method according to claim 40 or claim 41, wherein the oxidizing agent is inactivated by adding D-mannitol.

43. The method according to any one of claims 39 to 42, wherein the molar ratio of the oxidizing agent to the inactivator is from about 1:1 to about 1:

10.

44. The method according to any one of claims 26 to 43, wherein the method further comprises: Dialyze the medium molecular weight heparin solution in a dialysate to provide a dialyzed medium molecular weight heparin sample.

45. The method according to claims 26 to 44, wherein in dialysis step e., the dialysate is water.

46. The method according to claims 26 to 45, wherein dialysis step e. is carried out in a tubing with a 2 kD cut-off.

47. The method according to any one of claims 44 to 46, wherein the method further comprises: Isolate the medium molecular weight heparin from the dialyzed heparin sample.

48. The method according to claims 26 to 47, wherein the medium molecular weight heparin has an average molecular weight greater than about 8000 Da to about 13000 Da.

49. The method according to claims 26 to 48, wherein the medium molecular weight heparin has an average molecular weight of about 11000 Da.

50. The method according to claims 26 to 49, wherein the medium molecular weight heparin comprises at least three units of the IdoA2S-GlcNS6S disaccharide.

51. Use of reduced medium molecular weight heparin (MMWH-Red) for the treatment of endothelial diseases in a patient having a plasma von Willebrand factor antigen to ADAMTS13 ratio of at least about 2.

52. The MMWH-Red for such use according to claim 51, wherein the MMWH-Red inhibits von Willebrand factor, optionally wherein the MMWH-Red completely inhibits von Willebrand factor-induced platelet aggregation at a concentration of 15 μM as measured by the ristocetin-induced platelet aggregation assay.

53. The MMWH-Red for such use according to claim 52, wherein the MMWH-Red inhibits von Willebrand factor multimers, optionally wherein the von Willebrand factor is ultra-large von Willebrand factor.

54. The MMWH-Red for such use according to any one of claims 51 to 53, wherein the MMWH-Red inhibits the binding of platelets to von Willebrand factor.

55. The MMWH-Red for such use according to any one of claims 51 to 54, wherein the mass of the MMWH-Red is in the range greater than about 8000 Da (g / mol) to about 13000 Da (g / mol), optionally wherein the mass of the MMWH-Red is about 11000 Da (g / mol).

56. MMWH-Red for the use according to any one of claims 51 to 55, wherein the MMWH-Red comprises at least three units of the IdoA2S-25GlcNS6S disaccharide.

57. MMWH-Red for the use according to any one of claims 51 to 56, wherein the treatment of the endotheliopathy inhibits the hematogenous spread of cancer.

58. MMWH-Red for use in the treatment of a disease or disorder in a patient, wherein the patient has an endotheliopathy characterized by a ratio of plasma von Willebrand factor antigen to ADAMTS13 of at least about 2.

59. MMWH-Red for the use according to claim 58, wherein the disease or disorder is COVID-19, viral infection, acute respiratory distress syndrome, cancer, bacterial infection, sepsis, cardiovascular disease, diabetes, trauma, burn, inhalation injury, drug reaction, hematological disorder, subarachnoid hemorrhage or aneurysmal disease.

60. MMWH-Red for the use according to claim 59, wherein the endotheliopathy is caused by a viral infection, optionally wherein the viral infection is SARS-CoV-2.

61. MMWH-Red for the use according to claim 58, wherein the endotheliopathy is caused by cancer, optionally wherein the cancer is leukemia, lymphoma, myeloma or solid organ cancer.

62. MMWH-Red for the use according to any one of claims 51 to 61, wherein the medium molecular weight heparin is administered by an administration method selected from the group consisting of: parenteral, subcutaneous, subcutaneous, depot formulation, such as depot injection, intravenous, intramuscular, intrathecal, intradermal, intra-arterial, intra-articular, cutaneous, transdermal, intraosseous or inhalation.

63. MMWH-Red for the use according to claim 62, wherein the administration method is subcutaneous.

64. MMWH-Red for the use according to claim 62, wherein the administration method is intravenous.

65. MMWH-Red for the use according to claim 62, wherein the administration method is intramuscular.

66. MMWH-Red for the use according to claim 62, wherein the administration method is inhalation, optionally via a nebulizer.

67. MMWH-Red for the use according to any one of claims 51 to 66, wherein the MMWH-Red is administered at a dose of about 0.01 mg / kg to about 100 mg / kg, preferably about 0.01 mg / kg to about 10 mg / kg.

68. MMWH-Red for the use according to any one of claims 62 to 67, wherein the MMWH-Red is administered as a single dose or as a continuous dose. MMWH-Red for use in the use according to any one of claims 51 to 68 of the foregoing claims, wherein the MMWH-Red is comprised in a pharmaceutical formulation. MMWH-Red for use in the use according to claim 69, wherein the pharmaceutical formulation comprises an excipient. MMWH-Red for use in the use according to claim 70, wherein the excipient is selected from the group consisting of solvents, co-solvents, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavoring agents, lubricants, suspending agents, tonicity regulators, surfactants, solubilizers, suspending aids, dispersants, humectants, thickening agents, coloring agents, wetting agents, defoaming agents, viscosity regulators, sweetening agents, and combinations thereof. MMWH-Red for use in the use according to any one of claims 69 to 71, wherein the pharmaceutical formulation comprises an additional active agent, optionally wherein the additional active agent comprises low molecular weight heparin. MMWH-Red for use in the use according to any one of claims 51 to 72, wherein the MMWH-Red comprises a chemical modification. MMWH-Red for use in the use according to claim 73, wherein the chemical modification comprises N-acetylation, N-deacetylation, N-sulfation, O-sulfation, 2-O-desulfation, complete desulfation, or combinations thereof. A method of treating endotheliopathy, the method comprising administering to a patient in need thereof a therapeutically effective amount of MMWH-Red, wherein the ratio of plasma von Willebrand factor antigen to ADAMTS13 in the patient is at least about 2.

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