Preparation of intermediate molecular weight heparin

Abstract

The present invention provides for the synthesis of intermediate molecular weight heparin.

Description

[Technical Field]

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

[0002] Heparin is a naturally occurring, highly sulfated polysaccharide characterized by polysaccharide chains with a wide range of molecular weights. Heparin acts on a variety of different ligands with diverse activities. Heparin is a member of the glycosaminoglycan carbohydrate family and consists of repeating disaccharide units, GlcAβ1-4GlcNAcα1-4, with polydisperse sulfation, N-acetylation, and uronosyl epimerization. Heparin is highly heterogeneous. Heparin isolated from natural sources contains polysaccharide chains with molecular weights ranging from approximately 3,000 Da (g / mol) to approximately 30,000 Da (g / mol). This is known as unfractionated heparin (UFH or UF heparin). UFH can be enzymatically or chemically treated to produce shorter polysaccharide chains. Heparinase I cleaves at the α-1,4 bond between the unacetylated GlcNS6S and IdoA2S. Enzymatically or chemically treated UFH products can be affinity purified to obtain fractionated heparins, and the molecular weight of the polysaccharides in each fraction can be easily determined. Low molecular weight heparins (LMWHs) contain polysaccharide chains ranging from about 4,000 Da (g / mol) to about 8,000 Da (g / mol).

[0003] Few methods for preparing intermediate molecular weight heparins are known. One example of a method for preparing intermediate molecular weight heparins 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 wide range of products, ranging in molecular weight from 10,600 g / mol to 1,900 g / mol.

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

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

[0006] In a second aspect, the present invention provides a kit suitable for preparing intermediate molecular weight heparin, the kit comprising: (a) unfractionated heparin; (b) an aqueous buffer adjusted to a pH of about 5.0 to about 9.0; (c) an oxidizing agent; and (d) optionally, a deactivating agent.

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

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

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

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

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

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

[0014] In a tenth aspect, the present invention provides a method for treating vascular endotheliitis, the method comprising administering a therapeutically effective amount of intermediate molecular weight heparin to a subject in need of treatment, preferably when the patient has a plasma VWF:ADAMTS13 ratio of at least about 2.

[0015] In an eleventh aspect, the present invention provides use of the reduced intermediate molecular weight heparin as described above for the manufacture of a medicament for the treatment of vascular endotheliitis in a patient, preferably where the patient has a plasma VWF:ADAMTS13 ratio of at least about 2.

[0016] In a twelfth aspect, the present invention provides use of the reduced intermediate molecular weight heparin as described above for the manufacture of a medicament for the treatment of vascular endotheliitis in a patient, preferably where the patient has a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0017] For the avoidance of doubt, embodiments relating to each aspect of the invention apply mutatis mutandis to other aspects of the invention. Further aspects and embodiments of the invention will become apparent from the discussion that follows. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows MMWH prepared at 4° C. compared to heparin processed at 37° C., unprocessed heparin, and an 11 kDa standard. [Figure 2] FIG. 2 shows MMWH prepared at 4° C. with periodate alone compared to heparin treated with periodate and then NaOH at 4° C. [Figure 3] FIG. 3 shows a graph of the molecular weight distribution of the prepared intermediate molecular weight heparins. [Figure 4] FIG. 4 shows a graph of the activity of low molecular weight (LMW), unfractionated (UF) and medium molecular weight (MMW) heparins against factor IIa. [Figure 5] FIG. 5 shows a graph of the activity of LMW heparin, UF heparin, and MMW heparin against factor X. DETAILED DESCRIPTION OF THE INVENTION

[0019] Throughout this specification, one or more aspects of the present invention may be combined with one or more features described herein to define additional embodiments of the present invention.

[0020] In the discussion that follows, reference will be made to a number of terms, which should be understood to have the meanings set forth below, unless the context expressly indicates to the contrary.

[0021] As used herein, references to the singular form of a noun include the plural form of that noun and vice versa, unless the context indicates otherwise.

[0022] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a specified element or integer or group of elements or integers, but not the exclusion of other elements or integers or groups of elements or integers. The term "comprising" includes within its scope the terms "consisting" or "consisting essentially of."

[0023] The term "consisting" or variations thereof should be understood to imply the inclusion of a specified element or integer, or group of elements or integers, and the exclusion of other elements or integers, or groups of elements or integers.

[0024] The term "consisting essentially of" or variations thereof implies the inclusion of the specified elements, integers, or steps, or group of elements, integers, or steps; it should be understood that additional components may be present, but only those that do not materially affect the essential properties of the formulation, composition, or compound.

[0025] As used herein, the term "about," when specifying a number or value, is used to indicate a value that is within ±5% of the specified value.

[0026] The terms "treatment" and "therapy" define the therapeutic management of a patient to slow or stop the progression of, or to ameliorate or cure, a disorder or condition. Prevention of a disorder or condition as a result of treatment or therapy is also included.

[0027] As used herein, the term "patient" preferably refers to a mammal. Typically, the mammal is a human.

[0028] Von Willebrand factor (VWF) is a blood glycoprotein involved in hemostasis. VWF is a high-molecular-weight multimeric glycoprotein present in plasma and is constitutively produced as ultrahigh-molecular-weight VWF in endothelium (Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a 2050-amino acid protein.

[0029] A disaccharide is a sugar whose molecule contains two monosaccharide residues.

[0030] Low molecular weight heparin is defined herein as heparin having an average molecular weight of about 4000 Da (g / mol) to about 8000 Da (g / mol). Intermediate molecular weight heparin is defined herein as heparin having an average molecular weight of greater than about 8000 Da (g / mol) to about 13000 Da (g / mol).

[0031] Thus, in a first aspect, the present invention provides a method for the synthesis (or preparation) of a medium molecular weight heparin (MMWH).

[0032] The intermediate molecular weight heparin prepared by the method of the first aspect of the present invention may have a mass ranging from greater than about 8,000 Da (g / mol) to about 13,000 Da (g / mol), preferably from about 10,000 Da (g / mol) to about 12,000 Da (g / mol).The intermediate molecular weight heparin prepared by the method of the first aspect of the present invention may have a mass of about 11,000 Da (g / mol).

[0033] The intermediate molecular weight heparin prepared by the method of the first aspect of the present invention may comprise polysaccharide chains having an average molecular mass ranging from about 8,000 Da (g / mol) to about 13,000 Da (g / mol), preferably from about 10,000 Da (g / mol) to about 12,000 Da (g / mol).The intermediate molecular weight heparin prepared by the method of the first aspect of the present invention may comprise polysaccharide chains having an average molecular mass of about 11,000 Da (g / mol).

[0034] The molecular weight of the intermediate molecular weight heparin prepared by the method of the first aspect of the invention may be determined by size exclusion chromatography as described herein.

[0035] The intermediate molecular weight heparin prepared by the method of the first embodiment 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 linkage between GlcNS6S and IdoA2S, i.e., GlcNS6Sα1-4IdoA2S. For example, the intermediate molecular weight heparin prepared by the method of the first embodiment may contain at least four units, preferably at least five units, preferably at least six units, preferably at least eight units, and preferably at least 10 units of the GlcNS6S-IdoA2S disaccharide. The intermediate molecular weight heparin prepared by the method of the first embodiment may contain 25 or fewer units, e.g., 20 or fewer units, of the GlcNS6S-IdoA2S disaccharide. The presence of the GlcNS6S-IdoA2S disaccharide unit may be determined by antibody, mass spectrometry, or infrared light from chemical and enzymatic tests. The GlcNS6S-IdoA2S units may be arranged in order.

[0036] "IdoA" is α-L-iduronic acid. "IdoA2S" is IdoA modified by the addition of an O-sulfate group at carbon position 2 to produce 2-O-sulfo-α-L-iduronic acid. "GlcNS" is 2-deoxy-2-sulfamido-α-glucopyranosyl. "GlcNS6S" is 2-deoxy-2-sulfamido-α-glucopyranosyl-6-sulfate. An α1-4 linkage is an α-glycosidic bond between carbon-1 on one monosaccharide and carbon-4 on a second monosaccharide. A β1-4 linkage is a β-glycosidic bond between carbon-1 on one monosaccharide and carbon-4 on a second monosaccharide.

[0037] The intermediate molecular weight heparin prepared by the method of the first embodiment may contain UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc, where U can be iduronic acid (IdoA) or glucuronic acid (GlcA). The intermediate molecular weight heparin prepared by the method of the first embodiment may contain at least about 60% UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc. The intermediate molecular weight heparin prepared by the method of the first embodiment may contain at least about 45%, preferably at least about 48%, preferably at least about 49%, and preferably at least about 60% UA2S-GlcNS6S. The intermediate molecular weight heparin prepared by the method of the first embodiment may contain no more than about 60%, preferably no more than about 70%, and preferably no more than about 85% UA2S-GlcNS6S. The intermediate 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%, and preferably at least about 10% UA2S-GlcNS. The intermediate molecular weight heparin prepared by the method of the first aspect may contain no more than about 15%, preferably no more than about 20% UA2S-GlcNS. The intermediate 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%, and preferably at least about 10% UA-GlcNAc. The intermediate molecular weight heparin prepared by the method of the first aspect may contain no more than about 15%, preferably no more than about 20% UA-GlcNAc. In some embodiments, the intermediate molecular weight heparin prepared by the method of the first aspect may contain at least 49.2% UA2S-GlcNS6S, 5.4% UA2S-GlcN, and 5.4% UA-GlcNAc. In some embodiments, the intermediate molecular weight heparin prepared by the method of the first aspect may contain at least 82% UA2S-GlcNS6S, 9% UA2S-GlcNS, and 9% UA-GlcNAc. The percentage composition of UA-GlcNAc in the intermediate molecular weight heparin may be enriched compared to unfractionated heparin.

[0038] "UA" is uronic acid, a hexose with a negatively charged carboxylic acid at position 6. Uronic acids can independently be glucuronic acid or iduronic acid. "UA2S" is UA modified by the addition of an O-sulfate group at carbon position 2 to produce 2-O-sulfo-uronic acid. "GlcA" is β-D-glucuronic acid, and "GlcNAc" is 2-deoxy-2-acetamido-α-glucopyranosyl.

[0039] The method of the first embodiment includes the following steps: (a) dissolving unfractionated (UF) heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; and (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution.

[0040] UF heparin may be obtained from bovine or porcine tissue, for example, porcine intestine or bovine lung.

[0041] A "buffer" refers to a chemical that resists a change in pH in a solution when an acid or alkali is added to the solution. Typically, a buffer (or buffer system) contains a weak acid and its conjugate base, or a weak base and its conjugate acid.

[0042] Typically, a suitable buffer contains an acid with a pKa value that is 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 in the buffer has more than one pKa value (i.e., each molecule of the acid can donate more than one proton), at least one of the pKa values ​​should be within the desired pH range for the buffer to be suitable.

[0043] The weak acid and conjugate base (or weak base and conjugate acid) of a buffer solution are in equilibrium with each other. According to Le Châtelier's principle (when a constraint, such as a change in the concentration of a reactant, is applied to a system at equilibrium, the equilibrium will shift to oppose the effect of the constraint), adding an acid or base to the solution shifts the equilibrium position toward the conjugate base or weak acid, respectively. As a result, the concentration of free protons in the formulation (and therefore the pH) remains relatively unchanged.

[0044] Suitable buffer systems include acetate and acetic acid (pKa=4.75), citrate and citric acid (pKa=3.13, 4.76, and 6.40), and phosphate (pKa=2.14, 7.20, and 12.37), or mixtures thereof. Phosphate-buffered saline may also be used. The pKa values ​​quoted herein are reported in water at 25°C. Typically, buffers contain only one of the pairs listed above, i.e., one acid and its conjugate base. Buffers may contain acetate and acetic acid, citrate and citric acid, or phosphate and phosphoric acid.

[0045] Dissolution of unfractionated heparin can result in a pH change in the aqueous buffer, so adjust the pH of the first solution to ensure it is between about pH 5.0 and about pH 9.0.

[0046] If necessary, in step (a), the aqueous buffer solution is adjusted to a pH of about 6.0 to about 8.0, more preferably about 7.0. Typically, 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.

[0047] Typically, the aqueous buffer is a phosphate buffer, a citrate buffer, or an acetate buffer, i.e., the buffer system is a phosphate, a citrate, or an acetate. Preferably, the aqueous buffer is a phosphate buffer, and more preferably, the aqueous buffer is a sodium phosphate buffer or a potassium phosphate buffer, i.e., the buffer system is a sodium phosphate or a potassium phosphate.

[0048] The buffer system in the aqueous buffer 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 may be present at a concentration of about 50 mM.

[0049] 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, or more preferably about 3 mg / mL.

[0050] The oxidizing agent may be a periodate, such as sodium periodate or potassium periodate. Preferably, the oxidizing agent is sodium periodate. Alternatively, or in addition, the oxidizing agent may be a perchlorate, such as sodium perchlorate. Preferably, the oxidizing agent does not contain perchlorate. The use of perchloric acid in combination with perchlorate as an oxidizing agent results in an increased level of sample decomposition into smaller molecular weight species.

[0051] 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, and more preferably about 5 g / L to about 7 g / L. Preferably, the concentration of the oxidizing agent may be about 5.7 g / L. Preferably, the concentration of the oxidizing agent is about 5.7 g / L, and the oxidizing agent is sodium periodate.

[0052] The molar ratio of UF heparin to oxidizing agent can be about 1:1 to about 1:200, more preferably about 1:2 to 1:150, more preferably about 1:10 to 1:100, more preferably about 1:20 to 1:50, more preferably about 1:30 to 1:40. Typically, the molar ratio of UF heparin to oxidizing agent can be about 1:40.

[0053] Typically, the incubation temperature in step (c) is about 0°C to about 10°C, more preferably about 1°C to about 9°C, more preferably about 2°C to about 8°C, more preferably about 3°C ​​to about 7°C, and more preferably about 4°C to about 6°C. Preferably, the incubation temperature in step (c) is about 4°C. Typically, the incubation step (c) is carried out for about 1 hour to about 48 hours, more preferably about 4 hours to about 36 hours, more preferably about 8 hours to about 30 hours, more preferably about 12 hours to about 24 hours, more preferably about 15 hours to about 20 hours, and more preferably about 16 hours to about 18 hours. If necessary, step (c) may be carried out at about 20°C to about 25°C.

[0054] The incubation step (c) of the first aspect of the present invention may be carried out in a laboratory refrigerator set at the required temperature. The laboratory refrigerator may be set at a temperature of about 0°C to about 10°C, more preferably about 2°C to about 8°C, more preferably about 3°C ​​to about 7°C, more preferably about 4°C to about 6°C. Preferably, the laboratory refrigerator may be set at a temperature of about 4°C.

[0055] The method of the first aspect may further include step (d), which is a step of inactivating the oxidizing agent in the medium molecular weight heparin solution. The oxidizing agent may 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.

[0056] The molar ratio of oxidizing agent to deactivating agent may be from about 1:1 to about 1:10, more preferably from 1:2 to about 1:8, more preferably from 1:3 to about 1:6, more preferably from 1:4 to about 1:5. Typically, the molar ratio of oxidizing agent to deactivating agent may be about 1:2 or about 1:4.

[0057] The method of the first aspect may further comprise step (e), which is dialyzing the intermediate molecular weight heparin solution in the dialysate to obtain a dialyzed intermediate molecular weight heparin sample.

[0058] "Dialysis" or "dialysis" refers to a method of separating molecules in a solution by differences in their diffusion rates through a semipermeable membrane, e.g., dialysis tubing. A sample to be dialyzed and a dialysate (or buffer solution) are placed on opposite sides of the semipermeable membrane. Target sample molecules (e.g., proteins, DNA, or polysaccharides) larger than the membrane pores remain on the sample side of the membrane. Impurities such as small molecules and salts can pass through the membrane into the dialysate, thereby reducing the impurity concentration in the sample to a low level. The dialysate is replaced with fresh dialyzed solution to remove impurities that have passed from the sample into the dialysate. This allows more impurities to diffuse from the sample into the dialysate.

[0059] 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 by volume. Semipermeable membranes are typically made from thin films of regenerated cellulose or cellulose esters.

[0060] Dialysis may be performed by placing dialysis tubing containing the sample in dialysate. "Dialysate" is the liquid through which substances pass through the dialysis tubing. The dialysate may be refreshed as frequently as necessary to achieve optimal separation. Dialysis may be performed over a period of about 1 day to about 14 days, preferably about 5 days to about 10 days, and preferably about 7 days. The dialysate may be refreshed about once a day to about 10 times a day, preferably about twice a day to about 5 times a day, and preferably about three times a day. Typically, the dialysate is several times the volume of the sample, for example, about 2 to about 500 times the volume of the sample. The dialysate may be about 4 times the volume of the sample.

[0061] Typically, in step (e), the dialysate is water. Optionally, the dialysate may contain electrolytes such as sodium, potassium, magnesium, calcium, chloride, bicarbonate, lactate, glucose, amino acids, or combinations thereof.

[0062] The dialysis step (e) may be carried out, for example, with 2 kD cut-off tubing provided by Spectra / Por®. Those skilled in the art will know appropriate tubing cut-off sizes for different purposes. Alternatively, the dialysis step (e) may be carried out with a dialysis machine or dialyzer. Suitable dialyzers may be the Slide-A-Lyzer™, the Float-A-Lyzer, the Pur-A-lyzer, the D-Tube, and GeBAflex Dialyzers product lines.

[0063] The method of the first aspect may further comprise step (f), which is the step of isolating the intermediate molecular weight heparin from the dialyzed heparin sample. The intermediate molecular weight heparin may be isolated from the dialyzed heparin sample by lyophilization, centrifugation, or filtration. Preferably, the intermediate molecular weight heparin is isolated from the dialyzed heparin sample by lyophilization.

[0064] "Freeze drying" (also known as lyophilisation or cryodesiccation) is a drying method carried out at low temperatures. Freeze drying generally involves lowering the temperature and pressure below the triple point of the substance to remove the frozen solvent (e.g., water ice) by sublimation. For aqueous compositions such as those disclosed herein, freeze drying may be carried out at temperatures of about -20°C to about -80°C, preferably about -40°C, and at pressures of about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).

[0065] The MMWH may be purified by any suitable method known to those skilled in the art. Thus, the method may further comprise step (g), which is a step of purifying the intermediate molecular weight heparin. For example, the MMWH may be purified by exhaustive dialysis using phosphate buffer (pH=7.0) or saline, a desalting column (e.g., Sephadex G-25 using phosphate buffer (pH=7.0) or saline as the mobile phase), or precipitation of the MMWH.

[0066] The method may include an alkaline elimination step. Alternatively, the method may not include an alkaline elimination step. The alkaline elimination may be carried out using an alkaline salt such as sodium hydroxide, potassium hydroxide, or lithium hydroxide. The alkaline elimination step may be carried out by adding the alkaline salt to increase the pH of the medium molecular weight heparin solution to about pH 10 to about pH 14, preferably about pH 12, at about room temperature for about 10 minutes to about 3 hours, preferably about 30 minutes.

[0067] Preferably, the method does not include an alkaline elimination step. Preferably, the method does not include the addition of an alkali metal salt, such as NaOH, KOH, or LiOH. Preferably, the method does not include the addition of NaOH, KOH, or LiOH. Surprisingly, methods that do not include an alkaline elimination step or the addition of an alkali metal salt produce intermediate molecular weight heparins as defined herein that exhibit significantly lower activity against factors IIa and / or Xa compared to UF heparin and low molecular weight heparins.

[0068] MMWHs may be characterized by NMR, disaccharide analysis, ristocetin-induced platelet aggregation (RIPA), and factor X analysis.

[0069] This method is suitable for preparing intermediate molecular weight heparin on the milligram, gram, or kilogram scale.

[0070] Advantageously, the method of the first aspect reliably provides intermediate molecular weight heparin with excellent purity and reduced degradation.

[0071] The method may include a further step (h) comprising incubating the intermediate molecular weight heparin with a reducing agent to obtain reduced intermediate molecular weight heparin (MMMWH-Red). Preferably, the reducing agent is a mild reducing agent. The reducing agent may be sodium borohydride (NaBH), sodium cyanoborohydride (NaBHCN), sodium triacetylborohydride (NaBH(OAc)), or potassium borohydride (KBH).

[0072] 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. Typically, 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.

[0073] The solvent in step (h) can usually be selected from the group consisting of methanol, ethanol, water, THF, and combinations thereof. Preferably, the solvent in step (h) is water.

[0074] The method for synthesizing intermediate molecular weight heparin can consist of, or consist essentially of, the steps of: (a) dissolving unfractionated heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; and (d) purifying the intermediate molecular weight heparin.

[0075] The method for synthesizing intermediate molecular weight heparin can consist of or essentially of: (a) dissolving unfractionated heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; (d) inactivating the oxidizing agent in the intermediate molecular weight heparin solution; and (e) purifying the intermediate molecular weight heparin.

[0076] The method for synthesizing intermediate molecular weight heparin can consist of, or consist essentially of, the steps of: (a) dissolving unfractionated heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; (d) inactivating the oxidizing agent in the intermediate molecular weight heparin solution; and (e) dialyzing the intermediate molecular weight heparin solution in a dialysate to obtain a dialyzed intermediate molecular weight heparin sample.

[0077] The method for synthesizing intermediate molecular weight heparin may consist of, or may consist essentially of, the steps of: (a) dissolving unfractionated heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; (d) inactivating the oxidizing agent in the intermediate molecular weight heparin solution; (e) dialyzing the intermediate molecular weight heparin solution in a dialysate to obtain a dialyzed intermediate molecular weight heparin sample; and (f) isolating the intermediate molecular weight heparin from the dialyzed heparin sample.

[0078] In a second aspect, the present invention provides a kit suitable for preparing intermediate molecular weight heparin, the kit comprising: (a) unfractionated heparin; (b) an aqueous buffer adjusted to a pH of about 5.0 to about 9.0; (c) an oxidizing agent; and (d) optionally, a deactivating agent.

[0079] The MMWH produced by the method of the first aspect of the invention contains two aldehyde groups on the glucuronic acid, and in some cases, the activity of the aldehyde groups can result in their reaction with amine residues on proteins in the body via a Schiff base reaction.

[0080] Thus, in a third aspect, the present invention provides a method for preparing reduced intermediate molecular weight heparin (MMWH-Red). MMWH-Red contains no aldehyde groups or contains fewer aldehyde groups than MMWH. The method of the third aspect includes the following steps: (a) dissolving unfractionated (UF) heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; and (d) incubating the intermediate molecular weight heparin with a reducing agent to obtain reduced intermediate molecular weight heparin (MMWH-Red).

[0081] The reduced intermediate molecular weight heparin prepared by the method of the third aspect of the present invention may comprise polysaccharide chains having an average molecular mass ranging from about 9,000 Da (g / mol) to about 13,000 Da (g / mol), preferably from about 10,000 Da (g / mol) to about 12,000 Da (g / mol).The reduced intermediate molecular weight heparin prepared by the method of the third aspect of the present invention may comprise polysaccharide chains having an average molecular mass of about 11,000 Da (g / mol).

[0082] The molecular weight of MMWH-Red prepared by the method of the third aspect of the invention may be determined by size exclusion chromatography as described herein.

[0083] MMWH-Red prepared by the method of the third embodiment 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 linkage between GlcNS6S and IdoA2S, i.e., GlcNS6Sα1-4IdoA2S. For example, MMWH-Red prepared by the method of the third embodiment may contain at least four units, preferably at least five units, preferably at least six units, preferably at least eight units, and preferably at least 10 units of the GlcNS6S-IdoA2S disaccharide. MMWH-Red prepared by the method of the third embodiment may contain 25 or fewer units, e.g., 20 or fewer units, of the GlcNS6S-IdoA2S disaccharide. The presence of the GlcNS6S-IdoA2S disaccharide unit may be determined by infrared light from antibodies, mass spectrometry, or chemical and enzymatic assays. The GlcNS6S-IdoA2S units may be arranged in order.

[0084] MMWH-Red prepared by the method of the first embodiment may contain UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc, where U can be iduronic acid (IdoA) or glucuronic acid (GlcA). Medium-molecular-weight heparin prepared by the method of the third embodiment may contain at least about 60% UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc. MMWH-Red prepared by the method of the third embodiment may contain at least about 45%, preferably at least about 48%, preferably at least about 49%, and preferably at least about 60% UA2S-GlcNS6S. MMWH-Red prepared by the method of the third embodiment may contain no more than about 60%, preferably no more than about 70%, and preferably no more than about 85% UA2S-GlcNS6S. 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%, and preferably at least about 10% UA2S-GlcNS. MMWH-Red prepared by the method of the third aspect may contain no more than about 15%, preferably no more than about 20% UA2S-GlcNS. 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%, and preferably at least about 10% UA-GlcNAc. MMWH-Red prepared by the method of the third aspect may contain no more than about 15%, preferably no more than about 20% UA-GlcNAc. In some embodiments, MMWH-Red prepared by the method of the third aspect may contain at least 49.2% UA2S-GlcNS6S, 5.4% UA2S-GlcN, and 5.4% UA-GlcNAc. In some embodiments, MMWH-Red prepared by the method of the third aspect may contain at least 82% UA2S-GlcNS6S, 9% UA2S-GlcNS, and 9% UA-GlcNAc. The percentage composition of UA-GlcNAc in MMWH-Red may be enriched compared to unfractionated heparin.

[0085] The reducing agent can be sodium borohydride (NaBH4), sodium cyanoborohydride, sodium triacetylborohydride, or potassium borohydride (KBH4). Preferably, the oxidizing agent is sodium borohydride.

[0086] 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 intermediate 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 intermediate molecular weight heparin. The reducing agent may be used in an amount 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.

[0087] Typically, step (d) is carried out at about 0°C to about 30°C, or about 5°C to about 25°C, or 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. The solvent in step (d) can usually be selected from the group consisting of methanol, ethanol, water, THF, dichloroethane, and combinations thereof. Preferably, the solvent in step (d) is water.

[0088] The method of the first aspect may further comprise the step of inactivating the oxidizing agent in the MMWH-Red solution. The oxidizing agent may be inactivated by the addition of a deactivating agent selected from the group consisting of D-mannitol, glycerol, N-acetylmethionine, sodium sulfite, and combinations thereof. A particularly preferred deactivating agent is D-mannitol.

[0089] The molar ratio of oxidizing agent to deactivating agent may be from about 1:1 to about 1:10, more preferably from 1:2 to about 1:8, more preferably from 1:3 to about 1:6, more preferably from 1:4 to about 1:5. Typically, the molar ratio of oxidizing agent to deactivating agent may be about 1:2 or about 1:4.

[0090] MMWH-Red may be purified by any suitable method known to those skilled in the art. Thus, the method may further include a step of purifying the intermediate molecular weight heparin. For example, MMWH-Red may be purified by full dialysis using phosphate buffer (pH=7.0) or saline, a desalting column (e.g., Sephadex G-25 using phosphate buffer (pH=7.0) or saline as the mobile phase), or precipitation of MMWH-Red.

[0091] For example, the method may further include dialyzing the intermediate molecular weight heparin solution in a dialysate to obtain a dialyzed intermediate molecular weight heparin sample prior to the reduction step.

[0092] This method is suitable for preparing reduced intermediate molecular weight heparin on the milligram, gram, or kilogram scale.

[0093] Advantageously, the method of the third aspect provides reliably reduced intermediate molecular weight heparin with excellent purity and minimal degradation.

[0094] The method for synthesizing reduced intermediate molecular weight heparin can consist of, or consist essentially of, the steps of: (a) dissolving unfractionated (UF) heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; (d) incubating the intermediate molecular weight heparin with a reducing agent to obtain reduced intermediate molecular weight heparin (MMWH-Red); and (e) purifying the MMWH-Red.

[0095] The method for synthesizing reduced intermediate molecular weight heparin can consist of, or essentially consist of, the steps of: (a) dissolving unfractionated heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; (d) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; (e) incubating the intermediate molecular weight heparin with a reducing agent to obtain reduced intermediate molecular weight heparin (MMWH-Red); and (f) purifying the MMWH-Red.

[0096] The method for synthesizing reduced intermediate molecular weight heparin can consist of, or consist essentially of, the steps of: (a) dissolving unfractionated heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; (d) inactivating the oxidizing agent in the intermediate molecular weight heparin solution; (e) dialyzing the intermediate molecular weight heparin solution in a dialysate to obtain a dialyzed intermediate molecular weight heparin sample; (f) incubating the dialyzed intermediate molecular weight heparin with a reducing agent to obtain reduced intermediate molecular weight heparin (MMWH-Red); and (g) purifying the MMWH-Red.

[0097] The method for synthesizing reduced intermediate molecular weight heparin may consist of, or consist essentially of, the steps of: (a) dissolving unfractionated heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; (d) inactivating the oxidizing agent in the intermediate molecular weight heparin solution; (e) dialyzing the intermediate molecular weight heparin solution in a dialysate to obtain a dialyzed intermediate molecular weight heparin sample; (f) isolating the intermediate molecular weight heparin from the dialyzed heparin sample; (g) incubating the intermediate molecular weight heparin with a reducing agent to obtain reduced intermediate molecular weight heparin (MMWH-Red); and (h) purifying the MMWH-Red.

[0098] Preferably, the reducing agent is a mild reducing agent. The mild reducing agent can selectively reduce aldehydes and ketones to alcohols in the presence of esters. In other words, the mild reducing agent reduces aldehydes and ketones to alcohols at a faster rate than it reduces esters to alcohols. The mild reducing agent does not reduce carboxylic acids, nitriles, and amides under normal conditions. Preferably, the mild reducing agent can be sodium borohydride (NaBH4).

[0099] Preferably, the method does not include an alkaline elimination step. Preferably, the method does not include the addition of an alkali metal salt, such as NaOH, KOH, or LiOH. Preferably, the method does not include the addition of NaOH, KOH, or LiOH. Surprisingly, methods that do not include an alkaline elimination step or the addition of an alkali metal salt produce MMWH-Red as defined herein that exhibits significantly lower activity against factor IIa and / or Xa compared to UF heparin and low molecular weight heparins.

[0100] For the avoidance of doubt, embodiments relating to the first aspect of the invention apply mutatis mutandis to the third aspect of the invention.

[0101] Both MMWH and MMWH-Red inhibit von Willebrand factor and are therefore suitable active pharmaceutical ingredients (APIs) and pharmaceuticals for the purposes of the present invention.

[0102] Advantageously, MMWH-Red may exhibit greater stability as an API and as a pharmaceutical product compared to MMWH. Furthermore, MMWH-Red may reduce the potential for side reactions with excipients in pharmaceutical products, and MMWH-Red may reduce interactions with proteins in vivo.

[0103] In a fourth aspect, the present invention provides reduced intermediate molecular weight heparin (MMWH-Red). The MMWH-Red may be prepared according to the first or third aspect of the present invention.

[0104] MMWH-Red contains no aldehydes or contains fewer aldehydes than the MMWH produced according to the first embodiment. The presence or absence of aldehydes may be determined using the 2,4-dinitrophenylhydrazine test. MMWH-Red may not produce an orange-yellow precipitate upon reaction with 2,4-dinitrophenylhydrazine, or may produce less orange-yellow precipitate than the corresponding amount of starting material. Alternatively, the reduction of MMWH to MMWH-Red may be monitored by infrared spectroscopy.

[0105] MMWH-Red may be characterized by NMR, disaccharide analysis, ristocetin-induced platelet aggregation (RIPA), and factor X analysis.

[0106] In a fifth aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: (a) dissolving unfractionated (UF) heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; (b) adding an oxidizing agent to the first solution to obtain a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; (d) incubating the intermediate molecular weight heparin with a reducing agent to obtain reduced intermediate molecular weight heparin; The present invention provides MMWH-Red manufactured by

[0107] For the avoidance of doubt, embodiments relating to the first and third aspects of the invention apply mutatis mutandis to the fifth aspect of the invention.

[0108] In a sixth aspect, the present invention provides a composition comprising MMWH-Red, preferably a pharmaceutical composition.

[0109] For the avoidance of doubt, embodiments relating to the first and third aspects of the invention apply mutatis mutandis to the sixth aspect of the invention.

[0110] As described herein, vascular endotheliitis can be associated with several diseases. MMWH-Red inhibits von Willebrand factor. The present inventors have discovered that MMWH-Red can be used to treat vascular endotheliitis, particularly in patients with elevated plasma von Willebrand factor levels.

[0111] Although vascular endotheliitis can be caused by numerous diseases and / or conditions described herein, vascular endotheliitis associated with COVID-19 or SARS-CoV-2 will be primarily discussed herein. Those skilled in the art will appreciate that this discussion is merely for the purpose of providing examples and should not be construed as limiting the invention.

[0112] In late 2019, a novel betacoronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) was identified in Wuhan, China, as the cause of coronavirus disease 2019 (COVID-19). The rapid geographic progression of COVID-19 subsequently reached a peak, and the WHO declared it a pandemic in March 2020 (1). Clinical symptoms in those infected with SARS-CoV-2 range from asymptomatic cases to more severe pneumonia, which can lead to acute respiratory distress syndrome (ARDS) and multiple organ failure. Most symptomatic patients experience mild to moderate illness and most commonly do not require hospitalization (2-4). However, there is a cohort of patients who can progress to more severe disease. Symptom progression / clinical symptoms can manifest up to 2 weeks, beginning with an early prodromal phase and culminating in ARDS (3). The subgroup of patients who become critically ill and require ventilation or extracorporeal membrane oxygenation (ECMO) is now known to have a very poor outcome, with a high mortality rate approaching 90% (5).

[0113] Since its initial description, this disease has affected over 90 million individuals worldwide. The pathophysiological pathway remains unclear; therefore, management is symptomatic. Disease-modifying therapeutics that could be used while awaiting specific antiviral drugs or a vaccine are lacking. Several lines of evidence point toward endothelial dysfunction as a key pathophysiological mechanism in COVID-19. Prior to the current pandemic, markers of endothelial dysfunction were found to be associated with disease severity and mortality in patients with sepsis (6-9). In a postmortem examination of three COVID-19 patients, Varga et al. (10) demonstrated widespread endothelial injury affecting pulmonary, renal, gastrointestinal, and hepatic vessels. In one case, the authors reported that "most small blood vessels appeared congested," and in another, the patient died from intestinal ischemia with evidence of underlying endothelial injury.

[0114] Recently, two proposed hemostatic mechanisms have provided insights into an improved understanding of ARDS based on the molecular pathogenesis associated with vascular endotheliitis, which promotes inflammation and coagulation disorders in sepsis and other critical illnesses (11-14): One is the "two-path endothelial activation theory," which proposes that endothelial pathogenesis activates inflammatory pathways and microthrombosis. The other is the novel "two-path integrated theory" of surgical interventions that stop blood flow, which initiate thrombus formation and promote microthrombosis, leading to vascular microthrombosis (VMTD) (11, 13, 15). These two theories are consistent with each other because the endothelium contributes to the early stages of hemostasis and triggers the molecular mechanisms of thrombus formation. ARDS is often associated with sepsis from a variety of different causes and is seen in severe acute respiratory syndrome (SARS) caused by SARS-CoV (16), Middle East respiratory syndrome (MERS) caused by MERS-CoV (17), and the current COVID-19 outbreak. Sepsis-associated ARDS often occurs with other organ dysfunction, such as encephalopathy (18), liver failure (19)(20), acute renal failure, and acute necrotizing pancreatitis (21). This involvement of other organs suggests that ARDS may not be the primary disease but is part of an ongoing systemic pathogenesis caused by an infection or another serious illness.

[0115] Based on this, the physiological changes that contribute to multiple organ failure in sepsis and other critical illnesses are the same as the circulatory dysfunction that occurs as a result of vascular endotheliitis-associated VMTD (EA-VMTD) (14, 15). Therefore, infections cause endothelial injury, which leads to vascular endotheliitis. This then leads to disseminated microthrombosis (DIMT), which can cause, for example, local hypoxemia, systemic hypoxia, and / or ischemia. As previously mentioned, COVID-19 is now known to be associated with endothelial injury (10).

[0116] A case series of COVID-19 lung autopsies revealed the presence of numerous localized platelet-rich microthrombi and hemorrhagic foci in the lungs, paralleling diffuse alveolar damage (22). The authors hypothesized localized thrombotic microangiopathy in the lungs as key to the pathogenesis of COVID-19, and others have suggested that microthrombosis is an important driving force in the disease process (23). These microcirculatory changes were clearly demonstrated in the lungs, kidneys, and liver using contrast-enhanced ultrasound (24, 25). Similar findings were also seen in the brain (26). Thus, there is growing evidence that COVID-19 appears to cause endothelial injury and diffuse and widespread microthrombosis.

[0117] Hypercoagulability and COVID-19 are now widely accepted, and studies have demonstrated an increased odds ratio for in-hospital mortality and abnormal D-dimer levels, with higher levels associated with more severe disease (27-30). Several case reports have noted acute pulmonary thrombosis in patients with COVID-19 pneumonia in the absence of major predisposing factors for venous thromboembolism (27, 31, 32). More recently, Panigada et al. demonstrated that in addition to elevated D-dimer levels, there was a significant increase in factor VIII and von Willebrand factor (VWF) levels (33). A greater than 500% increase in VWF levels and a greater than 350% increase in factor VIII levels were reported by Escher et al. (34) in the setting of COVID-19. Furthermore, it has been demonstrated that thrombocytopenic patients have a greater than fivefold 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 predictors of severe illness and mortality.

[0118] Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a key role in hemostasis and thrombosis through platelet adhesion to injured and activated blood vessels. VWF is synthesized exclusively in megakaryocytes and endothelial cells (ECs), and interestingly, SARS-CoV can directly affect both of these cell types (22, 36).

[0119] The overwhelming majority of VWF found in plasma is derived from VWF synthesized within ECs and stored within Weibel-Palade bodies (WPBs). Although restricted to ECs, there are differences in VWF synthesis within different vascular beds of the body, with small blood vessels in the lung and brain expressing higher levels of VWF than similarly sized vessels in the liver or kidney, and higher levels in venous rather than arterial ECs (37). The majority of VWF stored in the WPBs of endothelial cells consists of ultrahigh molecular weight VWF (ULVWF). These ultrahigh molecular weight VWF multimers are more adhesive than the lower molecular weight VWF multimers in the circulation (38). Once secreted, ULVWF can spontaneously bind to platelets. Inflammatory cytokines such as interleukin-1 and tumor necrosis factor (TNF)-α can trigger exocytosis of WPBs, releasing their contents. Thus, plasma levels of VWF can be used as a marker of endothelial activation and vascular inflammation, and elevated levels of VWF have been found to be associated with ARDS and sepsis and independently correlated with mortality ( 39 , 40 ).

[0120] Upon secretion from EC, secreted VWF, which partially enters the circulation and partially binds to the endothelium, is sensitive to shear stress. This shear stress unfolds VWF, exposing sites for platelet binding, self-association, and cleavage by the enzyme ADAMTS13. These VWF molecules can self-assemble into long "strings" that bind platelets in both arterial and venous flow directions and have previously been shown to be adhesive to the endothelium (41-43). The protease ADAMTS13 cleaves VWF and ULVWF, and its perfusion over these platelet-VWF strings rapidly removes them from the circulation (41). ULVWF multimers released from WPBs have lower shear stress for unfolding and may therefore be the initiating molecules for this self-assembly process, resulting in highly adhesive strings that trap platelets. Platelet-VWF binding occurs via the GP Ib receptor. The binding site for this receptor is not normally exposed when VWF is in its globular form and therefore cannot bind platelets. Once VWF is spread, following shear stress, its binding sites become exposed and bind platelets with high affinity. Platelet-VWF binding induces a conformational change that leads to activation of the integrin GPIIbIIIa (also known as α2bβ3), potentially facilitating not only platelet-VWF binding but also platelet-platelet binding. For this reason, the use of standard antiplatelet drugs is likely to be ineffective (aspirin or P2Y12 inhibitors) or only partially effective in mitigating this pathological process, as suggested by the cohort study of Tremblay et al. (44).

[0121] This ability to form VWF-platelet-rich thrombi in the microvasculature is a hallmark of acquired thrombotic thrombocytopenic purpura (TTP), in which autoantibodies against ADAMTS13 are present. Interleukin-6 (IL-6) has also been shown to inhibit the cleavage of VWF-platelet strings (45). Furthermore, ADAMTS13 synthesis, at least in cultured cells, is dramatically inhibited by various cytokines, including IL-6 and TNF-α (46). This suggests that cytokine storms, and IL-6 in particular, may propagate microthrombosis. However, this also suggests that if intervention is performed early and without a spike in cytokine release, the disease may be more manageable and a rapid deterioration in the patient's clinical condition may be avoided.

[0122] There is now a significant body of evidence suggesting a significant imbalance in the VWF:ADAMTS13 ratio and levels of high-molecular-weight VWF multimers (equivalent to ULVWF) in COVID-19. As previously mentioned, very high levels of VWF were previously demonstrated, with the earliest case report noting this sudden increase in VWF levels being that of Escher et al. (34). Subsequently, Goshua et al. (47) documented a reported significant increase in plasma VWF concentrations in patients admitted to COVID-19, with increased levels associated with disease severity—mean VWF antigen levels of 565 ± 199% vs. 278 ± 133% (p < 0.0001) for patients admitted to the intensive care unit (ICU) compared with patients not treated there. Next, Rauch et al. (48) looked at the progression of COVID-19 patients in relation to these admission VWF levels. Patients with the highest VWF levels required more extensive oxygen support, while patients with normal VWF levels required neither hospitalization nor supplemental oxygen (n=10).

[0123] Shortly after Rauch et al.'s publication, Ladikou et al. (49) demonstrated increased VWF antigen levels in COVID-19 patients admitted to the ICU, with a positive correlation observed between VWF levels and patient age. They reported a median VWF antigen level of 350%; however, importantly, they also demonstrated significantly reduced ADAMTS13 levels (49.7%), suggesting a loss of VWF-cleaving proteases that normally degrade high-molecular-weight VWF multimers and reduce their activity. They speculated that the excessive release of VWF seen in COVID-19 patients could lead to ADAMTS13 depletion, contributing to the prothrombotic state. Furthermore, analysis of these data showed that median VWF levels were significantly higher (p = 0.015) in patients who died (477%) compared with those who survived (335%).

[0124] Helms et al. (50) recently published a multicenter, prospective cohort study in France evaluating the thrombosis risk of COVID-19 patients, which showed significant increases in VWF and factor VIII. Along with this data showing increased VWF and decreased ADAMTS13, further studies have demonstrated a significant derangement in the VWF:ADAMTS13 ratio. Huisman et al. (51) first demonstrated a mean VWF:ADAMTS13 ratio of 8.5 (normal, 0.5–2) in 12 patients admitted to the ICU. Subsequently, Mancini et al. (52) demonstrated similar findings, with elevated von Willebrand factor antigen (VWF:Ag) to ADAMTS13 activity ratios strongly associated with disease severity, with the worst ratio observed in patients requiring high-intensity care (intubation and mechanical ventilation) being 8.3 compared with 3.42 in patients requiring low-intensity care (p<0.001).

[0125] More recently, Philippe et al. (53) published their results from a cohort of 208 patients admitted to two centers in Paris, 23 of whom had mild symptoms and were treated as outpatients. They found that VWF:Ag levels scaled only with clinical severity, with levels being significantly higher in severely ill patients (median 507%, IQR 428-596) compared with non-severely ill patients (288%, 230-350, p<0.0001) or COVID-19 outpatients (144%, 133-198, p=0.007). In univariate analyses, 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 this remained highly significant in multivariate analyses adjusted for age, BMI, D-dimer levels, and C-reactive protein (CRP) (odds ratio, OR 25.6, 95% CI 5.6-198.2, p<0.001). More importantly, they showed that VWF high-molecular-weight multimers (HMWM) were significantly higher in severely ill patients (median ratio 1.18, IQR 0.86-1.09) compared with non-severely ill patients (0.96, IQR 1.04-1.39, p<0.001). Furthermore, HMWM level (ratio) (OR 116, 95% CI 10.2-1943, p<0.001) was one of the most significantly associated with hospital mortality.

[0126] A unifying theory is possible, in which vascular endothelial inflammation and endothelial injury trigger the release of VWF and ULVWF, leading to microthrombus formation. This, in turn, leads to hypoxia, which can intensify the process with a "cytokine storm" and the release of IL-6, which inhibits and reduces ADAMTS13 function, resulting in disseminated microthrombi and multiple organ dysfunction and failure. Microvascular thrombosis at the pulmonary level has also been suggested to be the cause of right ventricular dysfunction (54). This mechanism may be the primary cause of many of the currently observed findings, including high D-dimer levels (high because of the large amount of microthrombi), high levels of factor VIII and VWF (released from WPB in response to endothelial injury), and the atypical ARDS picture observed (55), as well as the widespread clinical picture of microthrombi and pulmonary, neurological, and gastrointestinal symptoms. Our suggested endothelial injury and microthrombosis may also explain why patients with pre-existing vascular endotheliitis and microarteriopathy (e.g., secondary to diabetes, hypertension, or obesity) are at increased risk for severe COVID-19 (29, 56). Similarly, rapidly growing evidence links low levels of ADAMTS13 and high levels of VWF with various diseases that predispose patients to poor outcomes and variable symptoms after SARS-CoV-2 infection (57-64). The use of standard antiplatelet drug therapy (aspirin or P2Y12 inhibitors) is also likely to be effective, assuming that VWF interaction with platelets activates the GP2b3a receptor. Inhibition of VWF-platelet binding via the GP1b receptor using either caplacizumab or amfibatide is an attractive option and has been proposed (65), but these drugs are not widely used and clinical experience with them is very limited. Similarly, they have a significant bleeding profile.

[0127] Therefore, there is a need for a treatment for vascular endotheliitis itself.

[0128] Heparin is a naturally occurring, highly sulfated polysaccharide characterized by polysaccharide chains with a wide range of molecular weights. Heparin acts on a variety of different ligands with diverse activities. 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 uronosyl epimerization. Heparin is highly heterogeneous. Heparin may be isolated from natural sources such as porcine intestine or bovine lung. Heparin isolated from natural sources contains polysaccharide chains with molecular weights ranging from approximately 3,000 Da to approximately 30,000 Da. This is known as unfractionated heparin (UFH). UFH can be enzymatically or chemically treated to produce shorter polysaccharide chains. Heparinase I cleaves at the α-1,4 bond between the unacetylated GlcNS6S and IdoA2S. Periodate treatment of UFH followed by alkaline cleavage cleaves the polysaccharide chains at unsulfated uronic acid units. The enzymatically or chemically treated UFH products can be affinity purified to obtain fractionated heparins, and the molecular weight of the polysaccharides in each fraction can be easily determined. Low molecular weight heparins (LMWHs) contain polysaccharide chains ranging from approximately 4,000 Da to approximately 8,000 Da.

[0129] In 1991, intravenous administration of heparin to patients undergoing open-heart surgery was first demonstrated to induce VWF-dependent platelet dysfunction without altering plasma VWF levels (66). This inhibitory effect of heparin on VWF-dependent platelet aggregation was independent of heparin's affinity for antithrombin III but depended on its molecular weight. Recent in vitro studies have shown that heparin binds to a specific amino acid sequence (residues 569–583) within the A1 domain of VWF, which contains a regular sequence of basic amino acids. Heparin binding induced a conformational change in the peptide at this binding site (67). Heparin bound equally to both activated and inactivated VWF but did not interfere with VWF binding to collagen. Because the platelet GpIb binding domain (residues 524–542) is also located in the A1 domain, it was suggested that heparin interferes with VWF binding to platelet GpIb both by steric hindrance and by inducing a conformational change in the domain that results in inhibition of platelet binding.

[0130] The structural specificity of heparin that contributes to VWF binding revolves around the key disaccharide units, GlcNS6S-IdoA2S and IdoA2S-GlcNS6S. This structural unit, which is normally degraded by heparinase I digestion, was successfully estimated by competitive binding assays using heparin fractions prepared by a specific method of heparin depolymerization that produces fragments of the predicted structure (68). Furthermore, tests with synthetic and structurally defined oligosaccharides demonstrated that assemblies of more than three disaccharide units are important for binding capacity. Similarly, low-molecular-weight (6100 Da (g / mol)) fractionated heparins showed higher affinity for VWF binding, but they were less able to inhibit VWF activity compared to UFH. This suggests that the minimum heparin molecular weight and molecular size are important for achieving steric hindrance.

[0131] Intermediate-molecular-weight heparins with a specific disaccharide unit (GlcNS6S-IdoA2S, also known as IdoA2S-GlcNS6S) can be produced from unfractionated heparin. These intermediate-molecular-weight heparins have specificity for inhibiting VWF-GPIb binding and therefore may halt microthrombosis, but because they have little effect on antithrombin III, they have little anticoagulant effect. Therefore, intermediate-molecular-weight heparins with a mass of approximately 11,000 Da (g / mol) may be an ideal therapeutic option when considering the treatment of patients with prothrombotic conditions dependent on elevated VWF levels and vascular endotheliitis. Furthermore, the results of these previous studies suggest that low-molecular-weight heparins are unlikely to work well, and while UFH may contain a sugar moiety capable of binding VWF, targeting the GPIb receptor is not optimal. Furthermore, monitoring UFH is difficult and other fractions of UFH, such as LMWH fractions, have anticoagulant effects that can lead to unpredictable and dangerous bleeding events.

[0132] Even more intriguing is the recent finding that SARS-CoV-2 binds to heparin sulfate, specifically requiring the IdoA2S-GlcNS6S sugar moiety (74, 75). This suggests that exogenous supply of these sugar moieties may inhibit endogenous heparin sulfate binding in the lung and thus act as a preventative treatment. Taken together, specialized intermediate-molecular-weight heparins (approximately 11,000 Da (g / mol) containing at least three GlcNS6S-IdoA2S disaccharide units inhibit viral attachment and replication, but may also inhibit microthrombosis caused by VWF release secondary to virus-induced vascular endotheliitis.

[0133] Heparin is resistant to certain oxidizing agents (e.g., IO 4-When exposed to oxidative stress (e.g., periodate ion), the diol moiety of glucuronic acid is degraded to generate two aldehyde groups, resulting in the MMWH described herein. The result is a significant loss of activity as measured by anti-Xa and anti-IIa tests routinely performed on heparin. Oxidation of glucuronic acid has been proposed to alter heparin's ability to bind antithrombin III (ATIII).

[0134] However, ristocetin-induced platelet aggregation (RIPA) tests show increased activity of MMWH, indicating that the heparin polysaccharide von Willebrand factor interaction is intact and possibly enhanced.

[0135] The two aldehyde groups on glucuronic acid may be reactive under certain circumstances. This reactivity may reduce the stability of oxidized polysaccharides over time. In addition, it may interact with free amines on proteins when injected into the body. The latter may undergo a Schiff base reaction with the free amines, similar to the reaction of glucose with hemoglobin to produce the well-known glycated hemoglobin commonly known as A1C.

[0136] One potential solution would be to reduce the aldehyde functionality to an alcohol functionality. This could be easily accomplished with a mild reducing agent such as sodium borohydride (NaBH). This reduction would not reform the cyclic ring of the glucuronic acid monosaccharide, and the binding of MMWH-Red to ATIII would remain unchanged. However, the reduced MMWH would still retain its interaction with von Willebrand factor and would be useful for the treatment of vascular endotheliitis.

[0137] Advantages of MMWH-Red include: maintenance of von Willebrand activity of the MMWH, greater stability as an active pharmaceutical ingredient (API) and as a pharmaceutical, minimal interaction with pharmaceutical excipients, reduced or no reaction with proteins in vivo, and minimal side effects. Thus, in a seventh aspect, the present invention provides an MMWH-Red according to the fourth or fifth aspect of the invention or a composition according to the sixth aspect of the invention for use in treating vascular endotheliitis.

[0138] MMWH-Red can inhibit von Willebrand factor (VWF), VWF multimers, preferably ultra-high molecular weight VWF, and platelet binding to VWF.

[0139] Preferably, the present invention provides MMWH-Red or a composition comprising MMWH-Red for use in treating vascular endotheliitis. Preferably, the present invention provides MMWH-Red or a composition comprising MMWH-Red for use in treating vascular endotheliitis in patients with 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 vascular endotheliitis in patients with a plasma von Willebrand factor to ADAMTS13 ratio of at least about 2.

[0140] Patients may have a VWF:ADAMTS13 ratio of at least about 2, at least about 4, at least about 8, or at least about 10. Patients may have a VWF:ADAMTS13 ratio of greater than about 2, greater than about 4, greater than about 8, or greater than about 10. Patients may have a VWF:ADAMTS13 ratio of about 2 to 16, about 4 to 12, or preferably about 6 to 10. Patients with a VWF:ADAMTS13 ratio greater than about 8 usually exhibit severe disease and often represent patients deteriorating toward death.

[0141] Patients may have a VWF antigen:ADAMTS13 ratio of at least about 2, at least about 4, at least about 8, or at least about 10. Patients may have a VWF antigen:ADAMTS13 ratio of greater than about 2, greater than about 4, greater than about 8, or greater than about 10. Patients may have a VWF antigen:ADAMTS13 ratio of about 2 to 16, about 4 to 12, or preferably about 6 to 10. Patients with a VWF antigen:ADAMTS13 ratio greater than about 8 usually exhibit severe disease and often represent patients deteriorating toward death.

[0142] VWF and ADAMTS13 levels in patients can be measured using ELISA. Ratios 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, VWF antigen levels can be determined in international units, ADAMTS13 levels can be determined in international units, and the VWF antigen:ADAMTS13 ratio can be determined.

[0143] A normal plasma VWF level ranges from about 50 IU / dL to about 200 IU / dL. In the general population, the average plasma VWF level is about 100 IU / dL. A high plasma VWF level is about 200 IU / dL or higher, for example, 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, or about 275 IU / dL to about 300 IU / dL.

[0144] The patient may have an elevated VWF antigen level of about 150% or more, about 175% or more, about 200% or more, about 300% or more, about 350% or more, about 400% or more, or about 500% or more. The patient may have a VWF antigen level of about 600% or less, about 700% or less, about 800% or less, or about 1000% or less.

[0145] Furthermore, plasma VWF levels can be temporarily elevated due to infection, inflammation, trauma, and physical and emotional stress. Thus, a patient can have elevated plasma von Willebrand factor levels for a non-temporary period, e.g., at least about 6 hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours. Preferably, a patient can have elevated plasma von Willebrand factor levels for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days. Even more preferably, a patient can have elevated plasma von Willebrand factor levels for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. Even more preferably, a patient can have elevated plasma von Willebrand factor levels for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year. The patient may have elevated plasma von Willebrand factor levels for up to about 1 week, up to about 4 weeks, up to about 2 months, up to about 4 months, up to about 6 months, or up to about 1 year.

[0146] MMWH-Red may have a mass of about 11,000 Da (g / mol). Reduced medium molecular weight heparin may contain at least three units of GlcNS6S-IdoA2S (or IdoA2S-GlcNS6S) disaccharide.

[0147] Vascular endotheliitis can be caused by any disease. Specifically, vascular endotheliitis can be caused by COVID-19 infection, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, septicemia, sepsis, cardiovascular disease, diabetes, trauma, particularly brain or head trauma, burns, inhalation injury, drug reaction, hematological condition, subarachnoid hemorrhage, aortic aneurysm disease, stroke, or cerebral parenchymal hemorrhage. Vascular endotheliitis can be caused by viral infection, which can be SARS-CoV-2. Vascular endotheliitis 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.

[0148] The infection may be bacterial, fungal, or parasitic. The infection may be bacterial. Bacterial infections include Actinomyces israelii, Bacillus anthracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelaii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, and Campylobacter jejuni. 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 tularensis, Haemophilus influenzae, Helicobacter pyloripylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira species, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroides, Rickettsia rickettsii The bacterial strain may be Salmonella rickettsii, Salmonella, Shigella, Spirochaetes Staphylococcus, Streptococcus, Treponema pallidum, Vibrio cholerae, or Yersinia pestis.

[0149] 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.

[0150] The infectious disease may be parasitic. The parasitic infection may be a protozoan eye infection, Chagas disease, leishmaniasis, toxoplasmosis, giardiasis, malaria, microsporidiosis, or rhinosporidiosis. Preferably, the parasitic infection is malaria.

[0151] The viral infection may be SARS-CoV-2. SARS-CoV-2 is the virus that causes the disease COVID-19. COVID-19 may result in ARDS. Vascular endotheliitis may be caused by SARS-CoV-2 infection. Vascular endotheliitis may be caused by COVID-19. Vascular endotheliitis may be caused by ARDS.

[0152] Vascular endotheliitis can be caused by cancer. The cancer can be leukemia, lymphoma, or myeloma. Alternatively, or in addition, the cancer can be a 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.

[0153] Vascular endotheliitis can be due to a hematological condition, such as thrombotic thrombocytopenic purpura, anemia, or sickle cell disease.

[0154] Endothelial cell dysfunction may allow tumor cells circulating in the blood to enter tissues. Therefore, treating vascular endotheliitis may prevent the hematogenous spread of blood-borne cancer. Treating vascular endotheliitis may inhibit the hematogenous spread of cancer. Medium molecular weight heparin may inhibit the hematogenous spread of cancer.

[0155] Biomarkers for vascular endotheliitis may include elevated von Willebrand factor (VWF), ultra-high molecular weight von Willebrand factor (ULVWF) levels, factor VIII levels, as well as syndecan-1, VWF antigen, VWF activity, VWF multimers, ADAMTS13 levels, platelet count, VCAM-1, ICAM-1, P-selectin levels, VWF:ADAMTS13 ratio, or VWF antigen:ADAMTS13 ratio. Preferably, the biomarker for vascular endotheliitis is the VWF:ADAMTS13 or VWF antigen:ADAMTS13 ratio.

[0156] The patient may have elevated plasma von Willebrand factor (VWF) levels compared to healthy control subjects. The patient may have persistently elevated plasma VWF levels compared to healthy controls. The plasma VWF levels may be elevated compared to healthy control subjects for a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or preferably at least about 1 week. The plasma VWF levels may be elevated compared to healthy control subjects for a period of up to about 1 week, up to about 4 weeks, up to about 2 months, up to about 4 months, up to about 6 months, or up to about 1 year.

[0157] For example, plasma VWF levels can be increased 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 more preferably at least about 90 nmol / L. Plasma VWF levels can be increased to about 130 nmol / L, about 150 nmol / L, or about 200 nmol / L. Plasma VWF levels can be increased to at least about 50 nmol / L for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 1 month, or at least about 1 year. Plasma VWF levels can be increased to at least about 60 nmol / L for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 1 month, or at least about 1 year. The plasma VWF level may be elevated to at least about 70 nmol / L for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 1 month, or at least about 1 year. The plasma VWF level may be elevated to at least about 90 nmol / L for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 1 month, or at least about 1 year. Plasma VWF levels may be measured using enzyme-linked immunosorbent assay (ELISA).

[0158] Alternatively or additionally, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or greater 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 may have a plasma von Willebrand factor level of about 200 IU / dL or greater for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days. Even more preferably, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or greater for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. Even more preferably, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or greater for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year.

[0159] Vascular endothelial function can be assessed in the coronary arteries and peripheral circulation. Noninvasive tests for assessing coronary endothelial function include Doppler echocardiography, which measures blood flow in response to pharmacological or physiological stimuli. Other tests include positron emission tomography and phase-contrast magnetic resonance imaging. However, the gold standard involves invasive quantitative coronary angiography, which examines changes in diameter 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.

[0160] The binding of MMWH-Red to VWF may be assessed by a competitive binding assay. Heparin-Sepharose beads are bound to labeled VWF, e.g. 125The labeled VWF may be incubated with I-vWF for a period of time to allow the labeled VWF to bind to the immobilized heparin. Various concentrations of MMWH-Red may then be added, and the amount of labeled VWF displaced may be determined. Other methods for determining MMWH-Red binding to VWF may include surface plasmon resonance, biolayer interferometry, isothermal titration calorimetry, fluorescence polarization binding assay, ELISA, and microscale thermophoresis.

[0161] Inhibition of platelet binding to VWF can be assessed by ristocetin-induced coagulation of fixed platelets. Platelets can be incubated with medium molecular weight heparin and citrated plasma (VWF source). Ristocetin can then be added, and platelet coagulation can then be determined. MMWH can completely inhibit VWF-induced platelet aggregation at a concentration of 15 μM, as measured by ristocetin-induced platelet aggregation assay. Other methods for determining inhibition of VWF binding to platelets can include ELISA, fluorescence-assisted cell sorting, dynamic light scattering, or flow chamber assay.

[0162] MMWH-Red may have a mass ranging from greater than about 8,000 Da (g / mol) to about 13,000 Da (g / mol), preferably about 10,000 Da (g / mol) to about 12,000 Da (g / mol). MMWH-Red may have a mass of about 11,000 Da (g / mol). MMWH-Red may comprise polysaccharide chains having an average molecular mass ranging from about 9,000 Da (g / mol) to about 13,000 Da (g / mol), preferably about 10,000 Da (g / mol) to about 12,000 Da (g / mol). MMWH-Red may comprise polysaccharide chains having an average molecular mass of about 11,000 Da (g / mol). The molecular weight of MMWH-Red may be determined, for example, by mass spectrometry or size exclusion chromatography.

[0163] MMWH-Red may contain at least 3 units of GlcNS6S-IdoA2S disaccharide, e.g., at least 4 units, at least 5 units, at least 6 units, at least 8 units, or at least 10 units. Medium molecular weight heparin may contain 25 or fewer units of GlcNS6S-IdoA2S disaccharide, e.g., 20 or fewer units. The presence of GlcNS6S-IdoA2S disaccharide units may be determined by antibody, mass spectrometry, or infrared light from chemical and enzyme tests. The GlcNS6S-IdoA2S units may be arranged in an orderly fashion.

[0164] MMWH-Red may contain at least 3 units of IdoA2S-GlcNS6S disaccharide, e.g., at least 4 units, at least 5 units, at least 6 units, at least 8 units, or at least 10 units. Reduced intermediate molecular weight heparin may contain 25 or fewer units of IdoA2S-GlcNS6S disaccharide, e.g., 20 or fewer units. The presence of IdoA2S-GlcNS6S disaccharide units may be determined by antibody, mass spectrometry, or infrared light from chemical and enzyme tests. The IdoA2S-GlcNS6S units may be arranged in an orderly fashion. The number of IdoA2S-GlcNS6S units may be adjusted to provide the desired anti-VWF activity and / or standard anticoagulant activity.

[0165] MMWH-Red may contain UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc. MMWH-Red may contain at least about 60% 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% UA2S-GlcNS6S. MMWH-Red may contain no more than about 60%, preferably no more than about 70%, preferably no more than about 85% 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% UA2S-GlcNS. MMWH-Red may contain no more than about 15%, preferably no more than about 20% UA2S-GlcNS. MMWH-Red may contain at least about 4%, preferably at least about 5%, preferably at least about 6%, and preferably at least about 10% UA-GlcNAc. Reduced intermediate molecular weight heparin may contain no more than about 15%, preferably no more than about 20% UA-GlcNAc. In some embodiments, MMWH-Red may contain at least 49.2% UA2S-GlcNS6S, 5.4% UA2S-GlcN, and 5.4% UA-GlcNAc. In some embodiments, MMWH-Red may contain at least 82% UA2S-GlcNS6S, 9% UA2S-GlcNS, and 9% UA-GlcNAc. The percentage composition of UA-GlcNAc contained in MMWH-Red may be enriched compared to unfractionated heparin.

[0166] Treatment of vascular endothelial inflammation with MMWH-Red may inhibit hematogenous spread of cancer. Human tumor cells, including both melanoma and colon cancer cells, demonstrate this ability by being able to bind VWF under high-shear flow conditions. Immobilized platelets bound to VWF were found to mediate the tethering, rolling, and strong adhesion of different cancer cell lines under flow shear stress. VWF played a key role in enabling this strong adhesion of tumor cells to immobilized platelets. Current data suggest that VWF plays an important role in cancer cell tethering. In addition, VWF-platelet binding, which occurs as part of the normal thrombosis pathway, may enable tumor cells to coalesce with VWF-platelets to generate VWF+platelet+cancer cell heteroaggregates, which may further aid in the blood-borne (hematogenous) spread of tumor cells. This process is driven, at least in part, by the ability of cancer cells to migrate to the blood vessel wall, where they can spread to other organs once initial binding to VWF and platelets occurs. In addition, various cancers have been shown to cause vascular endotheliitis through the resulting release of UL-VWF. Through this mechanism, tumors then release UL-VWF, which allows platelets to bind to tumor cells and cancer to spread hematogenously and metastatically. This cancer-induced vascular endotheliitis also results in an overall increased risk of thrombosis in patients with underlying malignant tumors. Therefore, any treatment aimed at treating vascular endotheliitis and inhibiting the binding of VWF to platelets and / or tumor cells will serve the dual purpose of reducing the risk of malignant tumor-associated thrombosis and also reducing the risk of hematogenous metastatic spread.

[0167] MMWH-Red may be administered by a method selected from the group consisting of parenteral, subcutaneous, intravenous, intramuscular, intrathecal, intradermal, intraarterial, intraarticular, cutaneous, transdermal, subcutaneous, depot form (e.g., depot injection), intraosseous, and inhalation. Preferably, the medium molecular weight heparin is administered subcutaneously, intravenously, or intramuscularly. The administration method may be inhalation, optionally with a nebulizer.

[0168] Previous studies have examined UFH as a nebulized agent in a variety of conditions. Small studies have shown that it limits pulmonary fibrin deposition, attenuates the progression of acute lung injury, and accelerates recovery (69). Early-phase clinical trials in patients with acute lung injury and related conditions have found that nebulized UFH reduces pulmonary dead space, coagulation activation, and microvascular thrombosis, improves lung injury, and increases time free of ventilatory support (70-73). In a pre-pandemic, double-blind, randomized trial of 256 critically ill, mechanically ventilated patients, nebulized UFH limited the progression of lung injury, including acute respiratory distress syndrome, and facilitated survivors' return home. Therefore, MMWH-Red may be administered by inhalation using a nebulizer.

[0169] Heparin dosage is typically measured in "Howell units." One unit of heparin ("Howell unit") is approximately equivalent to 0.002 mg of pure heparin, the amount required to keep 1 ml of feline blood fluid for 24 hours at 0°C. MMWH-Red may be administered at approximately 5,000 units per hour, followed by a bolus dose of approximately 1,200 to 1,600 units, which may be delivered by an infusion pump. MMWH-Red may be administered at a dose of approximately 18 units / kg to approximately 5,000 units / kg. Preferably, MMWH-Red may be administered at a dose of approximately 100 units / kg to approximately 800 units / kg. Alternatively, MMWH-Red may be administered at a dose of approximately 18 units / kg to approximately 75 units / kg. MMWH-Red may be administered at a dose of about 5000 units, about 4000 units, about 3000 units, about 2000 units, about 1000 units, or 500 units every 12 hours. MMWH-Red may be administered at a dose of about 5000 units every 12 hours.

[0170] The MMWH-Red may be administered at a dose of about 3 units to about 5,000 units, e.g., about 6 units to about 4,000 units, about 12 units to about 3,000 units, about 25 units to about 2,000 units, about 50 units to about 1,000 units, about 100 units to about 500 units, or about 125 units to about 250 units. The reduced intermediate molecular weight heparin may be administered at a dose of about 18 units / kg to about 5,000 units / kg, e.g., about 100 units / kg to about 4,000 units / kg, or about 200 units / kg to about 800 units / kg. The reduced intermediate molecular weight heparin may be administered at a dose of about 18 units / kg to about 75 units / kg. The dose may be administered as a single dose or continuously. The dose may be administered over a period of time. The period may be about 1 month to about 12 months, for example, 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, or about 6 months to about 7 months. The period may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, or about 4 days to about 5 days. The dose may be administered over about 1 hour to about 24 hours, about 2 hours to about 12 hours, or about 3 hours to about 6 hours. The dose may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0171] MMWH-Red may be administered at a dose of about 0.01 mg / kg to about 10 mg / kg, for example, about 0.05 mg / kg to about 9 mg / kg, about 0.5 mg / kg to about 8 mg / kg, about 1 mg / kg to about 7 mg / kg, about 1.5 mg / kg to about 6 mg / kg, or about 2 mg / kg to about 5 mg / kg. The dose may be administered as a single dose or continuously. The dose may be administered over a period of time. The period may be about 1 month to about 12 months, for example, 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, or about 6 months to about 7 months. The period may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, or about 4 days to about 5 days. The dose may be administered over a period of about 1 hour to about 24 hours, about 2 hours to about 12 hours, or about 3 hours to about 6 hours. The dose may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0172] MMWH-Red may be administered at a dose of about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 70 mg / kg, about 80 mg / kg, or about 100 mg / kg. MMWH-Red may 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 may be administered at a dose of about 0.01 mg / kg to about 10 mg / kg, preferably about 0.2 mg / kg to about 10 mg / kg, or about 0.2 mg / kg to about 1.6 mg / kg. MMWH-Red may be administered as a single dose or continuously. The MMWH-Red dosage may depend on the VWF antigen:ADAMTS13 ratio or overall VWF levels. One skilled in the art would be able to select an appropriate amount for a patient based on the VWF antigen:ADAMTS13 ratio or overall VWF levels.

[0173] MMWH-Red may 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, or about 1 mg / kg to about 1.5 mg / kg. The dose may be administered as a single dose or continuously. The dose may be administered over a period of about 1 month to about 12 months, for example, 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, or about 6 months to about 7 months. The period of time may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, or about 4 days to about 5 days. The dose may be administered over a period of about 1 hour to about 24 hours, about 2 hours to about 12 hours, or about 3 hours to about 6 hours. The dose may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0174] MMWH-Red may 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, or about 150 mg to about 200 mg. The dose may be administered as a single dose or continuously. The dose may be administered over a period of time. The period may be about 1 month to about 12 months, for example, 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, or about 6 months to about 7 months. The period may be about 1 to about 7 days, about 2 to about 6 days, about 3 to about 5 days, or about 4 to about 5 days. The dose may be administered over about 1 to about 24 hours, about 2 to about 12 hours, or about 3 to about 6 hours. The dose may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0175] 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. The dose can be administered as a single dose or continuous administration. The dose can be administered over a period of time. The period may be about 1 month to about 12 months, for example, 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, or about 6 months to about 7 months. The period may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, or about 4 days to about 5 days. The dose may be administered over about 1 hour to about 24 hours, about 2 hours to about 12 hours, or about 3 hours to about 6 hours. The dose may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0176] MMWH-Red may be administered at a dose of about 1 IU to about 50,000 IU, about 2 IU to about 25,000 IU, about 5 IU to about 20,000 IU, about 10 IU to about 10,000 IU, about 15 IU to about 5,000 IU, about 20 IU to about 2,500 IU, about 25 IU to about 2,000 IU, about 50 IU to about 1,500 IU, about 75 IU to about 1,000 IU, about 100 IU to about 500 IU, about 200 IU to about 400 IU, or about 250 IU to about 300 IU. The doses may be administered as a single dose or continuously. The doses may be administered over a period of time. The period may be about 1 month to about 12 months, for example, 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, or about 6 months to about 7 months. The period may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, or about 4 days to about 5 days. The dose may be administered over about 1 hour to about 24 hours, about 2 hours to about 12 hours, or about 3 hours to about 6 hours. The dose may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0177] An MMWH-Red equivalent to the VWF antigen:ADAMTS13 ratio may be administered. For example, patients with a high VWF antigen:ADAMTS13 ratio may be administered a higher dose of MMWH compared to patients with a lower VWF antigen:ADAMTS13 ratio.

[0178] The MMWH-Red may be contained in a pharmaceutical formulation. The pharmaceutical formulation may include an excipient. The excipient may be selected from the group including solvents, cosolvents, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavorings, lubricants, suspending agents, isotonicity agents, surfactants, solubilizers, dispersing aids, dispersants, humectants, thickeners, colorants, wetting agents, antifoaming agents, viscosity adjusters, sweeteners, and combinations thereof. The pharmaceutical formulation may include an additional active agent. The additional active agent may include medium molecular weight heparin or low molecular weight heparin.

[0179] The pharmaceutical formulation may include an additional active agent, including a composition of matter having a physiological effect, and may include low or medium molecular weight heparin or reduced medium molecular weight heparin of a different disaccharide composition. The additional active agent may be selected from the group comprising 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 analogues, apical sodium-bile acid cotransporter (ASBT) inhibitors, apoptosis signal-regulating kinase 1 (ASK1) inhibitors, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, chemokine receptor inhibitors, thiozolidinediones, GLP-1 analogues, biguanides, HIV replication inhibitors, metformin, opiates, anesthetics, HMG-CoA reductase inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), or any combination thereof.

[0180] MMWH-Red may comprise a chemical modification, which may be selected from the group including N-acetylation, de-N-acetylation, O-sulfation, de-2-O-sulfation, and complete desulfation.

[0181] In an eighth aspect, the present invention provides an MMWH-Red for use in the treatment of a disease or condition in a patient, wherein the patient has vascular endotheliitis characterized by a plasma von Willebrand factor to ADAMTS13 (VWF:ADAMTS13) ratio of at least about 2.

[0182] In a ninth aspect, the present invention provides an MMWH-Red for use in the treatment of a disease or condition in a patient, wherein the patient has vascular endotheliitis characterized by a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0183] Preferably, the disease or condition is selected from the group consisting of COVID-19, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, sepsis, cardiovascular disease, diabetes, trauma, particularly brain or head trauma, burns, inhalation injury, drug reaction, hematological condition, subarachnoid hemorrhage, aortic aneurysm disease, stroke, and cerebral parenchymal hemorrhage, and combinations thereof. Vascular endotheliitis may be caused by a viral infection, which may be SARS-CoV-2. Vascular endotheliitis may 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, vascular endotheliitis is caused by sepsis or septicaemia. Preferably, vascular endotheliitis is caused by sepsis.

[0184] In some embodiments, the invention provides an intermediate molecular weight heparin, MMWH-Red, for use in treating COVID-19 in a patient, wherein the patient has vascular endotheliitis 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.

[0185] In some embodiments, the present invention provides MMWH-Red for use in treating a viral infection in a patient, wherein the patient has vascular endotheliitis 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. The viral infection may be SARS-CoV-2.

[0186] In some embodiments, the present invention provides an MMWH-Red for use in the treatment of acute respiratory distress syndrome (ARDS) in a patient, wherein the patient has vascular endotheliitis 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.

[0187] In some embodiments, the present invention provides MMWH-Red for use in treating cancer in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen (VWF antigen:ADAMTS13) to ADAMTS13 ratio of at least about 2. The cancer can be leukemia, lymphoma, myeloma, or a 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.

[0188] In some embodiments, the present invention provides an MMWH-Red for use in treating a bacterial infection in a patient, wherein the patient has vascular endotheliitis 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.

[0189] In some embodiments, the present invention provides an MMWH-Red for use in the treatment of septicaemia in a patient, wherein the patient has vascular endotheliitis 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.

[0190] In some embodiments, the present invention provides an MMWH-Red for use in the treatment of sepsis in a patient, wherein the patient has vascular endotheliitis 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.

[0191] In some embodiments, the present invention provides an MMWH-Red for use in treating cardiovascular disease in a patient, wherein the patient has vascular endotheliitis 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.

[0192] In some embodiments, the present invention provides an MMWH-Red for use in the treatment of diabetes in a patient, wherein the patient has vascular endotheliitis 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.

[0193] In some embodiments, the present invention provides MMWH-Red for use in treating a traumatic injury in a patient, wherein the patient has vascular endotheliitis 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 brain trauma or head trauma.

[0194] In some embodiments, the present invention provides an MMWH-Red for use in the treatment of burns in a patient, wherein the patient has vascular endotheliitis 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 an MMWH-Red for use in treating an inhalation injury in a patient, wherein the patient has vascular endotheliitis 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.

[0196] In some embodiments, the present invention provides an MMWH-Red for use in treating a drug reaction in a patient, wherein the patient has vascular endotheliitis 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 an MMWH-Red for use in treating a hematological condition in a patient, wherein the patient has vascular endotheliitis 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.

[0198] In some embodiments, the present invention provides an MMWH-Red for use in the treatment of subarachnoid hemorrhage in a patient, wherein the patient has vascular endotheliitis 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 an MMWH-Red for use in the treatment of aortic aneurysm disease in a patient, wherein the patient has vascular endotheliitis 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] The MMWH-Red for use in the treatment of vascular endotheliitis as defined in the seventh, eighth or ninth aspect of the present invention is particularly advantageous because said heparin is capable of inhibiting microthrombosis caused by the release of VWF secondary to vascular endotheliitis due to any disease or pathology. In cases where the cause of the vascular endotheliitis is SARS-CoV-2, said MMWH-Red is additionally capable of inhibiting viral attachment and replication.

[0201] In a tenth aspect, the present invention provides a method for treating vascular endotheliitis, the method comprising administering a therapeutically effective amount of MMWH-Red to a subject in need of treatment. Preferably, the patient has a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio of at least about 2.

[0202] In an eleventh aspect, the present invention provides use of MMWH-Red for the manufacture of a medicament for the treatment of vascular endotheliitis in a patient, preferably having a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio of at least about 2.

[0203] For the avoidance of doubt, embodiments relating to each aspect of the invention apply mutatis mutandis to other aspects of the invention. Further aspects and embodiments of the invention will become apparent from the discussion herein.

[0204] Sepsis is the body's extreme response to an infection. It is characterized by a systemic inflammatory state in which the immune system responds to infection by attacking the body's own tissues and organs as a result of a dysregulated immune response. As a result, the body's cells are destroyed by apoptosis or necrosis, and intracellular material ends up in the bloodstream.

[0205] Histones are intracellular basic proteins found in the nucleus of cells. DNA wraps itself around histones to form chromatin, which then tightly coils around itself to facilitate the packing of long DNA molecules into the cell nucleus. While known for its role in packing DNA into the nucleus, chromatin (and its constituent histones) has also been found to play a role in innate immunity. During infection, neutrophils release granular proteins and chromatin, which together form extracellular fibers that bind Gram-positive and Gram-negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill the bacteria.

[0206] Circulating histones have been identified as mediators of damage in animal models and patients with sepsis. These histones can enter the bloodstream due to apoptosis or necrosis that occurs during sepsis, or as a result of intracellular material released via NET release in response to bacterial infection. Thus, histones can propagate 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 proinflammatory cytokines, thereby exacerbating an already dysregulated immune response. Specifically, TLR receptors on endothelial cells can be activated by circulating histones, leading to the induction of an immune response in endothelial cells. Consequently, the endothelium becomes inflamed, resulting in endothelial injury and vascular endotheliitis.

[0207] Thus, MMWH or MMWH-Red as defined herein can simultaneously inhibit VWF and bind to free histones, thereby treating sepsis. In embodiments, 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 subject has a plasma VWF:ADAMTS13 ratio of at least about 2.

[0208] An MMWH or MMWH-Red, as defined herein, can be a complement cascade modulator.

[0209] An MMWH or MMWH-Red as defined herein may be an immune modulator.

[0210] For the avoidance of doubt, embodiments relating to each aspect of the invention apply mutatis mutandis to other aspects of the invention. Further aspects and embodiments of the invention will become apparent from the discussion herein.

[0211] All documents cited herein, including any cross-references or related patents or applications, are hereby incorporated by reference in their entirety unless expressly excluded or otherwise limited.

[0212] It will be understood that various modifications may be made to the illustrated embodiments without departing from the spirit and scope of the present invention as defined by the appended claims. [Example]

[0213] The invention will now be demonstrated by reference to the following non-limiting examples.

[0214] Unless otherwise stated, room temperature and pressure are 20° C. (293.15 K, 68° F.) and 1 atm (standard pressure) (14.696 psi, 101.325 kPa), respectively. Experimental Method Size Exclusion Chromatography (SEC)

[0215] SEC was performed on a GEC Superdex 75 (10 / 100) column on an Agilent 1200 HPLC system equipped with a variable wavelength UV monitor or a GEC AKTA system equipped with a variable wavelength UV monitor. The mobile phase was 0.15 M NaCl in water, passed through the column at 0.4 mL / min. Optical density was measured at 232 nm. SEC was performed against an 11 kD standard. Freeze drying

[0216] Freeze drying (also known as lyophilisation or cryodesiccation) is a drying method carried out at low temperatures. Freeze drying generally involves lowering the temperature and pressure below the triple point of the substance to remove the frozen solvent (e.g., water ice) by sublimation. For aqueous compositions such as those disclosed herein, freeze drying may be carried out at temperatures of about -20°C to -80°C, preferably about -40°C, and pressures of about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar). MMW heparin manufacturing method 1. Development example 1.1 Optimization of periodate concentration (periodate method)

[0217] A batch of 80 mg unfractionated heparin was dissolved in 50 mM sodium phosphate buffer (30 mL) adjusted to pH 7.0.

[0218] Samples were prepared by adding (A) 43 mg, (B) 86 mg, (C) 173 mg, or (D) 200 mg of sodium periodate to heparin in sodium phosphate buffer. The resulting solution was incubated at 4°C for 16–18 hours or at 37°C for 6 hours.

[0219] A 200 μl aliquot was taken for SEC before dialysis. The remainder of the sample was dialyzed against water in 2 kd cutoff tubing (Spectra / Por®, product number 132109; 7 days: 3 changes / 4 volumes of water per day). The sample was then lyophilized at −40° C. and 10 Pa. The sample was then dissolved in water and analyzed by SEC on a Superdex® 75 SE (10 / 300) column in 0.15 M NaCl at 0.4 mL / min on either a GEC AKTA system or an Agilent 1200 system.

[0220] Aliquots taken before dialysis were analyzed by SEC on a Superdex® 75 SE (10 / 300) column in 0.15 M NaCl at 0.4 mL / min on either a GEC AKTA system or an Agilent 1200 system, showing a significant amount of disaccharide-like material mixed with the reagent peak.

[0221] SEC was performed on the samples and corrected for co-elution with a validated 11 KDa standard. SEC analysis of the post-dialysis samples provided the following results: a. Incubation at 37°C resulted in K av The sample ran as a broad peak centered at 0.12, resulting in degradation of the medium molecular weight heparin. b. Incubation at 4°C resulted in K av The sample ran as narrow peaks centered at molecular weights of 0.06 and 11 kD.

[0222] FIG. 1 shows MMWH prepared at 4° C. compared to heparin processed at 37° C., unprocessed heparin, and an 11 kDa standard.

[0223] Therefore, incubation at 4°C favors the production of intermediate molecular weight heparin, while incubation at 37°C leads primarily to other products.

[0224] All tested concentrations of sodium periodate resulted in the desired intermediate molecular weight heparin. The most effective concentration of sodium periodate was 173 mg per 30 mL of heparin in (C) sodium phosphate buffer (equivalent to the 8.6 g per 1.5 L used in the large-scale process sample below). 1.2 Perchlorate Addition

[0225] Samples were prepared by the periodate method (1.1) (A) and (B) described above. 732 mg of sodium perchlorate per 30 mL of sample (A) and (B) were added, and the resulting solutions were incubated at 4°C for 16–18 hours or at 37°C for 6 hours.

[0226] Samples were dialyzed against water in 2 kd cut-off tubing (Spectra / Por®, product number 132109; 7 days: 3 changes / 4 volumes of water per day). Samples were then lyophilized at -40°C and 10 Pa. Samples were then dissolved in water and analyzed by SEC on a Superdex® 75 SE (10 / 300) column in 0.15 M NaCl at 0.4 mL / min on either a GEC AKTA system or an Agilent 1200 system.

[0227] SEC was performed on the samples and corrected for co-elution with a validated 11 KDa standard. SEC analysis of the post-dialysis samples provided the following results:

[0228] All samples (incubated at 4°C and 37°C) were av This resulted in the production of material with a broader size range centered around 0.12. t There was also a significant increase in matter (i.e., smaller species).

[0229] This example demonstrates that oxidation with a mixture of periodate and perchlorate results in increased levels of decomposition to smaller species compared to oxidation with periodate alone, and therefore oxidation with periodate alone is a more effective method for preparing high-purity intermediate molecular weight heparin. 1.3 Alkali desorption

[0230] Samples were prepared by the periodate method (1.1) (A) and (B) above with incubation at 4°C and 37°C (1.1).

[0231] NaOH (2 M) was added to the samples to adjust the pH of the samples to pH 12. The samples were then incubated at room temperature for 30 minutes. Aliquots of these samples were analyzed by SEC on a Superdex® 75 SE (10 / 300) column in 0.15 M NaCl at 0.4 mL / min on either a GEC AKTA system or an Agilent 1200 system. Samples were subjected to SEC and corrected for coelution with a verified 11 KDa standard.

[0232] This alkali elimination occurs when K av More obvious peaks at 0.331, 0.35, 0.4, and 0.54, as well as K av Further resolution resulted with a large peak at 0.625.

[0233] Figure 2 shows MMWH prepared at 4°C using only periodate, compared to heparin treated with periodate and then NaOH at 4°C, which runs as a broad peak due to degradation. Thus, the preparation of high-purity intermediate molecular weight heparin is hindered by including alkaline elimination, as significant degradation is observed. 2. Example of large-scale process

[0234] UF heparin (porcine mucosal heparin; Iduron catalog number HEP001 / 100) (4 g; 2.7 mg / mL; average molecular weight 15 kDa) was dissolved in ice-cold (0°C–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 the addition of D-mannitol (30 g, 160 mmol).

[0235] The sample was then dialyzed against water in 2 kD cutoff tubing (Spectra / Por®, product number 132109; 7 days: 3 changes / 4 volumes of water per day). The sample was then lyophilized at -40°C and 10 Pa to give a yield of 2.1 g of medium molecular weight heparin.

[0236] The intermediate molecular weight heparin samples were then dissolved in water and analyzed by SEC on a Superdex® 75 SE (10 / 300) column in 0.15 M NaCl at 0.4 mL / min on either a GEC AKTA system or an Agilent 1200 system. The intermediate molecular weight heparin had an average molecular weight of 11 kDa referenced to an 11 kDa standard as shown in Figure 3.

[0237] As shown in Figures 4 and 5, the produced intermediate molecular weight heparins exhibited very low activity against factor IIa and / or factor Xa when compared to UF heparin and low molecular weight heparin. Thus, unlike UF heparin or LMW heparin, MMW heparin does not affect either factor IIa- or factor Xa-mediated coagulation.

[0238] Factor IIa (also known as thrombin) acts as a serine protease that converts soluble fibrogen into insoluble chains of fibrin, as well as catalyzing other coagulation-related reactions. Factor Xa is the activated form of clotting factor X. Factor X is a serine endopeptidase enzyme that plays a key role in several steps of the coagulation system.

[0239] Heparin (unfractionated heparin) and its derivatives, such as low-molecular-weight heparins, bind to the plasma cofactor antithrombin (AT) and inactivate several coagulation factors IIa, Xa, XIa, and XIIa. This inactivation of factor Xa by heparin is called "indirect" because it depends on the presence of AT and does not involve a direct interaction with factor Xa.

[0240] Activity against factor IIa and factor Xa can be measured using the Kinetichrome anti-IIa heparin kit and the Kinetichrome anti-Xa heparin kit, respectively.

[0241] Disaccharide analysis was performed on the medium molecular weight heparin, and the results are shown in Table 1 below. [Table 1]

[0242] UF heparin of porcine mucosa origin is used for purposes of synthesis herein and in the examples provided. For the avoidance of doubt, other sources of UF heparin are suitable for use in the methods disclosed herein. MMWH-Red manufacturing method Heparin oxidation

[0243] UFH heparin is oxidized as described above. Reduction of oxidized heparin

[0244] To a 10 mg / mL solution of oxidized heparin in demineralized water (110 mL) is added 20 mg of sodium borohydride. The reaction mixture is stirred for 3 hours at 25°C. The reduced medium molecular weight heparin is purified by exhaustive dialysis using phosphate buffer (pH = 7.0). References

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Claims

1. 1. A method for synthesizing a medium molecular weight heparin, said method comprising: a. dissolving unfractionated heparin in an aqueous buffer adjusted to a pH of about 5.0 to about 9.0 to obtain a first solution; b. adding an oxidizing agent to the first solution to obtain a second solution; c. incubating the second solution at a temperature of about 0°C to about 10°C to produce an intermediate molecular weight heparin solution; A method comprising:

2. 10. The method of claim 1, wherein the aqueous buffer is adjusted to a pH of about 7.

0.

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

4. The method of claim 3 , wherein the phosphate buffer is a sodium phosphate buffer.

5. 5. The method of any one of claims 1 to 4, wherein said incubating step c. is carried out for about 1 hour to about 48 hours.

6. The method of any one of claims 1 to 5, wherein the incubation temperature is about 4°C.

7. 7. The method of any one of claims 1 to 6, wherein the temperature of the aqueous buffer in step a. is about 0°C.

8. The method according to any one of claims 1 to 7, wherein the oxidizing agent is a periodate, preferably sodium periodate or potassium periodate.

9. The method comprises: d. deactivating the oxidizing agent in the medium molecular weight heparin solution; The method of any one of claims 1 to 8, further comprising:

10. 10. The method of claim 9, wherein the oxidizing agent is inactivated by the addition of a deactivating agent selected from D-mannitol, glycerol, N-acetylmethionine, sodium sulfite, and combinations thereof.

11. 11. The method of claim 9 or claim 10, wherein the oxidizing agent is inactivated by the addition of D-mannitol.

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

10.

13. The method comprises: e. dialyzing the intermediate molecular weight heparin solution in a dialysate to obtain a dialyzed intermediate molecular weight heparin sample; The method of any one of claims 1 to 12, further comprising:

14. The method according to any one of claims 1 to 13, wherein in the dialysis step e., the dialysate is water.

15. The method of any one of claims 1 to 14, wherein the dialysis step e. is performed in 2 kD cut-off tubing.

16. The method comprises: f. isolating the intermediate molecular weight heparin from the dialyzed heparin sample; The method of any one of claims 13 to 15, further comprising:

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

18. 18. The method of any one of claims 1 to 17, wherein the intermediate molecular weight heparin has an average molecular weight of greater than about 8000 Da up to about 13000 Da.

19. The method of any one of claims 1 to 18, wherein the medium molecular weight heparin has an average molecular weight of about 11,000 Da.

20. 20. The method of any one of claims 1 to 19, wherein the medium molecular weight heparin comprises at least three units of the IdoA2S-GlcNS6S disaccharide.

21. The method according to any one of claims 1 to 20, wherein the method does not include an alkaline elimination step.

22. The method according to any one of claims 1 to 21, wherein the method does not involve the addition of an alkali metal salt.

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

24. 23. The method of claim 22, wherein the alkaline salt is NaOH, KOH, or LiOH.

25. 1. A kit suitable for preparing intermediate molecular weight heparin, said kit comprising: a. unfractionated heparin; b. an aqueous buffer adjusted to a pH of about 5.0 to about pH 9.0; c. an oxidizing agent; d. optionally a deactivating agent; A kit comprising:

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

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

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

29. The method according to any one of claims 26 to 28, wherein the method does not include an alkaline elimination step.

30. 30. The method of any one of claims 26 to 29, wherein the method does not involve the addition of an alkali metal salt.

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

32. 32. The method of claim 31 , wherein the alkaline salt is NaOH, KOH, or LiOH.

33. 33. The method of any one of claims 26 to 32, wherein the aqueous buffer is adjusted to a pH of about 7.

0.

34. 34. The method of any one of claims 26 to 33, wherein the aqueous buffer is a phosphate buffer.

35. 35. The method of claim 34, wherein the phosphate buffer is a sodium phosphate buffer.

36. 36. The method of any one of claims 26 to 35, wherein said incubating step c. is carried out for about 1 hour to about 48 hours.

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

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

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

40. 41. The method of any one of claims 26 to 40, wherein the method further comprises inactivating the oxidizing agent in the intermediate molecular weight heparin solution.

41. 41. The method of claim 40, wherein the oxidizing agent is inactivated by the addition of a deactivating agent selected from D-mannitol, glycerol, N-acetylmethionine, sodium sulfite, and combinations thereof.

42. 42. The method of claim 40 or claim 41, wherein the oxidizing agent is inactivated by the addition of D-mannitol.

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

10.

44. 44. The method of any one of claims 26 to 43, wherein the method further comprises dialyzing the intermediate molecular weight heparin solution in a dialysate to obtain a dialyzed intermediate molecular weight heparin sample.

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

46. 46. ​​The method of any one of claims 26 to 45, wherein the dialysis step e. is performed in 2 kD cut-off tubing.

47. 47. The method of any one of claims 44 to 46, wherein the method further comprises isolating the intermediate molecular weight heparin from the dialyzed heparin sample.

48. 48. The method of any one of claims 26 to 47, wherein the intermediate molecular weight heparin has an average molecular weight of greater than about 8000 Da to about 13000 Da.

49. 49. The method of any one of claims 26 to 48, wherein the intermediate molecular weight heparin has an average molecular weight of about 11,000 Da.

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

51. 1. A reduced intermediate molecular weight heparin (MMWH-Red) for use in the treatment of vascular endotheliitis in patients with a plasma von Willebrand factor antigen to ADAMTS13 ratio of at least about 2.

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

53. MMWH-Red for use according to claim 52, wherein the MMWH-Red inhibits multimerization of von Willebrand factor, and optionally the von Willebrand factor is ultra-high molecular weight von Willebrand factor.

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

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

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

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

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

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

60. MMWH-Red for use according to claim 59, wherein the vascular endotheliitis is caused by a viral infection, and the viral infection may be SARS-CoV-2.

61. MMWH-Red for use according to claim 58, wherein the vascular endotheliitis is caused by cancer, and the cancer may be leukemia, lymphoma, myeloma, or solid organ cancer.

62. MMWH-Red for use according to any one of claims 51 to 61, wherein the medium molecular weight heparin is administered by a method of administration selected from the group consisting of parenteral, subcutaneous, subcutaneous, in depot form, e.g., depot injection, intravenous, intramuscular, intrathecal, intradermal, intraarterial, intraarticular, cutaneous, transdermal, intraosseous, and inhalation.

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

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

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

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

67. MMWH-Red for use according to any one of claims 51 to 66, wherein said 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 use according to any one of claims 62 to 67, wherein said MMWH-Red is administered as a single dose or as a continuous dose.

69. MMWH-Red for use according to any one of claims 51 to 68, wherein the MMWH-Red is contained in a pharmaceutical formulation.

70. MMWH-Red for use according to claim 69, wherein the pharmaceutical formulation comprises an excipient.

71. 71. The MMWH-Red for use according to claim 70, wherein the excipient is selected from the group consisting of solvents, co-solvents, buffering agents, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavorings, lubricants, suspending agents, isotonicity agents, surfactants, solubilizers, dispersing aids, dispersing agents, humectants, thickeners, colorants, wetting agents, antifoaming agents, viscosity modifiers, sweeteners, and combinations thereof.

72. MMWH-Red for 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.

73. MMWH-Red for use according to any one of claims 51 to 72, wherein said MMWH-Red comprises a chemical modification.

74. MMWH-Red for use according to claim 73, wherein the chemical modification comprises N-acetylation, de-N-acetylation, O-sulfation, de-2-O-sulfation, complete desulfation, or a combination thereof.

75. 1. A method for treating vascular endotheliitis, the method comprising administering a therapeutically effective amount of MMWH-Red to a patient in need of treatment, wherein the patient has a plasma von Willebrand factor antigen to ADAMTS13 ratio of at least about 2.

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