Medium molecular weight heparin

Abstract

The present invention relates to reduced heparin. The present invention further relates to the treatment of endothelial disorders, in particular reduced heparin for use in the treatment of endothelial disorders. The present invention also relates to the synthesis of reduced heparin.

Description

Technical Field

[0001] This invention relates to reduced medium molecular weight heparin. The invention further relates to the treatment of endotheliopathy, particularly reduced medium molecular weight heparin for the treatment of endotheliopathy. The invention also relates to the synthesis of reduced medium molecular weight heparin. Background Technology

[0002] The vascular system of vertebrates consists of arteries, veins, and capillaries. Blood flow through the vascular system is dynamic, maintaining homeostasis by delivering essential elements such as oxygen and leukocytes to the tissues that need them most. Blood flow is controlled by the vasodilation and vasoconstriction of blood vessels. Endothelial cells lining the lumen of blood vessels form a monolayer called the endothelium; this endothelium sits atop a layer of smooth muscle cells. These smooth muscle cells contract or relax, causing vasoconstriction or vasodilation, respectively. To reach the site of inflammation, leukocytes must travel from the blood through the endothelial cells to reach the inflamed tissue. In cases of hemorrhage, blood flow can be controlled by thrombus formation. Therefore, the regulation of vasoconstriction, vasodilation, vascular permeability, and thrombus formation is crucial for homeostasis.

[0003] Endothelial cells lining blood vessels are more than just simple components of the vessel wall. They produce and release vasoactive substances that dilate and constrict blood vessels. For example, endothelial cells produce nitric oxide (NO) in response to shear stress or stimuli such as acetylcholine, histamine, and thrombin. NO then diffuses to the smooth muscle cells surrounding the endothelium to initiate vasodilation. Reactive oxygen species released in response to inflammatory stimuli can increase endothelial permeability and promote leukocyte adhesion to endothelial cells through the expression of adhesion molecules. This helps drive leukocyte influx into sites of inflammation. Furthermore, the endothelium provides a surface for thrombus formation. Therefore, it is precisely endothelial cells that play a crucial role in homeostasis regulation.

[0004] Therefore, endothelial dysfunction or endothelial lesions can have serious consequences because blood flow, oxygen delivery, immune responses, and consequently homeostasis are all impaired. Endothelial lesions are characterized by decreased NO bioavailability. This can lead to increased expression of endothelial surface adhesion molecules, thereby initiating the recruitment of leukocytes to the vessel wall. Thus, endothelial inflammation, or endothelialitis, is observed in endothelial lesions. This can lead to defects in the endothelial lining of the vessel wall, exposing the subendothelial matrix to clotting factors in the blood. Consequently, platelet aggregation and thrombus formation occur, resulting in potentially fatal blood clots.

[0005] Endothelial lesions can be caused by a variety of diseases and are often considered a symptom rather than a cause. Therefore, treatment has focused on the underlying disease rather than the endothelial lesion itself. Consequently, there is a lack of treatments specifically targeting endothelial lesions. However, it has now been proposed that underlying endothelial lesions may actually drive disease severity and morbidity, and that the role of endothelial lesions is much larger than previously thought. Therefore, treating endothelial lesions, rather than just the underlying disease, could improve patient survival.

[0006] In this document, endothelial lesions and endotheliitis are used interchangeably. While endothelial lesions can be caused by a wide range of diseases and / or conditions described herein, this document will primarily discuss endothelial lesions associated with COVID-19 or SARS-CoV-2. Those skilled in the art will understand that this discussion is for illustrative purposes only and should not be construed as limiting the invention.

[0007] In late 2019, a novel beta-coronavirus (SARS-CoV-2) was discovered, which causes coronavirus disease 2019 (COVID-19). Subsequently, the rapid geographical spread of COVID-19 ultimately led the World Health Organization to declare it a pandemic in March 2020. The clinical presentation of patients infected with SARS-CoV-2 ranges from asymptomatic to more severe pneumonia, which can lead to acute respiratory distress syndrome (ARDS) and multiple organ failure. Most symptomatic patients will experience a mild to moderate form of illness, which usually does not require hospitalization. However, a subset of patients may progress to a more severe form of illness, where the evolution of symptoms / clinical presentation from the initial prodromal phase to ARDS can take up to 2 weeks. It is now known that the subgroup of patients who become critically ill and require ventilation or extracorporeal membrane oxygenation (ECMO) has a very poor prognosis, with a mortality rate approaching 90% (5).

[0008] Since its first description, the disease has affected more than 90 million people worldwide. Its pathophysiological pathways remain unclear; therefore, clinical management is supportive. Disease-modifying therapeutics that can be initiated while awaiting specific antiviral drugs or vaccines are lacking. Multiple pieces of evidence suggest that endothelial dysfunction is a key pathophysiological mechanism in COVID-19. Prior to the current pandemic, biomarkers of endothelial dysfunction had been shown to be associated with disease severity and mortality in patients with sepsis (6–9). Recently, Varga et al. (10) demonstrated extensive endotheliitis affecting blood vessels in the lungs, kidneys, gastrointestinal tract, and liver in autopsies of three COVID-19 patients. In one case, the authors reported that “most small vessels appeared congested,” while in another, the patient died of intestinal ischemia with evidence of underlying endotheliitis.

[0009] Recently, two proposed hemostatic mechanisms have provided insights into a better understanding of ARDS based on molecular pathogenesis associated with endothelial lesions that promote inflammation and coagulation disorders in sepsis and other critical illnesses (11–14): one is the “two-activation theory of the endothelium,” in which endothelial pathogenesis activates both inflammatory and microthrombus formation pathways; the other is a novel “two-path unifying theory” of hemostasis, in which hemostasis initiates thrombus formation and promotes microthrombus formation, leading to vascular microthrombus disease (VMTD) (11, 13, 15). These two theories are consistent because the endothelium contributes to the molecular mechanisms by which initial hemostasis triggers thrombus formation. ARDS is commonly associated with sepsis from a variety of different causes and has been observed in severe acute respiratory syndrome (SARS) caused by SARS-CoV (16), Middle East respiratory syndrome (MERS) caused by MERS-CoV (17), and now COVID-19. Sepsis-associated ARDS often develops alongside other organ dysfunctions, such as encephalopathy (18), liver failure (19)(20), acute renal failure, and acute necrotizing pancreatitis (21). This multi-organ involvement suggests that ARDS may not be the primary disease, but rather part of a persistent systemic pathogenesis triggered by infection or other critical illnesses.

[0010] Building on this, the underlying physiological alterations of multi-organ failure in sepsis and other critical illnesses have been identified as circulatory dysfunction caused by endothelial lesion-associated VMTD (EA-VMTD) (14, 15). Thus, infection triggers damage to the endothelium, leading to endothelial lesions. This subsequently results in disseminated microthrombosis (DIMT), which can trigger, for example, local hypoxemia, systemic hypoxia, and / or local ischemia, and as previously mentioned, it is now known that COVID-19 is associated with endotheliitis (10).

[0011] A case series of autopsies of the lungs in COVID-19 revealed numerous localized, platelet-rich microthrombi and hemorrhagic foci in the lungs, in addition to diffuse alveolar damage (22). The authors proposed that localized pulmonary thrombotic microangiopathy is key to the pathogenesis of COVID-19, and others have also suggested that microthrombus formation is a key driver of the disease process (23). These microcirculatory changes have been clearly demonstrated in the lungs, kidneys, and liver using contrast-enhanced ultrasound (24, 25). Similar findings have been observed in the brain (26). Thus, mounting evidence suggests that COVID-19 appears to cause endotheliitis and diffuse, widespread microthrombus formation.

[0012] The association between hypercoagulable states and COVID-19 is now widely accepted, and studies have shown that abnormal D-dimer levels, with higher levels associated with an increased odds ratio for more severe illness and in-hospital mortality (27, 28, 30). Several case reports have noted acute pulmonary embolism in COVID-19 pneumonia patients without a clear primary precipitating factor for venous thromboembolism (27, 31, 32). More recently, Panigada et al. showed that, in addition to elevated D-dimer levels, levels of coagulation factor VIII and von Willebrand factor (VWF) were also significantly elevated (33). Escher et al. (34) reported a COVID-19-related increase in VWF exceeding 500% and a coagulation factor VIII increase exceeding 350%. Furthermore, patients with thrombocytopenia have been shown to have a more than 5-fold increased risk of severe illness, and patients with the lowest platelet counts were associated with the highest mortality rates (33, 35, 36). Therefore, both hypercoagulable states and thrombocytopenia appear to be precursors to severe illness and mortality.

[0013] von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a crucial role in hemostasis and thrombosis, mediating platelet adhesion to damaged and activated blood vessels. It is synthesized only in megakaryocytes and endothelial cells (ECs), and interestingly, it has been noted that SARS-CoV can directly infect both of these cell types (22, 36).

[0014] Most of the vascular fibroblasts (VWFs) found in plasma originate from VWFs synthesized within the endocerebral cortex (EC) and stored in Weibel-Palade bodies (WPBs). Although confined to the EC, VWF synthesis varies across different vascular beds in the body, with higher levels of VWF expressed in small vessels of the lungs and brain than in similar-sized vessels in the liver or kidneys, and higher levels in venous ECs than in arterial ECs (37). The major component of VWFs stored in WPBs within endothelial cells consists of ultra-large VWFs (ULVWFs). These ultra-large VWF polymers are more adhesive than smaller VWF polymers in circulation (38). Upon secretion, ULVWFs can spontaneously bind to platelets. Inflammatory cytokines such as interleukin-1 and tumor necrosis factor (TNF)-α can trigger exocytosis of WPBs and release their contents. Therefore, plasma VWF levels can serve as a marker of endothelial activation and vascular inflammation, and elevated VWF levels have been shown to be associated with ARDS and sepsis, and independently associated with mortality (39, 40).

[0015] Following secretion from the EC, the secreted VWF (partially entering the circulation and partially binding to the endothelium) is sensitive to shear stress. This shear stress causes the VWF to unwind and expose sites for platelet binding, self-association, and cleavage by the enzyme ADAMTS13. These VWF molecules have previously been shown to self-associate and grow in "strings" along the flow direction (arteries and veins), binding platelets and adhering to the endothelium (41-43). The protease ADAMTS13 cleaves both VWF and ULVWF, perfusing these platelet-VWF strings onto them, leading to their rapid clearance from the circulation (41). The ULVWF polymers released from the WPB exhibit lower shear stress for unwinding and may therefore represent the molecules that initiate this self-assembly process, resulting in ultra-viscous strings capturing platelets. Platelet binding to VWF occurs via the GP Ib receptor at the A1 domain. When the VWF is in its globular form, the binding site of this receptor is typically not exposed, thus preventing platelet binding. Once the VWF unfolds due to shear stress, the binding site is exposed and binds to platelets with high affinity. Platelet binding to the VWF can induce a conformational change, leading to activation of integrin GPIIbIIIa (also known as α2bβ3) and promoting platelet-platelet and platelet-VWF cross-binding. Therefore, the use of standard antiplatelet drugs may be ineffective (aspirin or P2Y12 inhibitors) or only partially effective in alleviating this pathological process, as demonstrated by the cohort study by Tremblay et al. (44).

[0016] This ability to form VWF-rich platelet thrombi in microvessels 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 ULVWF-platelet chains (45). Furthermore, at least in cultured cells, ADAMTS13 synthesis was significantly inhibited by multiple cytokines, including IL-6 and TNF-α (46). This suggests that a cytokine storm, particularly IL-6, may propagate microthrombus formation. However, this also suggests that the disease may be more manageable and rapid deterioration of the patient's clinical condition may be avoided if early intervention is implemented and there is no surge in cytokine release.

[0017] There is now substantial evidence that there is a very significant imbalance in the VWF:ADAMTS13 ratio and the level of high molecular weight VWF polymers (equivalent to ULVWF) in COVID-19. As previously mentioned, very high VWF levels have been observed early on, with the earliest case report mentioning a surge in VWF levels being from Escher et al. (34). Subsequently, Goshua et al. (47) demonstrated that plasma VWF concentrations were significantly elevated in hospitalized COVID-19 patients, and that the elevation was correlated with disease severity—the mean VWF antigen level was 565 ± 199% in patients admitted to the intensive care unit (ICU) compared to 278 ± 133% in those not admitted to the ICU (p < 0.0001). Next, Rauch et al. (48) investigated the relationship between the progression of COVID-19 patients and their VWF levels at admission. Patients with the highest VWF levels required higher levels of oxygen support, while those with normal VWF levels did not require hospitalization or supplemental oxygen (n = 10).

[0018] Shortly after Rauch et al.'s work, Ladikou et al. (49) showed elevated VWF antigen levels in COVID-19 patients admitted to the ICU, with VWF levels positively correlated with patient age. They reported a median VWF antigen level of 350%, but crucially, they also showed a significant decrease in ADAMTS13 levels (49.7%), indicating a deficiency of the VWF-cleaving protease that typically degrades large VWF polymers and reduces their activity. They hypothesized that the excessive VWF release observed in COVID-19 patients led to ADAMTS13 depletion and contributed to a prothrombotic state. Further analysis of their data showed that the median VWF level in deceased patients (477%) was significantly higher than that in surviving patients (335%) (p=0.015).

[0019] Helms et al. (50) recently published a multicenter prospective cohort study in France assessing the risk of thrombosis in COVID-19 patients, showing a significant increase in VWF and coagulation factor VIII. In addition to these data showing increased VWF and decreased ADAMTS13, further research has shown a severely dysregulated VWF:ADAMTS13 ratio. Huisman et al. (51) first showed a mean VWF:ADAMTS13 ratio of 8.5 in 12 patients admitted to the ICU (normal range 0.5–2). Subsequently, Mancini et al. (52) demonstrated similar findings, showing an elevated ratio of von Willebrand factor antigen (VWF:Ag) to ADAMTS13 activity that was strongly correlated with disease severity, with the worst ratio of 8.3 in patients requiring high-intensity care (intubation and mechanical ventilation) compared to 3.42 in patients requiring low-intensity care (p<0.001).

[0020] Recently, Philippe et al. (53) published their findings on a cohort of 208 patients admitted to two centers in Paris, 23 of whom had only mild symptoms and were treated as outpatients. They found that only VWF:Ag was positively correlated with clinical severity, with significantly higher levels in critically ill patients (median 507%, IQR 428–596) than in non-critically ill patients (288%, 230–350, p < 0.0001) or COVID-19 outpatients (144%, 133–198, p = 0.007). In the univariate analysis model, a VWF:Ag level exceeding 423% at admission was significantly associated with higher in-hospital mortality (OR 89.7, 95% CI 25.9–567.4, p < 0.001), which remained highly significant in the multivariate analysis model adjusted for age, BMI, D-dimer, and C-reactive protein (CRP) (hazard ratio, OR 25.6, 95% CI 5.6–198.2, p < 0.001). More importantly, they showed that high molecular weight multimer (HMWM) VWF was significantly higher in critically ill patients than in non-critically ill patients (median ratio 1.18, IQR 0.86–1.09 vs. 0.96, 1.04–1.39, p < 0.001). In addition, HMWM level (ratio) (OR 116, 95% CI 10.2-1943, p < 0.001) was one of the factors most significantly associated with in-hospital mortality.

[0021] A unified theory could be developed that endothelial lesions and endotheliitis trigger the release of VWF and ULVWF, leading to microthrombus formation. This subsequently results in hypoxia, and the process can be exacerbated by a “cytokine storm” and the release of IL-6, which inhibits and reduces ADAMTS13 function, leading to a cascade of disseminated microthrombus formation and multiple organ dysfunction and failure. It has also been proposed that this microvascular thrombosis at the pulmonary level is the root cause of right ventricular dysfunction (54). This mechanism could explain many of the findings currently observed, including high D-dimer levels (elevated due to extensive microthrombus formation), high levels of coagulation factors VIII and VWF (released from WPB in response to endothelial injury), the observed microthrombus formation and atypical ARDS presentations (55), and the broad clinical presentation of pulmonary, neurological, and gastrointestinal symptoms. We believe that endotheliitis and microthrombosis may also explain why patients with pre-existing endothelial and microarterial lesions (e.g., secondary to diabetes, hypertension, or obesity) have an increased risk of severe COVID-19 (56). Similarly, mounting evidence links patients with low ADAMTS13 levels and high VWF levels to a variety of diseases and their different manifestations with poor prognosis after SARS-CoV-2 infection (57-64). Given that the interaction between VWF and platelets activates the GP2b3a receptor, standard antiplatelet drugs (aspirin or P2Y12 inhibitors) may be ineffective. Although inhibition of VWF-platelet binding via the GP1b receptor (using capsulbizumab or amphetamine) would be an attractive option and has been proposed (65), these drugs are not widely used and have very limited clinical experience. They also carry a significant risk of bleeding.

[0022] Therefore, treatment targeting the endothelial lesions themselves is necessary. Summary of the Invention

[0023] Heparin is a naturally occurring, highly sulfated polysaccharide characterized by a wide molecular weight range of its polysaccharide chains. Heparin acts on a variety of different ligands and exhibits diverse functions. A member of the glycosaminoglycan carbohydrate family, heparin consists of repeating disaccharide units of GlcAβ1-4GlcNAcα1-4, exhibiting polydisperse sulfated, N-acetylated, and uronic acid epimerization. Heparin is highly heterogeneous. Heparin can be isolated from natural sources, such as pig intestines or bovine lungs. Heparin isolated from natural sources contains polysaccharide chains with molecular weights ranging from about 3000 Da (g / mol) to about 30000 Da (g / mol). This is referred to as unfractionated heparin (UFH or UF heparin). UFH can be enzymatically or chemically treated to provide shorter polysaccharide chains. Heparinase I cleaves the polysaccharide chains at the α-1,4 bond between the unacetylated GlcNS6S and IdoA2S. Periodate treatment of UFH followed by base elimination cleaves the polysaccharide chains at the unsulfated uronic acid units. Products of chemically or enzymatically treated UFH can be affinity purified to produce fractionated heparin, in which the molecular weight of the polysaccharide in each fraction can be readily determined. Low molecular weight heparin (LMWH) contains polysaccharide chains with molecular weights ranging from about 4000 Da (g / mol) to about 8000 Da (g / mol).

[0024] In 1991, it was first demonstrated that intravenous administration of heparin during open-heart surgery induced 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 thrombin III, but rather dependent on the molecular weight of heparin. Subsequent in vitro experiments revealed that heparin binds to a specific amino acid sequence (residues 569-583) within the VWF A1 domain, where basic amino acids are regularly arranged. Heparin binding induces a conformational change in the peptide at this binding site (67). Heparin similarly binds to both activated and inactivated VWF, but does not interfere with VWF binding to collagen. Since the platelet GpIb binding domain (residues 524-542) is also located within the A1 domain, it was proposed that heparin inhibits platelet binding by interfering with VWF binding to platelet GpIb through steric hindrance and inducing conformational changes in this domain.

[0025] The structure-specificity of heparin responsible for binding VWF revolves around key disaccharide units—GlcNS6S-IdoA2S and IdoA2S-GlcNS6S. This structural unit is typically destroyed by heparinase I digestion, and its binding was successfully deduced using competitive binding assays of heparin fractions prepared by heparin-specific depolymerization methods, which produce fragments with predictable structures (68). Furthermore, studies using synthetic and structurally defined oligosaccharides have demonstrated that the assembly of more than three of these disaccharide units is crucial for binding efficacy. Similarly, although lower molecular weight (6100 Da(g / mol)) fractionated heparin has shown higher binding affinity for VWF, they are less effective at inhibiting VWF activity compared to UFH. This suggests that minimum heparin molecular weight and size are important for achieving steric hindrance.

[0026] Medium molecular weight heparin (MMWH) containing specific disaccharide units (GlcNS6S-IdoA2S, also written as IdoA2S-GlcNS6S) can be produced from unfractionated heparin. These MMWH fractions can have the specificity to inhibit VWF-GPIb binding, thereby preventing microthrombus formation, but they have little anticoagulant effect because they have almost no effect on thrombin III. Therefore, MMWH fractions of approximately 11,000 Da (g / mol) represent an ideal treatment option when considering treatment for patients with prothrombotic states dependent on elevated VWF levels and endothelial lesions. Furthermore, the results of these early studies suggest that low molecular weight heparin is unlikely to be effective and target GPIb receptors, and while UFH may contain a sugar moiety that can bind VWF, it is not optimal. In addition, monitoring UFH is difficult, and other fractions of UFH (e.g., LMWH fraction) have anticoagulant effects, which can lead to dangerous and unpredictable bleeding events.

[0027] Further interestingly, it has recently been shown that SARS-CoV-2 binds to heparan sulfate, and specifically requires the IdoA2S-GlcNS6S glycosyl moiety (74, 75). This suggests that exogenous supply of these glycosyl moieties can inhibit binding to endogenous heparan sulfate in the lungs, and thus could serve as a potential prophylactic treatment. In summary, a specialized medium molecular weight heparin (≈11000 Da(g / mol)) having at least three GlcNS6S-IdoA2S disaccharide units can inhibit viral adhesion and replication, and also inhibit microthrombus formation triggered by VWF release secondary to virus-induced endothelial lesions.

[0028] As described herein, endothelial lesions can be associated with a variety of diseases. As previously mentioned, the inventors have discovered that medium molecular weight heparin can be used to treat endothelial lesions, particularly those in patients with high plasma von Willebrand factor levels (WO2022 / 238587; incorporated herein by reference).

[0029] The inventors have now discovered that the reduced medium molecular weight heparin as described herein can be used to treat endothelial lesions, particularly endothelial lesions in patients with high plasma von Willebrand factor levels.

[0030] Therefore, in a first aspect, the present invention provides reduced medium molecular weight heparin (MMWH-Red). Preferably, MMWH-Red is prepared according to a second aspect of the present invention.

[0031] The medium molecular weight heparin can be used to treat endothelial diseases. The medium molecular weight heparin can inhibit von Willebrand factor (VWF). The medium molecular weight heparin can inhibit VWF polymers, preferably ultra-large VWF. The medium molecular weight heparin can inhibit the binding of platelets to VWF.

[0032] As described herein, the present invention also provides reduced medium molecular weight heparin for the treatment of endothelial lesions.

[0033] In a second aspect, the present invention provides a method for preparing reduced medium molecular weight heparin (MMWH-Red), comprising: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to a pH between about 5.0 and about 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of about 0°C to about 10°C to form a medium molecular weight heparin (MMWH) solution; and (d) incubating the medium molecular weight heparin with a reducing agent to produce MMWH-Red.

[0034] In a third aspect, the present invention provides a reduced medium molecular weight heparin produced according to the following steps: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to a pH between approximately 5.0 and approximately 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of approximately 0°C to approximately 10°C to form a medium molecular weight heparin solution; and (d) incubating the medium molecular weight heparin with a reducing agent to produce reduced medium molecular weight heparin.

[0035] In a fourth aspect, the present invention provides a composition comprising reduced medium molecular weight heparin.

[0036] In a fifth aspect, the present invention provides a reduced molecular weight heparin according to the first or third aspect, or MMWH-Red produced according to the second aspect, or a composition according to the fourth aspect, for the treatment of endothelial lesions. Preferably, the patient has endothelial lesions, characterized in that the ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen: ADAMTS13) is at least about 2. Alternatively or additionally, the patient with endothelial lesions may be characterized in that the ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen: ADAMTS13) is at least about 2.

[0037] In a sixth aspect, the present invention provides a reduced molecular weight heparin according to the first or third aspect, or MMWH-Red produced according to the second aspect, or a composition according to the fourth aspect, for treating a disease or condition of a patient, wherein the patient has endothelial lesions, characterized in that the ratio of plasma von Willebrand factor to ADAMTS13 (VWF: ADAMTS13) is at least about 2.

[0038] In a seventh aspect, the present invention provides a reduced molecular weight heparin according to the first or third aspect, or MMWH-Red produced according to the second aspect, or a composition according to the fourth aspect, for treating a patient with a disease or condition, wherein the patient has endothelial lesions, characterized in that the ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen: ADAMTS13) is at least about 2.

[0039] In an eighth aspect, the present invention provides a method for treating endothelial lesions, the method comprising administering a therapeutically effective amount of reduced medium molecular weight heparin to a subject (patient) in need of treatment. Preferably, the patient's plasma VWF:ADAMTS13 ratio is at least about 2. Preferably, the patient's plasma VWF antigen:ADAMTS13 ratio is at least about 2. Typically, the MMWH-Red may be an MMWH-Red according to the first or third aspect, an MMWH-Red produced according to the method of the second aspect, or a composition according to the fourth aspect.

[0040] In a ninth aspect, the present invention provides the use of reduced medium molecular weight heparin in the manufacture of a medicament for treating endothelial lesions in patients. Preferably, the patient's plasma VWF:ADAMTS13 ratio is at least about 2. Preferably, the MMWH-Red may be an MMWH-Red according to the first or third aspect, an MMWH-Red produced according to the method of the second aspect, or a composition according to the fourth aspect.

[0041] In a tenth aspect, the present invention provides the use of reduced medium molecular weight heparin in the manufacture of a medicament for treating endothelial lesions in patients. Preferably, the patient's plasma VWF antigen:ADAMTS13 ratio is at least about 2. Preferably, the MMWH-Red may be an MMWH-Red according to the first or third aspect, an MMWH-Red produced according to the method of the second aspect, or a composition according to the fourth aspect.

[0042] In an eleventh aspect, the present invention provides a kit suitable for preparing reduced medium molecular weight heparin, wherein the kit comprises: (a) unfractionated heparin; (b) an aqueous buffer solution adjusted to a pH between approximately 5.0 and approximately 9.0; (c) an oxidizing agent; (d) a reducing agent; and (e) an optional inactivating agent.

[0043] To avoid ambiguity, embodiments relating to each aspect of the invention are adapted to other aspects of the invention with necessary modifications. Further aspects and embodiments of the invention will become apparent from the following discussion. Attached Figure Description

[0044] Figure 1 The comparison shows that MMWH prepared at 4°C is compared with heparin treated at 37°C, untreated heparin, and 11 kDa standard.

[0045] Figure 2 The comparison shows that MMWH prepared using only periodate at 4°C is compared with heparin prepared first with periodate and then with NaOH at 4°C.

[0046] Figure 3 The molecular weight distribution diagram of the prepared medium molecular weight heparin is shown.

[0047] Figure 4 This is a graph showing the activity of low molecular weight (LMW) heparin, unfractionated (UF) heparin, and medium molecular weight (MMW) heparin against factor IIa.

[0048] Figure 5 The diagram shows the activity of LMW heparin, UF heparin, and MMW heparin against factor X.

[0049] Figure 6 For MMWH-Red and MMWH 1 H-NMR comparison.

[0050] Figure 7 Data on ritoxantrone-induced platelet aggregation (RIPA) of ungraded (UF) heparin samples (UF samples 1 and 2), three medium molecular weight heparins (MMWH samples 1, 2, and 3), and reduced medium molecular weight heparin (MMWH-Red) from three different blood donors. Mean values ​​were determined by experiments with at least technical reproducibility.

[0051] Figure 8 The mean amplitude percentages of UF heparin, three MMWH samples, and MMWH-Red measured in RIPA assays using blood from three different donors are given. The control group was sodium chloride solution. Each data point represents a biological replicate. Mean values ​​were calculated from three biological replicates. Numerical comparisons were performed using two-way ANOVA, and p-values ​​were calculated using Tukey's multiple comparison test. = P ≤ 0.05, = P ≤ 0.01, = P ≤ 0.001, = P ≤0.0001.

[0052] Figure 9 The mean slopes for UF heparin, three MMWH samples, and MMWH-Red measured in RIPA assays using blood from three different donors are given. The control group was sodium chloride solution. Each data point represents a biological replicate. The mean is calculated from the three biological replicates. Numerical comparisons were performed using two-way ANOVA, and p-values ​​were calculated using Tukey's multiple comparison test. = P ≤ 0.05, = P ≤ 0.01, = P ≤ 0.001, = P ≤ 0.0001.

[0053] Figure 10 The mean area under the curve for UF heparin, three MMWH samples, and MMWH-Red measured using blood from three different donors in a RIPA assay. The control group was sodium chloride solution. Each data point represents a biological replicate. The mean was calculated from the three biological replicates. Numerical comparisons were performed using two-way ANOVA, and p-values ​​were calculated using Tukey's multiple comparison test. = P ≤ 0.05, = P ≤ 0.01, = P ≤ 0.001, = P ≤0.0001.

[0054] Figure 11 The top graph shows the percentage inhibition of the mean amplitude of UF heparin, the three MMWH samples, and MMWH-Red measured in RIPA assays using blood from three different donors, compared to the control group. Each data point represents one biological replicate. The mean was calculated from three biological replicates. The bottom graph shows the percentage inhibition of the slope of the slope of UF heparin, the three MMWH samples, and MMWH-Red measured in RIPA assays using blood from three different donors, compared to the control group. Each data point represents one biological replicate. The mean was calculated from three biological replicates.

[0055] Figure 12 For -- the percentage inhibition of the area under the curve (AUC) of UF heparin, three MMWH samples, and MMWH-Red measured in RIPA assays using blood from three different blood donors, compared to the control group. Each data point represents a biological replicate. The mean was calculated from three biological replicates.

[0056] Detailed Implementation Plan Throughout this specification, one or more aspects of the invention may be combined with one or more features described in the specification to define different embodiments of the invention.

[0057] In the following discussion, many terms are referenced, and unless the context clearly indicates otherwise, these terms should be understood to have the meanings provided below.

[0058] References to singular nouns in this article include references to plural nouns, and vice versa, unless the context suggests otherwise.

[0059] Throughout this specification, the word “comprise” or variations thereof such as “comprises” or “comprising” will be understood to imply inclusion of the stated element or integer, or group of elements or integers, but does not exclude any other element or integer, or group of elements or integers. The term “comprising” includes, within its scope, the terms “consisting” or “consisting essentially of”.

[0060] The term “consisting” or variations thereof should be understood to imply inclusion of the stated element or integer, or group of elements or integers, and to exclude any other element or integer or group of elements or integers.

[0061] The term “consisting essentially of” or variations thereof should be understood to imply the inclusion of the stated element, integer or step, or group of elements, integers or steps, and that other components may be present, but only those that do not substantially affect the essential characteristics of the formulation, composition or compound.

[0062] The term "about" in this article, when used to modify numbers or values, refers to a value within ±5% of the specified value.

[0063] The terms "treatment" and "therapy" define therapeutic actions taken on a patient to slow or stop the progression of a disease or condition, or to improve or cure it. It also includes the prevention of disease or condition as a result of treatment or therapy.

[0064] As used in this article, the term "patient" preferably refers to a mammal. Typically, a mammal is a human.

[0065] Von von Willebrand factor (VWF) is a blood glycoprotein involved in hemostasis. VWF is a multimeric glycoprotein present in plasma, and is constitutively produced as super-large VWF in endothelial cells (Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and endothelial connective tissue. The basic VWF monomer is a protein of 2050 amino acids.

[0066] Disaccharides are sugars whose molecules contain two monosaccharide residues.

[0067] Low molecular weight heparin is defined in this paper as heparin with an average molecular weight of about 4,000 Da (g / mol) to about 8,000 Da (g / mol). Medium molecular weight heparin is defined in this paper as heparin with an average molecular weight greater than about 8,000 Da (g / mol) to about 13,500 Da (g / mol).

[0068] MMWH-Red as described herein contains no aldehyde group or contains fewer aldehyde groups than those produced during step (c) of the method in the second aspect. The presence or absence of an aldehyde group can be determined using a 2,4-dinitrophenylhydrazine test. MMWH-Red may not produce an orange-yellow precipitate upon reaction with 2,4-dinitrophenylhydrazine, or may produce an orange-yellow precipitate less than the corresponding amount of MMWH. Alternatively, the reduction of MMWH to MMWH-Red can be determined by infrared spectroscopy or... 1 H-NMR monitoring.

[0069] MMWH showed significantly reduced activity in routine anti-Xa and anti-IIa tests of heparin. It is speculated that the oxidation of glucuronide during MMWH preparation alters the binding affinity of heparin to antithrombin (ATIII). The ritoxromycin-induced platelet aggregation (RIPA) assay of MMWH showed increased activity, indicating that the interaction between heparin polysaccharides and von Willebrand factor remains intact.

[0070] The two aldehyde groups on the glucuronic acid of MMWH produced during the preparation process may be reactive under certain conditions. This reactivity may reduce the stability of the oxidized polysaccharide over time. Furthermore, when injected into the body, it may interact with free amines on proteins. The latter will react with the free amines via a Schiff base reaction, in the same manner as glucose reacts with hemoglobin to form the well-known glycated hemoglobin (commonly known as A1C).

[0071] Therefore, to mitigate these potential side reactions, in a first aspect, the present invention provides MMWH-Red, wherein these aldehyde groups are / have been reduced to hydroxyl groups. Preferably, MMWH-Red is prepared (produced) according to the method of the second aspect of the present invention. The method of the second aspect achieves this by using a reducing agent in the reduction step (d) to reduce MMWH (aldehyde form) to MMWH-Red (hydroxyl form).

[0072] The term "reduced" in the context of the molecular weight of heparin in the reduced form refers to the reduction of the aldehyde group formed in MMWH to a hydroxyl group by oxidizing one or more (or substantially all, or all) of the glucuronic acid moieties (in step (c) of the method). One or more (or substantially all, or all) of the glucuronic acid derivative moieties in MMWH-Red have the following structure: One or more (or substantially all, or all) glucuronic acid derivatives in MMWH have the following structure: Preferably, substantially all or all of the glucuronic acid portion in MMWH has this structure.

[0073] MMWH contains peaks corresponding to aldehyde groups in NMR spectra. MMWH contains peaks in the 9-10 ppm region of NMR spectra. MMWH contains peaks in the 9.20-9.30 ppm region of NMR spectra. MMWH exhibits activity comparable to UF heparin in ritoxormycin-induced platelet aggregation assays and has almost no activity in factor X analysis. MMWH reduces the amplitude observed in RIPA assays (compared to the control group) and has activity less than 5 IU / mg in factor X analysis. MMWH may have activity less than 5 IU / mg in factor X analysis.

[0074] MMWH can be characterized by molecular weight analysis. This can be performed using high-pressure size exclusion chromatography (HP-SEC). The ratio of M8000-16000 to M16000-24000 in MMWH can be greater than 2 and less than 10. MMWH can consist of less than 10%, preferably less than 8%, of polysaccharide chains with a mass greater than or equal to 24000 Da. MMWH can consist of 2% to 8% of polysaccharide chains with a mass greater than or equal to 24000 Da. MMWH can consist of 21% or less of polysaccharide chains with a mass between 16000 Da and 24000 Da. MMWH can consist of 14% to 21% of polysaccharide chains with a mass between 16000 Da and 24000 Da. MMWH can consist of 12% to 31% of polysaccharide chains with a mass equal to or less than 8000 Da.

[0075] MMWH-red may not contain an aldehyde peak in its NMR spectrum. MMWH-red may not contain a peak between approximately 9 and approximately 10 ppm corresponding to an aldehyde peak in its 1H-NMR spectrum. MMWH-red may not contain a peak above the background noise between approximately 9 and approximately 10 ppm corresponding to an aldehyde peak in its 1H-NMR spectrum.

[0076] MMWH-red may not contain the band corresponding to the aldehyde group (C=O) in its infrared spectrum. MMWH-red may not contain the bands from approximately 1740 to approximately 1720 cm⁻¹ corresponding to the aldehyde group (C=O) in its infrared spectrum. -1 The spectral bands between.

[0077] MMWH-red may contain an open-ring uronic acid having at least one hydroxyl group, preferably at least two hydroxyl groups. The medium molecular weight heparin may contain an open-ring uronic acid having one hydroxyl group, preferably two hydroxyl groups. The structure of MMWH-red can be analyzed using mass spectrometry, such as electrospray ionization-mass spectrometry.

[0078] MMWH-red can be characterized by molecular weight analysis. This can be performed using high-pressure size exclusion chromatography (HP-SEC). The ratio of M8000-16000 to M16000-24000 in MMWH-red can be greater than 2 and less than 10. MMWH-red can consist of less than 10%, preferably less than 8%, of polysaccharide chains with a mass greater than or equal to 24000 Da. MMWH-red can consist of 2% to 8% of polysaccharide chains with a mass greater than or equal to 24000 Da. MMWH-red can consist of 21% or less of polysaccharide chains with a mass between 16000 Da and 24000 Da. MMWH-red can consist of 14% to 21% of polysaccharide chains with a mass between 16000 Da and 24000 Da. MMWH-red can consist of 12% to 31% of polysaccharide chains with a mass equal to or less than 8000 Da.

[0079] The average molecular weight of the reduced heparin is greater than about 8,000 Da (g / mol) to about 13,500 Da (g / mol), more preferably greater than about 8,000 Da (g / mol) to about 13,000 Da (g / mol), more preferably about 9,000 Da (g / mol) to about 12,000 Da (g / mol), and more preferably about 10,000 Da (g / mol) to about 11,000 Da (g / mol).

[0080] MMWH-Red may contain polysaccharide chains with an average molecular weight ranging from about 8,000 Da (g / mol) to about 13,500 Da (g / mol), preferably from about 8,000 Da (g / mol) to about 13,000 Da (g / mol), and preferably from about 10,000 Da (g / mol) to about 12,000 Da (g / mol).

[0081] The average molecular weight of MMWH-Red can be determined by size exclusion chromatography as described herein.

[0082] MMWH-Red may contain at least three units of the GlcNS6S-IdoA2S (or IdoA2S-GlcNS6S) disaccharide. The GlcNS6S and IdoA2S monosaccharides are linked by an α1-4 bond, i.e., GlcNS6Sα1-4IdoA2S. For example, MMWH-Red may contain at least four, preferably at least five, preferably at least six, preferably at least eight, preferably at least ten units of the GlcNS6S-IdoA2S disaccharide. MMWH-Red may contain less than or equal to 25 units, for example, less than or equal to 20 units of the GlcNS6S-IdoA2S disaccharide. The presence of the GlcNS6S-IdoA2S disaccharide units can be determined by antibody testing, mass spectrometry, or inferred from chemical and enzymatic studies. The GlcNS6S-IdoA2S units may be arranged sequentially.

[0083] "IdoA" stands for α-L-iduronic acid. "IdoA2S" is IdoA modified by adding an O-sulfate group at carbon position 2 to form 2-O-sulfonyl-α-L-iduronic acid. "GlcNS" stands for 2-deoxy-2-sulfonamido-α-D-glucopyranosyl. "GlcNS6S" stands for 2-deoxy-2-sulfonamido-α-D-glucopyranosyl-6-O-sulfate. The α1-4 bond is an α-glycosidic bond between carbon-1 of a monosaccharide and carbon-4 of a second monosaccharide. The β1-4 bond is a β-glycosidic bond between carbon-1 of a monosaccharide and carbon-4 of a second monosaccharide.

[0084] MMWH-Red may contain UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc, wherein U may be iduronic acid (IdoA) or glucuronic acid (GlcA). 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%, and preferably at least about 60% UA2S-GlcNS6S. MMWH-Red may contain up to about 60%, preferably up to about 70%, and preferably up to about 85% UA2S-GlcNS6S. MMWH-Red 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 may contain up to about 15%, preferably up to about 20% UA2S-GlcNS. MMWH-Red may contain at least 4%, preferably at least 5%, preferably at least 6%, and preferably at least about 10% UA-GlcNAc. MMWH-Red may contain up to about 15%, preferably up to 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 more enriched than that of unfractionated heparin.

[0085] "UA" stands for uronic acid, which is a hexose sugar with a negatively charged carboxylic acid group at the 6-position. The uronic acid can be glucuronic acid or iduronic acid independently. "UA2S" is UA modified by adding an O-sulfate group at the 2-carbon position to form 2-O-sulfonyl-uronic acid. "GlcA" is β-D-glucuronic acid. "GlcNAc" is 2-deoxy-2-acetamido-α-D-glucopyranoyl.

[0086] MMWH-Red may include chemical modifications. The chemical modifications may be selected from the group consisting of N-acetylation, N-deacetylation, N-sulfation, O-sulfation, 2-O-desulfation, and complete desulfation.

[0087] There are few known methods for preparing MMWH and MMWH-Red. An example of a method for preparing medium molecular weight heparin is described in Poletti LF, Bird KE, Marques D, Harris RB, Suda Y, Sobel M. Structuralaspects 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 yields a variety of products with molecular weights ranging from 10600 g / mol to 1900 g / mol.

[0088] Therefore, a reliable method for preparing MMWH-Red is needed.

[0089] In a second aspect, the present invention provides a method for preparing MMWH-Red. MMWH-Red contains no aldehyde group, or contains fewer aldehyde groups than MMWH. The method in a third aspect comprises the steps of: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to a pH between approximately 5.0 and approximately 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of approximately 0°C to approximately 10°C to form an MMWH solution; and (d) incubating the MMWH with a reducing agent to produce MMWH-Red.

[0090] UF heparin can be obtained from bovine or swine tissues, such as swine intestines or bovine lungs.

[0091] A "buffer" is a chemical substance that resists changes in pH when an acid or base is added to the solution. Typically, a buffer solution (or buffer system) contains a weak acid and its conjugate base, or a weak base and its conjugate acid.

[0092] Typically, a suitable buffer contains an acid with a pKa value within ±1 of the required pH of the formulation. For example, if the required 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 acid molecule can contribute more than one proton), then for the buffer to be suitable, at least one pKa value should be within the required pH range.

[0093] A buffer is composed of a weak acid and its conjugate base (or a weak base and its conjugate acid) in equilibrium. According to Le Chatelier's principle (that if a constraint is imposed on a system in equilibrium, such as a change in reactant concentration, the equilibrium will shift to counteract the effect of that constraint), adding an acid or a base to the solution will shift the equilibrium towards the conjugate base or the weak acid, respectively. Therefore, the concentration of free protons in the formulation (and thus the pH) remains relatively constant.

[0094] Suitable buffering systems include acetate and acetic acid (pKa = 4.75), citrate and citric acid (pKa = 3.13, 4.76, and 6.40), and phosphoric acid (pKa = 2.14, 7.20, and 12.37), or mixtures thereof. Phosphate buffers can also be used. The pKa values ​​cited herein are reported values ​​measured in water at 25°C. Typically, the buffer contains only one of the above pairs, i.e., an acid and its conjugate base. The buffer may contain acetate and acetic acid, citrate and citric acid, or phosphate and phosphoric acid.

[0095] Adjust the pH to ensure that the pH of the first solution is between approximately pH 5.0 and approximately pH 9.0, as dissolving unfractionated heparin may cause pH changes in the aqueous buffer solution.

[0096] Optionally, in step (a), the aqueous buffer solution is adjusted to a pH between approximately 6.0 and approximately 8.0, more preferably to approximately 7.0. Typically, the temperature of the aqueous buffer solution in step (a) is approximately -2°C to approximately 4°C. Preferably, the temperature of the aqueous buffer solution in step (a) is approximately 0°C to approximately 2°C.

[0097] Typically, the aqueous buffer solution is a phosphate buffer, citrate buffer, or acetate buffer, meaning the buffer system is phosphate, citrate, or acetate. Preferably, the aqueous buffer solution is a phosphate buffer; more preferably, the aqueous buffer solution is a sodium phosphate buffer or potassium phosphate buffer, meaning the buffer system is sodium phosphate or potassium phosphate.

[0098] The buffer system in the aqueous buffer solution may be present at a concentration of about 10 mM to about 100 mM, more preferably about 20 mM to about 90 mM, more preferably about 30 mM to about 80 mM, more preferably about 40 mM to about 70 mM, and more preferably about 50 mM to about 60 mM. The buffer system in the aqueous buffer solution may be present at a concentration of about 50 mM.

[0099] The concentration of heparin in the aqueous buffer solution can be from about 0.5 mg / mL to about 10 mg / mL, more preferably from about 1 mg / mL to about 8 mg / mL, more preferably from about 1.5 mg / mL to about 6 mg / mL, more preferably from about 2 mg / mL to about 4 mg / mL. The concentration of heparin in the aqueous buffer solution can be from about 1.5 mg / mL, more preferably from about 1.8 mg / mL, more preferably from about 2 mg / mL, more preferably from about 2.5 mg / mL, more preferably from about 2.7 mg / mL, more preferably from about 3 mg / mL.

[0100] Alternatively, step (a) may include dissolving the UF heparin in an aqueous solution instead of an aqueous buffer solution. The aqueous solution may be water. The aqueous solution may consist of water or be substantially composed of water. Step (a) may include dissolving the unfractionated heparin (UF) in water. In this embodiment, the disclosure regarding aqueous buffer solutions also applies to aqueous solutions.

[0101] The oxidant may be periodate, such as sodium periodate or potassium periodate. Preferably, the oxidant is sodium periodate. Alternatively, the oxidant may be perchlorate, such as sodium perchlorate. Preferably, the oxidant does not contain perchlorate. Using a combination of perchlorate and periodate as an oxidant results in an increased level of sample decomposition into smaller molecular weight substances.

[0102] The concentration of the oxidant in the aqueous buffer solution can be from about 1 g / L to about 10 g / L. Preferably, the concentration of the oxidant can be from about 2 g / L to about 9 g / L, more preferably from about 4 g / L to about 8 g / L, and even more preferably from about 5 g / L to about 7 g / L. Preferably, the concentration of the oxidant is about 5.7 g / L. Preferably, the concentration of the oxidant is 5.7 g / L and the oxidant is sodium periodate.

[0103] The molar ratio of heparin to oxidant can be from about 1:1 to about 1:200, preferably from about 1:2 to about 1:150, more preferably from about 1:10 to about 1:100, more preferably from about 1:20 to about 1:50, and even more preferably from about 1:30 to about 1:40. Typically, the molar ratio of heparin to oxidant can be about 1:40 or typically about 1:150.

[0104] The molar ratio of UF heparin to oxidant can be determined based on the estimated molecular weight of typical heparin disaccharide, rather than the molecular weight of heparin. The estimated molecular weight of typical heparin disaccharide is 593.45 g / mol. This is used to approximate the number of moles of this disaccharide unit in the sample, thereby approximately calculating the number of moles of uronic acid residues present in the sample.

[0105] For example, if you want to add 1 equivalent of NaIO4 based on uronic acid and use 3 grams of heparin, the number of moles of NaIO4 to be added is 0.005 moles, which is 3 divided by 593.45.

[0106] Therefore, the ratio of UF heparin determined based on the estimated molecular weight of typical heparin disaccharide can be from about 1:0.5 to about 1:10, preferably from about 1:0.7 to about 1:8, more preferably from about 1:0.9 to about 1:5, more preferably from about 1:1 to about 1:2, and even more preferably from about 1:1.1 to about 1:2. Preferably, the ratio of the molar amount of uronic acid present in UF heparin to the molar amount of oxidant can be 1:1.1.

[0107] Typically, the incubation temperature in step (c) is from about 0°C to about 10°C, more preferably from about 1°C to about 9°C, more preferably from about 2°C to about 8°C, more preferably from about 3°C ​​to about 7°C, and more preferably from 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 from about 4 hours to about 36 hours, more preferably from about 8 hours to about 30 hours, more preferably from about 12 hours to about 24 hours, more preferably from about 15 hours to about 20 hours, and more preferably from about 16 hours to about 18 hours.

[0108] The incubation step (c) of the second aspect of the invention can be performed in a laboratory refrigerator set at the desired temperature. The laboratory refrigerator can 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, and even more preferably about 4°C to about 6°C. Preferably, the laboratory refrigerator can be set at a temperature of about 4°C.

[0109] The second aspect of the method may further include the step of inactivating the oxidant in the MMWH solution or MMWH-Red solution. The oxidant may be inactivated by adding an inactivating agent selected from the group consisting of: methanol, D-mannitol, glycerol, N-acetylmethionine, sodium sulfite, and combinations thereof. Particularly preferred inactivating agents are methanol or D-mannitol.

[0110] The molar ratio of oxidant to inactivator can be from about 1:1 to about 1:10, more preferably from about 1:2 to about 1:8, more preferably from about 1:3 to about 1:6, and even more preferably from about 1:4 to about 1:5. Typically, the molar ratio of oxidant to inactivator can be about 1:2 or about 1:4.

[0111] The reduction step (d) includes incubating MMWH with a reducing agent to produce MMWH-Red. Preferably, the reducing agent is a mild reducing agent. The reducing agent may be sodium borohydride (NaBH4), sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3), or potassium borohydride (KBH4).

[0112] 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).

[0113] Typically, the amount of the reducing agent used relative to medium molecular weight heparin is greater than about 0.5 molar equivalents, preferably greater than about 1 molar equivalent, more preferably greater than about 2 molar equivalents, more preferably greater than about 3 molar equivalents, and more preferably greater than about 5 molar equivalents. Typically, the amount of the reducing agent used relative to medium molecular weight heparin is less than about 15 molar equivalents, preferably less than about 12 molar equivalents, more preferably less than about 10 molar equivalents, more preferably less than about 8 molar equivalents, more preferably less than about 6 molar equivalents, and more preferably less than about 5 molar equivalents. The amount of the reducing agent used can be between about 0.5 molar equivalents and about 15 molar equivalents, preferably between about 1 molar equivalents and about 10 molar equivalents, more preferably between about 2 molar equivalents and about 6 molar equivalents. The amount of the reducing agent used can be between about 0.5 molar equivalents and about 15 molar equivalents, preferably between about 1 molar equivalents and about 10 molar equivalents, and more preferably between about 2 molar equivalents and about 6 molar equivalents.

[0114] Preferably, step (d) is performed at about 0°C to about 30°C, more preferably about 10°C to about 30°C, more preferably about 5°C to about 25°C, more preferably about 10°C to about 20°C, more preferably about 15°C to about 25°C, and more preferably about 20°C to about 25°C. Typically, step (d) is performed at about 20°C. Typically, step (d) is performed at room temperature. Typically, step (d) is performed for about 1 hour to about 24 hours, preferably about 2 hours to about 16 hours, more preferably about 3 hours to about 12 hours, and more preferably about 6 hours to about 10 hours.

[0115] The solvent in step (d) is typically selected from the group consisting of methanol, ethanol, water, THF, or combinations thereof. Preferably, the solvent in step (h) is water.

[0116] MMWH-Red can be purified by any suitable method known to those skilled in the art. Therefore, the method may further include a step of purifying MMWH-Red. For example, MMWH-Red can be purified by thorough dialysis using phosphate buffer (pH = 7.0) or saline, by using a desalting column (e.g., Sephadex G-25 with phosphate buffer (pH = 7.0) or saline as the mobile phase), or by precipitation of MMWH-Red.

[0117] Therefore, the method of the second aspect may further include dialyzing the MMWH solution in a dialysate to provide a dialyzed MMWH sample, and / or dialyzing the MMWH-Red in a dialysate to provide a dialyzed MMWH-Red sample.

[0118] Dialysis, or dialysis, is the process of separating molecules in solution by utilizing the difference in diffusion rates as molecules pass through a semipermeable membrane (such as a dialysis tube). The sample to be dialyzed and the dialysate (or buffer) are placed on opposite sides of the semipermeable membrane. Target sample molecules larger than the membrane pore size (such as proteins, DNA, or polysaccharides) remain on the sample side of the membrane. Small molecules and contaminants such as salts can pass through the membrane into the dialysate, thus reducing the concentration of contaminants in the sample to a low level. Replacing the dialysate with fresh dialysate removes contaminants that have transferred from the sample into the dialysate. This allows more contaminants to diffuse from the sample into the dialysate.

[0119] 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 molecular weight. The semipermeable membrane is typically made of regenerated cellulose or cellulose ester membrane.

[0120] Dialysis is performed by placing a dialysis tube containing a sample into a dialysis solution. The dialysis solution is the liquid through which substances pass from the dialysis tube. The dialysis solution can be changed frequently as needed to achieve optimal separation. Dialysis can be performed for approximately 1 to 14 days, preferably approximately 5 to 10 days, and more preferably approximately 7 days. The dialysis solution can be changed approximately once to 10 times per day, preferably approximately 2 to 5 times per day, and more preferably approximately 3 times per day. Typically, the volume of the dialysis solution is several times the volume of the sample, for example, approximately 2 to 500 times the sample volume. The volume of the dialysis solution can be approximately 4 times the sample volume.

[0121] Typically, the dialysate is water. Optionally, the dialysate may contain electrolytes such as sodium, potassium, magnesium, calcium, chloride ions, bicarbonate, lactate, glucose, amino acids, or combinations thereof.

[0122] The dialysis procedure can be performed in dialysis tubes with a 2 kD molecular weight cutoff, such as those made by Spectra / Por ® Provided. Those skilled in the art will recognize appropriate tube cut-off sizes for different purposes. Alternatively, the dialysis procedure can be performed in a dialysis apparatus or dialyzer. A suitable dialyzer may be a Slide-A-Lyzer. TM The product lines include Float-A-Lyzer, Pur-A-lyzer, D-Tube, and GeBAflex dialyzers.

[0123] The second aspect of the method may further include the steps of separating MMWH from a dialyzed heparin sample and / or separating MMWH-Red from a dialyzed MMWH-Red sample. The MMWH and / or MMWH-Red may be separated from the dialyzed sample by freeze-drying, centrifugation, or filtration. Preferably, the MMWH and / or MMWH-Red are separated from the dialyzed sample by freeze-drying.

[0124] Freeze drying (also known as lyophilization or cryogenic drying) is a drying process carried out at low temperatures. Freeze drying typically involves lowering the temperature and pressure below the triple point of the substance and removing the frozen solvent (e.g., water ice) by sublimation. For aqueous compositions, such as those disclosed herein, freeze drying can be carried out at temperatures of about -20°C to about -80°C, preferably about -40°C, and pressures of about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).

[0125] The second aspect of the method may include precipitating the reduced medium molecular weight heparin to provide a precipitate of reduced medium molecular weight heparin in the precipitation mixture. This precipitation step produces pure MMWH-Red. This precipitation step can be used as an alternative to the aforementioned dialysis.

[0126] Precipitating the reduced medium molecular weight heparin to provide a reduced medium molecular weight heparin precipitate in a precipitation mixture may include adding a precipitating agent to the reduced medium molecular weight heparin solution to provide a reduced medium molecular weight heparin precipitate in the precipitation mixture.

[0127] Preferably, the precipitant may contain or consist of alcohol or acetone.

[0128] Surprisingly, the precipitant precipitates the reduced medium molecular weight heparin. The precipitated reduced medium molecular weight heparin can then be separated from the precipitate mixture, for example, by filtration. Therefore, the method may further include the step of separating the precipitated reduced medium molecular weight heparin from the precipitate mixture.

[0129] This precipitation procedure advantageously enables the production and purification of reduced molecular weight heparin with higher efficiency and purity than known methods. This results in a highly purified material that does not require purification, for example, by column chromatography.

[0130] Preferably, the precipitant is added to the MMW-Red solution so that the final concentration of the precipitant in the precipitation mixture is about 50% v / v to about 90% v / v, preferably about 55% v / v to about 80% v / v, more preferably about 60% v / v to about 75% v / v. Preferably, the final concentration of the precipitant in the precipitation mixture is about 73% v / v. A precipitant concentration of about 73% v / v (e.g., methanol) results in good sedimentation of the precipitate, which can be separated into an easily filterable powder.

[0131] The alcohol may be a C1 to C3 alcohol. For example, the alcohol may be methanol, ethanol, propanol, or isopropanol. Preferably, the alcohol is methanol. Preferably, the alcohol is added to the MMWH-Red solution to achieve a final alcohol concentration in the precipitate mixture of about 50% v / v to about 90% v / v, preferably about 55% v / v to about 80% v / v, more preferably about 60% v / v to about 75% v / v. Preferably, the final alcohol concentration in the precipitate mixture is about 73% v / v. For example, adding 73 mL of alcohol (e.g., methanol) to 27 mL of MMWH-Red solution provides a final alcohol concentration of 73% v / v MeOH in the precipitate mixture. An alcohol concentration of about 73% v / v (e.g., methanol) results in good sedimentation of the precipitate, which can be separated into an easily filterable powder.

[0132] The resulting precipitate is a processable, filterable powder that can be easily separated by filtration and subsequently dried under vacuum.

[0133] During the addition of the precipitant, the temperature of the solution can be maintained between about 0°C and about 15°C, preferably between about 5°C and about 10°C. The precipitate mixture can be stirred for about 1 minute to about 30 minutes, preferably about 5 minutes to about 25 minutes, more preferably about 10 minutes to about 20 minutes. It is preferable to stir the precipitate mixture for about 10 minutes. After stirring, it is preferable to allow the precipitate to settle.

[0134] In some embodiments, a salt is added to the MMWH-Red solution prior to precipitation of MMWH-Red. The step of precipitating MMWH-Red may include adding a salt to the MMWH-Red solution, followed by the addition of a precipitating agent. As described above, the precipitating agent may consist of, or substantially consist of, an alcohol or acetone, or contain an alcohol or acetone.

[0135] The salt may be a lithium salt, sodium salt, or potassium salt. Preferably, the salt is a sodium salt. The salt may be lithium chloride, sodium chloride, or potassium chloride. Preferably, the salt is sodium chloride.

[0136] The mass of salt added to the MMWH-Red solution is chosen such that the final salt concentration in the MMWH-Red solution is from about 0.5% m / v to about 3% m / v, preferably from about 1% m / v to about 2.5% m / v, more preferably from about 1.5% to about 2% m / v. Preferably, the salt concentration is about 1.8% m / v. For example, adding 0.9 g of solid NaCl to 50 mL of MMWH-Red solution provides a NaCl concentration of 1.8% m / v. Most preferably, the salt solution contains sodium chloride at a concentration of 1.8% m / v.

[0137] After adding salt, the resulting solution can be cooled to about 5°C to about 20°C, preferably about 10°C to about 15°C. Preferably, the resulting solution can be cooled to about 15°C. Then a precipitant can be added.

[0138] If necessary, in the method of the second aspect, the MMWH intermediate in step (c) can be purified by precipitation to form precipitated MMWH, as described above. The addition of a precipitant can also be used to quench any remaining oxidant. In this embodiment, the reduction step (d) is performed on the precipitated MMWH.

[0139] The method may include an alkali elimination step after step (c). Alternatively, the method may exclude an alkali elimination step after step (c). Alkali elimination may be performed using an alkali metal salt such as sodium hydroxide, potassium hydroxide, or lithium hydroxide. The alkali elimination step may be performed by raising the pH of the medium molecular weight heparin solution to approximately pH 10 to approximately pH 14, preferably approximately pH 12, by adding an alkali metal salt for approximately 10 minutes to approximately 3 hours, preferably approximately 30 minutes, at approximately room temperature.

[0140] Preferably, the method does not include an alkali 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 alkali elimination step or the addition of an alkali metal salt produce medium molecular weight heparin to prepare reduced medium molecular weight heparin as defined herein, which exhibits very low activity against factors IIa and / or Xa compared to UF heparin and low molecular weight heparin.

[0141] MMWH and / or MMWH-Red can be characterized by NMR, disaccharide analysis, ritoxadixyl-induced platelet aggregation (RIPA), and factor X analysis. This method is applicable to the preparation of milligram, gram, or kilogram-level MMWH-Red.

[0142] Advantageously, the second method reliably provides MMWH-Red with good purity and reduced degradation.

[0143] The average molecular weight of the MMWH-Red prepared by the method of the second aspect is greater than about 8000 Da (g / mol) to about 13500 Da (g / mol), more preferably greater than about 8000 Da (g / mol) to about 13000 Da (g / mol), more preferably about 9000 Da (g / mol) to about 12000 Da (g / mol), and more preferably about 10000 Da (g / mol) to about 11000 Da (g / mol).

[0144] The MMWH-Red prepared by the method of the second aspect of the present invention may contain polysaccharide chains with an average molecular weight ranging from more than about 8000 Da (g / mol) to about 13500 Da (g / mol), preferably more than about 8000 Da (g / mol) to about 13000 Da (g / mol), and preferably from about 10000 Da (g / mol) to about 12000 Da (g / mol).

[0145] The average molecular weight of MMWH-Red prepared by the method of the second aspect of the present invention can be determined by size exclusion chromatography as described herein.

[0146] The MMWH-Red prepared by the method of the second aspect may contain at least three units of the GlcNS6S-IdoA2S (or IdoA2S-GlcNS6S) disaccharide. The GlcNS6S and IdoA2S monosaccharides are linked by an α1-4 bond between GlcNS6S and IdoA2S, i.e., GlcNS6Sα1-4IdoA2S. For example, the MMWH-Red prepared by the method of the second aspect may contain at least four units, preferably at least five units, preferably at least six units, preferably at least eight units, preferably at least ten units of the GlcNS6S-IdoA2S disaccharide. The MMWH-Red prepared by the method of the second aspect may contain less than or equal to 25 units of the GlcNS6S-IdoA2S disaccharide, for example, less than or equal to 20 units. The presence of the GlcNS6S-IdoA2S disaccharide units can be determined by antibody testing, mass spectrometry, or inferred from chemical and enzymatic studies. The GlcNS6S-IdoA2S units may be arranged sequentially.

[0147] The MMWH-Red prepared by the method of the second aspect may contain UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc, wherein U may be iduronic acid (IdoA) or glucuronic acid (GlcA). The medium molecular weight heparin prepared by the method of the second aspect may contain at least about 60% UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc. The MMWH-Red prepared by the method of the second aspect 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 MMWH-Red prepared by the method of the second aspect may contain up to about 60%, preferably up to about 70%, and preferably up to about 85% UA2S-GlcNS6S. The MMWH-Red prepared by the method of the second 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 MMWH-Red prepared by the method of the second aspect may contain up to about 15%, preferably up to about 20%, of UA2S-GlcNS. The MMWH-Red prepared by the method of the second aspect may contain at least 4%, preferably at least 5%, preferably at least 6%, preferably at least about 10%, of UA-GlcNAc. The MMWH-Red prepared by the method of the second aspect may contain up to about 15%, preferably up to about 20%, of UA-GlcNAc. In some embodiments, the MMWH-Red prepared by the method of the second aspect may contain at least 49.2% UA2S-GlcNS6S, 5.4% UA2S-GlcN, and 5.4% UA-GlcNAc. In some embodiments, the MMWH-Red prepared by the method of the second aspect may contain at least 82% UA2S-GlcNS6S, 9% UA2S-GlcNS, and 9% UA-GlcNAc. The percentage composition of UA-GlcNAc contained in MMWH-Red is comparable to that of ungraded heparin.

[0148] The synthesis of MMWH-Red may consist of or substantially consist of the following steps: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to a pH of approximately 5.0 to approximately 9.0 to provide a first solution; (b) adding an oxidizing agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of approximately 0°C to approximately 10°C to form an MMWH solution; (d) incubating the medium molecular weight heparin with a reducing agent to produce MMWH-Red; and (e) purifying MMWH-Red.

[0149] The synthesis method of MMWH-Red may consist of or substantially consist of the following steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to a pH between approximately 5.0 and approximately 9.0 to provide a first solution; (b) adding an oxidant to the first solution to provide a second solution; (c) incubating the second solution at a temperature of approximately 0°C to approximately 10°C to form a medium molecular weight heparin solution; (d) inactivating the oxidant in the MMWH solution; (e) incubating the medium molecular weight heparin with a reducing agent to produce MMWH-Red; and (f) purifying MMWH-Red.

[0150] The synthesis of MMWH-Red may consist of or substantially consist of the following steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to a pH of approximately 5.0 to approximately 9.0 to provide a first solution; (b) adding an oxidant to the first solution to provide a second solution; (c) incubating the second solution at a temperature of approximately 0°C to approximately 10°C to form a medium molecular weight heparin solution; (d) inactivating the oxidant in the medium molecular weight heparin solution; (e) dialyzing the MMWH solution in a dialysate to provide a dialyzed MMWH sample; (f) incubating the dialyzed MMWH with a reducing agent to produce MMWH-Red; and (g) purifying MMWH-Red.

[0151] The synthesis of MMWH-Red may consist of or substantially consist of the following steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to a pH of approximately 5.0 to approximately 9.0 to provide a first solution; (b) adding an oxidant to the first solution to provide a second solution; (c) incubating the second solution at a temperature of approximately 0°C to approximately 10°C to form an MMWH solution; (d) inactivating the oxidant in the MMWH solution; (e) dialyzing the MMWH solution in a dialysate to provide a dialyzed MMWH sample; (f) separating MMWH from the dialyzed heparin sample; (g) incubating MMWH with a reducing agent to produce MMWH-Red; and (h) purifying MMWH-Red.

[0152] To avoid ambiguity, embodiments relating to the first aspect of the invention, with necessary modifications, are applicable to the second aspect of the invention, and embodiments relating to the second aspect of the invention, with necessary modifications, are applicable to the first aspect of the invention.

[0153] As described herein, MMWH-Red inhibits von Willebrand factor and is therefore an active pharmaceutical ingredient (API) and medicine suitable for the purposes of this invention.

[0154] Advantageously, MMWH-Red exhibits higher stability as an API and in pharmaceutical products compared to MMWH. Furthermore, MMWH-Red reduces the likelihood of adverse reactions with excipients contained in pharmaceutical products, and it also reduces interactions with proteins and / or components in the bloodstream.

[0155] MMWH-Red contains no aldehyde group or contains fewer aldehyde groups than those produced during step (c) of the method in the second aspect. The presence or absence of aldehyde groups can be determined using a 2,4-dinitrophenylhydrazine test. MMWH-Red may not produce an orange-yellow precipitate when reacting with 2,4-dinitrophenylhydrazine, or may produce an orange-yellow precipitate less than the corresponding amount of starting material. Alternatively, the reduction of MMWH to MMWH-Red can be determined by infrared spectroscopy or 1 H-NMR monitoring.

[0156] MMWH-Red can be characterized by NMR, disaccharide analysis, ritoxadrine-induced platelet aggregation (RIPA), and factor Xa analysis.

[0157] In a third aspect, the present invention provides MMWH-Red produced according to the following steps: (a) Dissolve unfractionated (UF) heparin in an aqueous buffer solution adjusted to a pH between approximately 5.0 and approximately 9.0 to provide a first solution; (b) Add an oxidizing agent to the first solution to provide a second solution; (c) Incubate the second solution at a temperature of about 0°C to about 10°C to form an MMWH solution; and (d) Incubate the medium molecular weight heparin with a reducing agent to produce MMWH-Red.

[0158] To avoid ambiguity, embodiments related to the first and second aspects of the present invention are adapted to the third aspect of the present invention with necessary modifications.

[0159] In a fourth aspect, the present invention provides a composition comprising MMWH-Red. Preferably, the composition is a pharmaceutical composition.

[0160] The pharmaceutical composition may include excipients. The excipients may be selected from the group consisting of solvents, solubilizers, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavoring agents, lubricants, suspending agents, tension modifiers, surfactants, solubilizers, suspending agents, dispersants, humectants, thickeners, colorants, wetting agents, defoamers, viscosity modifiers, sweeteners, and combinations thereof.

[0161] The pharmaceutical composition may contain an additional active agent, which may be an API. The additional active agent may comprise a composition of substances with physiological effects. The additional active agent may comprise low molecular weight heparin, or MMWH or MMWH-Red with different disaccharide compositions. The additional active agent may be selected from the group comprising farnesoid X receptor (FXR) agonists, peroxisome proliferator-activated receptor (PPAR) agonists, arachnoidol, caspase inhibitors, galactoglobulin 3 inhibitors, mitogen-activated protein kinase 5 (MAPK5) inhibitors, fibroblast growth factor 19 (FGF19) agonists, FGF21 agonists, leukotriene D4 (LTD4) receptor antagonists, nicotinic acid analogs, apical sodium-dependent bile acid transporter (ASBT) inhibitors, apoptosis signal-regulated kinase 1 (ASK1) inhibitors, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, chemokine receptor inhibitors, thiazolidinediones, GLP-1 analogs, biguanides, HIV replication inhibitors, metformin, opioids, anesthetics, HMG-CoA reductase inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), or any combination thereof.

[0162] To avoid ambiguity, the embodiments related to the first to third aspects of the present invention are applied to the fourth aspect of the present invention after necessary modifications.

[0163] As described in this article, endothelial lesions can be associated with a variety of diseases. MMWH-Red inhibits von Willebrand factor. The inventors have discovered that MMWH-Red can be used to treat endothelial lesions, particularly in patients with high plasma von Willebrand factor levels.

[0164] When heparin is exposed to oxidants (e.g., IO4) - When periodate ions are present, the diol moiety of glucuronic acid is cleaved to form two aldehyde groups, leading to the formation of MMWH, as described herein. The result is a significant reduction in the activity shown in routine anti-Xa and anti-IIa tests of heparin. It is speculated that this oxidation of glucuronic acid alters the binding affinity of heparin to antithrombin III (ATIII).

[0165] However, ritoxadixyl-induced platelet aggregation (RIPA) assays showed no change or increase in MMWH activity. This suggests that the interaction between heparin polysaccharide and von Willebrand factor remains intact and may even be enhanced.

[0166] As described in this article, the reduction step (d) means that the cyclic ring of the glucuronide monosaccharide no longer forms, and the binding of MMWH-Red to ATIII remains unchanged. However, the reduced MMWH will still maintain its interaction with von Willebrand factor and can be used to treat endothelial lesions.

[0167] The advantages of MMWH-Red include: maintaining the activity of von Willebrand factor in MMWH, having higher stability as an active pharmaceutical ingredient (API) and drug, minimal interaction with drug excipients, reduced or no reaction with proteins and / or components in the blood, and minimizing side effects.

[0168] Therefore, in a fifth aspect, the present invention provides MMWH-Red according to the first or third aspect of the invention, MMWH-Red produced according to the second aspect, or a composition according to the fourth aspect of the invention, for treating endothelial lesions in patients.

[0169] MMWH-Red inhibits von Willebrand factor (VWF). MMWH-Red inhibits VWF multimers, preferably ultra-large VWF. MMWH-Red inhibits the binding of platelets to VWF.

[0170] Preferably, the present invention provides MMWH-Red or a composition comprising MMWH-Red for treating endothelial lesions in patients. Preferably, the present invention provides MMWH-Red according to the first or third aspect, or MMWH-Red produced according to the second aspect, or a composition according to the fourth aspect, for treating endothelial lesions in patients with a plasma von Willebrand factor antigen to ADAMTS13 ratio of at least about 2. Preferably, the present invention provides MMWH-Red according to the first or third aspect, or MMWH-Red according to the second aspect, or a composition according to the fourth aspect, for treating endothelial lesions in patients with a plasma von Willebrand factor to ADAMTS13 ratio of at least about 2.

[0171] The patient's VWF:ADAMTS13 ratio may be at least about 2, preferably at least about 4, more preferably at least about 8, and even more preferably at least about 10. The patient's VWF:ADAMTS13 ratio may be greater than about 2, preferably greater than about 4, more preferably greater than about 8, and even more preferably greater than about 10. The patient's VWF:ADAMTS13 ratio may be about 2-16, preferably about 4-12, and even more preferably about 6-10. Patients with a VWF:ADAMTS13 ratio greater than about 8 generally indicate severe disease and typically indicate that the patient is deteriorating and heading towards death.

[0172] The patient's VWF antigen:ADAMTS13 ratio may be at least about 2, preferably at least about 4, more preferably at least about 8, and even more preferably at least about 10. The patient's VWF antigen:ADAMTS13 ratio may be greater than about 2, preferably greater than about 4, more preferably greater than about 8, and even more preferably greater than about 10. The patient's VWF antigen:ADAMTS13 ratio may be about 2-16, preferably about 4-12, and even more preferably about 6-10. Patients with a VWF antigen:ADAMTS13 ratio greater than about 8 generally indicate severe disease and often indicate that the patient is deteriorating and heading towards death.

[0173] The levels of VWF and ADAMTS13 in patients can be measured using ELISA. The ratio can be calculated as described by Huisman et al. (Involvement of ADAMTS13 and von Willebrand factor in thromboembolic events in patients infected with SARS-CoV-2. Int J Lab Hematol. 2020 Oct;42(5):e211-2). In short, the levels of VWF antigen and ADAMTS13 can be measured in International Units (IU), and then the VWF antigen:ADAMTS13 ratio is determined.

[0174] The normal range of plasma VWF levels is approximately 50 IU / dL to approximately 200 IU / dL. The average plasma VWF level in the general population is approximately 100 IU / dL. High plasma VWF levels are approximately 200 IU / dL or higher, for example, approximately 200 IU / dL to approximately 400 IU / dL, approximately 225 IU / dL to approximately 375 IU / dL, approximately 250 IU / dL to approximately 350 IU / dL, and approximately 275 IU / dL to approximately 300 IU / dL.

[0175] The patient may have an elevated VWF antigen level of about 150% or higher, preferably about 175% or higher, more preferably about 200% or higher, more preferably about 300% or higher, more preferably about 350% or higher, more preferably about 400% or higher, and more preferably about 500% or higher. The patient's VWF antigen level may be as high as about 600%, preferably as high as about 700%, more preferably as high as about 800%, and more preferably as high as about 1000%.

[0176] It should be noted that plasma VWF levels can be temporarily elevated due to infection, inflammation, trauma, and physical and emotional stressors. Therefore, the patient may have a non-transiently elevated plasma von Willebrand factor (VWF) level, for example, lasting for at least about six hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours. Preferably, the patient may have an elevated plasma VWF level lasting for at least about one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least seven days. More preferably, the patient may have an elevated plasma VWF level lasting for at least one week, at least two weeks, at least three weeks, or at least four weeks. Even more preferably, the patient may have an elevated plasma VWF level lasting for at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, or at least one year. The patients may have elevated plasma von Willebrand factor levels for up to one week, up to four weeks, up to two months, up to four months, up to six months, or up to one year.

[0177] The mass of MMWH-Red can be approximately 11,000 Da (g / mol). The molecular weight heparin in the reduced form can contain at least three units of GlcNS6S-IdoA2S (or IdoA2S-GlcNS6S) disaccharide.

[0178] The endothelial lesions can be caused by any disease or condition. In particular, the endothelial lesions can be caused by COVID-19 (e.g., acute COVID-19 or post-COVID syndrome), infection, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, sepsis, septicemia, cardiovascular disease, diabetes, trauma, especially brain or head trauma (e.g., traumatic brain injury), burns, inhalation injury, drugs and drug reactions, hematological disorders, subarachnoid hemorrhage (e.g., aneurysmal subarachnoid hemorrhage), aneurysmal disease, stroke, cerebral hemorrhage, radiation-induced injury, ischemic stroke, pancreatitis, liver disease, kidney disease, or chronic obstructive pulmonary disease (COPD) or a combination thereof.

[0179] The endothelial lesions can be caused by viral infection, optionally SARS-CoV-2. The endothelial lesions can also be caused by cancer, particularly leukemia, lymphoma, myeloma, or solid organ cancers such as colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer, or skin cancer.

[0180] The infection can be bacterial, fungal, or parasitic. The infection can be bacterial. The bacterial infection can include *Actinomyces isatis*, *Bacillus anthracis*, *Bacteroides fragilis*, *Bordezoae pertussis*, *Bretorius burgdorferi*, *Bretorius gauze*, *Bretorius auriculata*, *Treponema pallidum*, *Brucella abortus*, *Brucella canis*, *Brucella martatus*, *Brucella suis*, *Campylobacter jejuni*, *Chlamydia pneumoniae*, *Chlamydia trachomatis*, *Chlamydia psittaci*, *Clostridium botulinum*, *Clostridium difficile*, *Clostridium perfringens*, *Clostridium tetani*, *Corynebacterium diphtheriae*, *Enterococcus faecalis*, and *Clostridium perfringens*. Enterococci, Escherichia coli, Tulafos, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroides, Rickettsia rickettsiae, Salmonella, Shigella, Spirochetes, Staphylococcus, Streptococcus, Treponema pallidum, Vibrio cholerae, or Yersinia pestis.

[0181] The infection may be fungal. The fungal infection may be caused by Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus neoformans, Cryptococcus gutterus, Histoplasma capsulatum, Mucor, tinea cruris, tinea corporis, or tinea pedis.

[0182] The infection may be parasitic. The parasitic infection may be a protozoan ocular infection, Chagas disease, leishmaniasis, toxoplasmosis, giardiasis, malaria, microsporidiosis, or rhinosporidiosis. Preferably, the parasitic infection is malaria.

[0183] The viral infection may be SARS-CoV-2. SARS-CoV-2 is the virus that causes COVID-19. COVID-19 can lead to ARDS. The endothelial lesions may be caused by SARS-CoV-2 infection. The endothelial lesions may be caused by COVID-19. The endothelial lesions may be caused by ARDS.

[0184] The endothelial lesions can be caused by cancer. The cancer can be leukemia, lymphoma, or myeloma. Alternatively, 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.

[0185] The endothelial lesions can be caused by hematological conditions such as thrombotic thrombocytopenic purpura, anemia, or sickle cell disease.

[0186] The endothelial lesions can be caused by liver diseases such as cirrhosis, hepatitis, non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), primary biliary cholangitis, and primary sclerosing cholangitis.

[0187] The endothelial lesions can be caused by sepsis.

[0188] Dysfunctional endothelial cells can allow tumor cells in the circulating bloodstream to enter tissues. Therefore, treating endothelial lesions can prevent the hematogenous spread of hematogenous cancers. Treating endothelial lesions can inhibit the hematogenous spread of cancer. The aforementioned medium molecular weight heparin can inhibit the hematogenous spread of cancer.

[0189] Biomarkers for endothelial lesions may include elevated levels of von Willebrand factor (VWF), ultra-large von Willebrand factor (ULVWF), coagulation factor VIII, and other markers such as multiligand proteoglycan-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 endothelial lesions is the VWF:ADAMTS13 ratio or the VWF antigen:ADAMTS13 ratio.

[0190] Compared with healthy control subjects, the patients may have elevated plasma von Willebrand factor (VWF) levels. Compared with healthy control subjects, the patients may have persistently high plasma VWF levels. Compared with healthy control subjects, plasma VWF levels may be elevated for at least about one day, at least two days, at least three days, at least four days, at least five days, at least six days, or preferably at least one week. Compared with healthy control subjects, plasma VWF levels may be elevated for up to one week, up to four weeks, up to two months, up to four months, up to six months, or up to one year.

[0191] For example, plasma VWF levels may rise 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 may rise to about 130 nmol / L, about 150 nmol / L, or about 200 nmol / L. Plasma VWF levels may rise to at least about 50 nmol / L for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about one week, at least about one month, or at least about one year. Plasma VWF levels may rise to at least about 60 nmol / L for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least one week, at least one month, or at least one year. Plasma VWF levels can rise to at least approximately 70 nmol / L for at least approximately one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least one week, at least one month, or at least one year. Plasma VWF levels can rise to at least approximately 90 nmol / L for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least one week, at least one month, or at least one year. Plasma VWF levels can be measured using enzyme-linked immunosorbent assay (ELISA).

[0192] Alternatively, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or higher for at least about six 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 higher for at least about one day, at least two days, at least three days, at least four days, at least five days, at least six days, or at least seven days. More preferably, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or higher for at least one week, at least two weeks, at least three weeks, or at least four weeks. Even more preferably, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or higher for at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, or at least one year.

[0193] Endothelial function can be assessed in both the coronary and peripheral circulation. Non-invasive tests for assessing coronary endothelial function include Doppler echocardiography, in which blood flow is measured in response to pharmacological or physiological stimulation. Other tests include positron emission tomography (PET) and phase-contrast magnetic resonance imaging (MRI). However, the gold standard involves invasive quantitative coronary angiography to examine changes in diameter in response to intracoronary infusion of endothelium-dependent vasodilators (such as acetylcholine). Assessment of the endothelium in the peripheral circulation includes brachial artery ultrasound and strain gauge venous impedance plethysmography.

[0194] The binding of MMWH-Red to VWF can be assessed using a competitive binding assay. Heparin-agarose beads can be incubated with labeled VWF (e.g., 125I-vWF) for a period of time to allow the labeled VWF to bind to immobilized heparin. Different concentrations of MMWH-Red can then be added, and the amount of labeled VWF displaced can be determined. Other methods for determining the binding of MMWH-Red to VWF may include surface plasmon resonance, biofilm interferometry, isothermal titration calorimetry, fluorescence polarization binding assay, ELISA, and microthermophoresis.

[0195] Inhibition of platelet binding to VWF can be assessed by ritoctomycin-induced agglutination of immobilized platelets. Platelets can be incubated with plasma treated with medium molecular weight heparin and citrate (VWF source). Ristoctomycin can then be added, followed by platelet aggregation assay. When measured by ritoctomycin-induced platelet aggregation assay, MMWH at a concentration of 15 µM completely inhibited VWF-induced platelet aggregation. Other methods to determine inhibition of VWF binding to platelets may include ELISA, fluorescence-assisted cell sorting, dynamic light scattering, or flow-through assay.

[0196] The average molecular weight of MMWH-Red can range from about 8000 Da (g / mol) to about 13500 Da (g / mol), or from about 8000 Da (g / mol) to about 13000 Da (g / mol), or from about 10000 Da (g / mol) to about 12000 Da (g / mol). MMWH-Red may contain polysaccharide chains with an average molecular weight ranging from about 8000 Da (g / mol) to about 13500 Da (g / mol), or from about 8000 Da (g / mol) to about 13000 Da (g / mol), or from about 9000 Da (g / mol) to about 13000 Da (g / mol), or from about 10000 Da (g / mol) to about 12000 Da (g / mol). The average molecular weight of MMWH-Red can be determined, for example, by mass spectrometry or size exclusion chromatography.

[0197] MMWH-Red may contain at least three units of the GlcNS6S-IdoA2S disaccharide, for example, at least four, five, six, eight, or ten units. The medium molecular weight heparin may contain less than or equal to 25 units of the GlcNS6S-IdoA2S disaccharide, for example, less than or equal to 20 units. The presence of the GlcNS6S-IdoA2S disaccharide units can be determined by antibody testing, mass spectrometry, or inferred from chemical and enzymatic studies. The GlcNS6S-IdoA2S units may be arranged sequentially.

[0198] MMWH-Red may contain at least three units of the IdoA2S-GlcNS6S disaccharide, for example, at least four, five, six, eight, or ten units. The reduced heparin may contain less than or equal to 25 units of the IdoA2S-GlcNS6S disaccharide, for example, less than or equal to 20 units. The presence of the IdoA2S-GlcNS6S disaccharide units can be determined by antibody testing, mass spectrometry, or inferred from chemical and enzymatic studies. The IdoA2S-GlcNS6S units may be arranged sequentially. The number of IdoA2S-GlcNS6S units may be adjusted to provide desired anti-VWF activity and / or standard anticoagulant activity.

[0199] MMWH-Red may contain UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc. MMWH-Red may contain at least about 60% of UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc. MMWH-Red may contain at least about 45%, preferably at least about 48%, preferably at least about 49%, and preferably at least about 60% of UA2S-GlcNS6S. MMWH-Red may contain up to about 60%, preferably up to about 70%, and preferably up to about 85% of UA2S-GlcNS6S. MMWH-Red may contain at least about 4%, preferably at least about 5%, preferably at least about 6%, and preferably at least about 10% of UA2S-GlcNS. MMWH-Red may contain up to about 15%, preferably up to about 20% of UA2S-GlcNS. MMWH-Red may contain at least 4%, preferably at least 5%, preferably at least 6%, and preferably at least about 10% of UA-GlcNAc. The reduced heparin may contain up to about 15%, preferably up to about 20%, of UA-GlcNAc. In some embodiments, MMWH-Red may contain at least 49.2% 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 is more enriched than that of unfractionated heparin.

[0200] Treatment of endothelial lesions with MMWH-Red can inhibit hematogenous spread of cancer. Human tumor cells can bind VWF under shear flow conditions; melanoma and colon cancer cells have demonstrated this ability. Platelets anchored to VWF have been shown to mediate tethering, rolling, and firm adhesion of different cancer cell lines under flow shear stress. VWF plays a crucial role in achieving this firm adhesion between tumor cells and anchored platelets. Existing data suggest that VWF plays an important role in tethered cancer cells. Furthermore, VWF-platelet binding, which occurs as part of the normal thrombosis pathway, can further promote the aggregation of tumor cells into VWF-platelets, forming heterogeneous aggregates of VWF + platelets + cancer cells, thereby facilitating the hematogenous spread of tumor cells. Once initial binding with VWF and platelets occurs, this process can be at least partially caused by the ability of cancer cells to metastasize to the vessel wall and thus spread to other organs. In addition, various cancers are known to cause endothelial lesions, leading to the release of UL-VWF. Through this mechanism, the tumor triggers the release of UL-VWF, which subsequently allows for embolization of platelets and tumor cells, as well as hematogenous and metastatic spread of cancer. This cancer-induced endothelial lesion also leads to an increased overall risk of thrombosis in patients with underlying malignancies. Therefore, any treatment aimed at treating endothelial lesions and inhibiting the binding of platelets and / or tumor cells to VWF would have a dual purpose: reducing the risk of malignancy-related thrombosis and reducing the risk of hematogenous metastasis.

[0201] MMWH-Red can be administered via methods selected from the group consisting of: parenteral, subcutaneous, intravenous, intramuscular, intrathecal, intradermal, intra-articular, intra-articular, transdermal, subcutaneous, and reservoir forms, such as reservoir injection, intraosseous, or inhalation. Preferably, the medium molecular weight heparin is administered subcutaneously, intravenously, or intramuscularly. The administration method may be inhalation, optionally via a nebulizer.

[0202] Previous studies have used UFH as a nebulizer for a variety of conditions. Small human studies have shown that nebulized UFH can limit fibrin deposition in the lungs, reduce the progression of acute lung injury, and accelerate recovery (69). Early trials in patients with acute lung injury and related conditions found that nebulized UFH reduced lung dead space, coagulation activation, microvascular thrombosis, improved lung injury, and increased time without ventilator support (70-73). In a pre-pandemic double-blind randomized study of 256 critically ill ventilated patients, nebulized UFH limited the progression of lung injury, including acute respiratory distress syndrome, and accelerated survivors' return home. Therefore, MMWH-Red can be administered via nebulizer inhalation.

[0203] Heparin dosage is typically measured in "Howell units". One unit of heparin ("Howell unit") is approximately equivalent to 0.002 mg of pure heparin, which is the amount required to keep 1 ml of cat blood in fluid for 24 hours at 0°C. MMWH-Red can be administered as a bolus dose of about 5000 units, followed by about 1200 to about 1600 units per hour, optionally delivered via an infusion pump. MMWH-Red can be administered at doses of about 18 units / kg to about 5000 units / kg. Preferably, MMWH-Red can be administered at doses of about 100 units / kg to about 800 units / kg. Alternatively, MMWH-Red can be administered at doses of about 18 units / kg to about 75 units / kg. MMWH-Red can be administered at doses of about 5000 units, about 4000 units, about 3000 units, about 2000 units, about 1000 units, or about 500 units every 12 hours. MMWH-Red can be administered at a dose of approximately 5000 units every 12 hours.

[0204] MMWH-Red can be administered in doses of about 3 units to about 5000 units, for example, about 6 units to about 4000 units, about 12 units to about 3000 units, about 25 units to about 2000 units, about 50 units to about 1000 units, about 100 units to about 500 units, or about 125 units to about 250 units. The reduced molecular weight heparin can be administered in doses of about 18 units / kg to about 5000 units / kg, for example, about 100 units / kg to about 4000 units / kg, or about 200 units / kg to about 800 units / kg. The reduced molecular weight heparin can be administered in doses of about 18 units / kg to about 75 units / kg. The doses can be given as a single dose or as a continuous dose. The doses can be given over a period of time. The period of time can 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 time period can be approximately 1 to 7 days, approximately 2 to 6 days, approximately 3 to 5 days, or approximately 4 to 5 days. The dosage can be administered approximately 1 hour to 24 hours, approximately 2 hours to 12 hours, or approximately 3 hours to 6 hours. The dosage can be administered during the duration of potential endothelial lesions and elevated VWF levels.

[0205] MMWH-Red can be administered at doses from about 0.01 mg / kg to about 10 mg / kg, such as 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 can be administered as a single dose or as a continuous dose. The dose can be administered over a period of time. The period of time can be from about 1 month to about 12 months, such as from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months to about 9 months, from about 5 months to about 8 months, or from about 6 months to about 7 months. The period of time can be from about 1 day to about 7 days, from about 2 days to about 6 days, from about 3 days to about 5 days, or from about 4 days to about 5 days. The dose can be administered from about 1 hour to about 24 hours, from about 2 hours to about 12 hours, or from about 3 hours to about 6 hours. The dose can be administered during the duration of potential endothelial lesions and elevated VWF levels.

[0206] MMWH-Red can be administered at doses 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 can be administered at doses of about 500 mg / kg or less, about 300 mg / kg or less, about 200 mg / kg or less, or about 100 mg / kg or less. MMWH-Red can be administered at doses 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 can be administered as a single dose or as a continuous dose. The dosage of MMWH-Red may depend on the VWF antigen:ADAMTS13 ratio or the overall VWF level. Those skilled in the art can select an appropriate dose for a patient based on the VWF antigen:ADAMTS13 ratio or the overall VWF level.

[0207] MMWH-Red can be administered at doses 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, and about 1 mg / kg to about 1.5 mg / kg. The doses can be administered as a single dose or as a continuous dose. The doses can be administered over a period of time. The period of time can 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, and about 6 months to about 7 months. The period of time can be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, and about 4 days to about 5 days. The doses can be administered in about 1 hour to about 24 hours, about 2 hours to about 12 hours, and about 3 hours to about 6 hours. The doses can be administered during the duration of potential endothelial lesions and elevated VWF levels.

[0208] MMWH-Red can be administered in doses 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, and about 150 mg to about 200 mg. The doses can be given as a single dose or as a continuous dose. The doses can be given over a period of time. The period of time can 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, and about 6 months to about 7 months. The period of time can also be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, and about 4 days to about 5 days. The dosage can be administered over approximately 1 hour to approximately 24 hours, approximately 2 hours to approximately 12 hours, and approximately 3 hours to approximately 6 hours. The dosage can be administered during the duration of potential endothelial lesions and elevated VWF levels.

[0209] MMWH-Red can be administered in doses 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 given as a single dose or as a continuous dose. The dose can be given over a period of time. The period of time can be from about 1 month to about 12 months, for example, from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months to about 9 months, from about 5 months to about 8 months, or from about 6 months to about 7 months. The time period can be from about 1 day to about 7 days, from about 2 days to about 6 days, from about 3 days to about 5 days, or from about 4 days to about 5 days. The dosage can be administered from about 1 hour to about 24 hours, from about 2 hours to about 12 hours, or from about 3 hours to about 6 hours. The dosage can be administered during the duration of potential endothelial lesions and elevated VWF levels.

[0210] MMWH-Red can be administered in doses 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, and about 250 IU to about 300 IU. The dose can be given as a single dose or as a continuous dose. The dose can be given over a period of time. The period of time can be 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, and about 6 months to about 7 months. The time period can be from about 1 day to about 7 days, from about 2 days to about 6 days, from about 3 days to about 5 days, or from about 4 days to about 5 days. The dosage can be administered from about 1 hour to about 24 hours, from about 2 hours to about 12 hours, or from about 3 hours to about 6 hours. The dosage can be administered during the duration of potential endothelial lesions and elevated VWF levels.

[0211] MMWH-Red can be administered at a rate comparable to the VWF antigen:ADAMTS13 ratio. For example, patients with a high VWF antigen:ADAMTS13 ratio can be administered a higher dose of MMWH compared to patients with a low VWF antigen:ADAMTS13 ratio.

[0212] MMWH-Red may be included in a pharmaceutical formulation. The pharmaceutical formulation may include excipients. The excipients may be selected from the group consisting of solvents, solubilizers, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavoring agents, lubricants, suspending agents, tension modifiers, surfactants, solubilizers, suspending agents, dispersants, humectants, thickeners, colorants, wetting agents, defoamers, viscosity modifiers, sweeteners, and combinations thereof. The pharmaceutical formulation may include additional active agents. The additional active agents may include medium molecular weight heparin or low molecular weight heparin.

[0213] The pharmaceutical formulation may contain additional active agents. These additional active agents comprise a composition of substances with physiological effects. The additional active agents may comprise low molecular weight heparin or medium molecular weight heparin or reduced medium molecular weight heparin with different disaccharide compositions. The additional active agent may be selected from the group comprising farnesoid X receptor (FXR) agonists, peroxisome proliferator-activated receptor (PPAR) agonists, alactal, caspase inhibitors, galactoglobulin 3 inhibitors, mitogen-activated protein kinase 5 (MAPK5) inhibitors, fibroblast growth factor 19 (FGF19) agonists, FGF21 agonists, leukotriene D4 (LTD4) receptor antagonists, nicotinic acid analogs, apical sodium-dependent bile acid transporter (ASBT) inhibitors, apoptosis signal-regulated kinase 1 (ASK1) inhibitors, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, chemokine receptor inhibitors, thiazolidinediones, GLP-1 analogs, biguanides, HIV replication inhibitors, metformin, opioids, anesthetics, HMG-CoA reductase inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), or any combination thereof.

[0214] MMWH-Red may include chemical modifications. The chemical modifications may be selected from the group consisting of N-acetylation, N-deacetylation, N-sulfation, O-sulfation, 2-O-desulfation, and complete desulfation.

[0215] In a sixth aspect, the present invention provides MMWH-Red according to the first or third aspect, or MMWH-Red produced according to the second aspect, or a composition according to the fourth aspect, for treating a disease or condition of a patient, wherein the patient has endothelial lesions, characterized in that the plasma von Willebrand factor to ADAMTS13 (VWF:ADAMTS13) ratio is at least about 2.

[0216] In a seventh aspect, the present invention provides MMWH-Red according to the first or third aspect, or MMWH-Red produced according to the second aspect, or a composition according to the fourth aspect, for treating a patient’s disease or condition, wherein the patient has endothelial lesions, characterized in that the ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen: ADAMTS13) is at least about 2.

[0217] Preferably, the disease or condition is selected from the group consisting of: COVID-19 (e.g., acute COVID-19 or post-COVID syndrome), infection, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, sepsis, septicemia, cardiovascular disease, diabetes, trauma (particularly brain or head trauma, such as traumatic brain injury), burns, inhalation injury, drugs and drug reactions, hematological disorders, subarachnoid hemorrhage (e.g., aneurysmal subarachnoid hemorrhage), aneurysmal disease, stroke, cerebral hemorrhage, radiation-induced injury, ischemic stroke, pancreatitis, liver disease, kidney disease, chronic obstructive pulmonary disease, or combinations thereof. The endothelial lesion may be caused by a viral infection, optionally SARS-CoV-2. The endothelial lesion 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, the endothelial lesion is caused by sepsis or septicemia. Preferably, the endothelial lesion is caused by sepsis.

[0218] In some embodiments, the present invention provides MMWH-Red for treating patients with COVID-19 (e.g., acute COVID-19 or post-COVID syndrome), wherein the patients have endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0219] In some embodiments, the present invention provides MMWH-Red for treating an infection in a patient with endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2. The infection may be SARS-CoV-2.

[0220] In some embodiments, the present invention provides MMWH-Red for treating a viral infection in a patient with endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2. The viral infection may be SARS-CoV-2.

[0221] In some embodiments, the present invention provides MMWH-Red for treating patients with acute respiratory distress syndrome (ARDS), wherein the patients have endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0222] In some embodiments, the present invention provides MMWH-Red for treating cancer in patients with endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2. The cancer may be leukemia, lymphoma, myeloma, or solid organ cancer, such as colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer, or skin cancer.

[0223] In some embodiments, the present invention provides MMWH-Red for treating bacterial infections in patients with endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0224] In some embodiments, the present invention provides MMWH-Red for treating patients with sepsis, wherein the patients have endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0225] In some embodiments, the present invention provides MMWH-Red for treating patients with sepsis, wherein the patients have endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0226] In some embodiments, the present invention provides MMWH-Red for treating cardiovascular disease in patients with endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0227] In some embodiments, the present invention provides MMWH-Red for treating patients with diabetes, wherein the patients have endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0228] In some embodiments, the present invention provides MMWH-Red for treating trauma in patients with endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2. Preferably, the trauma is brain trauma or head trauma, such as traumatic brain injury.

[0229] In some embodiments, the present invention provides MMWH-Red for treating burns in patients with endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0230] In some embodiments, the present invention provides MMWH-Red for treating inhalation injury in patients with endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0231] In some embodiments, the present invention provides MMWH-Red for treating patients with endothelial lesions and drug responses, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0232] In some embodiments, the present invention provides MMWH-Red for treating hematological conditions in patients with endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0233] In some embodiments, the present invention provides MMWH-Red for treating patients with subarachnoid hemorrhage (e.g., aneurysmal subarachnoid hemorrhage), wherein the patients have endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0234] In some embodiments, the present invention provides MMWH-Red for treating aneurysmal disease in patients with endothelial lesions, characterized in that the plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0235] In some embodiments, the present invention provides MMWH-Red for treating stroke patients, wherein the patients have endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0236] In some embodiments, the present invention provides MMWH-Red for treating patients with endothelial lesions of the brain parenchyma, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0237] In some embodiments, the present invention provides MMWH-Red for treating patients with radiation-induced damage, wherein the patients have endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0238] In some embodiments, the present invention provides MMWH-Red for treating patients with ischemic stroke, wherein the patients have endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0239] In some embodiments, the present invention provides MMWH-Red for treating patients with pancreatitis, wherein the patients have endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0240] In some embodiments, the present invention provides MMWH-Red for treating liver conditions in patients with endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0241] In some embodiments, the present invention provides MMWH-Red for treating patients with kidney disease, wherein the patients have endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0242] In some embodiments, the present invention provides MMWH-Red for treating patients with chronic obstructive pulmonary disease, wherein the patients have endothelial lesions, characterized in that the VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2.

[0243] MMWH-Red for treating endothelial lesions, as defined in the fifth, sixth, or seventh aspect of this invention, is particularly advantageous because the heparin can inhibit microthrombus formation triggered by VWF release secondary to endothelial lesions caused by any disease or condition. When the cause of the endothelial lesions is SARS-CoV-2, the MMWH-Red can additionally inhibit viral adhesion and replication.

[0244] In an eighth aspect, the present invention provides a method for treating endothelial lesions, the method comprising administering a therapeutically effective amount of MMWH-Red to a subject in need of treatment. Preferably, the patient's plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2. Typically, the MMWH-Red may be the MMWH-Red according to the first or third aspect, the MMWH-Red produced according to the method of the second aspect, or the composition according to the fourth aspect.

[0245] In a ninth aspect, the present invention provides the use of MMWH-Red in the manufacture of a medicament for treating endothelial lesions in patients. Preferably, the patient's plasma VWF:ADAMTS13 ratio is at least about 2 or the plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) ratio is at least about 2. Typically, the MMWH-Red may be the MMWH-Red according to the first or third aspect, the MMWH-Red produced according to the method of the second aspect, or the composition according to the fourth aspect.

[0246] Sepsis is an extreme response of the body to infection. It is characterized by a systemic inflammatory state, where the immune system responds to infection by attacking the body's own tissues and organs due to an immune dysregulation. As a result, cells in the body are destroyed through apoptosis or necrosis, and intracellular substances eventually enter the bloodstream.

[0247] Histones are basic intracellular proteins located in the cell nucleus. DNA wraps around histones to form chromatin, which then coils itself tightly to facilitate the packaging of long DNA molecules into the cell nucleus. While their role in packaging DNA into the nucleus is known, chromatin (and its constitutive histones) has also been shown to play a role in innate immunity. During infection, neutrophils release granule proteins and chromatin, which together form extracellular fibers that bind Gram-positive and Gram-negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill bacteria.

[0248] Circulating histones have been identified as mediators of damage in animal models and patients of sepsis. These histones enter the bloodstream through the release of intracellular material via apoptosis or necrosis (which occurs during sepsis) or through the release of NETs in response to bacterial infection. Thus, histones can perpetuate the immune system dysregulation observed during sepsis. For example, histones are damage-associated molecular patterns (DAMPs) that are sensed by Toll-like receptors (TLRs). Histone activation of TLRs on innate immune cells leads to the release of pro-inflammatory cytokines, thereby exacerbating an already dysregulated immune response. In particular, TLR receptors on endothelial cells can be activated by circulating histones, leading to the induction of an immune response in endothelial cells. Consequently, endothelial inflammation is observed, and endothelial lesions are observed.

[0249] Therefore, MMWH-Red, as defined herein, can bind to free histones and simultaneously inhibit VWF, thereby treating sepsis. In one embodiment, the present invention provides MMWH-Red for treating a subject (patient) with sepsis, systemic inflammatory response syndrome (SIRS), severe sepsis, or septic shock. In some embodiments, the subject has a plasma VWF:ADAMTS13 ratio of at least about 2. In some embodiments, the subject has a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0250] As defined in this paper, MMWH-Red can be a complement cascade modulator.

[0251] As defined in this article, MMWH-Red can be an immunomodulator.

[0252] To avoid ambiguity, embodiments relating to each aspect of the invention, with necessary modifications, are applicable to other aspects of the invention. Further aspects and embodiments of the invention will become apparent from the discussion herein.

[0253] Every reference cited in this article, including any cross-references or related patents or applications, is incorporated herein by reference in its entirety unless expressly excluded or otherwise restricted.

[0254] It should be understood that various modifications may be made to the illustrated embodiments without departing from the spirit and scope of the invention as defined by the appended claims.

[0255] Example The invention will now be illustrated by reference to the following non-limiting examples.

[0256] Unless otherwise specified, room temperature and atmospheric pressure are 20°C (293.15 K, 68 °F) and 1 atm (14.696 psi, 101.325 kPa), respectively.

[0257] Experimental methods Size exclusion chromatography (SEC) SEC was performed on a GEC Superdex 75 (10 / 100) column on an Agilent 1200 HPLC system equipped with a variable wavelength UV detector or a GEC AKTA system equipped with a variable wavelength UV detector. The mobile phase was 0.15 M NaCl dissolved in water, which was passed through the column at a rate of 0.4 mL / min. The optical density was measured at 232 nm. SEC was performed with reference to an 11 kD standard.

[0258] freeze-drying Freeze-drying (also known as lyophilization or cryogenic drying) is a drying process carried out at low temperatures. Freeze-drying typically involves lowering the temperature and pressure below the triple point of the substance and removing the frozen solvent (e.g., water ice) by sublimation. For aqueous compositions, such as those disclosed herein, freeze-drying can be carried out at temperatures from about -20°C to -80°C, preferably about -40°C, and pressures from about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).

[0259] MMW Heparin Manufacturing Method 1. Development Example 1.1 Optimization of periodate concentration (periodate method) Dissolve 80 mg of unfractionated heparin in 30 mL of 50 mM sodium phosphate buffer adjusted to pH 7.0.

[0260] Prepare samples by adding (A) 43 mg, (B) 86 mg, (C) 173 mg, or (D) 200 mg of sodium periodate to a heparin phosphate buffer solution. Incubate the resulting solutions at 4°C for 16 to 18 hours or at 37°C for 6 hours.

[0261] Before dialysis, take 200 µl of sample for SEC. Transfer the remaining sample to a dialysis tube with a 2 kDa molecular weight cutoff (Spectra / Por). ® Product No. 132109; 7 days: 3 times daily water replacement / 4 times volume of water) was dialyzed against water. The sample was then freeze-dried at -40°C and 10 Pa. The sample was then dissolved in water and, using a GEC AKTA system or an Agilent 1200 system with 0.15 M NaCl as the mobile phase, at a flow rate of 0.4 mL / min, was analyzed using Superdex. ® 75 SE (10 / 300) bars were analyzed via SEC.

[0262] Aliquots taken before dialysis were processed on a GEC AKTA system or an Agilent 1200 system using 0.15 M NaCl as the mobile phase at a rate of 0.4 mL / min in a Superdex atmosphere. ® SEC analysis on a 75 SE (10 / 300) column showed a large amount of disaccharide substances mixed with reagent peaks.

[0263] SEC analysis of the samples was performed and calibrated by 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 a broad peak centered at Kav 0.12, leading to the degradation of medium molecular weight heparin.

[0264] b. The sample obtained by incubation at 4˚C showed a narrow peak centered at Kav 0.06 and a molecular weight of 11 kD.

[0265] Figure 1 The comparison shows that MMWH prepared at 4°C is compared with heparin treated at 37°C, untreated heparin, and 11 kDa standard.

[0266] Therefore, incubation at 4°C favors the formation of medium molecular weight heparin. Incubation at 37°C primarily yields other products.

[0267] All periodate concentrations studied provided the required medium molecular weight heparin. The highest activity was achieved at a periodate concentration of 173 mg per 30 mL of heparin sodium phosphate buffer (C) (equivalent to 8.6 g / 1.5 L used in the large-scale process examples described below).

[0268] 1.2 Perchlorate Addition Samples were prepared according to (A) and (B) of the periodate method (1.1) described above. 732 mg / 30 mL sodium perchlorate was added to samples (A) and (B), and the resulting solutions were incubated at 4°C for 16 to 18 hours or at 37°C for 6 hours.

[0269] The sample was placed in a dialysis tube with a 2kD molecular weight cutoff (Spectra / Por). ® The sample (product number 132109) was dialyzed against water (7 days of dialysis: 3 times daily, 4 volumes of water changed). The sample was then freeze-dried at -40°C and 10 Pa. The sample was then dissolved in water and pumped at 0.4 mL / min using 0.15 M NaCl as the mobile phase on a GEC AKTA system or an Agilent 1200 system via Superdex. ® 75 SE (10 / 300) bars were analyzed via SEC.

[0270] SEC analysis of the samples was performed and calibrated by co-elution with a validated 11 kDa standard. SEC analysis of the post-dialysis samples provided the following results: All samples (incubated at 4˚C and 37˚C) resulted in a wider size range of material centered at Kav 0.12. There was also a significant increase in material at column Vt (i.e., smaller material).

[0271] This example demonstrates that oxidation using a mixture of periodate and perchlorate results in an increased level of breakdown into smaller substances compared to oxidation using periodate alone. Therefore, oxidation using periodate alone is a more efficient method for preparing high-purity, medium-molecular-weight heparin.

[0272] 1.3 Alkali Elimination Samples were prepared by incubation at 4˚C and 37˚C according to (A) and (B) of the periodate method (1.1) described above.

[0273] Add 2 M NaOH to the sample to adjust the pH to pH 12. Then incubate the sample at room temperature for 30 minutes. Analyze the sample using a GEC AKTA system or an Agilent 1200 system with 0.15 M NaCl as the mobile phase at a flow rate of 0.4 mL / min on a Superdex exchanger. ® Aliquots of these samples were analyzed by SEC on a 75 SE (10 / 300) column. The samples were SEC-analyzed and calibrated by co-elution with a validated 11 kDa standard.

[0274] The elimination of the base led to further degradation, with more defined peaks at Kav 0.331, 0.35, 0.4 and 0.54, and a large peak at Kav 0.625.

[0275] Figure 2 The comparison shows that MMWH prepared using only periodate at 4 ˚C is comparable to heparin treated with periodate followed by NaOH at 4 ˚C; the latter exhibits a broad peak due to degradation. Therefore, the preparation of high-purity medium molecular weight heparin is hindered by the inclusion of an alkaline elimination step, as significant degradation is observed.

[0276] 2. Large-scale process examples UF heparin (porcine mucosal heparin; Iduron catalog number HEP001 / 100) (4 g; 2.7 mg / mL; average molecular weight 15 kDa) was dissolved in 1.5 L of 50 mM sodium phosphate buffer (adjusted to pH 7.0) cooled in an ice bath (0°C to 2°C). Sodium periodate (8.56 g, 40 mmol) was added, and the sample was incubated overnight at 4°C. After incubation, sodium periodate was inactivated by adding D-mannitol (30 g, 160 mmol).

[0277] The sample was then placed in a dialysis tube with a 2kD molecular weight cutoff (Spectra / Por). ® The sample was dialyzed with water (product number 132109) for 7 days: 3 water changes per day / 4 volumes of water). The sample was then freeze-dried at -40°C and 10 Pa to provide medium molecular weight heparin in a yield of 2.1 g.

[0278] The medium molecular weight heparin sample was then dissolved in water and, using 0.15 M NaCl as the mobile phase, was pumped at a rate of 0.4 mL / min on a GEC AKTA system or an Agilent 1200 system via Superdex. ® 75 SE (10 / 300) bars were analyzed via SEC. For example... Figure 3 As shown, the average molecular weight of the medium molecular weight heparin is 11 kDa (refer to 11 kDa standard).

[0279] like Figure 4 and Figure 5 As shown, compared to UF heparin and low molecular weight heparin, the produced medium molecular weight heparin exhibits very low activity against factors IIa and / or Xa. Therefore, unlike UF heparin or LMW heparin, MMW heparin does not affect factor IIa or factor Xa-mediated coagulation.

[0280] Factor IIa (also known as thrombin) functions as a serine protease, converting soluble fibrinogen into insoluble fibrin chains and catalyzing other coagulation-related reactions. Factor Xa is the activated form of coagulation factor X. Factor X is a serine endopeptidase that plays a crucial role in multiple stages of the coagulation system.

[0281] Heparin (ungraded heparin) and its derivatives, such as low molecular weight heparin, bind to the plasma cofactor antithrombin (AT) to 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 rather than a direct interaction with factor Xa.

[0282] Activities against factor IIa and factor Xa can be measured using the Kinetichrome anti-IIa heparin kit and the Kinetichrome anti-Xa heparin kit, respectively. Activities against factor IIa and factor Xa can be measured using other USP-compliant kits. <208> Or the kit required by EP can be used for measurement.

[0283] The medium molecular weight heparin was analyzed for disaccharides. The results are shown in Table 1 below. The example synthesis provided herein uses porcine mucosal-derived UF heparin. For the avoidance of doubt, UF heparin from other sources is applicable to the methods disclosed herein.

[0284] MMWH-Red Manufacturing Method Heparin oxidation Oxidized UFH heparin as described in Part 2 of the above-mentioned MMW heparin manufacturing method.

[0285] Example 1 of the reduction of heparin oxide At room temperature, 300 mg of NaBH4 was added to a solution of heparin oxidase (6 g) dissolved in 100 mL of milli-Q. The resulting mixture was stirred at room temperature for 4 hours. The pH was then adjusted to pH 4 with hydrochloric acid, and the reaction mixture was stirred for 15 minutes. Subsequently, the pH was adjusted to pH 7 with sodium hydroxide. 2% m / v NaCl was added, followed by MeOH to bring the MeOH content in the reaction mixture to 73% v / v. The methanol reaction mixture was stirred for 10 minutes, and the precipitate was allowed to settle. The precipitate was then resuspended in methanol and collected by filtration. The product was dried under vacuum overnight at room temperature.

[0286] Example 2 of the reduction of heparin oxide Heparin oxidase (2.4 g) was dissolved in water (125 mL). NaBH4 (2.06 g) was added, and the reaction mixture was incubated at room temperature for 4 hours. The pH of the reaction mixture was then adjusted to 7.02 with glacial acetic acid.

[0287] The sample was then placed in a dialysis tube with a 2kD molecular weight cutoff (Spectra / Por). ® The sample was dialyzed with water (product number 132109) for 3 days: 2 water changes per day / 4 volumes of water. The sample was then freeze-dried at -40°C and 10 Pa to provide reduced heparin at medium molecular weight.

[0288] like Figure 6 As shown, through 1H-NMR monitoring of the reduction reaction showed that the aldehyde signal present in MMWH at ~9.25 ppm disappeared during the reduction reaction for the preparation of MMWH-Red (hydroxyl form), thus indicating the preparation of MMWH-Red.

[0289] The factor Xa activity of MMWH-Red prepared according to the above method is <10 IU / mg. The factor Xa activity of MMWH (an intermediate in this process) is <5 IU / mg. The factor Xa activity of unfractionated heparin is 169.24 IU / mg. Compared with unfractionated heparin and low molecular weight heparin, reduced medium molecular weight heparin shows very low activity against factor Xa. Therefore, unlike UF heparin or LMW heparin, MMWH-Red does not affect factor Xa-mediated coagulation.

[0290] Tests in ritoxime-induced platelet aggregation (RIPA) assays According to "Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the platelet physiology subcommittee of SSC / ISTH", Journal of Thrombosis and Haemostasis, 2013, 11:1183-1189.

[0291] Blood samples were collected from non-smokers who had never received any antiplatelet therapy (such as aspirin). Antiplatelet therapy is also known as platelet aggregation inhibitor or platelet aggregation depressant.

[0292] In the absence of venous stasis, draw blood from the donor into a 109 mM sodium citrate solution (VACUETTE, 3.5 mL #454327, batch number #A21013FQ). Discard the first 3 to 4 mL of blood drawn.

[0293] Allow blood samples to stand at room temperature for 15 minutes, then centrifuge. Platelet-rich plasma (PRP) is prepared by centrifuging the blood sample at 200 g for 10 minutes at 21°C without braking. Platelet-anemic plasma (PPP) is prepared by centrifuging the blood sample after PRP removal at 1500 g for 15 minutes at 21°C.

[0294] PRP quality was assessed by platelet counting. The platelet count in the PRP was 421 G / L. The platelet count in the PRP sample was not (and should not) adjusted to a standardized value using autologous PPP.

[0295] After centrifugation, PRP samples were allowed to stand at room temperature for 15 minutes before performing transmittance aggregation assays (LTA). PRP was used to set 0% transmittance in the aggregation apparatus. Autologous PPP was used to set 100% transmittance in the aggregation apparatus. LTA studies were performed at 37°C. Baseline tracing of the LTA was observed for at least 1 minute to check for oscillations and stability before adding the agonist. The agonist volume used for LTA was consistent and did not exceed 10% of the total volume.

[0296] Two UF heparin samples (UF heparin 1 and UF heparin 2), three MMWH samples (MMWH sample 1, MMWH sample 2 and MMWH sample 3) and one MMWH-Red sample (MMWH-Red) were prepared in 5.2 mM H2O solution.

[0297] Ristoxin working solution: -- 20 µL ristocetine stock solution (24 mg / mL ristocetine in saline).

[0298] working solution for samples: Mix 10 µL of 5.2 mM sample solution with 163 µL of saline to provide a 300 µM working solution for each sample.

[0299] RIPA scheme: Dilute 360 ​​µL of PRP with Agrastat (5 µg / mL; 1 / 10, GPIIIbIIIa antagonist). Add 20 µL of working sample solution or 20 µL of saline (control group) (to bring the final sample concentration to 15 µM). Stir the solution for 2 seconds, then incubate at 37°C without stirring for 10 minutes. Then incubate the solution at 37°C with stirring for 1 minute (baseline tracking). Add 20 µL of ristocetin working solution and measure platelet aggregation.

[0300] Each experiment was repeated twice. Amplitude, slope, and area under the curve (AUC) were calculated by software.

[0301] result: Both MMWH and MMWH-Red were found to reduce the measured amplitude, slope, and area under the curve to a greater extent than the control group (see [link to control group]). Figure 7-10 Therefore, MMWH-Red was also found to have an inhibitory effect on ristomycin-induced platelet aggregation (RIPA), although the inhibitory effect was lower than that of MMWH (see [link to study]). Figure 11-12This indicates that the interaction between heparin polysaccharide and von Willebrand factor remains intact. Although MMWH-Red has lower activity in RIPA than MMWH, this difference in RIPA activity between MMWH-Red and MMWH allows for therapeutic tunability.

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Claims

1. A reduced medium molecular weight heparin.

2. A reduced medium molecular weight heparin, wherein the average molecular weight of the reduced medium molecular weight heparin is greater than about 8000 Da (g / mol) to about 13500 Da (g / mol).

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

4. The method according to claim 3, wherein the reducing agent is a mild reducing agent.

5. The method according to claim 4, wherein the mild reducing agent is sodium borohydride.

6. The method according to any one of claims 3 to 5, wherein the method does not include an alkali elimination step.

7. The method according to any one of claims 3 to 6, wherein the method does not include the addition of alkali metal salts.

8. The method according to claim 7, wherein the alkali metal salt comprises NaOH, KOH or LiOH.

9. The method according to claim 8, wherein the alkali metal salt is NaOH, KOH or LiOH.

10. The method according to any one of claims 3 to 9, wherein the aqueous buffer solution is adjusted to approximately pH 7.

0.

11. The method according to any one of claims 3 to 10, wherein the aqueous buffer solution is a phosphate buffer.

12. The method of claim 11, wherein the phosphate buffer is a sodium phosphate buffer.

13. The method according to any one of claims 3 to 12, wherein the incubation step c is performed for about 1 hour to about 48 hours.

14. The method according to any one of claims 3 to 13, wherein the incubation temperature is about 4°C.

15. The method according to any one of claims 3 to 14, wherein the temperature of the aqueous buffer solution in step a is about 0°C.

16. The method according to any one of claims 3 to 15, wherein the oxidant is periodate, preferably sodium periodate or potassium periodate.

17. The method according to any one of claims 3 to 16, wherein the method further comprises inactivating the oxidant in the medium molecular weight heparin solution.

18. The method of claim 17, wherein the oxidant is inactivated by adding an inactivating agent selected from the group consisting of: D-mannitol, glycerol, N-acetylmethionine, sodium sulfite, and combinations thereof.

19. The method according to claim 17 or 18, wherein the oxidant is inactivated by adding D-mannitol.

20. The method according to any one of claims 18 or 19, wherein the molar ratio of the oxidant to the inactivator is about 1:1 to about 1:

10.

21. The method according to any one of claims 3 to 20, wherein the method further comprises dialyzing the medium molecular weight heparin solution in a dialysate to provide a dialyzed medium molecular weight heparin sample.

22. The method according to any one of claims 3 to 21, wherein in the dialysis step e, the dialysate is water.

23. The method according to any one of claims 3 to 22, wherein the dialysis step e is performed in a dialysis tube with a molecular weight cutoff of 2 kD.

24. The method according to any one of claims 21 to 23, wherein the method further comprises separating the medium molecular weight heparin from the dialyzed heparin sample.

25. The method according to any one of claims 3 to 23, wherein the average molecular weight of the medium molecular weight heparin is greater than about 8000 Da (g / mol) to about 13500 Da (g / mol).

26. The method according to any one of claims 3 to 25, wherein the average molecular weight of the medium molecular weight heparin is about 11,000 Da (g / mol).

27. The method according to any one of claims 3 to 26, wherein the medium molecular weight heparin comprises at least three units of IdoA2S-GlcNS6S disaccharide.

28. Reduced medium molecular weight heparin (MMWH-Red) for the treatment of patients with endothelial lesions, wherein the ratio of plasma von Willebrand factor antigen to ADAMTS13 is at least about 2.

29. The MMWH-Red of claim 28, wherein the MMWH-Red inhibits von Willebrand factor, optionally wherein, when measured by a ristocetin-induced platelet aggregation assay, the MMWH-Red completely inhibits von Willebrand factor-induced platelet aggregation at a concentration of 15 µM.

30. The MMWH-Red of claim 28, wherein the MMWH-Red inhibits the multimer of von Willebrand factor, optionally wherein the von Willebrand factor is ultra-large von Willebrand factor.

31. MMWH-Red for any one of claims 28 to 30, wherein MMWH-Red inhibits the binding of platelets to von Willebrand factor.

32. The MMWH-Red for the use of any one of claims 28 to 31, wherein the average molecular weight of the MMWH-Red is in the range of greater than about 8000 Da (g / mol) to about 13000 Da (g / mol), and optionally wherein the average molecular weight of the MMWH-Red is about 11000 Da (g / mol).

33. The MMWH-Red for use according to any one of claims 28 to 32, wherein the MMWH-Red comprises at least three units of the IdoA2S-GlcNS6S disaccharide.

34. MMWH-Red for any of the uses described in any one of claims 28 to 33, wherein the treatment of endothelial lesions inhibits hematogenous spread of cancer.

35. An MMWH-Red for treating a patient's disease or condition, wherein the patient has endothelial lesions, characterized in that... The plasma von Willebrand factor antigen to ADAMTS13 ratio is at least about 2.

36. The MMWH-Red for the use of claim 35, wherein the disease or condition is COVID-19, infection, viral infection, acute respiratory distress syndrome, cancer, bacterial infection, sepsis, septicemia, cardiovascular disease, diabetes, trauma, burns, inhalation injury, drugs and drug reactions, hematological disorders, subarachnoid hemorrhage, aneurysmal disease, stroke, cerebral hemorrhage, radiation-induced injury, ischemic stroke, pancreatitis, liver disease, kidney disease, chronic obstructive pulmonary disease, or a combination thereof.

37. The MMWH-Red of claim 36, wherein the endothelial lesion is caused by a viral infection, optionally wherein the viral infection is SARS-CoV-2.

38. The MMWH-Red of claim 36, wherein the endothelial lesion is caused by cancer, optionally wherein the cancer is leukemia, lymphoma, myeloma or solid organ cancer.

39. MMWH-Red for any one of claims 28 to 38, wherein the medium molecular weight heparin is administered by a method selected from the group consisting of: parenteral, subcutaneous, subcutaneous, reservoir form (e.g., reservoir injection), intravenous, intramuscular, intrathecal, intradermal, intra-articular, intra-articular, transdermal, intraosseous, or inhalation.

40. MMWH-Red for the use of claim 39, wherein the method of administration is subcutaneous.

41. MMWH-Red for the use of claim 39, wherein the method of administration is intravenous.

42. MMWH-Red for the use of claim 39, wherein the method of administration is intramuscular.

43. The MMWH-Red for the use of claim 39, wherein the method of administration is inhalation, optionally via a nebulizer.

44. The MMWH-Red for use according to any one of claims 28 to 43, wherein the MMWH-Red is administered at a dose of about 0.01 mg / kg to about 100 mg / kg, preferably about 0.01 mg / kg to about 10 mg / kg.

45. The MMWH-Red for any one of claims 39 to 44, wherein the MMWH-Red is administered as a single dose or a continuous dose.

46. ​​The MMWH-Red for any one of claims 28 to 45, wherein the MMWH-Red is contained in a pharmaceutical formulation.

47. MMWH-Red for the use of claim 46, wherein the pharmaceutical formulation comprises an excipient.

48. MMWH-Red for the use of claim 47, wherein the excipient is selected from the group consisting of solvents, cosolvents, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavoring agents, lubricants, suspending agents, tension modifiers, surfactants, solubilizers, suspending agents, dispersants, humectants, thickeners, colorants, wetting agents, defoamers, viscosity modifiers, sweeteners, and combinations thereof.

49. MMWH-Red for any of the uses described in any one of claims 46 to 48, wherein the pharmaceutical formulation comprises an additional active agent, optionally wherein the additional active agent comprises low molecular weight heparin.

50. The MMWH-Red for use according to any one of claims 28 to 49, wherein the MMWH-Red comprises chemical modification.

51. The MMWH-Red for use according to claim 50, wherein the chemical modification comprises N-acetylation, N-deacetylation, N-sulfation, O-sulfation, 2-O desulfation, complete desulfation, or a combination thereof.

52. A method for treating endothelial lesions, the method comprising administering a therapeutically effective amount of MMWH-Red to a patient in need of treatment, wherein the patient's plasma von Willebrand factor antigen to ADAMTS13 ratio is at least about 2.

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