Intermediate molecular weight heparin for use in the treatment of vascular endotheliitis - Patent Application 20070233334

Abstract

The present invention provides intermediate molecular weight heparins for use in the treatment of disease.

Description

[Technical field]

[0001] The present invention relates to the treatment of vascular endotheliitis. In particular, the present invention relates to intermediate molecular weight heparin for use in the treatment of vascular endotheliitis. [Background technology]

[0002] The vertebrate vascular system consists of arteries, veins and capillaries. Blood flow through the vascular system is a dynamic that allows the maintenance of homeostasis by delivering essential elements such as oxygen and white blood cells to tissues that need them most. Blood flow is controlled by the dilation and constriction of blood vessels. Endothelial cells lining the sides of the lumen of the vascular system function as a monolayer forming the endothelium; the endothelium rests on a layer of smooth muscle cells. These smooth muscle cells tighten or relax, resulting in the constriction of blood vessels (vasoconstriction) or the dilation of blood vessels (vasodilation), respectively. In the case of hemorrhage, blood flow can be controlled by the formation of thrombi. To reach the site of inflammation, white blood cells must pass from the blood through endothelial cells to reach the inflamed tissue. Thus, the regulation of vasoconstriction, vasodilation, vascular permeability and thrombus formation are important in regulating homeostasis.

[0003] Vascular endothelial cells are more than simple components of the vascular wall. They produce and release vasoactive substances that relax 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 serves to activate the influx of leukocytes to the site of inflammation. In addition, the endothelium provides a surface for thrombus formation. Thus, it is the endothelial cells that play a key role in regulating homeostasis.

[0004] Thus, endothelial dysfunction, or vascular endotheliitis, can have serious consequences on blood flow, oxygen delivery, immune response, and thus homeostasis does not function normally. Vascular endotheliitis is characterized by reduced NO bioavailability. This increases the expression of adhesion molecules on the endothelial surface, which can initiate leukocyte recruitment to the vessel wall. Thus, endothelial inflammation, or endothelial injury, is observed in vascular endotheliitis. This can lead to defective lining of the blood vessels by the endothelium, exposing the subendothelial matrix to clotting factors in the blood. As a result, platelet aggregation and thrombus formation occur, leading to potentially fatal thrombi.

[0005] Vascular endotheliitis may be the cause of several diseases and is usually seen as a symptom rather than a cause of disease. As a result, treatment has focused on targeting the underlying disease rather than the endotheliitis itself. As a result of this, treatment of endotheliitis is inadequate. It is possible that the underlying endotheliitis actually increases disease severity and mortality, and that endotheliitis plays a much larger role than previously thought. Thus, treatment of endotheliitis rather than just the underlying disease may increase patient survival.

[0006] Vascular endotheliitis and endothelial injury are used interchangeably herein. Vascular endotheliitis can be caused by a number of diseases and / or conditions described herein, but vascular endotheliitis associated with COVID-19 or SARS-CoV-2 is primarily discussed herein. Those skilled in the art will appreciate that this discussion is merely for the purpose of providing examples and should not be considered limiting of the present invention.

[0007] In late 2019, a novel betacoronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) was identified in Wuhan, China, as the cause of coronavirus disease 2019 (COVID-19). The rapid geographic progression of COVID-19 has since culminated in the WHO declaring it a pandemic in March 2020 (1). Clinical symptoms in those infected with SARS-CoV-2 range from asymptomatic patients to more severe pneumonia, which can lead to acute respiratory distress syndrome (ARDS) and multiple organ failure. Most symptomatic patients experience mild to moderate illness and most commonly do not require hospitalization (2–4). However, there is a cohort of patients who can progress to more severe disease, and the progression of symptoms / clinical symptoms can take up to 2 weeks, starting from an early prodromal phase and culminating in ARDS (3). The subgroup of patients who become critically ill and require ventilation or extracorporeal membrane oxygenation (ECMO) are now known to have a very poor outcome, with a high mortality rate approaching 90% (5).

[0008] Since the disease was first described, over 90 million individuals have been affected worldwide. The pathophysiological pathway remains unclear; therefore, management is symptomatic. There is a lack of disease-modifying therapeutics that could be pursued while awaiting specific antivirals or vaccines. Several lines of evidence point towards endothelial dysfunction as a major pathophysiological mechanism in COVID-19. Prior to the current pandemic, endothelial dysfunction markers were found to be associated with disease severity and mortality in septic patients (6-9). Recently, Varga et al. (10) demonstrated widespread endothelial injury affecting pulmonary, renal, gastrointestinal, and hepatic vasculature in postmortem examinations of three COVID-19 patients. In one of the cases, the authors reported that "most small vessels appeared to be congested," and in another, the patient died from intestinal ischemia with evidence of causative endothelial injury.

[0009] Recently, two proposed hemostatic mechanisms have provided insights into an improved understanding of ARDS based on the molecular pathology associated with vascular endotheliitis, which promotes inflammation and coagulopathy in sepsis and other critical illnesses (11-14): one is the “two-activation theory of endothelium,” in which endothelial pathogenesis activates inflammatory pathways and microthrombosis, while the other is the novel “two-path integration theory” in which surgical procedures to stop blood flow initiate thrombus formation and promote microthrombosis, leading to vascular microthrombosis (VMTD) (11, 13, 15). These two theories are consistent with each other, as the endothelium contributes early to hemostasis and triggers the molecular mechanisms of thrombus formation. ARDS is often associated with sepsis from a variety of different causes and is seen in severe acute respiratory syndrome (SARS) due to SARS-CoV (16), Middle East respiratory syndrome (MERS) due to MERS-CoV (17) and now COVID-19. Sepsis-associated ARDS often occurs in the presence of other organ dysfunction, such as encephalopathy (18), liver failure (19)(20), acute renal failure, and acute necrotizing pancreatitis (21). This involvement of other organs suggests that ARDS may not be the primary disease but is part of an ongoing systemic pathogenesis caused by the infection or another critical illness.

[0010] On this basis, the physiological changes responsible for multiple organ failure in sepsis and other critical illnesses are the same as the circulatory dysfunction that occurs as a result of vascular endotheliitis-associated VMTD (EA-VMTD) (14, 15). Thus, infection causes injury to the endothelium that leads to vascular endotheliitis. This then leads to disseminated microthrombosis (DIMT), which can cause, for example, local hypoxia, systemic hypoxemia, and / or ischemia, and, as mentioned above, COVID-19 is now known to be associated with endothelial injury (10).

[0011] A case series of COVID-19 lung autopsies revealed the presence of numerous localized platelet-rich microthrombi and hemorrhagic foci in the lungs in parallel with diffuse alveolar damage (22). The authors hypothesized pulmonary localized thrombotic microangiopathy as key to the pathogenesis of COVID-19, and others have suggested that microthrombosis is an important driving force in the disease process (23). These microcirculatory changes were clearly demonstrated in the lungs, kidneys, and liver using contrast ultrasound (24, 25). Similar findings were also seen in the brain (26). Thus, there is growing evidence that COVID-19 appears to cause endothelial injury and diffuse and widespread microthrombosis.

[0012] Hypercoagulability and COVID-19 are now widely accepted, and studies have shown abnormal levels of D-dimer with higher levels associated with more severe disease and increased odds ratios of in-hospital mortality (27-30). Several case reports have noted acute pulmonary thrombosis in patients with COVID-19 pneumonia in the absence of major predisposing factors for venous thromboembolism (27, 31, 32). More recently, Panigada et al. showed that in addition to elevated D-dimer levels, there was a significant increase in the levels of factor VIII and von Willebrand factor (VWF) (33). A more than 500% increase in VWF levels and a more than 350% increase in factor VIII levels were reported by Escher et al. (34) for COVID-19. Furthermore, it was demonstrated that in thrombocytopenic patients, the risk of severe disease was increased by more than five-fold, and patients with the lowest platelet counts were associated with the highest mortality (33, 35, 36). Thus, both hypercoagulability and thrombocytopenia appear to be predictors of severe morbidity and mortality.

[0013] Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a key role in hemostasis and thrombus formation by platelet adhesion to injured and activated vessels. VWF is synthesized exclusively in megakaryocytes and endothelial cells (ECs), and interestingly, SARS-CoV can directly affect both of these cell types (22, 36).

[0014] The vast majority of VWF found in plasma is derived from VWF synthesized in ECs and stored in Weibel-Palade bodies (WPBs). Although restricted to ECs, there are differences in VWF synthesis in different vascular beds of the body, with small blood vessels in the lung and brain expressing higher levels of VWF than similarly sized vessels in the liver or kidney and higher levels in venous rather than arterial ECs (37). The majority of VWF stored in the WPB of endothelial cells consists of ultra-high molecular weight VWF (ULVWF). These ultra-high molecular weight VWF multimers are more adhesive than the lower molecular weight VWF multimers in the circulation (38). Upon secretion, ULVWF can spontaneously bind to platelets. Inflammatory cytokines such as interleukin-1 and tumor necrosis factor (TNF)-α can release their contents and trigger exocytosis of the WPB. Thus, plasma levels of VWF can be used as a marker of endothelial activation and vascular inflammation, and elevated levels of VWF have been found to be associated with ARDS and sepsis and to correlate independently with mortality ( 39 , 40 ).

[0015] Secreted VWF, which partially enters the circulation upon secretion from EC and partially binds to the endothelium, is sensitive to shear stress. This shear stress unfolds VWF, exposing sites for platelet binding, self-association, and cleavage by the enzyme ADAMTS13. These VWF molecules can self-associate into long "strings" that bind platelets in both arterial and venous flow directions and have previously been found to be adhesive to the endothelium (41-43). The protease, ADAMTS13, cleaves VWF and ULVWF, and its perfusion over these platelet-VWF strings rapidly removed them from the circulation (41). ULVWF multimers released from the WPB have a lower shear stress to unfold and therefore may be the initiating molecules for this self-assembly process that results in highly adhesive strings that trap platelets. Binding of VWF to platelets occurs through the GP Ib receptor. The binding site for this receptor is not normally exposed when VWF is in its globular form and therefore cannot bind platelets. Once VWF spreads, following shear stress, the binding sites become exposed and bind platelets with high affinity. Binding of platelets to VWF induces a conformational change that leads to activation of the integrin GPIIbIIIa (also known as α2bβ3), which may facilitate platelet-platelet binding as well as platelet-VWF binding. For this reason, the use of standard antiplatelet drugs is likely to be ineffective (aspirin or P2Y12 inhibitors) or only partially effective in mitigating this pathological process, as suggested by the cohort study of Tremblay et al. (44).

[0016] This ability to form VWF-platelet-rich thrombi in the microvasculature is a hallmark of acquired thrombotic thrombocytopenic purpura (TTP), in which autoantibodies against ADAMTS13 are present. It has also been found that interleukin-6 (IL-6) can inhibit the cleavage of ULVWF-platelet strings (45). Furthermore, at least in cultured cells, the synthesis of ADAMTS13 is dramatically inhibited by various cytokines, including IL-6 and TNF-α (46). This suggests that cytokine storms, and especially IL-6, may propagate microthrombosis. However, this also suggests that if intervention is performed early and there is no spike in cytokine release, the disease may be more manageable and a rapid deterioration in the patient's clinical condition may be avoided.

[0017] There is now a significant body of evidence suggesting that there is a very significant imbalance in the VWF:ADAMTS13 ratio as well as the levels of high molecular weight VWF multimers (equivalent to ULVWF) in COVID-19. As mentioned earlier, very high levels of VWF were previously shown with the earliest case report mentioning this sudden increase in VWF levels being that of Escher et al. (34). Then, Goshua et al. (47) demonstrated a reported significant increase in plasma VWF concentrations in patients admitted with COVID-19 with increased levels associated with disease severity - mean VWF antigen levels of 565 ± 199% vs. 278 ± 133% (p < 0.0001) for patients admitted to the intensive care unit (ICU) compared to patients not treated in the ICU. Next, Rauch et al. (48) looked at the progression of COVID-19 patients in relation to these admitted VWF levels. Patients with the highest VWF levels required more extensive oxygen support, whereas patients with normal VWF levels did not require hospitalization or supplemental oxygen (n=10).

[0018] Shortly after the publication of Rauch et al., Ladikou et al. (49) showed increased VWF antigen levels in COVID-19 patients admitted to the ICU with a positive correlation seen in VWF levels and age of the patients. They reported a median VWF antigen level of 350%, however, importantly, they also showed a significantly reduced ADAMTS13 level (49.7%), suggesting a loss of VWF-cleaving proteases that normally degrade high molecular weight VWF multimers and reduce their activity. They speculated that the excessive release of VWF seen in COVID-19 patients could lead to depletion of ADAMTS13, contributing to a prothrombotic state. Furthermore, analysis of these data showed that the median VWF level was significantly higher (p=0.015) in patients who died (477%) compared to those who survived (335%).

[0019] Helms et al. (50) recently published a multicenter prospective cohort study in France evaluating the thrombosis risk in COVID-19 patients, which showed that VWF and factor VIII were considerably increased. In conjunction with this data showing an increase in VWF and a decrease in ADAMTS13, there are further studies showing that the VWF:ADAMTS13 ratio is significantly deranged. Huisman et al. (51) first showed that the mean VWF antigen:ADAMTS13 ratio was 8.5 (normal 0.5-2) from 12 patients admitted to the ICU. Later, Mancini et al. (52) demonstrated similar findings with an elevated ratio of von Willebrand factor antigen (VWF:Ag) to ADAMTS13 activity strongly associated with disease severity, with the worst ratio, 8.3, seen in patients requiring high-intensity care (intubation and mechanical ventilation), compared to patients requiring low-intensity care, 3.42 (p<0.001).

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

[0021] It is possible to develop a unifying theory that is caused by vascular endotheliitis and endothelial injury, which triggers the release of VWF and ULVWF, which leads to microthrombus formation. This then leads to hypoxia, which can intensify the process with a "cytokine storm" and the release of IL-6, which inhibits and reduces the function of ADAMTS13, with disseminated microthrombosis and multiple organ dysfunction and failure ensues. This microvascular thrombosis at the pulmonary level has also been suggested to be the cause of right ventricular dysfunction (54). This mechanism may be the primary cause of many of the currently observed findings, including high D-dimer levels (high levels due to the massive levels of microthrombosis), high levels of factor VIII and VWF (released from the WPB in response to endothelial injury) and the atypical ARDS picture seen (55), as well as the widespread clinical picture of microthrombosis and pulmonary, neurological and gastrointestinal symptoms. The endothelial injury and microthrombosis we suggest may also explain why patients with pre-existing vascular endotheliitis and microarteriopathy (e.g., secondary to diabetes, hypertension, or obesity) are at increased risk for severe COVID-19 (29, 56). Similarly, a rapidly growing body of evidence is linking patients with low levels of ADAMTS13 and high levels of VWF with a variety of diseases that predispose them to poor outcomes after SARS-CoV-2 infection and its variable symptoms (57-64). The use of standard antiplatelet drug treatments (aspirin or P2Y12 inhibitors) is also likely to be effective, assuming that VWF interaction with platelets activates the GP2b3a receptor. Inhibition of VWF-platelet binding by the GP1b receptor, using either caplacizumab or amfibatide, is an attractive option and has been proposed (65), but these drugs are not widely used and clinical experience with them is very limited. Similarly, they have a significant bleeding profile.

[0022] Thus, there is a need for a treatment for vascular endotheliitis itself. Summary of the Invention

[0023] Heparin is a naturally occurring highly sulfated polysaccharide characterized by polysaccharide chains of a wide range of molecular weights. Heparin acts on a variety of different ligands with diverse actions. Heparin is a member of the glycosaminoglycan carbohydrate family and consists of repeating disaccharide units of GlcAβ1-4GlcNAcα1-4 with polydisperse sulfation, N-acetylation and uronosyl epimerization. Heparin is highly heterogeneous. Heparin isolated from natural sources contains polysaccharide chains with molecular weights ranging from about 3000 Da to about 30,000 Da. This is known as unfractionated heparin (UFH). UFH can be enzymatically or chemically treated to provide shorter polysaccharide chains. The products of enzymatically or chemically treated UFH can be affinity purified to obtain fractionated heparins, and the molecular weights of the polysaccharides in each fraction can be easily determined. Low molecular weight heparins (LMWHs) contain polysaccharide chains ranging from about 4000 Da to about 8000 Da.

[0024] In 1991, it was first demonstrated that intravenous administration of heparin to patients undergoing open-heart surgery induced VWF-dependent platelet dysfunction without changes in plasma VWF levels (66). This inhibitory effect of heparin on VWF-dependent platelet coagulation was independent of the affinity of heparin for antithrombin III, but was dependent on the degree of heparin dissociation. Recent in vitro experiments have shown that heparin binds to a specific amino acid sequence (residues 569–583) in the A1 domain of VWF, which contains a regular sequence of basic amino acids. Heparin binding induced a conformational change in the peptide at this binding site (67). Heparin bound similarly to both activated and inactivated VWF, but did not interfere with VWF binding to collagen. Because the platelet GpIb-binding domain (residues 524–542) is also located in the A1 domain, it was suggested that heparin does not interfere with VWF binding to platelet GpIb both by steric hindrance and by inducing a conformational change in the domain that leads to inhibition of platelet binding.

[0025] The structural specificity of heparin that contributes to binding to VWF revolves around the major disaccharide units -GlcNS6S-IdoA2S and IdoA2S-GlcNS6S. Furthermore, it was demonstrated that the assembly of more than three units of disaccharide is important for the binding capacity. Similarly, low molecular weight (6100 Da (g / mol)) fractionated heparins showed higher affinity for binding to VWF, but they were less able to inhibit VWF activity compared to UFH. This suggests that the minimum heparin molecular weight and molecular size are important to obtain steric hindrance.

[0026] These intermediate molecular weight heparins have specificity for inhibiting VWF-GPIb binding and therefore may stop microthrombosis, but they have little anticoagulant effect because they have little effect on antithrombin III. Therefore, intermediate molecular weight heparins with a mass of about 11000 Da (g / mol) are the ideal therapeutic option when considering the treatment of patients with prothrombotic conditions dependent on increased VWF levels and vascular endotheliitis. Furthermore, the results of these previous studies suggest that low molecular weight heparins are unlikely to work well and do not target GPIb receptors, but while UFH may contain sugar moieties that can bind VWF, UFH is not optimal. Furthermore, monitoring of UFH is difficult, and other fractions of UFH, such as LMWH fractions, have anticoagulant effects that may lead to unpredictable and dangerous bleeding events.

[0027] Even more intriguing is the fact that SARS-CoV-2 has recently been found to bind heparin sulfate and, in particular, to require the IdoA2S-GlcNS6S sugar moiety (74, 75). This suggests that exogenous supply of these sugar moieties may inhibit binding to endogenous heparin sulfate in the lung and thus act as a preventative treatment. Taken together, specialized medium molecular weight heparins (~11,000 Da (g / mol) with at least three units of the GlcNS6S-IdoA2S disaccharide inhibit viral attachment and replication, but may also inhibit microthrombosis caused by the release of VWF secondary to viral-induced vascular endotheliitis.

[0028] As described herein, vascular endotheliitis may be associated with a number of diseases. The present inventors have discovered that intermediate molecular weight heparin can be used to treat vascular endotheliitis, particularly in patients with elevated plasma von Willebrand factor levels.

[0029] Therefore, the present invention provides a medium molecular weight heparin for use in treating vascular endotheliitis. The medium molecular weight heparin can inhibit von Willebrand factor (VWF). The medium molecular weight heparin can inhibit VWF multimers, preferably ultra-high molecular weight VWF. The medium molecular weight heparin can inhibit the binding of platelets to VWF.

[0030] Thus, in a first aspect, the present invention provides an intermediate molecular weight heparin for use in treating vascular endotheliitis in a patient, preferably where the patient has a plasma von Willebrand factor to ADAMTS13 ratio of at least about 2. Alternatively or additionally, the patient may have a von Willebrand factor antigen to ADAMTS13 ratio of at least about 2.

[0031] The intermediate molecular weight heparin can inhibit von Willebrand factor (VWF). The intermediate molecular weight heparin can inhibit VWF multimers, preferably ultra-high molecular weight VWF. The intermediate molecular weight heparin can inhibit the binding of platelets to VWF.

[0032] Patients may have a VWF:ADAMTS13 ratio of at least about 2, at least about 4, at least about 8, or at least about 10. Patients may have a VWF:ADAMTS13 ratio of greater than about 2, greater than about 4, or greater than about 8, or greater than about 10. Patients may have a VWF:ADAMTS13 ratio of about 2-16, about 4-12, or preferably about 6-10. Patients with a VWF:ADAMTS13 ratio of greater than about 8 usually exhibit severe disease and often represent patients who are deteriorating towards death.

[0033] Patients may have a VWF antigen:ADAMTS13 ratio of at least about 2, at least about 4, at least about 8, or at least about 10. Patients may have a VWF antigen:ADAMTS13 ratio of greater than about 2, greater than about 4, or greater than about 8, or greater than about 10. Patients may have a VWF antigen:ADAMTS13 ratio of about 2-16, about 4-12, or preferably about 6-10. Patients with a VWF antigen:ADAMTS13 ratio of greater than about 8 usually exhibit severe disease and often represent patients who are deteriorating towards death.

[0034] The levels of VWF and ADAMTS13 in patients can be measured using ELISA. The ratio can be calculated as described by Huisman et al. (51). Briefly, VWF antigen levels can be determined in international units, ADAMTS13 levels can be determined in international units, and the VWF antigen:ADAMTS13 ratio can be determined.

[0035] The normal level of plasma VWF is in the range of about 50 IU / dL to about 200 IU / dL. In the general population, the average level of plasma VWF is about 100 IU / dL. A high level of plasma VWF is about 200 IU / dL or more, for example, about 200 IU / dL to about 400 IU / dL, about 225 IU / dL to about 375 IU / dL, about 250 IU / dL to about 350 IU / dL, or about 275 IU / dL to about 300 IU / dL.

[0036] The patient may have an elevated VWF antigen level of about 150% or more, about 175% or more, about 200% or more, about 300% or more, about 350% or more, or about 400% or more, or about 500% or more. The patient may have a VWF antigen level of about 600% or less, about 700% or less, about 800% or less, or about 1000% or less.

[0037] However, plasma VWF levels may be temporarily elevated due to infection, inflammation, trauma, and physical and emotional stress. Thus, the patient may have elevated plasma von Willebrand factor levels for a non-temporary period, for example, at least about 6 hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours. Preferably, the patient may have elevated plasma von Willebrand factor levels for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days. Even more preferably, the patient may have elevated plasma von Willebrand factor levels for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. Even more preferably, the patient may have elevated plasma von Willebrand factor levels for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year. The patient may have elevated plasma von Willebrand factor levels for up to about 1 week, up to about 4 weeks, up to about 2 months, up to about 4 months, up to about 6 months, or up to about 1 year.

[0038] The medium molecular weight heparin may have a mass of about 11000 Da (g / mol). The medium molecular weight heparin may contain at least three units of the GlcNS6S-IdoA2S (or IdoA2S-GlcNS6S) disaccharide.

[0039] Vascular endotheliitis may be due to COVID-19, viral infections, acute respiratory distress syndrome (ARDS), cancer, bacterial infections, sepsis, cardiovascular disease, diabetes, trauma, especially brain or head trauma, burns, inhalation injury, drug reactions, hematological conditions, subarachnoid hemorrhage, aortic aneurysm disease, stroke, or cerebral parenchymal hemorrhage. Vascular endotheliitis may be due to viral infections, which may be SARS-CoV-2. Vascular endotheliitis may be due to cancer, especially 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.

[0040] Treatment of vascular endotheliitis with medium molecular weight heparin may inhibit hematogenous spread of cancer. Human tumor cells can and demonstrate this ability to bind VWF under high shear flow conditions, including both melanoma and colon cancer cells. Immobilized platelets bound to VWF were found to mediate tethering, rolling, and strong adhesion of different cancerous cell lines under flow shear stress. VWF played a key role in enabling this strong adhesion of tumor cells to immobilized platelets. Current data suggest that VWF plays a key role in tethering of cancerous cells. In addition, VWF-platelet binding, which occurs as part of the normal thrombosis pathway, may allow tumor cells to coalesce with VWF-platelets to generate heteroaggregates of VWF+platelets+cancer cells, which may further act to aid in the blood-borne (hematogenous) spread of tumor cells. This process is at least partially driven by the ability of cancer cells to migrate to the vessel wall, which may then spread to other organs once initial binding to VWF and platelets has occurred. In addition, various cancers have been found to cause vascular endotheliitis through the resulting release of UL-VWF. Through this mechanism, tumors then cause the release of UL-VWF, which allows platelets to bind to tumor cells and the hematogenous and metastatic spread of cancer. This cancer-induced vascular endotheliitis also leads to an overall increased risk of thrombosis in patients with underlying malignant tumors. Therefore, any treatment aimed at treating vascular endotheliitis and inhibiting the binding of platelets and / or tumor cells to VWF will serve the dual purpose of reducing the risk of malignant tumor-associated thrombosis and also reducing the risk of hematogenous metastatic spread.

[0041] The medium molecular weight heparin may be administered by a method of administration selected from the group consisting of parenteral, subcutaneous, intravenous, intramuscular, intrathecal, intradermal, intraarterial, intraarticular, cutaneous, transdermal, subcutaneous, depot form, e.g., depot injection, intraosseous, and inhalation.Preferably, the method of administration of the medium molecular weight heparin is subcutaneous, intravenous, or intramuscular.The method of administration may be inhalation, optionally by nebulizer.

[0042] The intermediate molecular weight heparin may be administered at a dose of about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 50 mg / kg, about 70 mg / kg, about 80 mg / kg, or about 100 mg / kg. The intermediate molecular weight heparin may be administered at a dose of about 500 mg / kg or less, about 300 mg / kg or less, about 200 mg / kg or less, or about 100 mg / kg or less. The intermediate molecular weight heparin may be administered at a dose of about 0.01 mg / kg to about 10 mg / kg, preferably about 0.2 mg / kg to about 10 mg / kg, about 0.2 mg / kg to about 1.6 mg / kg. The intermediate molecular weight heparin may be administered as a single dose or continuous administration. The dosage of medium molecular weight heparin may depend on the VWF antigen:ADAMTS13 ratio or overall VWF level. A person skilled in the art will be able to select the appropriate amount for a patient based on the VWF antigen:ADAMTS13 ratio or overall VWF level.

[0043] The medium molecular weight heparin may be included in a pharmaceutical formulation. The pharmaceutical formulation may include an excipient. The excipient may be selected from the group including solvents, cosolvents, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavorings, lubricants, suspending agents, isotonicity agents, surfactants, solubilizers, dispersing aids, dispersing agents, humectants, thickeners, colorants, wetting agents, antifoaming agents, viscosity adjusters, sweeteners, and combinations thereof. The pharmaceutical formulation may include an additional active agent. The additional active agent may include low molecular weight heparin.

[0044] The medium molecular weight heparin may comprise a chemical modification, which may be selected from the group including N-acetylation, N-deacetylation, N-sulfation, O-sulfation, de-2-O-sulfation, and complete desulfation.

[0045] In a second aspect, the present invention provides an intermediate molecular weight heparin for use in the treatment of a disease or condition in a patient, wherein the patient has vascular endotheliitis characterised by a ratio of plasma von Willebrand factor to ADAMTS13 (VWF:ADAMTS13) of at least about 2.

[0046] In some embodiments, the invention provides an intermediate molecular weight heparin for use in the treatment of COVID-19 in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0047] In some embodiments, the invention provides an intermediate molecular weight heparin for use in the treatment of a viral infection in a patient, wherein the patient has vascular endotheliitis characterized by a VWF:ADAMTS13 ratio of at least about 2. The viral infection may be SARS-CoV-2.

[0048] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of acute respiratory distress syndrome (ARDS) in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0049] In some embodiments, the invention provides an intermediate molecular weight heparin for use in treating cancer in a patient, wherein the patient has vascular endotheliitis characterized by a VWF:ADAMTS13 ratio of at least about 2. The cancer may be leukemia, lymphoma, myeloma, or a solid organ cancer, such as colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer, or skin cancer.

[0050] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of a bacterial infection in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0051] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of sepsis in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0052] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of cardiovascular disease in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0053] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of diabetes in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0054] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of trauma in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0055] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of burns in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0056] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in treating inhalation injury in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0057] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of a drug reaction in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0058] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of a hematological condition in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0059] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of subarachnoid hemorrhage in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0060] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of aortic aneurysmal disease in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF:ADAMTS13 ratio of at least about 2.

[0061] In a third aspect, the present invention provides an intermediate molecular weight heparin for use in the treatment of a disease or condition in a patient, wherein the patient has vascular endotheliitis characterised by a ratio of plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen:ADAMTS13) of at least about 2.

[0062] In some embodiments, the invention provides an intermediate molecular weight heparin for use in the treatment of COVID-19 in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0063] In some embodiments, the invention provides an intermediate molecular weight heparin for use in the treatment of a viral infection in a patient, wherein the patient has vascular endotheliitis characterized by a VWF antigen:ADAMTS13 ratio of at least about 2. The viral infection may be SARS-CoV-2.

[0064] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of acute respiratory distress syndrome (ARDS) in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0065] In some embodiments, the invention provides an intermediate molecular weight heparin for use in treating cancer in a patient, wherein the patient has vascular endotheliitis characterized by a VWF antigen:ADAMTS13 ratio of at least about 2. The cancer may be leukemia, lymphoma, myeloma, or a solid organ cancer, such as colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer, or skin cancer.

[0066] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of a bacterial infection in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0067] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of sepsis in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0068] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of cardiovascular disease in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0069] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of diabetes in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0070] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of trauma in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0071] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of burns in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0072] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in treating inhalation injury in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0073] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of a drug reaction in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0074] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of a hematological condition in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0075] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of subarachnoid hemorrhage in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0076] In some embodiments, the present invention provides an intermediate molecular weight heparin for use in the treatment of aortic aneurysmal disease in a patient, wherein the patient has vascular endotheliitis characterized by a plasma VWF antigen:ADAMTS13 ratio of at least about 2.

[0077] For the avoidance of doubt, the embodiments described herein relating to the intermediate molecular weight heparin of the first aspect of the invention also apply mutatis mutandis to the intermediate molecular weight heparin of the second and third aspects of the invention.

[0078] The medium molecular weight heparin for use in the treatment of vascular endotheliitis as defined in the first aspect of the present invention is particularly advantageous since said heparin is capable of inhibiting microthrombosis caused by the release of VWF secondary to vascular endotheliitis due to any disease or pathology, and in the case where the cause of vascular endotheliitis is SARS-CoV-2, said heparin is additionally capable of inhibiting viral attachment and replication.

[0079] In a fourth aspect, the present invention provides a kit comprising a medium molecular weight heparin for use in the treatment of vascular endotheliitis, particularly according to the first or second or third aspect of the invention.

[0080] In a fifth aspect, the present invention provides a method for treating vascular endotheliitis, the method comprising administering a therapeutically effective amount of intermediate molecular weight heparin to a subject in need of treatment, wherein the patient has a plasma VWF:ADAMTS13 ratio of at least about 2.

[0081] In a sixth aspect, the present invention provides the use of a medium molecular weight heparin as defined in the first or second or third aspect for the manufacture of a medicament for the treatment of vascular endotheliitis.

[0082] For the avoidance of doubt, the embodiments described herein relating to the intermediate molecular weight heparin of the first aspect of the invention also apply mutatis mutandis to the second to sixth aspects. [Brief description of the drawings]

[0083] The invention will now be described with reference to the following figures, which are intended to be non-limiting. [Figure 1] FIG. 1 shows a graph of the activity of low molecular weight (LMW), unfractionated (UF) and medium molecular weight (MMW) heparin against factor IIa. [Diagram 2] FIG. 2 shows a graph of the activity of LMW heparin, UF heparin and MMW heparin against factor X. [Diagram 3] FIG. 3 shows transmitted light aggregometry (LTA) tracings of inhibition of VWF-induced platelet aggregation with 5 μM, 10 μM and 15 μM doses of MMW heparin versus vehicle control. [Figure 4] FIG. 4 shows a transmitted light aggregometry (LTA) trace of the inhibition of VWF-induced platelet aggregation with a 15 μM dose of MMW heparin. [Diagram 5]5A and 5B show the effects of MMWH and LMWH on VWF-dependent platelet aggregation. [Figure 6] FIG. 6 shows LTA traces of inhibition of VWF-induced platelet aggregation with 5 μM, 10 μM and 20 μM MMWH. [Figure 7] FIG. 7 shows LTA traces of inhibition of VWF-induced platelet aggregation with 5 μM and 10 μM MMWH. [Figure 8] FIG. 8 shows LTA traces of inhibition of VWF-induced platelet aggregation with 10 μM MMWH and anti-VWF mAb. [Figure 9] FIG. 9 shows LTA traces of inhibition of VWF-induced platelet aggregation with 5 μM and 20 μM MMWH and anti-VWF mAb. [Figure 10] FIG. 10 shows the LTA traces of inhibition of VWF-induced platelet aggregation with 10 μM LMWH and anti-VWF mAb. [Figure 11] FIG. 11 shows the LTA traces of inhibition of VWF-induced platelet aggregation with 20 μM LMWH. [Figure 12] FIG. 12 shows the statistical analysis (area under the curve and slope) of MMWH, LMWH and mAb inhibition of VWF-induced platelet aggregation. [Figure 13A] FIG. 13 shows LTA traces of inhibition of VWF-induced platelet aggregation using 5 μM, 10 μM and 20 μM MMWH with non-ristocetin agonists: A. ADP as agonist; B: collagen as agonist; C. TRAP6 as agonist. [Figure 13B] FIG. 13 shows LTA traces of inhibition of VWF-induced platelet aggregation using 5 μM, 10 μM and 20 μM MMWH with non-ristocetin agonists: A. ADP as agonist; B: collagen as agonist; C. TRAP6 as agonist. [Figure 13C]FIG. 13 shows LTA traces of inhibition of VWF-induced platelet aggregation using 5 μM, 10 μM and 20 μM MMWH with non-ristocetin agonists: A. ADP as agonist; B: collagen as agonist; C. TRAP6 as agonist. [Figure 14] FIG. 14 shows graphs of MMWH (0 μM, 5 μM, 10 μM and 20 μM) inhibition of VWF-induced platelet aggregation in the presence of agonists: A. ADP; B. Collagen; C. TRAP-6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] Throughout this specification, one or more aspects of the present invention may be combined with one or more features described herein to define additional embodiments of the present invention.

[0085] In the discussion which follows, reference will be made to a number of terms, which should be understood to have the meanings set forth below, unless the context expressly indicates to the contrary.

[0086] As used herein, a reference to a singular noun includes a plural noun and vice versa, unless the context indicates otherwise.

[0087] Throughout this specification, the term "comprise" or variations such as "comprises" or "comprising" are understood to imply the inclusion of a specified element or integer, or group of elements or integers, but not the exclusion of other elements or integers, or groups of elements or integers. The term "comprising" includes within its scope the terms "consisting" or "consisting essentially of."

[0088] The term "consisting" or variations thereof should be understood to imply the inclusion of a specified element or integer, or group of elements or integers, as well as the exclusion of other elements or integers, or groups of elements or integers.

[0089] The term "consisting essentially of" or variations thereof implies the inclusion of the specified element, integer or step, or group of elements, integers or steps, and it should be understood that additional components may be present, but only those that do not materially affect the essential characteristics of the formulation, composition, or compound.

[0090] As used herein, the term "about" when specifying a number or value, is used to indicate a value that is within ±5% of the specified value.

[0091] The terms "treatment" and "therapy" define the therapeutic management of a patient to slow or stop the progression of, or to ameliorate or cure, a disorder or condition. Prevention of a disorder or condition as a result of treatment or therapy is also included.

[0092] As used herein, the term "patient" preferably refers to a mammal. Typically, the mammal is a human.

[0093] Von Willebrand factor (VWF) is a blood glycoprotein involved in hemostasis. VWF is a high molecular weight multimeric glycoprotein present in plasma and is constitutively produced as ultra-high molecular weight VWF in the endothelium (Weibel-Palade bodies), megakaryocytes (α-granules of platelets), and subendothelial connective tissue. The basic VWF monomer is a 2050 amino acid protein.

[0094] A disaccharide is a sugar whose molecule contains two monosaccharide residues.

[0095] Low molecular weight heparin is defined herein as heparin having an average molecular weight of about 4000 Da (g / mol) to about 8000 Da (g / mol). Medium molecular weight heparin is defined herein as heparin having an average molecular weight of greater than about 8000 Da (g / mol) to about 13000 Da (g / mol).

[0096] Vascular endotheliitis may be caused by any disease. In particular, vascular endotheliitis may be caused by COVID-19, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, sepsis, cardiovascular disease, diabetes, trauma, especially head trauma, burns, inhalation injury, drug reactions, hematological conditions, subarachnoid hemorrhage, aortic aneurysmal disease, stroke, or cerebral parenchymal hemorrhage.

[0097] The infection may be bacterial, fungal, or parasitic. The infection may be bacterial. Bacterial infections include Actinomyces israelii, Bacillus anthracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelaii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, and jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis tularensis, Haemophilus influenzae, Helicobacter pyloripylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira species, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroides, Rickettsia rickettsii The bacterial strain may be Salmonella rickettsii, Salmonella, Shigella, Spirochaetes Staphylococcus, Streptococcus, Treponema pallidum, Vibrio cholerae, or Yersinia pestis.

[0098] The infection may be fungal. The fungal infection may be Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, Histoplasma, mucormycetes, Tinea cruris, Tinea corporis, or Tinea pedis.

[0099] The infectious disease may be parasitic. The parasitic infectious disease may be a protozoan eye infection, Chagas disease, leishmaniasis, toxoplasmosis, giardiasis, malaria, microsporidiosis, or rhinosporidiosis. Preferably, the parasitic infectious disease is malaria.

[0100] The viral infection may be SARS-CoV-2. SARS-CoV-2 is the virus that causes the disease COVID-19. COVID-19 may result in ARDS. Vascular endotheliitis may be due to SARS-CoV-2 infection. Vascular endotheliitis may be due to COVID-19. Vascular endotheliitis may be due to ARDS.

[0101] Vascular endotheliitis may be caused by cancer.Cancer may be leukemia, lymphoma or myeloma.Alternatively or in addition, cancer may be solid organ cancer, such as colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer or skin cancer.

[0102] Vascular endotheliitis can be due to a hematological condition, such as thrombotic thrombocytopenic purpura, anemia, or sickle cell disease.

[0103] Endothelial cell dysfunction may allow a passageway for tumor cells circulating in the blood to enter tissues. Thus, treatment of vascular endotheliitis may prevent hematogenous spread of blood-borne cancer. Treatment of vascular endotheliitis may inhibit hematogenous spread of cancer. Medium molecular weight heparin may inhibit hematogenous spread of cancer.

[0104] Biomarkers of vascular endotheliitis may include elevated von Willebrand factor (VWF) levels, ultra-high molecular weight von Willebrand factor (ULVWF) levels, factor VIII levels, as well as syndecan-1, VWF antigen, VWF activity, VWF multimers, ADAMTS13 levels, platelet count, VCAM-1, ICAM-1, P-selectin levels, VWF:ADAMTS13 ratio or VWF antigen:ADAMTS13 ratio.Preferably, the biomarker of vascular endotheliitis is the ratio of VWF:ADAMTS13 or VWF antigen:ADAMTS13.

[0105] The patient may have elevated plasma von Willebrand factor (VWF) levels compared to healthy control subjects. The patient may have persistently elevated plasma VWF levels compared to healthy controls. The plasma VWF levels may be elevated compared to healthy control subjects for a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or preferably at least about 1 week. The plasma VWF levels may be elevated compared to healthy control subjects for a period of up to about 1 week, up to about 4 weeks, up to about 2 months, up to about 4 months, up to about 6 months, or up to about 1 year.

[0106] For example, the plasma VWF level may be increased to at least about 50 nmol / L, preferably at least about 60 nmol / L, even more preferably at least about 70 nmol / L, or even more preferably at least about 90 nmol / L. The plasma VWF level may be increased to about 130 nmol / L, about 150 nmol / L, or about 200 nmol / L. The plasma VWF level may be increased to at least about 50 nmol / L for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 1 month, or at least about 1 year. The plasma VWF level may be increased to at least about 60 nmol / L for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 1 month, or at least about 1 year. The plasma VWF level may be increased to at least about 70 nmol / L for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 1 month, or at least about 1 year. The plasma VWF level may be increased to at least about 90 nmol / L for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 1 month, or at least about 1 year. The plasma VWF level may be measured using an enzyme-linked immunosorbent assay (ELISA).

[0107] Alternatively or additionally, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or more for at least about 6 hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours. Preferably, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or more for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days. Even more preferably, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or more for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks. Even more preferably, the patient may have a plasma von Willebrand factor level of about 200 IU / dL or more for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year.

[0108] Vascular endothelial function can be assessed in the coronary arteries and in the peripheral circulation. Non-invasive tests for the assessment of coronary endothelial function include Doppler echocardiography, which measures blood flow in response to pharmacological or physiological stimuli. Other tests include positron emission tomography and phase contrast magnetic resonance imaging. However, the gold standard test involves invasive quantitative coronary angiography, which examines the change in diameter in response to intracoronary infusion of an endothelium-dependent vasodilator, such as acetylcholine. Assessment of the endothelium in the peripheral circulation includes brachial artery ultrasound and strain gauge venous impedance plethysmography.

[0109] The binding of intermediate molecular weight heparin to VWF may be assessed by a competitive binding assay. Heparin-Sepharose beads are bound to labeled VWF, e.g. 125I-vWF may be incubated for a period of time to allow the labeled VWF to bind to the immobilized heparin. Various concentrations of intermediate molecular weight heparin may then be added and the amount of labeled VWF displaced may be determined. Other methods for determining intermediate molecular weight heparin bound to VWF may include surface plasmon resonance, biolayer interferometry, isothermal titration calorimetry, fluorescence polarization binding assay, ELISA and microscale thermophoresis.

[0110] Inhibition of platelet binding to VWF may be evaluated by ristocetin-induced coagulation of fixed platelets. Platelets may be incubated with medium molecular weight heparin and citrated plasma (VWF source). Ristocetin may then be added and platelet coagulation may then be determined. MMWH can completely inhibit VWF-induced platelet aggregation at a concentration of 15 μM as measured by ristocetin-induced platelet aggregation assay. Other methods for determining inhibition of VWF binding to platelets may include ELISA, fluorescence-assisted cell sorting, dynamic light scattering, or flow chamber assay.

[0111] The intermediate molecular weight heparin may have a mass ranging from about 8000 Da (g / mol) to about 13000 Da (g / mol), preferably about 10000 Da (g / mol) to about 12000 Da (g / mol). The intermediate molecular weight heparin may have a mass of about 11000 Da (g / mol). The intermediate molecular weight heparin may comprise polysaccharide chains having an average molecular mass ranging from about 9000 Da (g / mol) to about 13000 Da (g / mol), preferably about 10000 Da (g / mol) to about 12000 Da (g / mol). The intermediate molecular weight heparin may comprise polysaccharide chains having an average molecular mass of about 11000 Da (g / mol). The molecular weight of the intermediate molecular weight heparin may be determined, for example, by mass spectrometry or size exclusion chromatography.

[0112] The intermediate molecular weight heparin may be chemically synthesized. The intermediate molecular weight heparin may be enzymatically synthesized. The intermediate molecular weight heparin may be purified using high pressure liquid chromatography.

[0113] The intermediate molecular weight heparin may comprise at least 3 units of GlcNS6S-IdoA2S disaccharide, e.g., at least 4 units, at least 5 units, at least 6 units, at least 8 units, or at least 10 units. The intermediate molecular weight heparin may comprise 25 units or less of GlcNS6S-IdoA2S disaccharide, e.g., 20 units or less. The presence of GlcNS6S-IdoA2S disaccharide units may be determined by antibodies, mass spectrometry, or infrared light from chemical and enzyme tests. The GlcNS6S-IdoA2S units may be arranged in order.

[0114] The medium molecular weight heparin may contain at least 3 units of IdoA2S-GlcNS6S disaccharide, e.g., at least 4 units, at least 5 units, at least 6 units, at least 8 units, or at least 10 units. The medium molecular weight heparin may contain 25 units or less of IdoA2S-GlcNS6S disaccharide, e.g., 20 units or less. The presence of IdoA2S-GlcNS6S disaccharide units may be determined by antibodies, mass spectrometry, or infrared light from chemical and enzyme tests. The IdoA2S-GlcNS6S units may be arranged in order. The number of IdoA2S-GlcNS6S units may be adjusted to provide the desired anti-VWF activity and / or standard anticoagulant activity.

[0115] The intermediate molecular weight heparin may comprise UA2S-GlcNS6S, UA2S-GlcNS, UA-GlcNAc. The intermediate molecular weight heparin may comprise at least about 60% UA2S-GlcNS6S, UA2S-GlcNS, and UA-GlcNAc. The intermediate molecular weight heparin may comprise at least about 45%, preferably at least about 48%, preferably at least about 49%, preferably at least about 60% UA2S-GlcNS6S. The intermediate molecular weight heparin may comprise no more than about 60%, preferably no more than about 70%, preferably no more than about 85% UA2S-GlcNS6S. The intermediate molecular weight heparin may comprise at least about 4%, preferably at least about 5%, preferably at least about 6%, preferably at least about 10% UA2S-GlcNS. The intermediate molecular weight heparin may comprise no more than about 15%, preferably no more than about 20% UA2S-GlcNS. The intermediate molecular weight heparin may comprise at least about 4%, preferably at least about 5%, preferably at least about 6%, preferably at least about 10% UA-GlcNAc. The intermediate molecular weight heparin may comprise no more than about 15%, preferably no more than about 20% UA-GlcNAc. In some embodiments, the intermediate molecular weight heparin may comprise at least 49.2% UA2S-GlcNS6S, 5.4% UA2S-GlcN, and 5.4% UA-GlcNAc. In some embodiments, the intermediate molecular weight heparin may comprise at least 82% UA2S-GlcNS6S, 9% UA2S-GlcNS, and 9% UA-GlcNAc. The percentage composition of UA-GlcNAc that comprises the intermediate molecular weight heparin may be enriched compared to unfractionated heparin.

[0116] The medium molecular weight heparin may be administered by a method of administration selected from parenteral, subcutaneous, intravenous, intramuscular, intrathecal, intradermal, intraarterial, or intra-articular, cutaneous, transdermal, subcutaneous, depot form, e.g., depot injection, intraosseous, and inhalation. Preferred methods of administration include subcutaneous, intravenous, intramuscular, or inhalation.

[0117] Previous studies have examined UFH as a nebulized agent in a variety of conditions. Small studies have shown that it limits pulmonary fibrin deposition, attenuates the progression of acute lung injury, and speeds recovery (69). Early phase trials in patients with acute lung injury and related conditions have found that nebulized UFH reduces pulmonary dead space, coagulation activation, and microvascular thrombosis, improves lung injury, and increases time free of ventilatory support (70-73). In a pre-pandemic double-blind randomized trial of 256 critically ill mechanically ventilated patients, nebulized UFH limited the progression of lung injury, including acute respiratory distress syndrome, and facilitated survivors returning home. Thus, intermediate molecular weight heparin may be administered by inhalation through a nebulizer.

[0118] Heparin dosage is usually measured in "Howell units". One unit of heparin ("Howell unit") is approximately equal to 0.002 mg of pure heparin, which is the amount required to keep 1 ml of feline blood fluid for 24 hours at 0°C. The intermediate molecular weight heparin may be administered at a bolus dose of about 5000 units per hour, which may be delivered by an infusion pump, followed by about 1200 to 1600 units. The intermediate molecular weight heparin may be administered at a dose of about 18 units / kg to about 5000 units / kg. Preferably, the intermediate molecular weight heparin may be administered at a dose of about 100 units / kg to about 800 units / kg. Alternatively, the intermediate molecular weight heparin may be administered at a dose of about 18 units / kg to about 75 units / kg. The intermediate molecular weight heparin may be administered at a dose of about 5000 units, about 4000 units, about 3000 units, about 2000 units, about 1000 units, or 500 units every 12 hours. The intermediate molecular weight heparin may be administered at a dose of 5000 units every 12 hours.

[0119] The intermediate molecular weight heparin may be administered at a dose 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 intermediate molecular weight heparin may be administered at a dose 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 intermediate molecular weight heparin may be administered at a dose of about 18 units / kg to about 75 units / kg. The dose may be given as a single dose or as a continuous dose. The dose may be given over a period of time. The period may be about 1 month to about 12 months, for example, about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, about 6 months to about 7 months. The period may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, about 4 days to about 5 days. The dose may be administered for about 1 hour to about 24 hours, about 2 hours to about 12 hours, about 3 hours to about 6 hours. The dose may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0120] The medium molecular weight heparin may be administered at a dose of about 0.01 mg / kg to about 10 mg / kg, for example, at a dose of about 0.05 mg / kg to about 9 mg / kg, about 0.5 mg / kg to about 8 mg / kg, about 1 mg / kg to about 7 mg / kg, about 1.5 mg / kg to about 6 mg / kg, or about 2 mg / kg to about 5 mg / kg. The dose may be given as a single dose or as a continuous dose. The dose may be given over a period of time. The period of time may be about 1 month to about 12 months, for example, about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, about 6 months to about 7 months. The period of time may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, about 4 days to about 5 days. The dose may be administered over a period of about 1 hour to about 24 hours, about 2 hours to about 12 hours, about 3 hours to about 6 hours, for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0121] The medium molecular weight heparin may be administered at a dose of about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 8 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 2 mg / kg, or about 1 mg / kg to about 1.5 mg / kg. The dose may be given as a single dose or as a continuous dose. The dose may be given over a period of time. The period of time may be about 1 month to about 12 months, for example, about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, or about 6 months to about 7 months. The period of time may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, or about 4 days to about 5 days. The dose may be administered over a period of about 1 hour to about 24 hours, about 2 hours to about 12 hours, or about 3 hours to about 6 hours. Said doses may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0122] The medium molecular weight heparin may be administered at a dose of about 0.1 mg to about 5000 mg, about 0.5 mg to about 2000 mg, about 1 mg to about 1000 mg, about 5 mg to about 900 mg, about 10 mg to about 800 mg, about 20 mg to about 700 mg, about 30 mg to about 600 mg, about 50 mg to about 500 mg, about 75 mg to about 400 mg, about 100 mg to about 300 mg, about 125 mg to about 250 mg, or about 150 mg to about 200 mg. The dose may be given as a single dose or continuous administration. The dose may be given over a period of time. The period of time may be about 1 month to about 12 months, for example, about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, or about 6 months to about 7 months. The period may be from about 1 to about 7 days, from about 2 to about 6 days, from about 3 to about 5 days, or from about 4 to about 5 days. The dose may be administered for 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 may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0123] The medium molecular weight heparin may be administered in a dose of, for example, about 1 international unit (IU), about 2 IU, about 5 IU, about 10 IU, about 15 IU, about 20 IU, about 25 IU, about 50 IU, about 75 IU, about 100 IU, about 200 IU, about 300 IU, about 400 IU, about 500 IU, about 1000 IU, about 1500 IU, about 2000 IU, about 2500 IU, about 5000 IU, about 10000 IU, about 20000 IU, or about 25000 IU. The dose may be given as a single dose or continuous doses. The dose may be given over a period of time. The period may be about 1 month to about 12 months, for example, about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, about 6 months to about 7 months. The period may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, about 4 days to about 5 days. The dose may be administered for about 1 hour to about 24 hours, about 2 hours to about 12 hours, about 3 hours to about 6 hours. The dose may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0124] The medium molecular weight heparin may be administered in a dose of about 1 IU to about 50000 IU, about 2 IU to about 25000 IU, about 5 IU to about 20000 IU, about 10 IU to about 10000 IU, about 15 IU to about 5000 IU, about 20 IU to about 2500 IU, about 25 IU to about 2000 IU, about 50 IU to about 1500 IU, about 75 IU to about 1000 IU, about 100 IU to about 500 IU, about 200 IU to about 400 IU, or about 250 IU to about 300 IU. The dose may be given as a single dose or as a continuous dose. The dose may be given over a period of time. The period may be about 1 month to about 12 months, for example, about 2 months to about 11 months, about 3 months to about 10 months, about 4 months to about 9 months, about 5 months to about 8 months, about 6 months to about 7 months. The period may be about 1 day to about 7 days, about 2 days to about 6 days, about 3 days to about 5 days, about 4 days to about 5 days. The dose may be administered for about 1 hour to about 24 hours, about 2 hours to about 12 hours, about 3 hours to about 6 hours. The dose may be administered for the duration of the underlying vascular endotheliitis and elevated VWF levels.

[0125] A medium molecular weight heparin may be administered commensurate with the VWF antigen:ADAMTS13 ratio. For example, patients with a high VWF antigen:ADAMTS13 ratio may be administered a higher dose of MMWH compared to patients with a lower VWF antigen:ADAMTS13 ratio.

[0126] The medium molecular weight heparin may be included in a pharmaceutical formulation. The pharmaceutical formulation comprises a composition of matter suitable for administration to a subject. The pharmaceutical formulation may be in liquid, solid, colloidal or aerosol form. The excipient may be selected from the group consisting of solvents, cosolvents, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavorings, lubricants, suspending agents, isotonicity agents, surfactants, solubilizers, dispersing aids, dispersants, humectants, thickeners, colorants, wetting agents, antifoaming agents, viscosity adjusters, sweeteners and combinations thereof. The pharmaceutical formulation may include glucose. The pharmaceutical formulation may include sodium chloride. The pharmaceutical formulation may include phosphate buffered saline.

[0127] The pharmaceutical formulation may include an additional active agent, which may include a composition of matter having a physiological effect, which may include a low molecular weight heparin or a medium molecular weight heparin composition of different disaccharides. The additional active agent may be selected from the group comprising farnesoid X receptor (FXR) agonists, peroxisome proliferator-activated receptor (PPAR) agonists, aramchol, caspase inhibitors, galectin 3 inhibitors, mitogen-activated protein kinase 5 (MAPK5) inhibitors, fibroblast growth factor 19 (FGF19) agonists, FGF21 agonists, leukotriene D4 (LTD4) receptor antagonists, niacin analogues, apical sodium bile acid cotransporter (ASBT) inhibitors, apoptosis signal-regulating kinase 1 (ASK1) inhibitors, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers, chemokine receptor inhibitors, thiozolidinediones, GLP-1 analogues, biguanides, HIV replication inhibitors, metformin, opiates, anesthetics, HMG-CoA reductase inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), or any combination thereof.

[0128] The intermediate molecular weight heparin may include chemical modifications. The chemical modifications include any chemical changes to the intermediate molecular weight heparin. Thus, the chemical modifications may include N-acetylation, de-N-acetylation, N-sulfation, O-sulfation, de-2-O-sulfation, complete desulfation, or a combination thereof.

[0129] The present invention provides a kit comprising an intermediate molecular weight heparin as defined herein for use in the treatment of vascular endotheliitis. The kit may comprise intermediate molecular weight heparin in a unit dosage form at a dosage amount as defined herein. The kit may comprise a pharmaceutical package. The kit may comprise the necessary reagents for synthesizing intermediate molecular weight heparin for use according to the present invention.

[0130] The present invention provides a method for treating vascular endotheliitis, the method comprising administering to a subject in need of treatment a therapeutically effective amount of intermediate molecular weight heparin as defined herein, the therapeutically effective amount being any amount of intermediate molecular weight heparin necessary to treat vascular endotheliitis to some extent.

[0131] The present invention provides the use of a medium molecular weight heparin as defined herein for the manufacture of a medicament for the treatment of vascular endotheliitis.

[0132] All documents cited herein, including any cross-references or related patents or applications, are hereby incorporated by reference in their entirety unless expressly excluded or limited.

[0133] It will be understood that various modifications can be made to the embodiments shown without departing from the spirit and scope of the invention, as defined by the appended claims. EXAMPLES

[0134] The invention will now be demonstrated by reference to the following non-limiting examples.

[0135] Unless otherwise stated, room temperature and pressure are 20° C. (293.15 K, 68° F.) and 1 atm (standard pressure) (14.696 psi, 101.325 kPa), respectively.

[0136] Disaccharide analysis Disaccharide analysis was performed on MMW heparin and the results are shown in Table 1 below. [Table 1]

[0137] Factor IIa and factor Xa activity Factor IIa (also known as thrombin) acts as a serine protease that converts soluble fibrogen into insoluble chains of fibrin as well as catalyzing other coagulation-related reactions. Factor Xa is the activated form of clotting factor X. Factor X is a serine endopeptidase enzyme that plays an important role in several steps of the coagulation system.

[0138] Heparin (unfractionated heparin) and its derivatives, such as low molecular weight heparins, bind to the plasma cofactor antithrombin (AT) and inactivate several clotting factors IIa, Xa, XIa, and XIIa. This inactivation of factor Xa by heparin is called "indirect" because it depends on the presence of AT and does not interact directly with factor Xa.

[0139] As shown in Figures 1 and 2, MMW heparin exhibits very low activity towards factor IIa and factor Xa when compared to UF heparin and LMW (low molecular weight) heparin, respectively. Thus, unlike UF or LMW heparin, MMW heparin does not affect either factor IIa or factor Xa mediated coagulation.

[0140] Analysis of MMW heparin in the ristocetin-induced platelet aggregation assay. Protocol according to "Promoting standardization of transmitted light platelet aggregation testing: Working group consensus from the SSC / ISTH Subcommittee on Platelet Physiology", Journal of Thrombosis and Haemostasis, 2013, 11:1183-1189.

[0141] Preparation of ristocetin working solution: Ristocetin stock solution (50 mg / mL) was diluted with saline to 24 mg / mL. 20 μL from a 400 μL solution of 24 mg / mL ristocetin in saline provides a solution with a ristocetin concentration of 1.2 mg / mL.

[0142] Preparation of MMW heparin working solution: MMW heparin (11 kDa, 0.569 g) was dissolved in HO (10 mL) to give a 5.2 mM solution of MMW heparin in HO. A 5.2 mM stock solution of MMW heparin in HO was frozen at -20°C.

[0143] MMW heparin was diluted in saline as follows: [Table 2]

[0144] Protocol Blood samples were taken from non-smokers who were not taking any antiplatelet therapy (e.g., aspirin), which may also be known as platelet coagulation inhibitors or platelet aggregation inhibitors.

[0145] Venous stasis-free blood was collected from donors into 109 mM sodium citrate solution (VACUETTE, 3.5 mL #454327, Lot #A21013FQ). The first 3-4 mL of collected blood was discarded.

[0146] The blood samples were allowed to stand at room temperature for 15 minutes before centrifugation. Platelet rich plasma (PRP) was prepared by centrifuging the blood samples at 200 g for 10 minutes at 21° C. without the brake. Platelet poor plasma (PPP) was prepared by removing the PRP from the blood samples by centrifuging the blood samples at 1500 g for 15 minutes at 21° C.

[0147] PRP quality was assessed by performing a platelet count of the PRP. The platelet count in the PRP was 421 G / L. The platelet count of the PRP sample was not (and should not be) adjusted to a standardized value using autologous PPP.

[0148] After centrifugation, the PRP samples were left at room temperature for 15 minutes before performing the Transmitted Light Platelet Aggregation Assay (LTA) test. With PRP, the aggregometer was set at 0% transmitted light. With autologous PPP, the aggregometer was set at 100% transmitted light.

[0149] For each of the three MMW heparin working solution concentrations prepared (5 μM, 10 μM and 15 μM), the following steps were performed.

[0150] 20 μL of MMW Heparin diluted in saline (described above under "Preparation of MMW Heparin Working Solution") was added to 360 μL of PRP. The resulting solution was vortexed for 2 seconds and then incubated at 37° C. for 5 minutes without stirring. The solution was then incubated at 37° C. for 1 minute with stirring. The baseline trace of LTA was observed for oscillation and stability for at least 1 minute prior to addition of agonist (ristocetin).

[0151] 20 μL of ristocetin working solution, i.e., ristocetin diluted in saline (described above under "Preparation of ristocetin working solution"), was added to the MMW heparin and PRP solution to give a final solution volume of 400 μL.

[0152] The LTA test was performed at 37° C. During the LTA test, the PRP samples were constantly stirred at 1000 rpm using a disposable stirrer.

[0153] - The volume of agonist (ristocetin) added for LTA testing should be consistent and not exceed 10% of the total sample volume. In this example, the volume of agonist should not exceed 40 μL.

[0154] Three doses of MMW heparin were tested: 5 μM, 10 μM, and 15 μM, and compared to a saline vehicle control. As shown in Figure 3, all tested concentrations (5 μM, 10 μM, and 15 μM) completely inhibited VWF-induced platelet aggregation.

[0155] The highest MMW heparin concentration, 15 μM, was retested, which gave similar results, i.e., complete inhibition of VWF-induced platelet aggregation, as shown in FIG.

[0156] Thus, when MMW heparin was added to PRP samples, a complete inhibition of VWF-induced platelet aggregation was observed.

[0157] Dose-dependent MMWH inhibition of von Willebrand factor platelet binding. Protocol according to "Promoting standardization of transmitted light platelet aggregation testing: Working group consensus from the SSC / ISTH Subcommittee on Platelet Physiology", Journal of Thrombosis and Haemostasis, 2013, 11:1183-1189.

[0158] Preparation of ristocetin working solution: Ristocetin stock solution (50 mg / mL) was diluted with saline to 48 mg / mL.

[0159] Preparation of MMW heparin working solution: MMW heparin (11 kDa, 0.569 g) was dissolved in HO (10 mL) to give a 5.2 mM MMW heparin solution in HO. The 5.2 mM MMW heparin stock solution in HO was frozen at -20°C.

[0160] 20 μL was used in each aggregation test, corresponding to 20 dilutions. MMWH was diluted in saline as follows: [Table 3]

[0161] Monoclonal anti-VWF As a positive control, an anti-VWF monoclonal antibody that blocks ristocetin-induced platelet aggregation was used. Briefly, Ab #701 5.5 mg / mL was diluted to 200 μg / mL in saline. 20 μL of 200 μg / mL Ab #701 in saline was further diluted in 400 μL PRP to give a final antibody concentration of 10 μg / mL. Preparation of LMWH: Lovenox (enoxaparin) solution Storage solution 8000UI: 100mg / mL; Molecular weight: 4500Da; Lovenox storage solution concentration: 22mM To prepare a final concentration of 20 μM LMWH: Dilute 18 μL of Lovenox 22 mM stock in 1000 μL saline to obtain a 400 μM stock. 20 μL of 400 μM stock in a final volume of 400 μL provides a final concentration of LMWH of 20 μM. To prepare a final concentration of 10 μM LMWH: Dilute 9 μL of Lovenox 22 mM stock in 1000 μL saline to obtain a 200 μM stock. 20 μL of the 200 μM stock in a final volume of 400 μL provides a final concentration of 10 μM LMWH. Agonist-induced aggregation ADP: Stock concentration 5 mM, diluted to 40 μM in saline. 20 μL of 40 μM stock in a final volume of 400 μL provides a final concentration of 2 μM ADP. Collagen: Stock concentration 1 mg / mL, diluted to 40 μg / mL in saline. 20 μL of 40 μg / mL collagen stock in a final volume of 400 μL provides a final concentration of 2 μg / mL collagen. TRAP6: Stock concentration 20 mM, diluted to 200 μM in saline. 20 μL of 200 μM ADP stock in a final volume of 400 μL provides a final concentration of 10 μM ADP.

[0162] Protocol Blood samples were taken from non-smokers not receiving any antiplatelet therapy (eg, aspirin).

[0163] Venous stasis-free blood was collected from donors into 109 mM sodium citrate solution (VACUETTE, 3.5 mL #454327, Lot #A21013FQ). The first 3-4 mL of collected blood was discarded.

[0164] The blood samples were allowed to stand at room temperature for 15 minutes before centrifugation. Platelet rich plasma (PRP) was prepared by centrifuging the blood samples at 200 g for 10 minutes at 21° C. without the brake. Platelet poor plasma (PPP) was prepared by removing the PRP from the blood samples by centrifuging the blood samples at 1500 g for 15 minutes at 21° C.

[0165] PRP quality was assessed by performing a platelet count of the PRP. The platelet count of the PRP samples was not (and should not be) adjusted to a standardized value using autologous PPP.

[0166] -After centrifugation, the PRP samples were left at room temperature for 15 minutes before performing the transmitted light platelet aggregation assay (LTA) test. With PRP, the aggregometer was set at 0% transmitted light. With autologous PPP, the aggregometer was set at 100% transmitted light. The LTA test was performed at 37°C. During the LTA test, the PRP samples were constantly stirred at 1000 rpm using a disposable stirrer. -The volume of agonist added for LTA should be consistent and never exceed 10% of the total sample volume.

[0167] MMWH test inhibition 20 μL MMWH (5, 10 or 20 μM) and 10 μL ReoPro (final concentration of 20 μg / mL) were added to 360 μL PRP. The resulting solution was vortexed for 2 seconds and then incubated at 37° C. for 5 minutes without stirring. The solution was then incubated at 37° C. for 1 minute with stirring. The baseline trace of LTA was observed for oscillations and stability for at least 1 minute prior to addition of agonist (ristocetin).

[0168] 10 μL of ristocetin working solution, i.e., ristocetin diluted in saline (described above under "Preparation of ristocetin working solution"), was added to the MMWH, ReoPro and PRP solutions to give a final solution volume of 400 μL and a final ristocetin concentration of 1.2 mg / mL.

[0169] A control experiment was also performed using the above method, but using 20 μL of saline instead of MMWH.

[0170] LMWH test inhibition 20 μL LMWH (10 or 20 μM) and 10 μL ReoPro (final concentration of 20 μg / mL) were added to 360 μL PRP. The resulting solution was vortexed for 2 seconds and then incubated at 37° C. for 5 minutes without stirring. The solution was then incubated at 37° C. for 1 minute with stirring. The baseline trace of LTA was observed for oscillations and stability for at least 1 minute prior to addition of agonist (ristocetin).

[0171] 10 μL of ristocetin working solution, i.e., ristocetin diluted in saline (described above under "Preparation of ristocetin working solution"), was added to the LMWH, ReoPro and PRP solutions to give a final solution volume of 400 μL and a final ristocetin concentration of 1.2 mg / mL.

[0172] A control experiment was also performed using the above method, but using 10 μL of saline instead of ReoPro.

[0173] Monoclonal Anti-VWF Test Inhibitor 20 μL Ab#701 and 10 μL ReoPro (final concentration of 20 μg / mL) were added to 360 μL PRP. The resulting solution was vortexed for 2 seconds and then incubated at 37° C. for 5 minutes without stirring. The solution was then incubated at 37° C. for 1 minute with stirring. The baseline trace of LTA was observed for oscillation and stability for at least 1 minute prior to addition of agonist (ristocetin).

[0174] 10 μL of ristocetin working solution, i.e., ristocetin diluted in saline (described above under "Preparation of ristocetin working solution"), was added to the monoclonal anti-VWF, ReoPro and PRP solutions to give a final solution volume of 400 μL and a final ristocetin concentration of 1.2 mg / mL.

[0175] MMWH test inhibition in agonist-induced aggregation The agonists investigated were ADP, collagen and TRAP6.

[0176] 20 μL MMWH (5, 10 or 20 μM) or 20 μL saline was added to 360 μL PRP. The resulting solution was vortexed for 2 seconds and then incubated at 37° C. for 5 minutes without stirring. The solution was then incubated at 37° C. for 1 minute with stirring. The baseline trace of LTA was observed for oscillation and stability for at least 1 minute prior to addition of agonist.

[0177] 20 μL agonist (ADP, collagen or TRAP6) was added to the MMWH and PRP solution to give a final solution volume of 400 μL and the following final agonist concentrations: ADP: 2 μM; Collagen: 2 μg / mL; or TRAP6: 10 μM.

[0178] result To selectively analyze the coagulation phase, experiments were performed in the presence of ReoPro (an inhibitor of αIIbβ3). Results are presented as slopes and areas under the curve (AUC). 1) MMWH dose response (0, 5, 10 and 20 μM) [Table 4] The dose-response curves are shown in FIG. 2) MMWH dose response (0, 5 and 10 μM) [Table 5] The dose-response curves are shown in FIG. 3) mAb and MMWH 10 μM [Table 6] The dose response curves are shown in FIG. 4) Dose response of mAb (anti-VWF) and MMWH (20μM x 2 and 5μM) [Table 7] The dose response curves are shown in FIG. 5) Dose response of mAb (anti-VWF) and LMWH (10 μM × 3) [Table 8] The dose response curves are shown in FIG. 6) LMWH (20μM×3) dose response [Table 9] The dose response curves are shown in FIG. 7) Statistical analysis One-way analysis of variance (AUC) [Table 10] [Table 11] One-way ANOVA (slope) [Table 12] [Table 13] 8) Agonist-induced platelet aggregation As shown in Figures 13 and 14, no effect on platelet aggregation was observed when ADP, collagen or TRAP6 were used as agonists.

[0179] conclusion MMWHs were tested in von Willebrand factor-dependent platelet clotting experiments and in platelet aggregation tests induced by ADP, collagen, or thrombin receptor-activating peptide 6 (TRAP6). In the platelet clotting experiments, low molecular weight heparin (LMWH) and monoclonal anti-VWF antibody were used as negative and positive controls, respectively.

[0180] As shown in Figures 5A and 5B, VWF-dependent platelet aggregation was effectively inhibited by MMWH in a dose-dependent manner, with no significant inhibition observed in the presence of LMWH. In contrast, anti-VWF monoclonal antibodies (known to interfere with VWF-platelet interactions) completely inhibited platelet aggregation. A 50% inhibition was obtained at MMWH concentrations between 3.4 and 3.7 μM.

[0181] As shown in Figures 13 and 14, no effect on platelet aggregation was observed when other agonists were used. The present invention may be further understood with reference to the following non-limiting provisions: Article 1. A medium molecular weight heparin for use in the treatment of vascular endotheliitis. Article 2. 2. An intermediate molecular weight heparin for use according to clause 1, wherein said intermediate molecular weight heparin inhibits von Willebrand factor. Article 3. 3. The medium molecular weight heparin for use according to clause 2, wherein said medium molecular weight heparin inhibits multimerization of von Willebrand factor, and optionally said von Willebrand factor is ultra-high molecular weight von Willebrand factor. Article 4. 4. The intermediate molecular weight heparin for use according to any one of clauses 1 to 3, wherein the intermediate molecular weight heparin inhibits binding between the platelets and von Willebrand factor. Article 5. 5. The intermediate molecular weight heparin for use according to any one of clauses 1 to 4, wherein the intermediate molecular weight heparin has a mass in the range of more than about 8000 Da (g / mol) to about 13000 Da (g / mol), optionally the intermediate molecular weight heparin has a mass of about 11000 Da (g / mol). Article 6. 6. The medium molecular weight heparin for use according to any one of clauses 1 to 5, wherein said medium molecular weight heparin comprises at least 3 units of IdoA2S-GlcNS6S disaccharide. Article 7. 7. The medium molecular weight heparin for use according to any one of clauses 1 to 6, wherein the vascular endotheliitis is due to COVID-19, a viral infection, acute respiratory distress syndrome, cancer, an infectious disease, sepsis, a cardiovascular disease, diabetes, trauma, in particular brain or head trauma, burns, inhalation injury, a drug reaction, a hematological condition, subarachnoid hemorrhage, aortic aneurysmal disease, stroke, or cerebral parenchymal hemorrhage. Article 8. 8. The intermediate molecular weight heparin for use according to clause 7, wherein said vascular endotheliitis is caused by a viral infection, and said viral infection may be SARS-CoV-2. Article 9. 8. The medium molecular weight heparin for use according to clause 7, wherein said vascular endotheliitis is caused by cancer, and said cancer may be leukemia, lymphoma, myeloma, or solid organ cancer. Article 10. 7. The medium molecular weight heparin for use according to any one of clauses 1 to 6, wherein the treatment of vascular endotheliitis inhibits hematogenous spread of cancer. Article 11. 11. The medium molecular weight heparin for use according to any one of clauses 1 to 10, wherein the medium molecular weight heparin is administered by a method of administration selected from the group consisting of parenteral, subcutaneous, depot form, e.g., depot injection, intravenous, intramuscular, intrathecal, intradermal, intraarterial, intraarticular, cutaneous, transdermal, intraosseous, and inhalation. Article 12. 12. The medium molecular weight heparin for use according to clause 11, wherein the method of administration is subcutaneous. Article 13. 12. The medium molecular weight heparin for use according to clause 11, wherein the method of administration is intravenous. Article 14. 12. The medium molecular weight heparin for use according to clause 11, wherein the method of administration is intramuscular. Article 15. 12. The medium molecular weight heparin for use according to clause 11, wherein said method of administration is inhalation, optionally by nebulizer. Article 16. 16. The intermediate molecular weight heparin for use according to any one of claims 1 to 15, wherein the intermediate molecular weight heparin is administered at a dose of about 0.01 mg / kg to about 10 mg / kg. Article 17. 17. The intermediate molecular weight heparin for use according to any one of clauses 11 to 16, wherein said intermediate molecular weight heparin is administered as a single dose or as a continuous dose. Article 18. 18. The medium molecular weight heparin for use according to any one of clauses 1 to 17, wherein the medium molecular weight heparin is comprised in a pharmaceutical formulation. Article 19. 19. The medium molecular weight heparin for use according to clause 18, wherein the pharmaceutical formulation comprises an excipient. Article 20. 20. The medium molecular weight heparin for use according to clause 19, wherein the excipient is selected from the group consisting of solvents, co-solvents, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, flavorings, lubricants, suspending agents, isotonicity agents, surfactants, solubilizers, dispersion aids, dispersing agents, humectants, thickening agents, colorants, humectants, antifoaming agents, viscosity modifiers, sweeteners and combinations thereof. Article 21. 21. The medium molecular weight heparin for use according to any one of clauses 18 to 20, wherein the pharmaceutical formulation comprises an additional active agent, optionally wherein the additional active agent comprises a low molecular weight heparin or a medium molecular weight heparin of different disaccharide composition. Article 22. 22. The medium molecular weight heparin for use according to any one of clauses 1 to 21, wherein the medium molecular weight heparin comprises a chemical modification. Article 23. 23. The medium molecular weight heparin for use according to clause 22, wherein said chemical modification comprises N-acetylation, N-deacetylation, N-sulfation, O-sulfation, de-2-O-sulfation, complete desulfation, or a combination thereof. Article 24. 24. A kit comprising a medium molecular weight heparin for use according to any one of clauses 1 to 23. Article 25. 13. A method for treating vascular endotheliitis, the method comprising administering a therapeutically effective amount of medium molecular weight heparin to a subject in need of treatment. References 1. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al.A Novel Coronavirus from Patients with Pneumonia in China,2019.N Engl J Med.2020 20;382(8):727‐33. 2. 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Claims

1. An intermediate molecular weight heparin for use in the treatment of endothelial vasculitis in a patient having a ratio of von Willebrand factor antigen in plasma to ADAMTS13 of at least 2 ± 5%.

2. The intermediate molecular weight heparin according to claim 1, which inhibits von Willebrand factor, for the use described above.

3. The intermediate molecular weight heparin according to claim 2, which completely inhibits von Willebrand factor-induced platelet aggregation at concentrations of 5 μM, 10 μM, and 15 μM as measured by the ristocetin-induced platelet aggregation assay.

4. The intermediate molecular weight heparin according to claim 2, which inhibits von Willebrand factor multimers, for the use described above.

5. The intermediate molecular weight heparin according to claim 4, wherein the von Willebrand factor is ultra-high molecular weight von Willebrand factor.

6. (i) The intermediate molecular weight heparin inhibits the binding of the platelets to von Willebrand factor, (ii) The intermediate molecular weight heparin has a mass in the range exceeding 8000 ± 5% Da (g / mol) and up to 13000 ± 5% Da (g / mol), (iii) The intermediate molecular weight heparin has a mass of 11000 ± 5% Da (g / mol), (iv) The intermediate molecular weight heparin contains at least 3 units of the IdoA2S-GlcNS6S disaccharide; and / or, (v) The treatment of the endothelial vasculitis inhibits hematogenous spread of cancer, The intermediate molecular weight heparin for the use described in any one of claims 1 to 5.

7. An intermediate molecular weight heparin for use in the treatment of a disease or condition in a patient, wherein the patient has endothelial vasculitis characterized by a ratio of von Willebrand factor antigen in plasma to ADAMTS13 of at least 2 ± 5%, for the use described above.

8. The disease or condition is COVID-19, viral infection, acute respiratory distress syndrome, cancer, bacterial infection, sepsis, cardiovascular disease, diabetes, mental trauma, burns, inhalation injury, drug reaction, hematological condition, subarachnoid hemorrhage, aortic aneurysm disease; or, the disease or condition is a fungal infection, the intermediate molecular weight heparin for the use described in claim 7.

9. (i) The endothelial vasculitis is caused by a viral infection, (ii) The endothelial vasculitis is caused by a viral infection, and the viral infection is SARS-CoV-2. (iii) The endothelial vasculitis is caused by cancer, and the cancer is leukemia, lymphoma, myeloma, or solid organ cancer. (iv) The endothelial vasculitis is caused by cancer, and the cancer is a solid organ cancer selected from colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer, or skin cancer. (v) The endothelial vasculitis is caused by a hematological disorder; or (vi) The endothelial vasculitis is caused by a hematological disorder, and the hematological disorder is thrombotic thrombocytopenic purpura, anemia, or sickle cell disease. Intermediate molecular weight heparin for use according to claim 8.

10. Administering the intermediate molecular weight heparin by an administration method selected from the group consisting of parenteral, subcutaneous, subcutaneous, depot form, for example, depot injection, intravenous, intramuscular, intrathecal, intradermal, intraarterial, intraarticular, cutaneous, transdermal, intraosseous, and inhalation, the intermediate molecular weight heparin for use according to any one of claims 1 to 5 and claims 7 to 9.

11. (i) The administration method is subcutaneous. (ii) The administration method is intravenous. (iii) The administration method is intramuscular. (iv) The administration method is inhalation; or (v) The administration method is by nebulizer. Intermediate molecular weight heparin for use according to claim 10.

12. Administering the intermediate molecular weight heparin as a single dose or continuous dose, the intermediate molecular weight heparin for use according to claim 10.

13. The intermediate molecular weight heparin is contained in a pharmaceutical preparation, the intermediate molecular weight heparin for use according to any one of claims 1 to 5 and claims 7 to 9.

14. The pharmaceutical preparation contains an excipient, the intermediate molecular weight heparin for use according to claim 13.

15. The excipient is selected from the group consisting of solvents, co-solvents, buffers, stabilizers, antioxidants, preservatives, chelating agents, emulsifiers, fragrances, lubricants, suspending agents, isotonic agents, surfactants, solubilizing agents, dispersion aids, dispersants, humectants, thickeners, colorants, wetting agents, defoaming agents, viscosity modifiers, sweeteners, and combinations thereof, the intermediate molecular weight heparin for use according to claim 14.

16. The pharmaceutical preparation contains an additional active agent, and the additional active agent is intermediate molecular weight heparin for use according to claim 13, which contains low molecular weight heparin.

17. (i) The intermediate molecular weight heparin contains chemical modification. (ii) The intermediate molecular weight heparin contains chemical modification, and the chemical modification includes N-acetylation, de-N-acetylation, N-sulfation, O-sulfation, de-2-O sulfation, complete desulfation, or a combination thereof; and / or (iii) The intermediate molecular weight heparin is administered at a dose of 0.01 mg / kg to 100 mg / kg. (iv) The intermediate molecular weight heparin is administered at a dose of 0.01 mg / kg to 10 mg / kg. Intermediate molecular weight heparin for use according to any one of claims 1 to 5 and claims 7 to 9.

18. A kit, which contains intermediate molecular weight heparin for use according to any one of claims 1 to 5 and claims 7 to 9.

19. The intermediate molecular weight heparin is administered by an administration method selected from the group consisting of parenteral, subcutaneous, subcutaneous, depot form, for example, depot injection, intravenous, intramuscular, intrathecal, intradermal, intra-arterial, intra-articular, skin, transdermal, intraosseous, and inhalation, for use according to claim 6.

20. The intermediate molecular weight heparin is contained in a pharmaceutical preparation, for use according to claim 6.

21. The intermediate molecular weight heparin is contained in a pharmaceutical preparation, for use according to claim 10.

22. (i) The intermediate molecular weight heparin contains chemical modification. (ii) The intermediate molecular weight heparin contains chemical modification, and the chemical modification includes N-acetylation, de-N-acetylation, N-sulfation, O-sulfation, de-2-O sulfation, complete desulfation, or a combination thereof; and / or (iii) The intermediate molecular weight heparin is administered at a dose of 0.01 mg / kg to 100 mg / kg. (iv) The intermediate molecular weight heparin is administered at a dose of 0.01 mg / kg to 10 mg / kg. Intermediate molecular weight heparin for use according to claim 6.

23. (i) The intermediate molecular weight heparin contains chemical modification. (ii) The intermediate molecular weight heparin includes chemical modifications, and the chemical modifications include N-acetylation, de-N-acetylation, N-sulfation, O-sulfation, de-2-O sulfation, complete desulfation, or combinations thereof; and / or (iii) The intermediate molecular weight heparin is administered at a dose of 0.01 mg / kg to 100 mg / kg, (iv) The intermediate molecular weight heparin is administered at a dose of 0.01 mg / kg to 10 mg / kg, Intermediate molecular weight heparin for use according to claim 10.

24. (i) The intermediate molecular weight heparin includes chemical modifications, (ii) The intermediate molecular weight heparin includes chemical modifications, and the chemical modifications include N-acetylation, de-N-acetylation, N-sulfation, O-sulfation, de-2-O sulfation, complete desulfation, or combinations thereof; and / or (iii) The intermediate molecular weight heparin is administered at a dose of 0.01 mg / kg to 100 mg / kg, (iv) The intermediate molecular weight heparin is administered at a dose of 0.01 mg / kg to 10 mg / kg, Intermediate molecular weight heparin for use according to claim 13.

25. A kit comprising the intermediate molecular weight heparin for use according to claim 6.

26. A kit comprising the intermediate molecular weight heparin for use according to claim 10.

27. A kit comprising the intermediate molecular weight heparin for use according to claim 13.

28. A kit comprising the intermediate molecular weight heparin for use according to claim 17.

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