Functionalised medium molecular weight heparin

Abstract

A functionalised medium molecular weight heparin includes a linker and a functional group. The functional group may be a polar functional group, an apolar functional group, an acidic functional group, or a basic functional group. The functional group may be an aryl, a phenyl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, an alcohol, a thiol, an ether, a sulphide, an amine, a carboxylic acid, and / or a fluoroalkyl. The linker may be an oxime linker or an amine linker.

Description

FUNCTIONALISED MEDIUM MOLECULAR WEIGHT HEPARINFIELD OF THE INVENTION

[0001] The present invention relates to functionalised medium molecular weight heparin, preparation of functionalised medium molecular weight heparin, and the treatment of endotheliopathy, particularly, functionalised medium molecular weight heparin for use in the treatment of endotheliopathy.BACKGROUND OF THE INVENTION

[0002] The vertebrate vasculature system consists of arteries, veins and capillaries. Blood flow through the vasculature is dynamic allowing for the maintenance of homeostasis through the delivery of essential elements such as oxygen and leukocytes to the tissues most in need. Blood flow is controlled through the dilation and constriction of the blood vessels. The endothelial cells which line the inside of the lumen of the vasculature act as a monolayer to form the endothelium ; the endothelium sits upon a layer of smooth muscle cells. These smooth muscle cells either constrict or relax which results in constriction of the blood vessels (vasoconstriction) or dilation of the blood vessels (vasodilation), respectively. In the case of haemorrhage, blood flow can be controlled through thrombus formation. To reach sites of inflammation, leukocytes must pass from the blood through the endothelial cells to reach inflamed tissue. The regulation of vasoconstriction, vasodilation, vascular permeability, and thrombus formation are therefore crucial for the regulation of homeostasis.

[0003] The endothelial cells lining the blood vessel are more than simple constituents of the vessel wall. Endothelial cells produce and release vasoactive substances that relax and constrict blood vessels. For example, endothelial cells produce nitric oxide (NO) in response to sheer stress or stimuli, such as acetylcholine, histamine and thrombin. The NO then diffuses to the smooth muscle cells surrounding the endothelium to initiate vasodilation. Reactive oxygen species, which are released in response to inflammatory stimuli, can increase endothelial permeability and promote leukocyte adhesion to the endothelial cells via the expression of adhesion molecules. This serves to drive the influx of leukocytes to sites of inflammation. Moreover, the endothelium provides a surface for thrombus formation. Thus, it is the endothelial cells that play a crucial role in the regulation of homeostasis.

[0004] Endothelial dysfunction, or endotheliopathy, can therefore have dire consequences as blood flow, oxygen delivery, the immune response and, therefore, homeostasis are impaired. Endotheliopathy is characterised by decreased NO bioavailability. This can result in the increased expression of adhesion molecules on the endothelial surface, thereby initiating leukocyte recruitment to the vascular wall. Thus, inflammation of the endothelium, or endothelialitis, is observed in endotheliopathy. This can result in a defective lining of the blood vessels by the endothelium, resulting in the exposure of the subendothelial matrix to clotting factors in the blood. Consequently, platelet aggregation and thrombus formation occur, resulting in a potentially lethal blood clot.

[0005] Endotheliopathy may be caused by a number of diseases and is typically viewed as a symptom rather than a cause of disease. Consequently, treatments have focussed on targeting the causative disease rather than the endotheliopathy itself. As a result of this, treatments for endotheliopathy are lacking. However, it is now suggested that an underlying endotheliopathy may actually drive disease severity and morbidity and endotheliopathy plays a much larger role than previously thought. Therefore, treating the endotheliopathy and not just the causative disease may increase patient survival.

[0006] Herein, endotheliopathy and endothelialitis are used interchangeably. While endotheliopathy may be caused by a large number of diseases and / or conditions as described herein, endotheliopathy with reference to COVID-19 or SARS-CoV-2 will primarily be discussed herein. The skilled person will understand that this discussion is merely to provide an example and should not be considered limiting on the present invention.

[0007] In late 2019, in Wuhan, China a new beta-coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) was identified, causing coronavirus disease 2019 (COVID-19). Thereafter, a rapid geographical progression of COVID-19 culminated in the WHO declaring a pandemic in March 2020 (1). The clinical manifestations of those infected with SARS-CoV-2 range from the asymptomatic patient to a more severe pneumonia, which can lead to acute respiratory distress syndrome (ARDS) and multi-organ failure. The majority of symptomatic patients will experience a mild-to-moderate form of the disease, which most commonly does not require hospitalisation (2-4). However, there is a cohort of patients that can progress to the more severe form of the disease, where evolution of symptoms / clinical manifestations can take up to 2 weeks, starting from an initial prodromal stage through to ARDS (3). It is now known that the subgroup of patients that become critical and require ventilation or extracorporeal membrane oxygenation (ECMO) have very poor outcomes with high rates of mortality that approach 90% (5).

[0008] Since the disease was first described it has affected more than 90 million individuals globally. The pathophysiological pathways remain unclear; accordingly, management is supportive. Disease-modifying therapeutics which could be instigated whilst awaiting a specific anti-viral drug or vaccination are lacking. Several lines of evidence point towards endothelial dysfunction as a key pathophysiological mechanism in COVID-19. Prior to the current pandemic, markers of endothelial dysfunction have been shown to be correlated with disease severity and mortality in patients with sepsis (6-9). Recently Varga et al. (10) demonstrated a widespread endothelialitis that affected pulmonary, renal, gastrointestinal and hepatic vessels on post mortem examination of three patients with COVID-19. In one of the cases the authors reported 'most of the small vessels appeared congested' and in another case the patient died from bowel ischaemia with evidence of underlying endothelialitis.

[0009] Recently, two proposed haemostatic mechanisms have provided insight into an improved understanding of ARDS based on a molecular pathogenesis associated with endotheliopathy that promotes inflammation and coagulation disorder in sepsis and other critical illnesses (11-14) : one is the "two-activation theory of the endothelium" in which an endothelial pathogenesis activates the inflammatory pathway and microthrombotic pathway, whilst the other is a novel "two-path unifying theory" of haemostasis in which haemostasis initiates thrombogenesis and promotes micro-thrombogenesis, leading to vascular microthrombotic disease (VMTD) (11,13,15). These two theories are in congruity with one another since the endothelium contributes to initial haemostasis and triggers the molecular mechanisms of thrombogenesis. ARDS is often associated with sepsis from a variety of different causes and has been 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 develops with other organ dysfunction such as encephalopathy (18), hepatic failure (19)(20), acute renal failure, and acute necrotizing pancreatitis (21). This multi-organ involvement suggests ARDS may not be the primary disease but is part of an on-going systemic pathogenic mechanism triggered by an infection or another critical illness.

[0010] On this basis the underlying physiologic alteration of multi-organ failure in sepsis and other critical illnesses is identified as circulatory dysfunction occurring as a result of an endotheliopathy associated VMTD (EA-VMTD) (14,15). Therefore, the infection triggers an insult to the endothelium, causing an endotheliopathy. This then results in disseminated microthrombosis (DIMT), which may trigger, for example, local hypoxaemia, systemichypoxia, and / or ischaemia, and as mentioned earlier COVID-19 is now known to be associated with an endothelialitis (10).

[0011] A case series of COVID-19 pulmonary autopsies revealed that, alongside diffuse alveolar damage, numerous localised platelet-rich micro-thrombi and foci of haemorrhage were present in the lungs (22). The authors posited a pulmonary-localised thrombotic microangiopathy as key to the pathogenesis of COVID-19 with others also suggesting the micro-thrombosis is a critical driver in the disease process (23). These microcirculatory changes have been clearly demonstrated in the lungs, kidneys and the liver using contrast enhanced ultrasound (24,25). Similar findings have also been seen in the brain (26). Therefore, there is a growing body of evidence that COVID-19 appears to cause an endothelialitis and a diffuse and widespread microthrombosis.

[0012] Hypercoagulability and COVID-19 is now widely accepted and studies have shown abnormal levels of D-Dimers with higher levels associated with more severe disease and increased odds ratio of in-hospital mortality (27-30). Several case reports have noted acute pulmonary emboli in patients with COVID-19 pneumonia in the absence of major predisposing factors for venous thromboembolism formation (27,31,32). More recently Panigada et al. have shown that, in addition to raised D-Dimer levels, there was a marked increase in the levels of Factor VIII and von Willebrand factor (VWF) (33). An increase of over 500% in VWF and >350% increase in Factor VIII levels were reported by Escher et al (34) in relation to COVID-19. Furthermore, it has been demonstrated that patients with a thrombocytopaenia were at over 5-fold increased risk of severe disease and those with the lowest platelet counts were associated with the highest mortality (33,35,36) Therefore, both hypercoagulability and thrombocytopaenia appear to be harbingers of severe disease and mortality.

[0013] Von Willebrand Factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in haemostasis and thrombosis mediating platelet adhesion to injured and activated vessels. It is synthesized only in megakaryocytes and endothelial cells (ECs) and it is interesting to note that SARS-CoV can directly infect both of these cell types (22,36).

[0014] The vast majority of VWF found in the plasma is derived from the VWF synthesised within the ECs, where it is stored within the Weibel Palade Bodies (WPB). Although restricted to ECs there are differences in the synthesis of VWF within the different vascular beds of the body, with the small vessels of the lung and brain expressing higher levels of VWF than similar sized vessels of the liver or kidney and higher levels in venous rather than arterial ECs (37). A major portion of the VWF stored in the WPBs of endothelial cellsis made up of ultra-large VWF (ULVWF). These ultra-large VWF multimers are more adhesive than the smaller VWF multimers in the circulation (38). Upon secretion, ULVWF can spontaneously bind platelets. Inflammatory cytokines such as Interleukin-1 and tumour necrosis factor (TNF)-alpha can trigger the exocytosis of WPBs with release of their contents. Thus, plasma level of VWF can be used as a marker of endothelial activation and vascular inflammation and raised levels of VWF have been shown to associate with ARDS and sepsis, and to correlate independently to mortality (39,40).

[0015] Upon secretion from ECs, the secreted VWF, which partly enters the circulation and partly binds to the endothelium, is sensitive to shear stress. This shear stress unfolds the VWF and exposes sites for platelet binding, self-association as well as for cleavage via the enzyme ADAMTS13. It has previously been shown these VWF molecules can self-associate into long 'strings' in the direction of flow, both arterial and venous, that bind to platelets and are adherent to the endothelium (41-43). A protease, ADAMTS13, cleaves VWF and ULVWF, perfusion of which over these platelet-VWF strings led to them being rapidly removed from the circulation (41). The ULVWF multimers released from the WPBs have a lower shear stress for unfolding and therefore may represent the initiating molecules for this self-assembly process which leads to hyper-adhesive strings capturing platelets. The binding of platelets to the VWF occurs via the GP lb receptor at the Al domain. The binding site for this receptor is usually not exposed when the VWF is in its globular form and therefore cannot bind to platelets. Once VWF unfurls, secondary to shear stress, the binding site is exposed and binds with high affinity to platelets. The binding of platelets to VWF may cause a conformational change leading to activation of the integrin GPIIbllla (also known as a2bp3) and promoting platelet-platelet as well as platelet-VWF cross binding. For this reason, the use of standard anti-platelet agents is likely to be ineffective (Aspirin or P2Y12 inhibitors) or only partially effective in mitigating this pathological process as was suggested by the cohort study of Tremblay et al (44).

[0016] This ability to form VWF-platelet rich thrombi in the microvasculature is the hallmark of acquired thrombotic thrombocytopaenic purpura (TTP) in which autoantibodies to ADAMTS13 are present. It has also been shown that Interleukin-6 (IL-6) can inhibit the cleavage of ULVWF - platelet strings (45). Furthermore, the synthesis of ADAMTS13, at least in cultured cells, is dramatically inhibited by a variety of cytokines including IL-6 and TNF-alpha (46). This suggests that the cytokine storm, and particularly IL-6, may propagate the microthrombosis. However, this also suggests that if intervention is implemented early and there is no spike in the release of cytokines the disease may be more manageable and the rapid deterioration in the clinical status of patients can be averted.

[0017] There is now a significant body of evidence to suggest that there is very marked imbalance in the VWF:ADAMTS13 ratio as well as in the levels of high molecular weight VWF multimers (equivalent to ULVWF) in COVID-19. As mentioned earlier very high levels of VWF have been shown previously with the earliest case report to mention this surge in the levels of VWF being that of Escher et al (34). Subsequently, Goshua et al. (47) demonstrated that reported marked elevations 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 for those admitted to an intensive care unit (ICU) compared to those not admitted to ICU (pcO.OOOl). Next, Rauch et al. (48) looked at the progression of patients with COVID-19 in relationship to their admission VWF. Those with the highest VWF levels required greater levels of oxygen support whereas those patients that had normal VWF levels did not require admission to hospital nor supplementary oxygen (n = 10).

[0018] Shortly after the Rauch et al. publication Ladikou et al. (49) showed an increase in the VWF antigen levels of patients with COVID-19 admitted to the ICU with a positive correlation seen in the VWF levels and the age of the patients. They reported a median VWF Antigen level of 350% however, and crucially, they also showed a markedly reduced level of ADAMTS13 (49.7%), suggesting loss of the VWF cleaving protease that ordinarily degrades large VWF multimers and reduces its activity. They speculated that excess release of VWF seen in COVID-19 patients leads to depletion of ADAMTS13 and contributes to the prothrombotic state. Further analysis of their data showed that median VWF levels were significantly higher in patients that died (477%) compared to the ones that remained alive (335%) (p = 0.015).

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

[0020] Most recently, Philippe et al. (53) published their results from a cohort of 208 patients admitted to two centres in Paris of whom 23 had only mild symptoms and were treated as outpatients. They found that only VWF:Ag scaled according to clinical severity, with levels significantly higher in critical patients (median 507%, IQR 428-596) compared to non-critical patients (288%, 230-350, p < 0.0001) or COVID-19 outpatients (144%, 133-198, p = 0.007). In a univariable analysis model a VWF:Ag level over 423% at admission was significantly associated with higher in-hospital mortality (OR 89.7 95% CI 25.9-567.4, p < 0.001) which remained very significant in a multivariable analysis model adjusted on 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 critical patients (median ratio 1.18, IQR 0.86-1.09) compared to non-critical patients (0.96, 1.04-1.39, p < 0.001). Furthermore, the levels of HMWM (ratio) (OR 116, 95% CI 10.2-1943, p < 0.001) was one of the most significantly associated with in-hospital mortality.

[0021] It is possible to develop a unifying theory that is triggered by an endotheliopathy and endothelial itis, which causes the release of VWF and ULVWF resulting in the formation of microthrombi. This then leads to hypoxia and the process can be accentuated by the 'cytokine storm' and release of IL-6, which inhibits and reduces the functions of ADAMTS13, resulting in a cascade of disseminated microthrombosis and multi-organ dysfunction and failure. It has also been suggested that this microvascular thrombosis at the pulmonary level is the origin of right ventricular dysfunction (54). This mechanism can account for many of the findings currently being observed including the high D-Dimer levels (high because of the huge levels of microthrombosis), high levels of Factor VIII and VWF (released from the WPBs in response to an endothelial insult), the microthrombosis and atypical ARDS picture being seen (55), as well as the widespread clinical picture of pulmonary, neurological and gastrointestinal symptoms. The endothelialitis and microthrombosis we suggest may also explain why patients with a pre-existing endotheliopathy and micro-arteriopathy (e.g., secondary to diabetes mellitus, hypertension, or obesity) are at increased risk of severe COVID-19 (29,56). Similarly, there is a rapidly growing body of evidence linking patients with low levels of ADAMTS13 and high levels of VWF with a variety of diseases that pre-dispose to a poor outcome after infection with SARS-CoV-2 and to its variable presentation (57-64). The use of standard anti-platelet medication (aspirin or P2Y12 inhibitors) is also likely to be ineffective given that the interaction between VWF and platelets activates the GP2b3a receptor. Although the inhibition of VWF-platelet binding via the GPlb receptor, using either caplacizumab or anfibatide, would be an attractive option and has been suggested (65) these drugs are notin widespread use and clinical experience with them is extremely limited. Similarly, they carry a significant haemorrhage profile.

[0022] Thus, there is a need for a treatment of endotheliopathy per se.SUMMARY OF THE INVENTION

[0023] Heparin is a naturally occurring, highly sulphated polysaccharide characterised by a wide molecular weight range of polysaccharide chains. Heparin acts at a variety of different ligands with varied actions. Heparin is a member of the glycosaminoglycan carbohydrate family and consists of repeating disaccharide units of GlcApl-4GlcNAcal-4 with poly-disperse sulfation, N-acetylation and uronosyl epimerization. Heparin is highly heterogenous. 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 deliver shorter polysaccharide chains. The products of the chemically or enzymatically treated UFH can be affinity purified to yield fractionated heparin where the molecular weight of the polysaccharides in each fraction can be readily determined. Low molecular weight heparin (LMWH) contains polysaccharide chains in the range of about 4000 Da to about 8000 Da.

[0024] In 1991, it was first demonstrated that the intravenous administration of heparin to patients during open heart surgery induced the impairment of VWF-dependent platelet function, without changes in plasma VWF levels (66). This inhibitory effect of heparin on VWF-dependent platelet agglutination was not dependent on the heparin's affinity for antithrombin III, but was dependent upon the molecular weight of heparin. From later in vitro experiments, it was found that heparin bound to a specific amino acid sequence within the Al domain of VWF (residues 569 - 583), in which basic amino acids are regularly arranged. Heparin binding induced conformational changes in a peptide of this binding site (67). Heparin bound to both activated and inactivated VWF similarly, but did not interfere with VWF binding to collagen. Since the platelet Gplb-binding domain (residues 524 - 542) is also located in the Al domain, it was suggested that heparin interferes with VWF binding to platelet Gplb both by steric hindrance and by inducing a conformational change of the domain that results in inhibition of platelets binding.

[0025] The structural specificity of the heparin that is responsible for binding to VWF, revolves around key disaccharide units - GlcNS6S-IdoA2S and IdoA2S-GlcNS6S. Further, it has been demonstrated that the assembly of more than 3 units of the disaccharide wascrucial for the binding potency. Similarly, although fractionated heparins of lower molecular weight (6100 Da (g / mol)) have shown a higher affinity to binding to the VWF they were less able to inhibit VWF activity compared to UFH. This suggests that a minimum heparin molecular weight and molecular size is important in order to achieve steric hindrance.

[0026] These medium molecular weight heparins may have specificity towards inhibiting VWF-GPIb binding, hence stopping microthrombosis, but as they have little effect on antithrombin III they have little anti-coagulant effect. Thus, medium molecular weight heparins with a mass of about 11 000 Da (g / mol) represent an ideal treatment option when considering the treatment of patients with pro-thrombotic states that are dependent upon increased VWF levels and endotheliopathies. Furthermore, the results of these earlier studies suggests that low molecular weight heparins are unlikely to work and do not target the GPIb receptor and that UFH, whilst it may contain the sugar moieties that can bind to VWF, is sub-optimal. Moreover, monitoring of UFH is difficult and the other fractions of UFH, e.g. the LMWH fractions, have anticoagulant effects which can result in dangerous bleeding events, which are unpredictable.

[0027] Of further interest is the fact that it has recently been shown that SARS-CoV-2 binds to heparin sulphates and in particular requires the IdoA2S-GlcNS6S sugar moiety (74,75). This suggests that exogenous supply of these sugar moieties may inhibit binding to the endogenous heparan sulphates in the lungs and hence act as a potential prophylactic treatment. Taken together, a specialized medium molecular weight heparin (~ 11000 Da (g / mol)) with at least 3 units of the GlcNS6S-IdoA2S disaccharide may inhibit viral adherence and replication but also inhibit the microthrombosis triggered by the release of VWF secondary to the endotheliopathy caused by the virus.

[0028] As described herein, endotheliopathy may be associated with a number of diseases. As previously described the Inventors have found that medium molecular weight heparin can be used to treat endotheliopathy, particularly endotheliopathy in a patient having a high plasma von Willebrand factor level (WO 2022 / 238587; incorporated by reference herein).

[0029] The Inventors have now found that functionalised medium molecular weight heparin as described herein can be used to treat endotheliopathy, particularly endotheliopathy in a patient having a high plasma von Willebrand factor level.

[0030] Thus, in a first aspect the invention provides a functionalised medium molecular weight heparin (FMMWH). The FMMWH comprises a functional group.

[0031] In a second aspect, the invention provides a method of synthesising a FMMWH comprising I. providing a medium molecular weight heparin (MMWH) comprising a carbonyl; and, II reacting the MMWH with a functionalising agent to provide the FMMWH; wherein the functionalising agent comprises an oxyamine.

[0032] In a third aspect, the invention provides a FMMWH comprising one or more of Structure I, wherein structure I comprises:wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2; wherein L is selected from the group consisting of -N(H)-, N(R.2)-, and -O-C(=O)-;R2 is selected from the group consisting of an alkyl, fluoroalkyl, carboxylic acid, sulfonate, aryl, heteroaryl, heteroalkyl, and heterocycle; wherein at least one X is -CH2-L-R2.

[0033] In a fourth aspect, the invention provides a composition comprising the FMMWH according to the first aspect of the invention.

[0034] In a fifth aspect, the invention provides a composition comprising the FMMWH according to the third aspect of the invention.

[0035] In a sixth aspect, the invention provides FMMWH for use in the treatment of endotheliopathy in a patient. The FMMWH may inhibit von Willebrand factor (VWF). The FMMWH may inhibit multimers of VWF, preferably ultra-large VWF. The FMMWH may inhibit the binding of platelets to VWF. Preferably, the patient has an endotheliopathy characterised by a plasma von Willebrand factor to ADAMTS13 (VWF:ADAMTS13) ratio of at least about 2. Alternatively or additionally, the patient may have an endotheliopathy characterised by a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2. The FMMWH may be the FMMWH accordingto the first aspect, the third aspect, FMMWH comprised in the composition of the fourth aspect or FMMWH comprised in the composition of the fifth aspect.

[0036] In a seventh aspect, the invention provides functionalised medium molecular weight heparin for use in the treatment of a disease or condition in a patient, wherein the patient has an endotheliopathy characterised by a plasma von Willebrand factor to ADAMTS13 (VWF: ADAMTS13) ratio of at least about 2. The FMMWH may be the FMMWH according to the first aspect, the third aspect, FMMWH comprised in the composition of the fourth aspect or FMMWH comprised in the composition of the fifth aspect.

[0037] In an eighth aspect, the invention provides FMMWH for use in the treatment of a disease or condition in a patient, wherein the patient has an endotheliopathy characterised by a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2. The FMMWH may be the FMMWH according to the first aspect, the third aspect, FMMWH comprised in the composition of the fourth aspect or FMMWH comprised in the composition of the fifth aspect

[0038] In a ninth aspect, the invention provides a method of synthesis of functionalised medium molecular weight heparin, the method comprising the steps of: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; and (d) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin.

[0039] In a tenth aspect, the invention provides a method of synthesis of functionalised medium molecular weight heparin, the method comprising the steps of: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 0 °C to about 10 °C to form a medium molecular weight heparin solution; and (d) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin.

[0040] The methods of the ninth or tenth aspect may be used to prepare the FMMWH of the third aspect of the invention. The methods of the second, ninth or tenth aspect may be used to prepare the FMMWH of the first aspect of the invention.

[0041] In an eleventh aspect, the invention provides functionalised medium molecular weight heparin produced according to the method of the ninth aspect.

[0042] In a twelfth aspect, the invention provides functionalised medium molecular weight heparin produced according to method of the tenth aspect.

[0043] In a thirteenth aspect, the invention provides a functionalised medium molecular wight heparin produced according to the second aspect.

[0044] In a fourteenth aspect, the invention provides a method of treating endotheliopathy, the method comprising administering to a subject (patient) in need of treatment a therapeutically effective amount of FMMWH. Preferably, wherein the patient has plasma VWF:ADAMTS13 ratio of at least about 2 or wherein the patient has plasma VWF antigen :ADAMTS13 ratio of at least about 2.

[0045] In a fifteenth aspect, the invention provides the use of the FMMWH for the manufacture of a medicament for the treatment of endotheliopathy in a patient. Preferably, wherein the patient has plasma VWF:ADAMTS13 ratio of at least about 2 or wherein the patient has plasma VWF antigen :ADAMTS13 ratio of at least about 2.

[0046] In a sixteenth aspect, the invention provides a kit suitable for preparing FMMWH, wherein the kit comprises: (a) unfractionated heparin; (b) an aqueous solvent (c) an oxidising agent; (d) a functionalising agent; and (e) optionally, an inactivating agent.

[0047] In a seventeenth aspect, the invention provides a kit suitable for preparing functionalised medium molecular weight heparin, wherein the kit comprises: (a) unfractionated heparin; (b) an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0; (c) an oxidising agent; (d) a functionalising agent; and (e) optionally, an inactivating agent.

[0048] For the avoidance of doubt, embodiments related to each aspect of the invention apply mutatis mutandis to the other aspects of the invention. Further aspects and embodiments of the present invention will be evident from the discussion that follows below.DESCRIPTION OF THE FIGURES

[0049] FIG. 1 - Scheme of the structures of the functionalised medium molecular weight heparins G18086 (A), G18087 (B) and G18088 (C). Asterisks indicate the newly formed CH = N and CH2 that can represent markers of the functionalised structure in NMR analysis.

[0050] FIG. 2 - 1H-NMR spectra of all the analysed samples, G18088 (FIG. 2A), G18087 (FIG. 2B), G18086 (FIG. C), and medium molecular weight heparin (G18085; FIG. 2D).

[0051] FIG. 3 -1H-13C-HSQC-DEPT NMR spectra of medium molecular weight heparin (G18085) with the acetal signal labelled.

[0052] FIG. 4 -1H-13C-HSQC-DEPT NMR spectra of G18086 with the CH = N signal labelled and the area where the acetal signal that is now absent labelled.

[0053] FIG. 5 -1H-13C-HSQC-DEPT NMR spectra of G18087 with the CH = N signal labelled and the area where the acetal signal that is now absent labelled.

[0054] FIG. 6 -1H-13C-HSQC-DEPT NMR spectra of G18088 with the CH = N signal labelled and the area where the acetal signal that is now absent labelled.

[0055] FIG. 7 - ^-“C-HSQC-DEPT NMR spectra of G18086 (grey) in superimposition with reference G18085 (MMWH; black) shown in the full (top) and heparin (bottom) window.

[0056] FIG. 8 - ^-“C-HSQC-DEPT NMR spectra of G18087 (grey) in superimposition with reference G18085 (MMWH; black) shown in the full (top) and heparin (bottom) window.FIG. 9- ^-^C-HSQC-DEPT NMR spectra of G18088 (grey) in superimposition with reference G18085 (MMWH; black) shown in the full (top) and heparin (bottom) window.DETAILED DESCRIPTION OF THE INVENTION

[0057] Throughout this specification, one or more aspects of the invention may be combined with one or more features described in the specification to define distinct embodiments of the invention.

[0058] In the discussion that follows, reference is made to a number of terms, which are to be understood to have the meanings provided below, unless a context expressly indicates to the contrary.

[0059] References herein to a singular of a noun encompass the plural of the noun, and vice-versa, unless the context implies otherwise.

[0060] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer, or group of elements or integers, but not the exclusion of any other element or integer, or group of elements or integers. The term "comprising" includes within its ambit the term "consisting" or "consisting essentially of".

[0061] The term "consisting" or variants thereof is to be understood to imply the inclusion of a stated element or integer, or group of elements or integers, and the exclusion of any other element or integer or group of elements or integers.

[0062] The term "consisting essentially of" or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and that further components may be present, but only those not materially affecting the essential characteristics of the formulation, composition, or compound.

[0063] The term "about" herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified.

[0064] The terms "treatment" and "therapy" define the therapeutic treatment of a patient, in order to reduce or halt the rate of progression of a disorder or condition, or to ameliorate or cure the disorder or condition. Prophylaxis of a disorder or condition as a result of treatment or therapy is also included.

[0065] As used herein, the term "patient" preferably refers to a mammal. Typically, the mammal is a human.

[0066] von Willebrand factor (VWF) is a blood glycoprotein involved in haemostasis. VWF is a large multimeric glycoprotein present in blood plasma and produced constitutively as ultra-large VWF in endothelium (in the Weibel-Palade bodies), megakaryocytes (flgranules of platelets), and subendothelial connective tissue. The basic VWF monomer is a 2050-amino acid protein.

[0067] A disaccharide is a sugar whose molecules contain two monosaccharide residues.

[0068] A low molecular weight heparin is defined herein as a heparin with an average molecular weight of from about 4000 Da (g / mol) to about 8000 Da (g / mol). A medium molecular weight heparin is defined herein as a heparin with an average molecular weight of from greater than about 8000 Da (g / mol) to about 13500 Da (g / mol).

[0069] Chemical structures may have been drawn showing acid or basic groups in their neutral state. However, it should be appreciated that at physiological pH some of these groups may be protonated or deprotonated as appropriate. For example, a carboxylic acid and a sulphate may be deprotonated at physiological pH and an amine may be protonated at physiological pH

[0070] As described herein, there is a need for treatments of endotheliopathy per se. As previously described the Inventors have found that medium molecular weight heparin (MMWH) can be used to treat endotheliopathy, particularly endotheliopathy in a patient having a high plasma von Willebrand factor level (WO 2022 / 238587; incorporated by reference herein).

[0071] Medium molecular weight heparin (MMWH) displays a significant reduction in the activity as performed by the anti-Xa and anti-IIa tests routinely performed on heparin. It is proposed that the oxidation of the glucuronic acid during the preparation of MMWH changes the binding capability of heparin to antithrombin (ATIII). Ristocetin-induced platelet aggregation (RIPA) tests of MMWH show an increased activity of MMWH, indicating that the von Willebrand Factor interaction of the heparin polysaccharide remains intact.

[0072] The two aldehyde groups on the glucuronic acid of the MMWH produced during preparation may in some circumstances be reactive. This reactivity can potentially decrease the stability of the oxidized polysaccharide over time. Additionally, it may interact with free amines on proteins or components in blood when injected into the body. The latter would be through a Schiff base reaction with the free amines in the same way as glucose reacts with haemoglobin to form the well-known glycated haemoglobin commonly known as A1C.

[0073] Thus, in order to mitigate these potential side reactions, in a first aspect, the invention provides a functionalised medium molecular weight heparin (FMMWH).

[0074] The FMMWH comprises a functional group. The functional group may replace the aldehyde groups on the glucuronic acid of the MMWH produced during oxidation. The functional group may replace at least two aldehyde groups on the glucuronic acid of theMMWH produced during oxidation. The functional group may replace the majority of the aldehydes groups on the glucuronic acid of the MMWH produced during oxidation.

[0075] The functional group may be attached to the FMMWH. The FMMWH is derived from heparin and in particular MMWH. Heparin and MMWH may be sulphated polysaccharides. A polysaccharide is a polymer of saccharides. The FMMWH may comprise a polysaccharide and the functional group. The polysaccharide may be sulphated. The FMMWH may comprise a sulphated saccharide. The functional group may be attached to the polysaccharide comprising the FMMWH. The polysaccharide may be sulphated. The functional group may be attached to a uronic acid residue of the FMMWH. Preferably, the FMMWH comprises at least one functional group that is not attached to a terminal monomer. A terminal monomer is a monomer of the polysaccharide that is at the end of the polysaccharide chain.

[0076] The functional group may be any kind of functional group. The functional group modifies the properties of the FMMWH when compared to MMWH. For example, the FMMWH may have improved or altered binding to VWF when compared to MMWH. Alternatively or additionally, the FMMWH may have improved or altered biocompatibility when compared to MMWH.

[0077] The functional group may comprise a polar functional group, an apolar functional group, an acid functional group, or a basic functional group. The functional group may comprise a functional group selected from a polar functional group, an apolar functional group, an acidic functional group, a basic functional group or a combination thereof. The functional group may comprise at least one carbon atom. The functional group may comprise at least two carbon atoms.

[0078] The functional group may comprise an aryl, a phenyl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, an alcohol, a thiol, an ether, a sulphide, an amine, an amide, a fluoroalkyl and / or a ketone. The functional group may comprise a benzyl, a carboxylic acid or a trifluoro alkyl. Preferably the functional group comprises an aryl, a carboxylic acid or a trifluoro alkyl. Even more preferably, the functional group is an aryl, a carboxylic acid or a trifluoro alkyl.

[0079] The alkyl may comprise C1-C5 alkyl. The Ci-Cs alkyl may be methyl, ethyl, n-propyl, i-propyl, n-butyl or t-butyl. The fluoroalkyl may comprises C1-C5 fluoroalkyl. Preferably, the C1-C5 fluoroalkyl is fluoromethyl or fluoroethyl. Preferably, the C1-C5 fluoroalkyl is trifluoromethyl or trifluoroethyl.

[0080] It will be appreciated that heparin and MMWH may be considered to comprise functional groups. For example, heparin (or MMWH) usually comprise a carboxylic acid functional group and / or a sulphate functional group. A functional group as described herein should not be understood as a functional group that is already present in heparin or MMWH. A functional group as described herein is an additional functional group to those already present in heparin or MMWH. A functional group as described herein may be the same as a functional group in heparin or MMWH. If the functional group as described herein is the same as a functional group in heparin or MMWH then it will have different connectivity to the functional group already present in heparin or MMWH. Alternatively or additionally, the functional group may be different to a functional group already present in heparin or MMWH.

[0081] The functional group may be a heterologous functional group. The term "heterologous" in this context means that the functional group is introduced into MMWH. "Heterologous" should therefore be understood to mean that the functional group is in addition to those functional groups present in heparin or MMWH.

[0082] The heterologous functional group may comprise a functional group that is not present in heparin or MMWH. Alternatively or additionally, a heterologous functional group may comprise a functional group that is present in heparin or MMWH. If the heterologous functional group comprises a functional group that is present in heparin or MMWH then it has different connectivity to the functional group that is present in heparin or MMWH. For example, the heterologous functional group may be connected to a different carbon atom in the polysaccharide chain when compared to a functional group that is present in heparin or MMWH. A heterologous functional group may comprise a functional group that is added to MMWH.

[0083] The FMMWH may be prepared by treating unfractionated heparin with an oxidising agent, for example periodate and in particular sodium periodate. As a result, the diol of the uronic acid present in unfractionated heparin is oxidised to produce two aldehydes, thereby breaking the carbon-carbon bond between carbons 2 and 3 of the uronic acid. These aldehydes may then be reacted with a functionalising agent to introduce the functional group. The functional group may therefore be attached to carbons 2 and 3 of the uronic acid. This is shown in schematic I below with the carbons numbered. In schematic I below, X comprises the functional group and each X is the same functional group. The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH or to a chain terminating group. The carbon-X bond may be any kindof chemical bond, for example, a single or double chemical bond. Preferably, the FMMWH comprises at least one uronic acid comprising a functional group wherein the at least one uronic acid is not attached to a chain terminating group. Preferably, the FMMWH comprises at least one uronic acid comprising a functional group wherein the at least one uronic acid is attached to two other saccharides.Schematic I:

[0084] The functional group may comprise a heterologous functional group wherein the heterologous functional group is attached to carbons 2 and 3 of the uronic acid of the FMMWH. The functional group may comprise a heterologous functional group wherein the heterologous functional group is attached to carbons 2 and 3 of the uronic acid of the FMMWH and there is no carbon-carbon bond between carbons 2 and 3 of the uronic acid of the FMMWH. The heterologous functional group attached to carbons 2 and 3 are the same.

[0085] The FMMWH may comprise a linker. The linker may link the functional group. The linker may be any kind of linker. The linker may be only a chemical bond. For example, in schematic I above the linker may be the carbon-X bond. The linker may link the functional group to the FMMWH. The linker may link the functional group to the polysaccharide comprising the FMMWH. The linker may link the functional group to a uronic acid residue of the FMMWH. The linker may link the functional group to carbons 2 and 3 of the uronic acid of the FMMWH. The linker may be attached to carbons two and three of the uronic acid of the FMMWH and there may be no carbon-carbon bond between carbons 2 and 3 of the uronic acid of the FMMWH.

[0086] The linker may comprise a chemical bond. The linker may comprise an amine, an ester, an amide, an ether, an ether, an alkyl, a ketone, a cycloalkyl, or an oxime. The linker may comprise an amine linker, an ester linker, an amide linker, an alkyl linker, , a cycloalkyl linker, or an oxime linker. Preferably the linker is an amine linker or an oxime linker. Most preferably the linker is an oxime linker.

[0087] The FMMWH may comprise one more of structure A, wherein structure A comprises:wherein X is CHn-L-Ri; wherein L is the linker, Ri is the functional group and n= 1 or 2.

[0088] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure A is at the end of the polysaccharide chain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide. Preferably, the FMMWH comprises at least one structure A wherein structure A is not attached to a chain terminal moiety. Preferably, the FMMWH comprises at least one structure A wherein structure A is attached to two other saccharides of the FMMWH polysaccharide chain. The COOH group shown in structure A may be deprotonated, for example at physiological pH. The COOH group of carbon 6 of structure A may be deprotonated. The OH group attached to carbon 6 of structure A may be deprotonated.

[0089] In structure A, both X are the same. In structure A, both L and Ri are the same. Preferably, the linker is an amine linker or an oxime linker. X may be CHn-L-Ri wherein L and n are selected from the group consisting of -N(H)- and n = 2, and = N-O- and n = l.

[0090] Most preferably, the linker is an oxime linker. When the linker is an oxime linker, X of structure A is HC=N-O-Ri; wherein Ri comprises the functional group.

[0091] An oxime linker may be obtained by the reaction of a carbonyl group (either an aldehyde or a ketone) and an oxyamine (Ulrich, Sebastien, et al. "Oxime ligation: a chemoselective click-type reaction for accessing multifunctional biomolecular constructs." Chemistry-A European Journal 20.1 (2014) : 34-4)).

[0092] For example, the aldehyde formed on carbons 2 and 3 of the uronic acid following treatment of heparin with the oxidising agent may be reacted with an oxyamine to produce an oxime linker. An oxyamine suitable for forming the oxime linker may comprise the structure:wherein Ri comprises the functional group.

[0093] Alternatively or additionally, an oxyamine suitable for forming the oxime linker may comprise the structure:wherein Ri comprises the functional group.

[0094] An oxyamine suitable for forming the oxime linker may comprise the structure:

[0095] Wherein Ri comprises the functional group and n= 0-10, preferably 0-5, even more preferably 1-5 and most preferably 1. Without wishing to be bound by theory, by varying the number of CH 2 (i.e. n) present in the linker, the spatial presentation of the functional group can be varied.

[0096] The FMMWH may comprise a sulphated structure. For example, the FMMWH may comprise one or more of structure Al, wherein structure Al comprises:wherein X is CHn-L-Ri; wherein L is the linker, Ri is the functional group and n= 1 or 2.

[0097] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure Al is at the end of the polysaccharidechain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide. Preferably, the FMMWH comprises at least one structure Al wherein structure Al is not attached to a chain terminal moiety. Preferably, the FMMWH comprises at one least structure Al wherein structure Al is attached to two other saccharides of the FMMWH polysaccharide chain. The COOH group and SO3H groups shown in structure Al may be deprotonated, for example at physiological pH. The COOH group and SO3H groups shown in structure Al may be deprotonated and the amine (NH) shown in structure Al may be protonated, for example at physiological pH.

[0098] In structure Al, both X are the same. In structure Al, both L and Rli are the same. Preferably, the linker is an amine linker or an oxime linker. X may be CHn-L-Ri wherein L and n are selected from the group consisting of -N(H)- and n = 2, and =N-O- and n = l.

[0099] Most preferably, the linker is an oxime linker. When the linker is an oxime linker, X of structure Al is HC=N-O-Ri; wherein Ri comprises the functional group.

[0100] The FMMWH may comprise one or more of structure B, wherein structure B comprises:wherein Ri comprises the functional group. In structure B, the linker is the oxime linker. Ri is the same functional group.

[0101] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure B is at the end of the polysaccharide chain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide. Preferably, the FMMWH comprises at least one structure B wherein structure B is not attached to a chain terminal moiety. Preferably, the FMMWH comprises at least one structure B wherein structure B is attached to two other saccharides of the FMMWH polysaccharide chain. The COOH group shown in structure B may be deprotonated, for example at physiological pH.The COOH group of carbon 6 of structure B may be deprotonated. The OH group attached to carbon 6 of structure B may be deprotonated.

[0102] The FMMWH may comprise a sulphated structure. For example, the FMMWH may comprise one or more of structure Bl, wherein structure Bl comprises:wherein Ri comprises the functional group. In structure Bl, the linker is the oxime linker. Ri is the same functional group.

[0103] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure Bl is at the end of the polysaccharide chain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide. Preferably, the FMMWH comprises at least one structure Bl wherein structure Bl is not attached to a chain terminal moiety. Preferably, the FMMWH comprises at one least structure Bl wherein structure Bl is attached to two other saccharides of the FMMWH polysaccharide chain. The COOH group and SO3H groups shown in structure Bl may be deprotonated, for example at physiological pH. The COOH group and SO3H groups shown in structure Bl may be deprotonated and the amine (NH) shown in structure Bl may be protonated, for example at physiological pH.

[0104] The FMMWH may comprise one of more of structure C, wherein structure C comprises:wherein Ri comprises the functional group. In structure C, the linker is the oxime linker. Ri is the same functional group.

[0105] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure C is at the end of the polysaccharide chain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide. Preferably, the FMMWH comprises at least one structure C wherein structure C is not attached to a chain terminal moiety. Preferably, the FMMWH comprises at least structure C wherein structure C is attached to two other saccharides of the FMMWH polysaccharide chain. The COOH group shown in structure C may be deprotonated, for example at physiological pH. The COOH group of carbon 6 of structure C may be deprotonated. The OH group attached to carbon 6 of structure C may be deprotonated.

[0106] The FMMWH may comprise a sulphated structure. For example, the FMMWH may comprise one or more of structure Cl, wherein structure Cl comprises:wherein Ri comprises the functional group. In structure Cl, the linker is the oxime linker. Ri is the same functional group.

[0107] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure Cl is at the end of the polysaccharide chain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide. Preferably, the FMMWH comprises at least one structure Cl wherein structure Cl is not attached to a chain terminal moiety. Preferably, the FMMWH comprises at least one structure Cl wherein structure C is attached to two other saccharides. The COOH group and SO3H groups shown in structure Cl may be deprotonated, for example at physiological pH. The COOH groupand SO3H groups shown in structure Cl may be deprotonated and the amine (NH) shown in structure Cl may be protonated, for example at physiological pH.

[0108] Preferably, Ri comprises an aryl. For example, the FMMWH may comprise the structure one or more of structure D, wherein structure D comprises

[0109] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure D is at the end of the polysaccharide chain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide. Preferably, the FMMWH comprises at least one structure D wherein structure D is not attached to a chain terminating group. Preferably, the FMMWH comprises at least one structure D wherein structure D is attached to two other saccharides. The COOH group shown in structure D may be deprotonated, for example at physiological pH. The COOH group of carbon 6 of structure D may be deprotonated. The OH group attached to carbon 6 of structure D may be deprotonated.

[0110] Preferably, Ri comprises a carboxylic acid. Even more preferably, Ri is a carboxylic acid. For example, the FMMWH may comprise one or more of structure E wherein structure E comprises

[0111] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure E is at the end of the polysaccharide chain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide. Preferably, the FMMWH comprises at least one structure E wherein structure E is not attached to a chain terminating group. Preferably, the FMMWH comprises at least one structure Ewherein structure E is attached to two other saccharides. The COOH groups shown in structure E may be deprotonated, for example at physiological pH.

[0112] Preferably, Ri comprises trifluoroalkyl. Even more preferably, Ri is trifluoroalkyl. For example, the FMMWH may comprise one or more of structure F wherein structure F comprises

[0113] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure F is at the end of the polysaccharide chain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide. Preferably, the FMMWH comprises at least one structure F wherein structure F is not attached to a chain terminating group. Preferably, the FMMWH comprises at least one structure F wherein structure F is attached to two other saccharides. The COOH group shown in structure F may be deprotonated, for example at physiological pH. The COOH group of carbon 6 of structure F may be deprotonated. The OH group attached to carbon 6 of structure F may be deprotonated.

[0114] In a second aspect, the invention provides a method of synthesising a functionalised medium molecular weight heparin. The method comprises:I. providing a medium molecular weight heparin comprising a carbonyl; and,II. reacting the medium molecular weight heparin with a functionalising agent to provide the functionalised medium molecular weight heparin; wherein the functionalising agent comprises an oxyamine.

[0115] Preferably the carbonyl is an aldehyde.

[0116] Step I may comprise dissolving the MMWH in water.

[0117] Providing a medium molecular weight heparin comprising a carbonyl may comprise oxidising unfractionated heparin with an oxidising agent. Accordingly, Step I and Step II of the method may comprise step (i) and step (ii) respectively wherein step (i) comprises incubating unfractionated heparin with the oxidising agent to provide the MMWH; and step (ii) comprises reacting the medium molecular weight heparin with the functionalising agent to provide functionalised medium molecular weight heparin.

[0118] Unfractionated heparin may be obtained from bovine or porcine tissue, for example porcine intestine or bovine lung.

[0119] Step (i) may be performed in an aqueous solvent. Typically the aqueous solvent is water. Preferably, step (i) is performed in water. Step (i) may comprise dissolving the unfractionated heparin in an aqueous solvent to provide a first solution. The first solution may consist or consist essentially of unfractionated heparin and water. The aqueous solvent may be water.

[0120] The concentration of unfractionated heparin in the first solution may be from about 0.5 mg / mL to about 100 mg / mL, more preferably from about 1 mg / mLto about 85 mg / mL, more preferably from about 1.5 mg / mL to about 80 mg / mL, more preferably from about 2 mg / mL to about 75 mg / mL, more preferably from about 2 mg / mL to about 70 mg / mL, more preferably from about 10 mg / L to about 65 mg / mL, more preferably from about 25 mg / mL to about 65 mg / mL, most preferably from about 30 mg / mL to about 60 mg / mL.

[0121] The concentration of UF heparin in the first solution may be about 1.5 mg / mL, more preferably about 1.8 mg / mL, more preferably about 2 mg / mL, more preferably about 2.5 mg / mL, more preferably about 2.7 mg / mL, more preferably about 3 mg / mL, more preferably 8 mg / mL, more preferably about 16 mg / ml, more preferably about 30 mg / mL and most preferably about 60 mg / mL. The concentration of UF heparin is most preferably about 60 mg / mL.

[0122] The oxidising agent may be added to the first solution to provide a second solution. Step (i) may comprise dissolving the unfractionated heparin in an aqueous solvent to provide a first solution and adding the oxidising agent to the first solution to provide a second solution. The second solution may consist or consist essentially of unfractionated heparin, water and the oxidising agent. Preferably the unfractionated heparin and the oxidising agent are incubated at room temperature. The aqueous solvent may be water. Preferably, the aqueous solvent is water.

[0123] The oxidising agent may be a periodate, for example sodium periodate or potassium periodate. Preferably, the oxidising agent is sodium periodate. Alternatively, or additionally, the oxidising agent may be a perchlorate, for example sodium perchlorate. Preferably, the oxidising agent does not comprise a perchlorate. Use of a perchlorate in combination with a periodate as the oxidising agent results in an increased level of sample breakdown to smaller molecular weight species.

[0124] The concentration of the oxidising agent in the second solution may be from about 1 g / L to about 40 g / L. Preferably, the concentration of the oxidising agent may be from about 2g / L to about 35 g / L, more preferably from about 4 g / L to about 30 g / L, more preferably from about 20 g / L to about 25 g / L. The concentration of the oxidising agent in the second solution may be about 23 g / L or greater. Preferably the concentration of oxidising agent is about 23 g / L. Preferably the concentration of the oxidising agent is about 23 g / L and the oxidising agent is sodium periodate.

[0125] The molar ratio of UF heparin to oxidising agent may be from about 1 : 1 to about 1:200, more preferably from about 1 :2 to about 1: 150, more preferably from about 1: 10 to about 1: 100, more preferably from about 1:20 to about 1 : 50, more preferably from about 1 :20 to about 1 :30, most preferably from about 1:25-1:30. Typically, the molar ratio of UF heparin to oxidising agent may be about 1 :27.

[0126] The molar ratio of UF heparin to oxidising agent may be determined based on the estimated molecular weight of the canonical heparin disaccharide as opposed to the molecular weight of heparin. The estimated molecular weight of the canonical heparin disaccharide is 593.45 g / mol. This is used to approximate the number of moles of this disaccharide and therefore the uronic acid residues present in the sample.

[0127] For example, if it is desired to add 1 equivalent of NalCh based on uronic acid and 3 grams of heparin is used, the number of moles NaICk to add is 0.005 moles i.e. 3 divided by 593.45.

[0128] Thus, the ratio of UF heparin determined based on the estimated molecular weight of the canonical heparin disaccharide may be from about 1 :0.5 to about 1 : 10, preferably from about 1 :0.7 to about 1 :8, more preferably from about 1:0.9 to about 1 : 5, more preferably from about 1 : 1 to about 1 :2, more preferably from about 1: 1.1 to about 1 :2. Preferably, the ratio of moles of uronic acid present in the UF heparin to moles of oxidising agent may be 1 : 1.1.

[0129] Preferably, step (i) is carried out room temperature. Step (i) may be carried out at a temperature of from about 15 °C to about 30 °C. Preferably, the temperature is from about 16 °C to about 25 °C, preferably from about 17 °C to about 22 °C, preferably from about 18 °C to about 21 °C, preferably about 19 °C to about 20 °C. By performing the reaction at or around room temperature, costs to produce the product are reduced due to shorter reaction times.

[0130] Step (i) may comprise dissolving unfractionated heparin in an aqueous solvent to provide a first solution; adding an oxidising agent to the first solution to provide a second solution; and incubating the second solution to form medium molecular weight heparin. The unfractionated heparin may be dissolved in water. The aqueous solvent may be water.

[0131] Typically, incubating the second solution is carried out at room temperature. The incubation may be carried out at from about 15 °C to about 30 °C, more preferably from about 16 °C to about 28 °C, more preferably from about 17 °C to about 27 °C, more preferably from about 18 °C to about 25 °C. Preferably, the incubation temperature is about 18 °C to about 20 °C. Typically, the incubating is carried out for from about 0.5 hour to about 40 hours, more preferably from about 1 hours to about 24 hours, more preferably from about 2 hours to about 20 hours, more preferably from about 3 hours to about 18 hours. Preferably, the incubating may be carried out for about 3 hours.

[0132] The aqueous solvent may be an aqueous solution. The aqueous solution may be an aqueous buffer solution. When the aqueous solution is an aqueous buffer solution, in step (a) unfractionated heparin is dissolved in an aqueous buffer solution adjusted to between about pH 4.0 and pH 9.0 to provide a first solution.

[0133] "Buffer" refers to a chemical which, in a solution, resists a change in pH when acid or alkali is added to the solution. Typically, a buffer solution (or buffer system) comprises a weak acid and its conjugate base, or a weak base and its conjugate acid.

[0134] Typically, a suitable buffer comprises an acid with a pKa value that lies within ±1 of the desired pH of the formulation. For example, if the desired pH of the formulation is about 7.0, a suitable buffer comprises a weak acid with a pKa value of from about 6.0 to about 8.0. If the acid of a buffer has more than one pKa value (i.e. each molecule of the acid is able to donate more than one proton), in order for the buffer to be suitable, at least one of the pKa values should lie within the desired pH range.

[0135] The weak acid and conjugate base (or weak base and conjugate acid) of the buffer are in equilibrium with one another. In accordance with Le Chatelier's principle (if a constraint, such as a change in concentration of a reactant, is applied to a system in equilibrium, the equilibrium will shift so as to counteract the effect of the constraint), addition of acid or base to the solution shifts the position of equilibrium in favour of the conjugate base or weak acid, respectively. Consequently, the concentration of free protons in the formulation (and thus the pH) is relatively unchanged.

[0136] Suitable buffer systems comprise an acetate salt and acetic acid (pKa = 4.75), a citrate salt and citric acid (pKa = 3.13, 4.76 and 6.40), and phosphoric acid (pKa = 2.14, 7.20 and 12.37), or mixtures thereof. Phosphate buffered saline may also be used. The pKa values cited herein are those reported at 25 °C in water. Typically, the buffer comprises only one of the pairs listed above, i.e. one acid and its conjugate base. The buffer may comprise an acetate salt and acetic acid, a citrate salt and citric acid, or a phosphate salt and phosphoric acid.

[0137] The pH may be adjusted to ensure that the pH of the first solution is between from about pH 4.0 to about pH 9.0 because dissolving unfractionated heparin may result in the pH of the aqueous buffer solution changing. However, it has been surprisingly found that the reaction can be carried out in water and a buffer is not needed.

[0138] Optionally, the aqueous buffer solution is adjusted to between about pH 5.0 and about pH 8.0.

[0139] Step (i) may comprise purifying the medium molecular weight heparin. Step (i) may comprise isolating the MMWH. Isolating the MMWH may comprise isolating the MMWH as a solid.

[0140] Isolating the medium molecular weight heparin may comprise precipitating and filtering the medium molecular weight heparin. For example, following the treatment of the unfractionated heparin with the oxidising agent, a precipitating agent may be added. The precipitating agent may comprise or consist of an alcohol or acetone. The alcohol may be a Ci to C3 alcohol. For example, the alcohol may be methanol, ethanol, propanol, or isopropanol. Preferably the alcohol is methanol. Preferably, the alcohol is added to the medium molecular weight heparin solution to give a final concentration of the alcohol in the precipitation mixture of from about 50% v / v to about 90% v / v, more preferably from about 55% v / v to about 80% v / v, and even more preferably from about 60% v / v to about 75% v / v. Preferably the final alcohol concentration in the precipitation mixture is about73% v / v. For example, addition of 73 mL of an alcohol, e.g. methanol, to a 27 mL medium molecular weight heparin solution provides a final alcohol concentration of 73% v / v MeOH in the precipitation mixture. Conveniently, the alcohol precipitates the medium molecular weight heparin, as well as quenches the excess oxidising agent. The precipitated medium molecular weight heparin may then be isolated, for example by filtration.

[0141] During the addition of the alcohol, the temperature of the solution may be kept between about 0 °C and about 15 °C, preferably between about 5 °C and about 10 °C.

[0142] A salt may be added prior to addition of the precipitating agent (e.g. the alcohol).

[0143] The salt may be a lithium salt, a sodium salt, or a potassium salt. Preferably, the salt is a sodium salt. The salt may be lithium chloride, sodium chloride, potassium chloride. Preferably the salt is sodium chloride.

[0144] The mass of salt added to the medium molecular weight heparin solution is selected to result in a final salt concentration in the medium molecular weight heparin solution of from about 0.5% m / v to about 3% m / v, preferably from about 1% m / v to about 2.5% m / v, and even more preferably from about 1.5% to about 2% m / v. Preferably the concentration of the salt is about 1.8% m / v. For example, addition of addition of 0.9 g solid NaCI to a 50 mL medium molecular weight heparin solution provides a NaCI concentration of 1.8% m / v. Most preferably, the salt solution comprises sodium chloride at a concentration of 1.8% m / v.

[0145] Following addition of the salt, the resulting solution may be cooled to from about 5 °C to about 20 °C , preferably from about 10 °C to about 15 °C . Preferably, the resulting solution may be cooled to about 15 °C. The precipitating agent may then be added.

[0146] Purifying the medium molecular weight heparin may comprise dialysis. For example, following incubating the unfractionated heparin and the oxidising agent in solution, the oxidising agent may be inactivated by the addition of an inactivating agent. The inactivating agent may be methanol, D-mannitol, glycerol, AZ-acetylmethionine, sodium sulfite, and combinations thereof. A particularly preferred inactivating agent is methanol or D-mannitol. The molar ratio of the oxidising agent to the inactivating agent may be from about 1 : 1 to about 1 : 10, more preferably from about 1:2 to about 1:8, more preferably from about 1 :3 to about 1:6, more preferably from about 1 :4 to about 1 : 5. Typically, the molar ratio of the oxidising agent to the inactivating agent may be about 1:2 or about 1:4.

[0147] Following addition of the inactivating agent to the solution, the resulting solution may be dialysed to provide a dialysed medium molecular weight heparin sample.

[0148] Dialysis" or "dialysing" means the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, for example dialysis tubing. The sample for dialysis and a dialysate (or buffer) are placed on opposite sides of the semipermeable membrane. Target sample molecules (e.g. proteins, DNA or polysaccharides) that are larger than the membrane pores remain on the sample side of the membrane. Contaminants such as small molecules and salts can pass through the membrane into the dialysate, thereby reducing the concentration of contaminants in the sample to low levels. Changing the dialysate for fresh dialysate removes the contaminants that have passed from the sample to the dialysate. This allows more contaminants to diffuse from the sample into the dialysate.

[0149] Dialysis can separate small molecules such as salts, reducing agents, or dyes, from larger macromolecules such as proteins, DNA or polysaccharides. Dialysis can also be used to separate polysaccharides by molecular weight. The semipermeable membrane is typically made of a film of regenerated cellulose or cellulose esters.

[0150] Dialysis may be carried out by placing a dialysis tubing containing a sample in a dialysate. A "dialysate" is the fluid into which material passes from the dialysis tubing. The dialysate may be refreshed as often as necessary to achieve optimum separation. The dialysis may be carried out over a period of about 1 day to about 14 days, preferably about 5 days to about 10 days, preferably about 7 days. The dialysate may be refreshed about 1 time a day to about 10 times a day, preferably about 2 times a day to about 5 times a day, preferably about 3 times a day. Typically, the dialysate is many times the volume of the sample, for example from about 2 to about 500 times the volume of the sample. The dialysate may be about 4 times the volume of the sample.

[0151] Typically, the dialysate is water. Optionally, the dialysate may comprise electrolytes such as sodium, potassium, magnesium, calcium, chloride, bicarbonate, lactate, glucose, amino acids, or combinations thereof.

[0152] The dialysing may be carried out in 2 kD cut off tubing, for example provided by Spectra / Por®. The skilled person is aware of the appropriate tubing cut off size for different purposes. Alternatively, the dialysing step may be carried out in a dialysis deviceor dialyzer. Suitable dialyzers may be the Slide-A-Lyzer™, the Float-A-Lyzer, the Pur-A- lyzer, the D-Tube, and GeBAflex Dialyzers product lines.

[0153] The method may further comprise the step of isolating the medium molecular weight heparin from the dialysed heparin sample. The medium molecular weight heparin may be isolated from the dialysed heparin sample by freeze-drying, centrifuging, or filtration. Preferably, the medium molecular weight heparin is isolated from the dialysed heparin sample by freeze-drying.

[0154] "Freeze drying" (also known as lyophilisation or cryodesiccation) is a drying process carried out at low temperature. Freeze drying generally involves reducing temperature and pressure to below the substance's triple point and removing the frozen solvent (e.g. water ice) by sublimation. For aqueous compositions, such as those disclosed herein, freeze drying may be carried out at temperatures of from about -20 °C to about -80 °C, preferably about -40 °C, and pressures of from about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).

[0155] The MMWH provided in step (i) following incubation with the oxidising agent may be used immediately in step (ii). The MMWH heparin provided in step (i) may be used in step (ii). The MMWH provided following incubation with the oxidising agent may not be subject to any further chemical treatment prior to step (ii). For example, the MMWH provided in step (i) is not treated with alkali or acid. The MMWH provided in step (i) may not undergo an alkaline elimination step and / or an acid hydrolysis step.

[0156] Alternatively, the MMWH provided in step (i) may undergo further chemical treatment prior to step (ii).

[0157] The MMWH may be provided in water at room temperature. For example, the MMWH may be dissolved in water at room temperature. Providing the medium molecular weight heparin comprising a carbonyl may comprise dissolving the MMWH in water at room temperature.

[0158] Step II (or step (ii)) may be carried out in water. Step II may be carried out in an aqueous solvent. Preferably, step II is carried out in water. Step II may be carried out in a solvent, wherein the solvent comprises water. For example, the MMWH provided in step I may be prepared in water and the functionalising agent added. For example, Step I may comprise dissolving the MMWH in water to provide a first solution and Step II maycomprise adding the functionalising agent to the first solution to provide a second solution and incubating the second solution.

[0159] Preferably, step II is carried out an acidic pH. Step II may comprise reacting the MMWH with the functionalising agent at an acidic pH to provide FMMWH. The pH may be from about 3 to about less than 7 or from about 4 to about less than 7. The pH may be less than about 7, less than about 6, less than about 5, or less than about 4. Preferably, the pH is less than about 7. Preferably, the pH is about 4.

[0160] Preferably, the functionalising agent is added in excess over the amount of MMWH present.

[0161] The stoichiometry may be determined by using the mass of tri-sulphated disaccharide and 100% oxidation of the uronic acids. The molar ratio of UF heparin to functionalising agent may be determined based on the estimated molecular weight of the canonical heparin disaccharide as opposed to the molecular weight of heparin. The estimated molecular weight of the canonical heparin disaccharide is approximately 593 g / mol. This is used to approximate the number of moles of this disaccharide and therefore the uronic acid residues present in the sample. Preferably, the molar ratio of UF heparin to functionalising agent is determined based on the estimated molecular weight of the canonical heparin disaccharide as opposed to the molecular weight of heparin.

[0162] For example, if it is desired to add 2 equivalents of functionalising agent based on uronic acid and 3 grams of heparin is used, the number of moles functionalising agent to add is 0.010 moles i.e. (3 - 593.45) x 2.

[0163] Step II may comprise reacting the MMWH with at least about two, at least about three, at least about four or at least about five equivalents of the functionalising agent. Step II may comprise reacting the MMWH with about two, about three, about four, or about five equivalents of the functionalising agent. Step II may comprise reacting the MMWH with about two equivalents of the functionalising agent. Preferably, step II comprises reacting the MMWH with at least about two equivalents of the functionalising agent.

[0164] Step II may be carried out at room temperature. Step II may be carried out at from about 15 °C to about 30 °C, more preferably from about 16 °C to about 28 °C, more preferably from about 17 °C to about 27 °C, more preferably from about 18 °C to about 25 °C. Preferably, the incubation temperature is about 18 °C to about 20 °C.

[0165] Typical ly, step II is carried out for from about 0.5 hour to about 24 hours, more preferably from about 0.5 hours to about 12 hours, more preferably from about 0.5 hours to about 6 hours, more preferably from about 1 hours to about 3 hours. Preferably, the incubating may be carried out for about 1 hour.

[0166] Preferably, step II is carried out in water, at room temperature, with two equivalents of functionalising agent, for 1 hour.

[0167] The functionalising agent comprises an oxyamine. A hydroxylamine is HO-NH2. The hydrogen may be replaced to produce an oxyamine. A hydroxylamine may be a suitable functionalising agent if the desired functional group is a hydrogen. When a different functional group is required, an oxyamine is a suitable functionalising agent.

[0168] The oxyamine may comprisewherein Ri comprises a functional group.

[0169] Alternatively or additionally, the oxyamine may comprise:wherein Ri comprises a functional group.

[0170] The oxyamine may comprise the structure:wherein Ri comprises a functional group and n= 0-10, preferably 0-5, even more preferably 1-5 and most preferably 1.

[0171] Without wishing to be bound by theory, the nitrogen of the oxyamine is nucleophilic and attacks the carbonyl of the MMWH, for example the aldehyde formed following oxidation of unfractionated heparin with an oxidising agent. Water is eliminated and a new carbon-nitrogen double bond is formed. The oxyamine may comprise a functional group. As a result of this reaction, the functional group can be readily introduced into the MMWH to provide a FMMWH. It has been found that by reacting MMWH with an oxyamine as described herein, several varied functional groups may be introduced into the MMWH.

[0172] Step I may comprise dissolving the MMWH in an aqueous solvent (for example water) to provide a first solution and step II may comprise adding the functionalising agent to the first solution to produce a second solution and incubating the second solution to provide the FMMWH. The incubation may be for one hour at room temperature. By incubating the second solution, the MMWH and the functionalising agent react together.

[0173] The functional group may comprise a polar functional group, an apolar functional group, an acidic functional group, or a basic functional group.

[0174] The functional group may be selected from the group comprising an aryl, a phenyl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, an alcohol, a thiol, an ether, a sulphide, an amine, a carboxylic acid, and a fluoroalkyl. The functional group may comprise a combination thereof.

[0175] The functional group may comprise a functional group selected from the group comprising a benzyl, a carboxylic acid or a trifluoro alkyl. Preferably, the functional group comprises a functional group selected from the group consisting of a benzyl, a carboxylic acid or a trifluoro alkyl.

[0176] The method may further comprise purifying the FMMWH. The method may further comprise step III, wherein step III comprises purifying the FMMWH. Purifying the FMMWH may comprise precipitation, dialysis, chromatography, solvent exchange, and / or lyophilization.

[0177] Precipitation may comprise the addition of a precipitation agent. For example, following reacting the medium molecular weight heparin with the functionalising agent to provide FMMWH the precipitation agent may be added. The precipitation agent may comprise a precipitation agent as described herein. The precipitation agent may be an alcohol. The alcohol may be a Ci to C3 alcohol. For example, the alcohol may be methanol,ethanol, propanol, or isopropanol. Preferably the alcohol is methanol. Preferably, the precipitation agent is methanol. The precipitation agent may be added to give a final concentration of 50% of the precipitation agent. For example, methanol may be added to perform a 50% methanol precipitation. The method may therefore comprise the steps of I. dissolving the MMWH in an aqueous solvent to provide a first solution, II adding the functionalising agent to the first solution to provide a second solution and incubating the second solution to produce a FMMWH solution, and III adding the precipitation agent to the FMMWH solution.

[0178] Following addition of the precipitation agent, the FMMWH may be isolated. The FMWWH may be isolated by filtration. Alternatively, following precipitation the FMWWH may be isolated by lyophilization. The lyophilization may be performed as described herein. For example, following addition of the precipitation agent, the mother liquor may be poured off and the remaining sample lyophilised to remove any remaining mother liquor. The method may therefore comprise the steps of I. dissolving the MMWH in an aqueous solvent to provide a first solution, II. adding the functionalising agent to the first solution to provide a second solution and incubating the second solution to produce a FMMWH solution, III. adding a precipitation agent to the FMMWH solution to precipitate the FMMWH, and IV. isolating the FMMWH. Isolating the FMMWH may comprise filtration and / or lyophilization.

[0179] Following reacting the MMWH with the functionalising agent, dialysis may be carried out to purify the FMMWH. For example, following reacting the MMWH with the functionalising agent in water, the reaction mixture may be placed in dialysis tubing and the dialysis tubing placed in a dialysate. The dialysate may be refreshed as often as necessary to achieve optimum separation. The dialysis may be carried out over a period of about 1 day to about 14 days, preferably about 5 days to about 10 days, preferably about 7 days. The dialysate may be refreshed about 1 time a day to about 10 times a day, preferably about 2 times a day to about 5 times a day, preferably about 3 times a day. Typically, the dialysate is many times the volume of the sample, for example from about 2 to about 500 times the volume of the sample. The dialysate may be about 4 times the volume of the sample.

[0180] Typically, the dialysate is water. Optionally, the dialysate may comprise electrolytes such as sodium, potassium, magnesium, calcium, chloride, bicarbonate, lactate, glucose, amino acids, or combinations thereof.

[0181] The dialysing may be carried out in 2 kD cut off tubing, for example provided by Spectra / Por®. The skilled person is aware of the appropriate tubing cut off size for different purposes. Alternatively, the dialysing step may be carried out in a dialysis device or dialyzer. Suitable dialyzers may be the Slide-A-Lyzer™, the Float-A-Lyzer, the Pur-A- lyzer, the D-Tube, and GeBAflex Dialyzers product lines.

[0182] The method may therefore comprise the steps of I. dissolving the MMWH in an aqueous solvent to provide a first solution, II. adding the functionalising agent to the first solution to provide a second solution and incubating the second solution to produce a FMMWH solution, and III. dialysing the FMMWH solution.

[0183] The method may further comprise the step of isolating the FMMWH following dialysis. The FMMWH may be isolated by freeze-drying, centrifuging, or filtration. Preferably, the FMMWH is isolated from the dialysed FMMWH sample by freeze-drying. The method may therefore comprise the steps of I. dissolving the MMWH in an aqueous solvent to provide a first solution, II. adding the functionalising agent to the first solution to provide a second solution and incubating the second solution to produce a FMMWH solution, III. dialysing the FMMWH solution, and IV. isolating the FMMWH. Isolating the FMMWH may comprise freeze-drying, centrifuging, and / or filtration.

[0184] Chromatography may be used to purify the FMMWH. Preferably, the chromatography is liquid chromatography. For example, the liquid chromatography may be size exclusion chromatography or ion exchange chromatography. The ion exchange chromatography may be anion exchange chromatography. The anion exchange chromatography may be weak anion exchange chromatography or strong anion exchange chromatography. The method may therefore comprise the steps of I. dissolving the MMWH in an aqueous solvent to provide a first solution, II. adding the functionalising agent to the first solution to provide a second solution and incubating the second solution to produce a FMMWH solution, III. purifying the FMMWH by chromatography.

[0185] The method may further comprise the step of isolating the FMMWH following chromatography. The FMMWH may be isolated by freeze-drying, centrifuging, or filtration. Preferably, the FMMWH is isolated by freeze-drying. The method may therefore comprise the steps of I. dissolving the MMWH in an aqueous solvent to provide a first solution, II. adding the functionalising agent to the first solution to provide a second solution and incubating the second solution to produce a FMMWH solution, III. purifying the FMMWH by chromatography, and IV. isolating the FMMWH. Isolating the FMMWH may comprise freeze-drying, centrifuging, and / or filtration.

[0186] Following reacting the MMWH and the functionalising agent, the FMMWH may be purified by solvent exchange. For example, following incubation of the MMWH and the functionalising agent in an aqueous solvent, the method may comprise centrifuging the aqueous solution in a centrifuge tube with a filter membrane with, for example, a 3 kDa cut off. The cut off may be of an appropriate size to ensure that the FMMWH does not pass through the filter membrane. The aqueous solution is then concentrated. The permeate may then be poured away and the concentrate may then be diluted with water and centrifuged in the centrifuge tube again. This may be repeated several times to exchange the solvent for water. Excess functionalising agent will pass through the filter membrane, thereby purifying the FMMWH. The method may therefore comprise the steps of I. dissolving the MMWH in an aqueous solvent to provide a first solution, II. adding the functionalising agent to the first solution to provide a second solution and incubating the second solution to produce a FMMWH solution, and III. purifying the FMMWH by solvent exchange.

[0187] Following solvent exchange, the FMMWH may be isolated. For example, the FMMWH may be isolated by lyophilization. The method may therefore comprise the steps of I. dissolving the MMWH in an aqueous solvent to provide a first solution, II. adding the functionalising agent to the first solution to provide a second solution and incubating the second solution to produce a FMMWH solution, III. purifying the FMMWH by solvent exchange, and IV. isolating the FMMWH.

[0188] The FMMWH may be isolated by direct lyophilization. For example, following reacting the MMWH and the functionalising agent in water, the reaction mixture may be directly lyophilized. This provides a rapid purification method. The method may therefore comprise I. dissolving the MMWH in an aqueous solvent to provide a first solution, II. adding the functionalising agent to the first solution to provide a second solution and incubating the second solution to produce a FMMWH solution, III. lyophilising the FMMWH solution.

[0189] In some embodiments, the invention provides a FMMWH comprising one or more of Structure I, wherein structure I comprises:wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2; wherein L is selected from the group consisting of -N(H)-, -N(R.2)- and -O-C(=O)-;R2 is selected from the group consisting of alkyl, fluoroalkyl, carboxylic acid, sulfonate, aryl, heteroaryl, heteroalkyl, and heterocycle; wherein at least one X is -CH2-L-R2.

[0190] The dotted bonds may represent a bond to another saccharide in the polysaccharide chain of the FMMWH. Alternatively, when structure I is at the end of the polysaccharide chain, one dotted bond may represent a bond to hydrogen or some other chain terminal moiety and the other dotted bond may represent a bond to another saccharide.

[0191] In some embodiments, one X is -C(=O)H. In some embodiments, both X are -CH2- L-R2. In some embodiments, L is -N(H)-. In some embodiments L is -N(R2)-. In some embodiments, L is -O-C(=O)-. In some embodiments, when both X are -CH2-L-R2, the R2 groups may be linked.

[0192] Preferably, both X are -CH2-L-R2. Preferably, both X are -CH2-L-R2 and L is -N(H)-. Preferably, both X are -CH2-I.-R2.and both L is -O-C(=O)-. Preferably, both X are -CH2-L- R2 and L is -N(R2)-.

[0193] In some embodiments, the alkyl comprises C1-C5 alkyl. In some embodiments, the C1-C5 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl or t-butyl. In some embodiments, the fluoroalkyl comprises C1-C5 fluoroalkyl. Preferably, the C1-C5 fluoroalkyl is fluoromethyl. Preferably, the C1-C5 fluoroalkyl is trifluoromethyl.

[0194] In some embodiments, R2 may be a carboxylic acid and may be -C(=O)-OH. In some embodiments, R2 may comprise a carboxylic acid and may be -C(R3)(H)-C(=O)-OH, wherein R3 may be any side chain of the 20 natural amino acids, for example the side chain of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0195] In some embodiments, R2 may be sulfonate, in this embodiment the sulfonate may be -S(=O)2-OH. In some embodiments, R2 may be aryl, in this embodiment the aryl may be phenyl or phenol. In some embodiments, R2 may be heteroaryl, in this embodiment, the heteroaryl may be imidazole, furan, oxazole, thiazole, pyridine, pyrimidine or thiophene.

[0196] In some embodiments, the C1-C5 alkyl is methyl and L is -N(H)- . In this embodiment, the FMMWH comprises one or more of structure II:wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2.

[0197] In this embodiment, when X is -C(=O)H the FMMWH comprises one or more of structure III:

[0198] Alternatively, in this embodiment both X are -CH2-L-R2 wherein L is -N(H)- and R2 is methyl. In this embodiment, the FMMWH comprises one or more of structure IV:

[0199] In some embodiments, R2 is fluoroalkyl, the fluoroalkyl comprises C1-C5 fluoroalkyl. In some embodiments, the C1-C5 fluoroalkyl is trifluoromethyl and L is -N(H)-. In this embodiment, the FMMWH comprises one or more of structure V:wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2.

[0200] In this embodiment, when X is -C(=O)H the FMMWH comprises one or more of structure VI:

[0201] Alternatively, in this embodiment both X are -CH2-L-R2 wherein L is -N(H)- and R2 is CF3. In this embodiment, the FMMWH comprises one or more of structure VII:

[0202] In some embodiments, R2 is -C(=O)-OH and L is -N(H)-. In this embodiment, the FMMWH comprises one or more of structure VIII:wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2.

[0203] In this embodiment, when X is -C(=O)H the FMMWH comprises one or more of structure IX:

[0204] Alternatively, in this embodiment both X are-CH2-L-R.2 and the FMMWH comprises one or more of structure X:

[0205] In some embodiments, R.2 is a sulfonate, the sulfonate is -S(=O)2-OH and L is CH2-N(H)-. In this embodiment, the FMMWH comprises one or more of structure XI:wherein X is selected from the group consisting of -C(=O)H and CH2-L-R2.

[0206] In this embodiment, when X is -C(=O)H the FMMWH comprises one or more of structure XII:

[0207] Alternatively, in this embodiment both X are -CH2-L-R2 and the FMMWH comprises one or more of structure XIV:

[0208] In some embodiments, R2 is an aryl, the aryl is phenyl and L is -N(H)-. In this embodiment, the FMMWH comprises one or more of structure XV:wherein X is selected from the group consisting of -C(=O)H and CH2-L-R2.

[0209] In this embodiment, when X is -C(=O)H the FMMWH comprises one or more of structure XVI

[0210] Alternatively, in this embodiment both X are -CH2-L-R2 and the FMMWH comprises one or more of structure XVII:

[0211] In some embodiments, R2 is an aryl, the aryl is phenol and L is -N(H)-. In this embodiment, the FMMWH comprises one or more of structure XVIII:wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2.

[0212] In this embodiment, when X is -C(=O)H the FMMWH comprises one or more of structure XIX:

[0213] Alternatively, in this embodiment both X are-CH2-L-R.2 and the FMMWH comprises one or more of structure XX:

[0214] It will be appreciated that whilst the hydroxyl group is shown in the para position in structures XVII-XX, the hydroxyl group may alternatively be in the meta or ortho position. The phenol group may also comprise more than one hydroxyl group. For example, 2, 3, 4 or 5 hydroxy groups. The hydroxyl group may also act as a tether for further functionalisation. For example, an ether bond may be formed to an alkyl chain, such as a methyl or ethyl alkyl chain. Alternatively, the hydroxyl group may be esterified to link to another group such as an alkyl group, for example a methyl or ethyl alkyl chain.

[0215] In some embodiments, R2 is a heteroaryl, the heteroaryl is imidazole and L is - N(H)-. In this embodiment, the FMMWH comprises one or more of structure XXI:wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2.

[0216] In this embodiment, when X is -C(=O)H the FMMWH comprises one or more of structure XXII:

[0217] Alternatively, in this embodiment both X are -CH2-L-R2 and the FMMWH comprises one or more of structure XXIII:

[0218] In some embodiments, R2 is a heteroalkyl, the heteroalkyl is a C1-C5 heteroalkyl. In some embodiments the heteroalkyl is a C4 alkyl, the heteroatom comprises nitrogen and L is -N(H)-. For example, the FMMWH may comprise one or more of structure XXIV:wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2.

[0219] In this example, when X is -C(=O)H the FMMWH comprises one or more of structureXXV:

[0220] In this example, the free amine may react with the aldehyde. In this instance, the FMMWH comprises one or more structure XXVI :

[0222] Alternatively, in this example both X are -CH2-L-R2 and the FMMWH comprises one or more of structure XXVII :

[0223] In some embodiments, R2 is fluoroalkyl, the fluoroalkyl is fluoromethyl and L is - N(H)-. In this embodiment, the FMMWH comprises one or more of structure XXVIII :wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2.

[0224] In this embodiment, when X is -C(=O)H the FMMWH comprises one or more of structure XXIX:

[0225] Alternatively, in this embodiment both X are -CH2-L-R2 wherein L is -N(H)- and R2 is CH2F. In this embodiment, the FMMWH comprises one or more of structure XXX:

[0226] Without wishing to be bound by theory, it is believed that functionalising MMWH with hydrophobic groups, such as methyl groups, may facilitate binding to hydrophobic regions in the VWF receptor or even prevent a particular region of the functionalised heparin from binding in this area. Furthermore, it is believed that functionalising groups containing one or more fluorine atoms have the ability to hydrogen bond. This may contribute to the activation of the VWF without activating ATIII.

[0227] Without wishing to be bound by theory, it is believed VWF activation may be due to a negative charge on the heparin molecule. Thus, functionalising groups having a negative charge (e.g. -N(H)-C(O)OH or -N(H)-CH2-S(O)2OH) associated with them atphysiological pH would enhance this activation. Furthermore, -N(H)-CH2-S(O)2OH mimics sulfonation of the heparin molecule.

[0228] The FMMWH does not comprise an aldehyde moiety or comprises fewer aldehyde moieties than MMWH produced during step (c) of the methods of the ninth and tenth aspects. The presence of an aldehyde or lack thereof may be determined using 2,4- Dinitrophenyl Hydrazine test. FMMWH may not produce an orange-yellow precipitate with reacted with 2,4-dinitrophenylhydrazine or less orange-yellow precipitate than a corresponding amount of MMWH. Alternatively, the conversion of MMWH to FMMWH may be monitored by infra-red spectroscopy or ^-NMR.

[0229] The FMMWH may have an average molecular weight in the range of greater than about 8000 Da (g / mol) to about 13500 Da (g / mol), preferably greater than about 8000 Da (g / mol) to about 13 000 Da (g / mol), preferably about 10 000 Da (g / mol) to about 12 000 Da (g / mol). The FMMWH may comprise polysaccharide chains with an average molecular weight in the range of greater than about 8000 Da (g / mol) to about 13500 Da (g / mol), preferably about 9000 Da (g / mol) to about 13 000 Da (g / mol), preferably about 10 000 Da (g / mol) to about 12 000 Da (g / mol).

[0230] The FMMWH may comprise polysaccharide chains with an average molecular weight in the range of greater than about 8000 Da (g / mol) to about 13500 Da (g / mol), preferably greater than about 8000 Da (g / mol) to about 13 000 Da (g / mol), preferably about 10 000 Da (g / mol) to about 12 000 Da (g / mol).

[0231] The average molecular weight of the FMMWH may be determined by size exclusion chromatography as described herein.

[0232] The FMMWH may comprise at least three units of a GlcNS6S-IdoA2S (or IdoA2S- GlcNS6S) disaccharide. The GlcNS6S and IdoA2S monosaccharides are linked by an al-4 linkage between GlcNS6S and IdoA2S, i.e. GlcNS6Sal-4IdoA2S. For example, the FMMWH may comprise at least four units, preferably at least five units, preferably at least six units, preferably at least eight units, preferably at least ten units of a GlcNS6S-IdoA2S disaccharide. The FMMWH may comprise less than or equal to 25 units of the GlcNS6S- IdoA2S disaccharide, for example less than or equal to 20 units. The presence of the units of the GlcNS6S-IdoA2S disaccharide may be determined by an antibody, mass spectrometry, or inferred from chemical and enzymatical studies. The GlcNS6S-IdoA2S units may be ordered in succession.

[0233] "IdoA" is a-L-iduronic acid. "IdoA2S" is IdoA modified by the addition of an 0- sulfate group at carbon position 2 to form 2-O-sulfo-a-L-iduronic acid. "GIcNS" is 2-deoxy- 2-sulfamido-a-D-glucopyranosyl. "GlcNS6S" is 2-deoxy-2-sulfamido-o-D-glucopyranosyl- 6-0-sulfate. An al-4 linkage is an alpha glycosidic bond between carbon-1 on one monosaccharide and carbon-4 on a second monosaccharide. A pl-4 linkage is beta glycosidic bond between carbon-1 on one monosaccharide and carbon-4 on a second monosaccharide.

[0234] The FMMWH may comprise UA2S-GlcNS6S, UA2S-GlcNS, UA-GIcNAc, wherein U may be iduronic acid (IdoA) or glucuronic acid (GlcA). The FMMWH may comprise at least about 60% UA2S-GlcNS6S, UA2S-GlcNS, and UA-GIcNAc. The FMMWH may comprise at least about 45%, preferably at least about 48%, preferably at least about 49%, preferably at least about 60% UA2S-GlcNS6S. The FMMWH may comprise up to about 60%, preferably up to about 70%, preferably up to about 85% UA2S-GlcNS6S. The FMMWH may comprise at least about 4%, preferably at least about 5%, preferably at least about 6%, preferably at least about 10% UA2S-GlcNS. The FMMWH may comprise up to about 15%, preferably up to about 20% UA2S-GlcNS. The FMMWH may comprise at least 4%, preferably at least 5%, preferably at least 6%, preferably at least about 10% UA-GIcNAc. The FMMWH may comprise up to about 15%, preferably up to about 20% UA-GIcNAc. In some embodiments the FMMWH may comprise at least 49.2% UA2S-GlcNS6S, 5.4% UA2S-GlcN and 5.4% UA-GIcNAc. In some embodiments the FMMWH may comprise at least 82% UA2S-GlcNS6S, 9% UA2S-GlcNS and 9% UA-GIcNAc. The percentage composition of UA-GIcNAc comprised in the FMMWH may be enriched compared to unfractionated heparin.

[0235] "UA" is a uronic acid, which is a hexose with a negatively charged carboxylate at the 6-position. The uronic acid may independently be glucuronic acid or iduronic acid. "UA2S" is UA modified by the addition of an O-sulfate group at carbon position 2 to form 2-O-sulfo-uronic acid. "GlcA" is 0-D-glucuronic acid "GIcNAc" is 2-deoxy-2-acetamido-a- D-glucopyranosyl.

[0236] The FMMWH may comprise a further chemical modification. The chemical modification comprises any chemical change to the medium molecular weight heparin. Accordingly, the chemical change may comprise N-acetylation, N-deacetylation, N- sulfation, O-sulfation, 2-0 desulfation, complete desulfation, or any combination of these.

[0237] FMMWH may be characterised by NMR, disaccharide analysis, ristocetin-induced platelet aggregation (RIPA), Factor Xa analysis.

[0238] Advantageously, FMMWH may display reduced Factor Xa activity and may display inhibition in ristocetin-induced platelet aggregation. Thus, FMMWH advantageously may be used in treating endotheliopathy.

[0239] In a fourth aspect, the invention provides a composition comprising FMMWH. Preferably, the composition is a pharmaceutical composition.

[0240] The pharmaceutical composition may comprise an excipient. The excipient may be selected from the group comprising solvents, co-solvents, buffers, stabilisers, antioxidants, preservatives, chelating agents, emulsifiers, flavourings, lubricants, suspending agents, tonicity adjusting agents, surfactants, solubilising agents, suspending aids, dispersion agents, humectants, thickeners, colouring agent, wetting agent, antifoaming agent, viscosity modifier, sweeteners and combinations thereof.

[0241] The pharmaceutical co may comprise an additional active agent, which may be an API. The additional active agent may comprise a composition of matter that has a physiological effect. The additional active agent may comprise low molecular weight heparin, or a MMWH or a FMMWH of a different disaccharide composition. The additional active agent may be selected from the group comprising farnesoid X receptor (FXR) agonist, a peroxisome proliferator-activator receptor (PPAR) agonist, aramchol, a caspase inhibitor, a galectin 3 inhibitor, a mitogen-activated protein kinase 5 (MAPK5) inhibitor, a fibroblast growth factor 19 (FGF19) agonist, a FGF21 agonist, a leukotriene D4 (LTD4) receptor antagonist, a niacin analog, an apical sodium bile acid cotransporter (ASBT) inhibitor, apoptosis signal regulating kinase 1 (ASK1) inhibitor, an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, a chemokine receptor inhibitor, a thiozolidinedione, a GLP-1 analog, a biguanide, an HIV replication inhibitor, metoformin, an opiate, an anaesthetic, HMG-CoA reductase inhibitor, a nonsteroidal anti-inflammatory drug (NSAID), or any combination of these, p

[0242] For the avoidance of doubt, embodiments related to first or third aspect of the invention apply mutatis mutandis to the fourth aspect of the invention.

[0243] The Inventors have now found that functionalised medium molecular weight heparin as described herein can be used to treat endotheliopathy, particularly endotheliopathy in a patient having a high plasma von Willebrand factor level.

[0244] Accordingly, in a sixth aspect, the invention provides functionalised medium molecular weight heparin (FMMWH) for use in treating endotheliopathy in a patient.Preferably, the patient has an endotheliopathy characterised by a plasma von Willebrand factor to ADAMTS13 (VWF: ADAMTS13) ratio of at least about 2. Alternatively or additionally, the patient may have an endotheliopathy characterised by a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0245] The FMMWH may inhibit von Willebrand factor (VWF). The FMMWH may inhibit multimers of VWF, preferably ultra-large VWF. The FMMWH may inhibit the binding of platelets to VWF.

[0246] The patient may have a VWF:ADAMTS13 ratio of at least about 2, preferably of at least about 4, more preferably of at least about 8, more preferably of at least about 10. The patient may have a VWF:ADAMTS13 ratio greater than about 2, preferably greater than about 4, more preferably greater than about 8, more preferably greater than about 10. The patient may have a VWF:ADAMTS13 ratio of about 2-16, preferably about 4-12, more preferably about 6-10. A patient having a VWF to ADAMTS13 ratio of greater than about 8 typically indicates severe illness and often is indicative of a patient deteriorating towards death.

[0247] The patient may have a VWF antigen :ADAMTS13 ratio of at least about 2, preferably of at least about 4, more preferably of at least about 8, more preferably of at least about 10. The patient may have a VWF antigen :ADAMTS13 ratio greater than about 2, preferably greater than about 4, more preferably greater than about 8, more preferably greater than about 10. The patient may have a VWF antigen:ADAMTS13 ratio of about 2- 16, preferably about 4-12, more preferably about 6-10. A patient having a VWF antigen to ADAMTS13 ratio of greater than about 8 typically indicates severe illness and often is indicative of a patient deteriorating towards death.

[0248] The level of VWF and ADAMTS13 in the patient may be measured using an ELISA. The ratio may be calculated as described by Huisman et al (Involvement of ADAMTS13 and von Willebrand factor in thromboembolic events in patients infected with SARS-CoV- 2. Int J Lab Hematol. 2020 Oct;42(5):e211-2). Briefly, the level of the VWF antigen may be determined in international units and the level of ADAMTS13 may be determined in international units and then the ratio of VWF antigen : ADAMTS13 determined.

[0249] Normal levels of plasma VWF are in the range of from about 50 IU per dL to about 200 IU per dL. The mean level of plasma VWF in the general population is about 100 IU per dL. High levels of plasma VWF are those of about 200 IU per dL or more, for examplefrom about 200 IU per dL to about 400 IU per d L, from about 225 IU per dL to about 375 IU per dL, from about 250 IU per dL to about 350 IU per dL, from about 275 IU per dL to about 300 IU per dL.

[0250] The patient may have a raised VWF antigen level of about 150% or more, preferably of about 175% or more, more preferably of about 200% or more, more preferably of about 300% or more, more preferably of about 350% or more, more preferably about 400% or more, more preferably of about 500% or more. The patient may have a VWF antigen level of up to about 600%, preferably up to about 700%, more preferably up to about 800%, more preferably up to about 1000%.

[0251] It is noted that levels of plasma VWF may be temporarily raised by infections, inflammation, trauma, and with physical and emotional stressors. Accordingly, the patient may have a non-temporarily raised plasma von Willebrand factor level, for example for at least about six hours, at least about 12 hours, at least about 18 hours or at least about 24 hours. Preferably, the patient may have a raised plasma von Willebrand factor level for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, or at least about seven days. Even more preferably the patient may have a raised plasma von Willebrand factor level for at least about one week, at least about two weeks, at least about three weeks or at least about four weeks. Yet even more preferably the patient may have a raised plasma von Willebrand factor level for at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months or at least about one year. The patient may have a raised plasma von Willebrand factor level for up to about one week, up to about four weeks, up to about two months, up to about four months, up to about six months, or up to about one year.

[0252] The endotheliopathy may be caused by any disease or condition. In particular, the endotheliopathy may be caused by COVID-19 (for example, acute COVID-19 or post- COVID-19 syndrome), infection, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, septicaemia, sepsis, cardiovascular disease, diabetes mellitus, trauma, in particular brain or head trauma e.g. traumatic brain injury, burns, inhalational injury, drugs and drug reactions, haematological conditions, subarachnoid haemorrhage (for example aneurysmal subarachnoid hemorrhage), aneurysmal diseases, stroke, brain parenchymal haemorrhage, radiation induced injury, ischemic stroke, pancreatitis, hepatological conditions, renal diseases, or chronic obstruction pulmonary disease (COPD), or combinations thereof. The endotheliopathy may be caused by a viral infection, optionally wherein the viral infection is SARS-CoV-2. The endotheliopathy maybe caused by cancer, in particular leukaemia, 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.

[0253] The infection may be bacterial, fungal, or parasitic. The infection may be bacterial. The bacterial infection may be Actinomyces israelii, Bacillus anthracis, Bacteroides fragilis, Bordetella pertussis, Borrelia burgdoferi, Borrelia garinii, Borrelia afzelaii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophilia psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira species, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Nocardia asteroides, Rickettsia rickettsii, Salmonella, Shigella, Spirochaetes Staphylococcus, Streptococcus, Treponema pallidum, Vibrio cholerae, or Yersinia pestis.

[0254] 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.

[0255] The infection may be parasitic. The parasitic infection may be protozoan eye infection, Chagas' disease, leishmaniasis, toxoplasmosis, giardiasis, malaria, microsporidiosis, or Rhinosporidiosis. Preferably, the parasitic infection is malaria.

[0256] The viral infection may be SARS-CoV-2. SARS-CoV-2 is the virus responsible for the disease COVID-19. COVID-19 can result in ARDS. The endotheliopathy may be caused by SARS-CoV-2 infection. The endotheliopathy may be caused by COVID-19. The endotheliopathy may be caused by ARDS.

[0257] The endotheliopathy may be caused by cancer. The cancer may be leukaemia, lymphoma or myeloma. Alternatively, oradditionally the cancer may be solid organ cancer, for example colon cancer, breast cancer, brain cancer, lung cancer, pancreatic cancer, testicular cancer, prostate cancer, cervical cancer, liver cancer, or skin cancer.

[0258] The endotheliopathy may be caused by haematological conditions, for example Thrombotic thrombocytopenic purpura, anaemia, or sickle cell disease.

[0259] The endotheliopathy may be caused by hepatological conditions, for example cirrhosis, hepatitis, nonalcoholic fatty liver disease (NAFL), nonalcoholic steatohepatitis (NASH), primary biliary cholangitis, and primary sclerosing cholangitis.

[0260] The endotheliopathy may be caused by sepsis.

[0261] In a seventh aspect, the invention provides functionalised medium molecular weight heparin for use in the treatment of a disease or condition in a patient, wherein the patient has an endotheliopathy characterised by a plasma von Willebrand factor to ADAMTS13 (VWF:ADAMTS13) ratio of at least about 2.

[0262] In an eighth aspect, the invention provides functionalised medium molecular weight heparin for use in the treatment of a disease or condition in a patient, wherein the patient has an endotheliopathy characterised by a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0263] Preferably, the disease or condition is selected from the group consisting of: COVID-19 (for example, acute COVID-19 or post-COVID-19 syndrome), infection, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, septicaemia, sepsis, cardiovascular disease, diabetes mellitus, trauma, in particular brain or head trauma e.g. traumatic brain injury, burns, inhalational injury, drugs and drug reactions, haematological conditions, subarachnoid haemorrhage (for example aneurysmal subarachnoid hemorrhage), aneurysmal diseases, stroke, brain parenchymal haemorrhage, radiation induced injury, ischemic stroke, pancreatitis, hepatological conditions, renal diseases, chronic obstruction pulmonary disease or combinations thereof. The endotheliopathy may be caused by a viral infection, optionally wherein the viral infection is SARS-CoV-2. The endotheliopathy may be caused by cancer, in particular leukaemia, 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. Preferably, the endotheliopathy is caused by sepsis or septicaemia. Preferably, the endotheliopathy is caused by sepsis.

[0264] In some embodiments, the present invention provides MMWH-Red for use in the treatment of COVID-19 (for example, acute COVID-19 or post-COVID-19 syndrome) in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0265] In some embodiments, the present invention provides MMWH-Red for use in the treatment of infection in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2. The infection may be SARS-CoV-2.

[0266] In some embodiments, the present invention provides MMWH-Red for use in the treatment of viral infection in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2. The viral infection may be SARS-CoV-2.

[0267] In some embodiments, the present invention provides MMWH-Red for use in the treatment of acute respiratory distress syndrome (ARDS) in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0268] In some embodiments, the present invention provides MMWH-Red for use in the treatment of cancer in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2. The cancer may be leukaemia, 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.

[0269] In some embodiments, the present invention provides MMWH-Red for use in the treatment of bacterial infection in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0270] In some embodiments, the present invention provides MMWH-Red for use in the treatment of septicaemia in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0271] In some embodiments, the present invention provides MMWH-Red for use in the treatment of sepsis in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0272] In some embodiments, the present invention provides MMWH-Red for use in the treatment of cardiovascular disease in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0273] In some embodiments, the present invention provides MMWH-Red for use in the treatment of diabetes mellitus in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0274] In some embodiments, the present invention provides MMWH-Red for use in the treatment of trauma in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2. Preferably, the trauma is brain trauma or head trauma, e.g. traumatic brain injury.

[0275] In some embodiments, the present invention provides MMWH-Red for use in the treatment of burns in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0276] In some embodiments, the present invention provides MMWH-Red for use in the treatment of inhalational injury in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0277] In some embodiments, the present invention provides MMWH-Red for use in the treatment of drugs and drug reactions in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or aplasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0278] In some embodiments, the present invention provides MMWH-Red for use in the treatment of haematological conditions in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0279] In some embodiments, the present invention provides MMWH-Red for use in the treatment of subarachnoid haemorrhage (for example aneurysmal subarachnoid hemorrhage) in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF: ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0280] In some embodiments, the present invention provides MMWH-Red for use in the treatment of aneurysmal diseases in a patient, wherein the patient has an endotheliopathy characterised by a plasma VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0281] In some embodiments, the present invention provides MMWH-Red for use in the treatment of stroke in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0282] In some embodiments, the present invention provides MMWH-Red for use in the treatment of brain parenchymal haemorrhage in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0283] In some embodiments, the present invention provides MMWH-Red for use in the treatment of radiation induced injury in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0284] In some embodiments, the present invention provides MMWH-Red for use in the treatment of ischemic stroke in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0285] In some embodiments, the present invention provides MMWH-Red for use in the treatment of pancreatitis in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0286] In some embodiments, the present invention provides MMWH-Red for use in the treatment of hepatological conditions in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0287] In some embodiments, the present invention provides MMWH-Red for use in the treatment of renal diseases in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0288] In some embodiments, the present invention provides MMWH-Red for use in the treatment of chronic obstruction pulmonary disease in a patient, wherein the patient has an endotheliopathy characterised by a VWF:ADAMTS13 ratio of at least about 2 or a plasma von Willebrand factor antigen to ADAMTS13 (VWF antigen :ADAMTS13) ratio of at least about 2.

[0289] FMMWH for use in the treatment of endotheliopathy as defined herein are particularly advantageous as said heparins can inhibit the microthrombosis triggered by the release of VWF secondary to the endotheliopathy caused by any disease or condition. When the cause of the endotheliopathy is SARS-CoV-2, said FMMWH can additionally inhibit viral adherence and replication.

[0290] Dysfunctional endothelial cells may allow for the passage of tumour cells circulating in the blood to pass into the tissues. Thus, treating the endotheliopathy may prevent the haematogenous spread of blood borne cancers. The treatment of the endotheliopathy may inhibit the haematogenous spread of cancer. The medium molecular weight heparin may inhibit the haematogenous spread of cancer.

[0291] Biomarkers of endotheliopathy may include raised von Willebrand factor (VWF) levels, ultra-large von Willebrand factor (ULVWF) levels, Factor VIII levels as well as other markers such as Syndecan 1, VWF antigen, VWF activity, VWF multimers, ADAMTS13 levels, platelet counts, VCAM-1, ICAM-1, P-selectin levels, VWF:ADAMTS13 ratio or VWF antigen :ADAMTS13 ratio. Preferably, a biomarker of endotheliopathy is the ratio of VWF:ADAMTS13 or VWF antigen : ADAMTS13.

[0292] The patient may have raised plasma von Willebrand factor (VWF) levels compared to a healthy control subject. The patient may have sustained high levels of plasma VWF compared to a healthy control. The levels of plasma VWF may be raised compared to a healthy control subject over a period of at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, or preferably at least about one week. The levels of plasma VWF may be raised compared to a healthy control subject for a period of up to about one week, up to about four weeks, up to about two months, up to about four months, up to about six months, or up to about one year.

[0293] For example, the level of plasma VWF may be raised to at least about 50 nmol / L, preferably at least about 60 nmol / L, even more preferably at least about 70 nmol / L or yet even more preferably at least about 90 nmol / L. The level of plasma VWF may be raised to about 130 nmol / L, to about 150 nmol / L, or to about 200 nmol / L. The level of plasma VWF may be raised to at least about 50 nmol / L for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about one week, at least about one month or at least about one year. The level of plasma VWF may be raised to at least about 60 nmol / L for at least about one day, at least about two days, at least about three, days, at least about four days, at least about five days, at least about six days, at least about one week, at least about one month or at least about one year. The level of plasma VWF may be raised to at least about 70 nmol / L for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about one week, at least about one month or at least about one year. The level of plasma VWF may be raised to at least about 90 nmol / L for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about one week, at least about one month or at least about one year. The plasma VWF level may be measured using an Enzyme-Linked Immunosorbent Assay (ELISA).

[0294] Alternatively or additionally, the patient may have a plasma von Willebrand factor level of about 200 IU pr dl_ or more for at least about six hours, at least about 12 hours, at least about 18 hours or at least about 24 hours. Preferably, the patient may have a plasma von Willebrand factor level of about 200 IU pr dl_ or more for at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, or at least about seven days. Even more preferably the patient may have a plasma von Willebrand factor level of about 200 IU pr dL or more for at least about one week, at least about two weeks, at least about three weeks or at least about four weeks. Yet even more preferably the patient may have a plasma von Willebrand factor level of about 200 IU pr dL or more for at least about one month, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months or at least about one year.

[0295] Vascular endothelial function can be assessed in the coronary and peripheral circulations. Non-invasive tests for the assessment of coronary endothelial function include Doppler echocardiography where blood flow is measured in response to pharmacological or physiological stimuli. Other tests include positron emission tomography and phasecontrast magnetic resonance imaging. However, the gold standard test involves invasive quantitative coronary angiography to examine changes in diameter in response to intracoronary infusions of endothelium-dependent vasodilators such as acetylcholine. Assessment of the endothelium in the peripheral circulation includes brachial artery ultrasound and strain-gauge venous impedance plethysmography.

[0296] Binding of FMMWH to VWF may be assessed by a competitive binding assay. Heparin-Sepharose beads may be incubated with labelled VWF, for example125I-vWF, for a period of time to allow the labelled VWF to bind to the immobilized heparin. Varying concentrations of the FMMWH may then be added and the amount of displaced labelled VWF determined. Other methods to determine FMMWH binding to VWF may include surface plasmon resonance, biolayer interferometry, isothermal titration calorimetry, fluorescence polarisation binding assays, ELISA and microscale thermophoresis.

[0297] The inhibition of platelets binding to VWF may be assessed by ristocetin-induced agglutination of fixed platelets. Platelets may be incubated with the FMMWH and citrate treated plasma (a VWF source). Ristocetin may then be added, and platelet agglutination then determined. The FMMWH may fully inhibit VWF-induced platelet aggregation at a concentration of 15 pM when measured by a ristocetin-induced platelet aggregation assay. Other methods to determine the inhibition of VWF binding to platelets may include ELISA, fluorescence assisted cell sorting, dynamic light scattering, or flow chamber assays.

[0298] The treatment of endotheliopathy by FMMWH may inhibit the haematogenous spread of cancer. Human tumour cells can bind to VWF under shear flow conditions with both melanoma and colon cancer cells demonstrating this ability. The immobilized platelets, bound to the VWF, have been shown to mediate tethering, rolling, and the firm adhesion of different cancerous cell lines under flow shear stress. The VWF played a critical role in enabling this firm adhesion of the tumour cells to the immobilized platelets. The existing data suggests that VWF plays an important role in tethering cancerous cells. In addition, the VWF-Platelet binding that occurs as part of the normal thrombosis pathways may further act to allow the coalescence of tumour cells into the VWF-Platelet to form heteroaggregates of VWF+platelets+cancer cells which thereby help in the blood borne (haematogenous) spread of tumour cells. This process may at least in part be caused by the ability of cancer cells to translocate to the vessel wall and thereby spread to other organs once the initial binding to VWF and Platelets has occurred. In addition, various cancers are known to cause an endotheliopathy with the resultant release of UL-VWF. By this mechanism, the tumour triggers the release of UL-VWF that then allows the tethering of platelets and tumour cells and the haematogenous spread of the cancer and the metastatic spread. This cancer induced endotheliopathy also results in an overall increase in the risk of thrombosis in patients with underlying malignancy. Therefore, any treatment aimed at treating an endotheliopathy and inhibiting the binding of platelets and / or tumour cells to VWF would serve a dual purpose of decreasing the risk of malignancy associated thrombosis and also reduce the risk of haematogenous metastatic spread. The FMMWH may be administered by an administration method selected from parenteral, subcutaneous, intravenous, intramuscular, intrathecal, intradermal, intraarterial, or intraarticular, cutaneous, transcutaneous, subcutaneous, depot form, for example depot injection, intra-osseus, or inhalation. Preferred methods of administration comprise subcutaneous, intravenous, intramuscular or inhalation. Administration by inhalation may optionally be via a nebuliser.

[0299] Previous studies have looked at UFH as a nebulised agent in a variety of conditions. Small human studies indicate that nebulised UFH limits pulmonary fibrin deposition, attenuates progression of acute lung injury and hastens recovery (69). Early-phase trials in patients with acute lung injury and related conditions found that nebulised UFH reduced pulmonary dead space, coagulation activation, microvascular thrombosis, improved lung injury and increased time free of ventilatory support (70-73). In a pre-pandemic doubleblind randomised study in 256 critically ill ventilated patients, nebulised UFH limited progression of lung injury including acute respiratory distress syndrome and acceleratedreturn to home in survivors. Thus, the FMMWH may be administered by inhalation via a nebuliser.

[0300] Heparin dosage is typically measured in "Howell Units". One unit of heparin (the "Howell unit") is an amount approximately equivalent to 0.002 mg of pure heparin, which is the quantity required to keep 1 ml of cat's blood fluid for 24 hours at 0 °C. The FMMWH may be administered at a bolus does of about 5000 units, followed by about 1200 to about 1600 units per hour optionally delivered by an infusion pump. The FMMWH may be administered at a dose of about 18 units / kg to about 5000 units / kg. Preferably, the FMMWH may be administered at a dose of about 100 units / kg to about 800 units / kg. Alternatively, the FMMWH may be administered at a dose of about 18 units / kg to about 75 units / kg. The FMMWH may be administered at a dose of about 5000 units, about 4000 units, about 3000 units, about 2000 units, about 1000 units or about 500 units every 12 hours. The FMMWH may be administered at a dose of about 5000 units every 12 hours.

[0301] The FMMWH may be administered at a dose of about 3 units to about 5000 units, for example from about 6 units to about 4000 units, from about 12 units to about 3000 units, from about 25 units to about 2000 units, from about 50 units to about 1000 units, from about 100 units to about 500 units, or from about 125 units to about 250 units. The FMMWH may be administered at a dose of about 18 unit / kg to about 5000 units / kg, for example from about 100 units / kg to about 4000 units per / kg, or from about 200 units / kg to about 800 units / kg. The FMMWH 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 of time may be from about 1 month to about 12 months, for example from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months to about 9 months, from about 5 months to about 8 months, from about 6 months to about 7 months. The period of time may be about 1 day to 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 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 endotheliopathy and raised VWF levels.

[0302] The FMMWH may be administered at a dose of about 3 units to about 5000 units, for example from about 6 units to about 4000 units, from about 12 units to about 3000 units, from about 25 units to about 2000 units, from about 50 units to about 1000 units, from about 100 units to about 500 units, or from about 125 units to about 250 units. The FMMWH may be administered at a dose of about 18 unit / kg to about 5000 units / kg, forexample from about 100 units / kg to about 4000 units per / kg, or from about 200 units / kg to about 800 units / kg. The FMMWH 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 of time may be from about 1 month to about 12 months, for example from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months to about 9 months, from about 5 months to about 8 months, from about 6 months to about 7 months. The period of time may be about 1 day to 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 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 endotheliopathy and raised VWF levels.

[0303] The FMMWH 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 from about 1 month to about 12 months, for example from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months to about 9 months, from about 5 months to about 8 months, from 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 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 endotheliopathy and raised VWF levels.

[0304] The FMMWH may be administered at a dose of from about 0.01 mg / kg, from about 0.1 mg / kg, from about 1 mg / kg, from about 5 mg / kg, from about 10 mg / kg, from about 20 mg / kg, from about 30 mg / kg, from about 50 mg / kg, from about 70 mg / kg, from about 80 mg / kg, or from about 100 mg / kg. The FMMWH 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 FMMWH may be administered at a dose of from about 0.01 mg / kg to about 10 mg / kg, preferably from about 0.2 mg / kg to about 10 mg / kg, from about 0.2 mg / kg to about 1.6 mg / kg. The FMMWH may be administered as a single dose or as a continuous dose. The FMMWH dosage amount may be dependent on the VWF antigen : ADAMTS13 ratio or the overall VWF levels. The skilled person would be able select a suitable amount for a patient based on the VWF antigen : ADAMTS13 ratio or the overall VWF levels.

[0305] The FMMWH may be administered at a dose of from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 8 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.5 mg / kg to about 2 mg / kg, from 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 from about 1 month to about 12 months, for example from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months to about 9 months, from about 5 months to about 8 months, from about 6 months to about 7 months. The period of time may be from about 1 day to about 7 days, from about 2 days to about 6 days, from about 3 days to about 5 days, from about 4 days to about 5 days. The dose may be administered over 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 endotheliopathy and raised VWF levels.

[0306] The FMMWH may be administered at a dose of from about 0.1 mg to about 5000 mg, from about 0.5 mg to about 2000 mg, from about 1 mg to about 1000 mg, from about 5 mg to about 900 mg, from about 10 mg to about 800 mg, from about 20 mg to about 700 mg, from about 30 mg to about 600 mg, from about 50 mg to about 500 mg, from about 75 mg to about 400 mg, from about 100 mg to about 300 mg, from about 125 mg to about 250 mg, from about 150 mg to about 200 mg. 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 from about 1 month to about 12 months, for example from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months to about 9 months, from about 5 months to about 8 months, from about 6 months to about 7 months. The period of time may be from about 1 day to about 7 days, from about 2 days to about 6 days, from about 3 days to about 5 days, from about 4 days to about 5 days. The dose may be administered over 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 endotheliopathy and raised VWF levels.

[0307] The FMMWH may be administered at a dose of, for example about 1 international units (IU), about 2 IU, about 5 IU, about 10 ILJ, about 15 ILJ, about 20 IU, about 25 ILJ, 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 10 000 IU, about 20 000 IU, or about 25 000 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 of time may be from about 1 month to about 12 months, for example from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months toabout 9 months, from about 5 months to about 8 months, from about 6 months to about 7 months. The period of time may be from about 1 day to about 7 days, from about 2 days to about 6 days, from about 3 days to about 5 days, from about 4 days to about 5 days. The dose may be administered over 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 endotheliopathy and raised VWF levels.

[0308] The FMMWH may be administered at a dose of from about 1 IU to about 50 000 IU, from about 2 IU to about 25 000 IU, from about 5 IU to about 20 000 IU, from about 10 IU to about 10 000 IU, from about 15 IU to about 5000 IU, from about 20 IU to about 2500 IU, from about 25 IU to about 2000 IU, from about 50 IU to about 1500 IU, from about 75 IU to about 1000 IU, from about 100 IU to about 500 IU, from about 200 IU to about 400 IU, from 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 of time may be from about 1 month to about 12 months, for example from about 2 months to about 11 months, from about 3 months to about 10 months, from about 4 months to about 9 months, from about 5 months to about 8 months, from about 6 months to about 7 months. The period of time may be from about 1 day to about 7 days, from about 2 days to about 6 days, from about 3 days to about 5 days, from about 4 days to about 5 days. The dose may be administered over 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 endotheliopathy and raised VWF levels.

[0309] The FMMWH may be administered that is commensurate with the VWF antigen : ADAMTS13 ratio. For example, a patient with a high VWF antigen : ADAMTS13 ratio may be administered a higher dose of FMMWH compared to a patient with a VWF antigen : ADAMTS13 ratio that is lower.

[0310] The FMMWH may be comprised 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, co-solvents, buffers, stabilisers, antioxidants, preservatives, chelating agents, emulsifiers, flavourings, lubricants, suspending agents, tonicity adjusting agents, surfactants, solubilising agents, suspending aids, dispersion agents, humectants, thickeners, colouring agent, wetting agent, anti-foaming agent, viscosity modifier, sweeteners and combinations thereof. The pharmaceutical formulation may comprise glucose. The pharmaceutical formulation maycomprise sodium chloride. The pharmaceutical formulation may comprise phosphate buffered saline.

[0311] The pharmaceutical formulation may comprise an additional active agent. The additional active agent comprises a composition of matter that has a physiological effect. The additional active agent may comprise low molecular weight heparin, a medium molecular weight heparin, or a FMMWH of a different disaccharide composition. The additional active agent may be selected from the group comprising farnesoid X receptor (FXR) agonist, a peroxisome proliferator-activator receptor (PPAR) agonist, aramchol, a caspase inhibitor, a galectin 3 inhibitor, a mitogen-activated protein kinase 5 (MAPK5) inhibitor, a fibroblast growth factor 19 (FGF19) agonist, a FGF21 agonist, a leukotriene D4 (LTD4) receptor antagonist, a niacin analog, an apical sodium bile acid cotransporter (ASBT) inhibitor, an apoptosis signal regulating kinase 1 (ASK1) inhibitor, an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker, a chemokine receptor inhibitor, a thiozolidinedione, a GLP-1 analog, a biguanide, an HIV replication inhibitor, metoformin, an opiate, an anaesthetic, HMG-CoA reductase inhibitor, a nonsteroidal antiinflammatory drug (NSAID), or any combination of these.

[0312] For the avoidance of doubt, embodiments related to the sixth aspect of the invention apply mutatis mutandis to the seventh and eighth aspects of the invention. Likewise, embodiments related to the second and eighth aspects of the invention apply mutatis mutandis to the sixth aspect of the invention

[0313] The FMWWH may be chemically synthesised. The FMMWH may be enzymatically synthesised. High pressure liquid chromatography may be used to purify the FMMWH. The FMMWH of the first or third aspect may be synthesised via the method of the ninth aspect or the method of the tenth aspect.

[0314] In an ninth aspect, the invention provides a method of synthesis of functionalised medium molecular weight heparin, the method comprising the steps of: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; and (d) reacting the medium molecular weight heparin in the medium molecular weight heparin solution with a functionalising agent to provide functionalised medium molecular weight heparin solution.

[0315] UF heparin may be obtained from bovine or porcine tissue, for example porcine intestine or bovine lung.

[0316] The aqueous solvent is typically water. The aqueous solvent may consist of or consist essentially of water. Step (a) may comprise dissolving the unfractionated heparin (UF) in water. The first solution may consist of or consist essentially of unfractionated heparin and water. The second solution may consist of or consist essentially of unfractionated heparin, water and the oxidising agent. Preferably, the reaction is carried out in water. Preferably, the aqueous solvent consists of water.

[0317] The aqueous solvent may be an aqueous solution. The aqueous solution may be an aqueous buffer solution. When the aqueous solution is an aqueous buffer solution, in step (a) unfractionated heparin is dissolved in an aqueous buffer solution adjusted to between about pH 4.0 and pH 9.0 to provide a first solution.

[0318] "Buffer" refers to a chemical which, in a solution, resists a change in pH when acid or alkali is added to the solution. Typically, a buffer solution (or buffer system) comprises a weak acid and its conjugate base, or a weak base and its conjugate acid.

[0319] Typically, a suitable buffer comprises an acid with a pKa value that lies within ±1 of the desired pH of the formulation. For example, if the desired pH of the formulation is about 7.0, a suitable buffer comprises a weak acid with a pKa value of from about 6.0 to about 8.0. If the acid of a buffer has more than one pKa value (i.e. each molecule of the acid is able to donate more than one proton), in order for the buffer to be suitable, at least one of the pKa values should lie within the desired pH range.

[0320] The weak acid and conjugate base (or weak base and conjugate acid) of the buffer are in equilibrium with one another. In accordance with Le Chatelier's principle (if a constraint, such as a change in concentration of a reactant, is applied to a system in equilibrium, the equilibrium will shift so as to counteract the effect of the constraint), addition of acid or base to the solution shifts the position of equilibrium in favour of the conjugate base or weak acid, respectively. Consequently, the concentration of free protons in the formulation (and thus the pH) is relatively unchanged.

[0321] Suitable buffer systems comprise an acetate salt and acetic acid (pKa = 4.75), a citrate salt and citric acid (pKa = 3.13, 4.76 and 6.40), and phosphoric acid (pKa = 2.14, 7.20 and 12.37), or mixtures thereof. Phosphate buffered saline may also be used. The pKa values cited herein are those reported at 25 °C in water. Typically, the buffer comprises only one of the pairs listed above, i.e. one acid and its conjugate base. Thebuffer may comprise an acetate salt and acetic acid, a citrate salt and citric acid, or a phosphate salt and phosphoric acid.

[0322] The pH may be adjusted to ensure that the pH of the first solution is between from about pH 4.0 to about pH 9.0 because dissolving unfractionated heparin may result in the pH of the aqueous buffer solution changing. However, it has been surprisingly found that the reaction can be carried out in water and a buffer is not needed.

[0323] Optionally, in step (a), the aqueous buffer solution is adjusted to between about pH 5.0 and about pH 8.0.

[0324] Typically, the temperature of the aqueous solvent in step (a) is from about 15 °C to about 30 °C. Preferably, the temperature of the aqueous solvent in step (a) is from about 16 °C to about 25 °C, preferably from about 17 °C to about 22 °C, preferably from about 18 °C to about 21 °C, preferably about 19 °C to about 20 °C. Preferably, the temperature of the aqueous solvent in step (a) is room temperature. By performing the reaction at or around room temperature, costs to produce the product are reduced due to shorter reaction times.

[0325] Typically, the aqueous buffer solution is a phosphate buffer, a citrate buffer or an acetate buffer, i.e. the buffering system is phosphate, citrate or acetate. Preferably, the aqueous buffer solution is a phosphate buffer, more preferably the aqueous buffer solution is a sodium phosphate buffer or a potassium phosphate buffer, i.e. the buffering system is sodium phosphate or potassium phosphate.

[0326] The buffering system in the aqueous buffer solution may be present at a concentration of from about 10 mM to about 100 mM, more preferably from about 20 mM to about 90 mM, more preferably from about 30 mM to about 80 mM, more preferably from about 40 mM to about 70 mM, more preferably from about 50 mM to about 60 mM. The buffering system in the aqueous buffer solution may be present at a concentration of about 50 mM.

[0327] The concentration of UF heparin in the aqueous solution may be from about 0.5 mg / mL to about 100 mg / mL, more preferably from about 1 mg / mL to about 85 mg / mL, more preferably from about 1.5 mg / mL to about 80 mg / mL, more preferably from about 2 mg / mL to about 75 mg / mL, more preferably from about 2 mg / mL to about 70 mg / mL, more preferably from about 10 mg / L to about 65 mg / mL, more preferably from about 25 mg / mL to about 65 mg / mL, most preferably from about 30 mg / mL to about 60 mg / mL.

[0328] The concentration of UF heparin in the aqueous solution may be about 1.5 mg / mL, more preferably about 1.8 mg / mL, more preferably about 2 mg / mL, more preferably about 2.5 mg / mL, more preferably about 2.7 mg / mL, more preferably about 3 mg / mL, more preferably 8 mg / mL, more preferably about 16 mg / ml, more preferably about 30 mg / mL and most preferably about 60 mg / mL. The concentration of UF heparin in the aqueous solution is most preferably about 60 mg / mL.

[0329] The oxidising agent may be a periodate, for example sodium periodate or potassium periodate. Preferably, the oxidising agent is sodium periodate. Alternatively, or additionally, the oxidising agent may be a perchlorate, for example sodium perchlorate. Preferably, the oxidising agent does not comprise a perchlorate. Use of a perchlorate in combination with a periodate as the oxidising agent results in an increased level of sample breakdown to smaller molecular weight species.

[0330] The concentration of the oxidising agent in the aqueous solution may be from about 1 g / L to about 40 g / L. Preferably, the concentration of the oxidising agent may be from about 2g / L to about 35 g / L, more preferably from about 4 g / L to about 30 g / L, more preferably from about 20 g / L to about 25 g / L. The concentration of the oxidising agent in the aqueous solution may be about 23 g / L or greater. Preferably the concentration of oxidising agent is about 23 g / L. Preferably the concentration of the oxidising agent is about 23 g / L and the oxidising agent is sodium periodate.

[0331] The molar ratio of UF heparin to oxidising agent may be from about 1 : 1 to about 1:200, more preferably from about 1 :2 to about 1: 150, more preferably from about 1: 10 to about 1: 100, more preferably from about 1:20 to about 1 : 50, more preferably from about 1 :20 to about 1 :30, most preferably from about 1:25-1:30. Typically, the molar ratio of UF heparin to oxidising agent may be about 1 :27.

[0332] The molar ratio of UF heparin to oxidising agent may be determined based on the estimated molecular weight of the canonical heparin disaccharide as opposed to the molecular weight of heparin. The estimated molecular weight of the canonical heparin disaccharide is 593.45 g / mol. This is used to approximate the number of moles of this disaccharide and therefore the uronic acid residues present in the sample.

[0333] For example, if it is desired to add 1 equivalent of NaICk based on uronic acid and 3 grams of heparin is used, the number of moles NaICk to add is 0.005 moles i.e. 3 divided by 593.45.

[0334] Thus, the ratio of UF heparin determined based on the estimated molecular weight of the canonical heparin disaccharide may be from about 1 :0.5 to about 1 : 10, preferably from about 1 :0.7 to about 1 :8, more preferably from about 1:0.9 to about 1 : 5, more preferably from about 1 : 1 to about 1:2, more preferably from about 1: 1.1 to about 1 :2. Preferably, the ratio of moles of uronic acid present in the UF heparin to moles of oxidising agent may be 1 : 1.1.

[0335] Typically, the incubation temperature in step (c) is carried out at room temperature. The incubation in step (c) may be carried out at from about 15 °C to about 30 °C, more preferably from about 16 °C to about 28 °C, more preferably from about 17 °C to about 27 °C, more preferably from about 18 °C to about 25 °C. Preferably, the incubation temperature in step (c) is about 18 °C to about 20 °C. Typically, the incubating step (c) is carried out for from about 0.5 hour to about 40 hours, more preferably from about 1 hours to about 24 hours, more preferably from about 2 hours to about 20 hours, more preferably from about 3 hours to about 18 hours. Preferably, the incubating step may be carried out for about 3 hours.

[0336] Incubation step (c) of the ninth aspect of the invention may be carried out on a laboratory bench at ambient room temperature.

[0337] In step (d), the functionalising agent may comprise (i) an oxyamine as described herein, (ii) an amine, preferably a primary or secondary amine, or (iii) a reducing agent and a carboxylic acid or an activated carboxylic acid. When the functionalising agent is an amine, step (d) is a reductive amination. When the functionalising agent is a reducing agent and carboxylic acid or activated carboxylic acid, step (d) is a reduction followed by esterification. Most preferably the functionalising agent is an oxyamine.

[0338] When step (d) is a reductive amination, the functionalising agent may further comprise an acid and a reducing agent.

[0339] When step (d) is a reductive amination, step (d) may comprise the following steps:(i) adding an amine to the MMWH solution, optionally in the presence of an acid or a metal catalyst, to form an MMWH imine solution;(ii) adding a reducing agent to the MMWH imine solution to form a functionalised medium molecular weight heparin.

[0340] Preferably, the solvent for the reductive amination reaction is methanol, ethanol, formic acid, dichloromethane, tetrahydrofuran, dichloroethane, trifluoroethanol. Preferably the reducing agent is sodium triacetoxyborohydride, sodium cyanoborohydride or sodium borohydride. Preferably, the reducing agent is sodium triacetoxyborohydride or sodium cyanoborohydride. The reducing agent may be used at greater than about 0.5 molar equivalents, or greater than about 1 molar equivalent, or greater than about 2 molar equivalents, or greater than about 3 molar equivalents, or greater than about 5 molar equivalents relative to medium molecular weight heparin. Typically the reducing agent is used at less than about 15 molar equivalents, or less than about 12 molar equivalents, or less than about 10 molar equivalents, or less than about 8 molar equivalents, or less than about 6 molar equivalents, or less than about 5 molar equivalents relative to medium molecular weight heparin. The reducing agent may be used in amount of between about 0.5 molar equivalents and about 15 molar equivalents, or between about 1 molar equivalents and about 10 molar equivalents, or between about 2 molar equivalents and about 6 molar equivalents.

[0341] The amine may be R2NH2 or (R.2)2NH. R2 is as defined herein. Typically, R2 is selected from the group consisting of alkyl, fluoroalkyl, -C(=O)-OH, sulfonate, aryl, heteroaryl, heteroalkyl, and heterocycle. For example, the amine may be methylamine, fluoromethylamine, or carbamic acid. The amine may be used at greater than about 0.5 molar equivalents, or greater than about 1 molar equivalent, or greater than about 2 molar equivalents, or greater than about 3 molar equivalents, or greater than about 5 molar equivalents relative to medium molecular weight heparin. Typically the amine is used at less than about 15 molar equivalents, or less than about 12 molar equivalents, or less than about 10 molar equivalents, or less than about 8 molar equivalents, or less than about 6 molar equivalents, or less than about 5 molar equivalents relative to medium molecular weight heparin. The amine may be used in amount of between about 0.5 molar equivalents and about 15 molar equivalents, or between about 1 molar equivalents and about 10 molar equivalents, or between about 2 molar equivalents and about 6 molar equivalents.

[0342] Preferably, the reductive amination is carried at a temperature of from about 15 °C to about 30 °C, preferably from 20 °C to about 25 °C. Preferably, the reaction is carried out for from about 30 minutes to about 24 hours, preferably from about 1 hour to about 18 hours, preferably from about 2 hours to about 10 hours.

[0343] The above disclosure should not be considered limiting on the reductive amination step. The skilled person is aware of appropriate reaction conditions suitable for carryingout the reductive amination reaction. For example, the reductive amination may be metal, e.g. nickel, catalysed.

[0344] When step (d) is a reduction followed by esterification, step (d) may comprise the following steps:(i) adding a reducing agent to the MMWH solution to form a reduced MMWH solution;(ii) adding a carboxylic acid to the reduced MMWH solution, optionally in the presence of an acid catalyst or dimethylaminopyridine, to form a functionalised medium molecular weight heparin.

[0345] In step (ii) the carboxylic acid may alternatively be replaced with an activated carboxylic acid, such as an acid chloride. In this embodiment, a base such as triethylamine may be included in the reaction.

[0346] The term "reduced" in the context of reduced medium molecular weight heparin means that the aldehyde groups formed in MMWH by oxidation of one or more of (or a plurality of, or substantially all of, or all of) the glucuronic acid moieties (in step (c) of the method) are reduced to hydroxyl groups. The one or more (or a plurality, or substantially all, or all) glucuronic acid derived moieties in MMWH-Red have the following structure:

[0347] The one or more (or a plurality, or substantially all, or all) glucuronic acid derived moieties in MMWH have the following structure:Preferably, substantially all or all of the glucuronic acid moieties in MMWH are have this structure.

[0348] Preferably, the reducing agent is a mild reducing agent. The reducing agent may be sodium borohydride (NaBFU), sodium cyanoborohydride (NaBH CN), sodium triacetoxyborohydride (NaBH(OAc)3) or potassium borohydride (KBF ).

[0349] The mild reducing agent may selectively reduce aldehydes and ketones to alcohols in the presence of esters. In other words, the mild reducing agent reduces aldehydes and ketones to alcohols at a faster rate than it reduces esters to alcohols. The mild reducing agent does not reduce carboxylic acids, nitriles, and amides under normal conditions.

[0350] Typically the reducing agent is used at greater than about 0.5 molar equivalents, or greater than about 1 molar equivalent, or greater than about 2 molar equivalents, or greater than about 3 molar equivalents, or greater than about 5 molar equivalents relative to medium molecular weight heparin. Typically the reducing agent is used at less than about 15 molar equivalents, or less than about 12 molar equivalents, or less than about 10 molar equivalents, or less than about 8 molar equivalents, or less than about 6 molar equivalents, or less than about 5 molar equivalents relative to medium molecular weight heparin. The reducing agent may be used in amount of between about 0.5 molar equivalents and about 15 molar equivalents, or between about 1 molar equivalents and about 10 molar equivalents, or between about 2 molar equivalents and about 6 molar equivalents.

[0351] Preferably, the reducing step is carried out at from about 0 °C to about 30 °C, preferably from about 5 °C to about 30 °C, preferably from about 10 °C to about 25 °C, preferably from about 10 °C to about 20 °C, preferably from about 15 °C to about 25 °C, preferably from about 20 °C to about 25 °C. Typically, the reduction step is carried out at about 20 °C. typically, the reduction step is carried out at room temperature. Typically, the reduction step is carried out for from about 1 hour to about 24 hours, preferably from about 2 hours to about 16 hours, more preferably from about 3 hours to about 12 hours, more preferably from about 6 hours to about 10 hours.

[0352] The solvent for this reducing step may typically be selected from the group consisting of methanol, ethanol, water, THF, or combinations thereof. Preferably, the solvent is water.

[0353] In the esterification step, the acid catalyst may be sulfuric acid, tosic acid or hydrochloric acid. To drive the esterification reaction, water may be removed from the reaction mixture during the esterification reaction, for example by use of a drying agent, such as molecular sieves or magnesium sulfate, or with use of a Dean-Stark apparatus.

[0354] The carboxylic acid may be R.2C(O)OH, wherein R? may be as defined herein. Typically, 2 is selected from the group consisting of an alkyl, a fluoroalkyl, -C(=O)-OH, a sulfonate, an aryl, heteroaryl, a heteroalkyl, and a heterocycle. For example, the carboxylic acid may be acetic acid, fluoroacetic acid. Typically the carboxylic acid is used at greater than about 0.5 molar equivalents, or greater than about 1 molar equivalent, or greater than about 2 molar equivalents, or greater than about 3 molar equivalents, or greater than about 5 molar equivalents relative to medium molecular weight heparin. Typically the carboxylic acid is used at less than about 15 molar equivalents, or less than about 12 molar equivalents, or less than about 10 molar equivalents, or less than about 8 molar equivalents, or less than about 6 molar equivalents, or less than about 5 molar equivalents relative to medium molecular weight heparin. The carboxylic acid may be used in amount of between about 0.5 molar equivalents and about 15 molar equivalents, or between about 1 molar equivalents and about 10 molar equivalents, or between about 2 molar equivalents and about 6 molar equivalents.

[0355] The acid chloride may be RiC(O)OH, wherein R2 may be as defined herein. Typically, 2 is selected from the group consisting of an alkyl, a fluoroalkyl, -C(=O)-OH, a sulfonate, an aryl, heteroaryl, a heteroalkyl, and a heterocycle. For example, the acid chloride may be acetyl chloride, fluoroacetyl chloride. Typically the acid chloride is used at greater than about 0.5 molar equivalents, or greater than about 1 molar equivalent, or greater than about 2 molar equivalents, or greater than about 3 molar equivalents, or greater than about 5 molar equivalents relative to medium molecular weight heparin. Typically the acid chloride is used at less than about 15 molar equivalents, or less than about 12 molar equivalents, or less than about 10 molar equivalents, or less than about 8 molar equivalents, or less than about 6 molar equivalents, or less than about 5 molar equivalents relative to medium molecular weight heparin. The acid chloride may be used in amount of between about 0.5 molar equivalents and about 15 molar equivalents, or between about 1 molar equivalents and about 10 molar equivalents, or between about 2 molar equivalents and about 6 molar equivalents.

[0356] The solvent for the esterification step may be acetonitrile, dimethylformamide, toluene, dichloroethane, or dichloromethane.

[0357] Preferably, the esterification is carried at a temperature of from about 15 °C to about 80 °C, preferably from about 20 °C to about 60 °C, preferably from about 20 °C to about 25 °C. Preferably, the reaction is carried out for from about 30 minutes to about 24hours, preferably from about 1 hour to about 18 hours, preferably from about 2 hours to about 10 hours.

[0358] The above disclosure should not be considered limiting on the esterification step. The skilled person is aware of appropriate reaction conditions suitable for carrying out the esterification reaction.

[0359] The method may further comprise the step of inactivating the oxidising agent in the MMWH solution. The oxidising agent may be inactivated by the addition of an inactivating agent selected from the group consisting of: methanol, D-mannitol, glycerol, / V-acetylmethionine, sodium sulfite, and combinations thereof. A particularly preferred inactivating agent is methanol or D-mannitol. Methanol is preferred.

[0360] The molar ratio of the oxidising agent to the inactivating agent may be from about 1: 1 to about 1 : 10, more preferably from about 1 :2 to about 1:8, more preferably from about 1 :3 to about 1 :6, more preferably from about 1:4 to about 1 :5. Typically, the molar ratio of the oxidising agent to the inactivating agent may be about 1 : 2 or about 1 :4.

[0361] The method may further comprise precipitating the functionalised medium molecular weight heparin to provide a functionalised medium molecular weight heparin precipitate in a precipitation mixture. This precipitations step results in pure FMMWH.

[0362] Precipitating the functionalised medium molecular weight heparin to provide a functionalised medium molecular weight heparin precipitate in a precipitation mixture may comprise adding a precipitating agent to the functionalised medium molecular weight heparin solution to provide a functionalised medium molecular weight heparin precipitate in a precipitation mixture.

[0363] Preferably, the precipitating agent may comprise or consist of an alcohol or acetone.

[0364] Surprisingly, the precipitating agent precipitates the functionalised medium molecular weight heparin. The precipitated functionalised medium molecular weight heparin may then be isolated from the precipitation mixture, for example by filtration. Thus, the method may further comprise the step of isolating the precipitated functionalised medium molecular weight heparin from the precipitation mixture.

[0365] This precipitation procedure advantageously results in a more efficient and high purity method for the production and purification of functionalised medium molecular weight heparin than known methods. This results in highly pure material that does not need to be purified, for example, via a column. The precipitate obtained is a handleable, filtrable powder which can readily be isolated by filtration and subsequently dried under vacuum.

[0366] Preferably, the precipitating agent is added to the FMMWH solution to give a final concentration of the precipitating agent in the precipitation mixture of from about 50% v / v to about 90% v / v, preferably from about 55% v / v to about 80% v / v, and more preferably from about 60% v / v to about 75% v / v. Preferably the final concentration of the precipitating agent in the precipitation mixture is about 73% v / v. A precipitating agent (e.g. methanol) concentration of about 73% v / v results in a precipitate that settles well and can be isolated as an easily filtrable powder.

[0367] The alcohol may be a Ci to C3 alcohol. For example, the alcohol may be methanol, ethanol, propanol, or isopropanol. Preferably the alcohol is methanol. Preferably, the alcohol is added to the FMMWH solution to give a final concentration of the alcohol in the precipitation mixture of from about 50% v / v to about 90% v / v, more preferably from about 55% v / v to about 80% v / v, and even more preferably from about 60% v / v to about 75% v / v. Preferably the final alcohol concentration in the precipitation mixture is about 73% v / v. For example, addition of 73 mL of an alcohol, e.g. methanol, to a 27 mL FFMWH solution provides a final alcohol concentration of 73% v / v MeOH in the precipitation mixture. An alcohol (e.g. methanol) concentration of about 73% v / v results in a precipitate that settles well and can be isolated as an easily filtrable powder.

[0368] During the addition of the precipitating agent, the temperature of the solution may be kept between about 0 °C and about 15 °C, preferably between about 5 °C and about 10 °C.

[0369] In some embodiments, a salt is added to the FMMWH solution prior to precipitating the FMMWH. The step of precipitating the FMMWH may comprise adding a salt to the FMMWH solution followed by addition of the precipitating agent. As described above, the precipitating agent may consist of, consist essentially of or comprise an alcohol or acetone. The salt may be a lithium salt, a sodium salt, or a potassium salt. Preferably, the salt is a sodium salt. The salt may be lithium chloride, sodium chloride, potassium chloride. Preferably the salt is sodium chloride.

[0370] The mass of salt added to the FMMWH solution is selected to result in a final salt concentration in the FMMWH solution of from about 0.5% m / v to about 3% m / v, preferably from about 1% m / v to about 2.5% m / v, and even more preferably from about 1.5% to about 2% m / v. Preferably the concentration of the salt is about 1.8% m / v. For example, addition of addition of 0.9 g solid NaCI to a 50 mL FMMWH solution provides a NaCI concentration of 1.8% m / v. Most preferably, the salt solution comprises sodium chloride at a concentration of 1.8% m / v.

[0371] Following addition of the salt, the resulting solution may be cooled to from about 5 °C to about 20 °C , preferably from about 10 °C to about 15 °C . Preferably, the resulting solution may be cooled to about 15 °C. The precipitating agent may then be added.

[0372] Alternatively to purification by precipitation, the method may comprise the step of dialysing the functionalised medium molecular weight heparin solution in a dialysate to provide a dialysed functionalised medium molecular weight heparin sample.

[0373] "Dialysis" or "dialysing" means the process of separating molecules in solution by the difference in their rates of diffusion through a semipermeable membrane, for example dialysis tubing. The sample for dialysis and a dialysate (or buffer) are placed on opposite sides of the semipermeable membrane. Target sample molecules (e.g. proteins, DNA or polysaccharides) that are larger than the membrane pores remain on the sample side of the membrane. Contaminants such as small molecules and salts can pass through the membrane into the dialysate, thereby reducing the concentration of contaminants in the sample to low levels. Changing the dialysate for fresh dialysate removes the contaminants that have passed from the sample to the dialysate. This allows more contaminants to diffuse from the sample into the dialysate.

[0374] Dialysis can separate small molecules such as salts, reducing agents, or dyes, from larger macromolecules such as proteins, DNA or polysaccharides. Dialysis can also be used to separate polysaccharides by molecular weight. The semipermeable membrane is typically made of a film of regenerated cellulose or cellulose esters.

[0375] Dialysis may be carried out by placing a dialysis tubing containing a sample in a dialysate. A "dialysate" is the fluid into which material passes from the dialysis tubing. The dialysate may be refreshed as often as necessary to achieve optimum separation. The dialysis may be carried out over a period of about 1 day to about 14 days, preferably about 5 days to about 10 days, preferably about 7 days. The dialysate may be refreshed about 1 time a day to about 10 times a day, preferably about 2 times a day to about 5 times aday, preferably about 3 times a day. Typically, the dialysate is many times the volume of the sample, for example from about 2 to about 500 times the volume of the sample. The dialysate may be about 4 times the volume of the sample.

[0376] Typically, the dialysate is water. Optionally, the dialysate may comprise electrolytes such as sodium, potassium, magnesium, calcium, chloride, bicarbonate, lactate, glucose, amino acids, or combinations thereof.

[0377] The dialysing step may be carried out in 2 kD cut off tubing, for example provided by Spectra / Por®. The skilled person is aware of the appropriate tubing cut off size for different purposes. Alternatively, the dialysing step may be carried out in a dialysis device or dialyzer. Suitable dialyzers may be the Slide-A-Lyzer™, the Float-A-Lyzer, the Pur-A- lyzer, the D-Tube, and GeBAflex Dialyzers product lines.

[0378] In this embodiment, the method may further comprise the step of isolating the functionalised medium molecular weight heparin from the dialysed heparin sample. The functionalised medium molecular weight heparin may be isolated from the dialysed heparin sample by freeze-drying, centrifuging, or filtration. Preferably, the functionalised medium molecular weight heparin is isolated from the dialysed heparin sample by freeze-drying.

[0379] "Freeze drying" (also known as lyophilisation or cryodesiccation) is a drying process carried out at low temperature. Freeze drying generally involves reducing temperature and pressure to below the substance's triple point and removing the frozen solvent (e.g. water ice) by sublimation. For aqueous compositions, such as those disclosed herein, freeze drying may be carried out at temperatures of from about -20 °C to about -80 °C, preferably about -40 °C, and pressures of from about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).

[0380] The FMMWH may be purified by any suitable method known to the skilled person. Thus, in this embodiment, the method may further comprise the step of purifying the medium molecular weight heparin. For example, the FMMWH may be purified by exhaustive dialysis using phosphate buffer (pH = 7.0) or saline, a desalting column (e.g. Sephadex G-25 with phosphate buffer (pH = 7.0) or saline used as the mobile phase) or precipitation of the MMWH.

[0381] In general, the method may include an alkaline elimination step. Alternatively, the method may not include an alkaline elimination step. Alkaline elimination may be carried out using an alkali salt such as, for example, sodium hydroxide, potassium hydroxide orlithium hydroxide. The alkaline elimination step may be carried out by addition of an alkali salt to increase the pH of the medium molecular weight heparin solution to about pH 10 to about pH 14, preferably about pH 12, for about 10 minutes to about 3 hours, preferably about 30 minutes, at about room temperature.

[0382] Preferably, the method does not include an alkaline elimination step. Preferably, the method does not include the addition of an alkali metal salt, for example an alkali metal salt such as NaOH, KOH or LiOH. Preferably, the method does not include the addition of NaOH, KOH or LiOH. Surprisingly, a method that does not include an alkaline elimination step or the addition of an alkali metal salt produces functionalised medium molecular weight heparin as defined herein that displays very low activity against Factor Ila and / or Xa as compared to UF heparin and low molecular weight heparin.

[0383] FMMWH may be characterised by NMR, disaccharide analysis, ristocetin-induced platelet aggregation (RIPA), Factor X analysis. The methods of the invention are suitable for preparing medium molecular weight heparin on the milligram, gram or kilogram scale.

[0384] The method of synthesis of FMMWH may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin; and (e) purifying the FMMWH. Step (e) may comprise precipitating the FMMWH and isolating the FMMWH.

[0385] The method of synthesis of FMMWH may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin and (f) purifying the FMMWH. Step (f) may comprise quenching the oxidising agent, precipitating the FMMWH and isolating the FMMWH.

[0386] The method of synthesis of FMMWH may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a firstsolution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin; and (f) dialysing the FMMWH solution in a dialysate to provide a dialysed FMMWH sample.

[0387] The method of synthesis of FMMWH may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous solvent to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin; (f) dialysing the FMMWH solution in a dialysate to provide a dialysed MMWH sample; and (f) Isolating the FMMWH from the dialysed heparin sample.

[0388] For the avoidance of doubt, embodiments related to the first and third to eighth aspects of the invention apply mutatis mutandis to ninth aspect of the invention.

[0389] In a tenth aspect, the invention provides a method of synthesis of functionalised medium molecular weight heparin, the method comprising the steps of: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 0 °C to about 10 °C to form a medium molecular weight heparin solution; and (d) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin.

[0390] For the avoidance of doubt, embodiments related to the ninth aspect of the invention apply mutatis mutandis to the tenth aspect of the invention. More generally, embodiments related to all aspects of the invention apply mutatis mutandis to the tenth aspect of the invention.

[0391] Further embodiments of the tenth aspect of the invention are as follows.

[0392] The pH is adjusted to ensure that the pH of the first solution is between from about pH 5.0 to about pH 9.0 because dissolving unfractionated heparin may result in the pH of the aqueous buffer solution changing.

[0393] Optionally, in step (a), the aqueous buffer solution is adjusted to between about pH 6.0 and about pH 8.0, more preferably to about pH 7.0. Typically, the temperature of the aqueous buffer solution in step (a) is from about -2 °C to about 4 °C. Preferably, the temperature of the aqueous buffer solution in step (a) is from about 0 °C to about 2 °C.

[0394] Typically, the aqueous buffer solution is a phosphate buffer, a citrate buffer or an acetate buffer, i.e. the buffering system is phosphate, citrate or acetate. Preferably, the aqueous buffer solution is a phosphate buffer, more preferably the aqueous buffer solution is a sodium phosphate buffer or a potassium phosphate buffer, i.e. the buffering system is sodium phosphate or potassium phosphate.

[0395] The buffering system in the aqueous buffer solution may be present at a concentration of from about 10 mM to about 100 mM, more preferably from about 20 mM to about 90 mM, more preferably from about 30 mM to about 80 mM, more preferably from about 40 mM to about 70 mM, more preferably from about 50 mM to about 60 mM. The buffering system in the aqueous buffer solution may be present at a concentration of about 50 mM.

[0396] The concentration of UF heparin in the aqueous buffer solution may be from about 0.5 mg / mL to about 10 mg / mL, more preferably from about 1 mg / mL to about 8 mg / mL, more preferably from about 1.5 mg / mL to about 6 mg / mL, more preferably from about 2 mg / mL to about 4 mg / mL. The concentration of UF heparin in the aqueous buffer solution may be about 1.5 mg / mL, more preferably about 1.8 mg / mL, more preferably about 2 mg / mL, more preferably about 2.5 mg / mL, more preferably about 2.7 mg / mL, more preferably about 3 mg / mL.

[0397] Typically, the incubation temperature in step (c) is from about 0 °C to about 10 °C, more preferably from about 1 °C to about 9 °C, more preferably from about 2 °C to about 8 °C, more preferably from about 3 °C to about 7 °C, more preferably from about 4 °C to about 6 °C. Preferably, the incubation temperature in step (c) is about 4 °C. Typically, the incubating step (c) is carried out for from about 1 hour to about 48 hours, more preferably from about 4 hours to about 36 hours, more preferably from about 8 hours to about 30 hours, more preferably from about 12 hours to about 24 hours, more preferably from about 15 hours to about 20 hours, more preferably from about 16 to about 18 hours.Preferably, the incubation step is carried out for about 3 hours. Optionally, step (c) may be carried out at from about 20 to about 25 °C.

[0398] Incubation step (c) of the tenth aspect of the invention may be carried out in a laboratory fridge set at the required temperature. The laboratory fridge may be set at a temperature of from about 0 °C to about 10 °C, more preferably from about 2 °C to about 8 °C, more preferably from about 3 °C to about 7 °C, more preferably from about 4 °C to about 6 °C. Preferably, the laboratory fridge may be set at a temperature of about 4 °C.

[0399] The method of synthesis of FMMWH may consist of or consist essentially of the steps: (a) dissolving unfractionated (UF) heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 0 °C to about 10 °C to form a MMWH solution; (d) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin; and (e) purifying the FMMWH. Step (e) may comprise precipitating the FMMWH and isolating the FMMWH.

[0400] The method of synthesis of FMMWH may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 0 °C to about 10 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the MMWH solution; (e) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin; and (f) purifying the FMMWH. Step (f) may comprise precipitating the FMMWH and isolating the FMMWH.

[0401] The method of synthesis of FMMWH may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 0 °C to about 10 °C to form a medium molecular weight heparin solution; (d) inactivating the oxidising agent in the medium molecular weight heparin solution; (e) dialysing the MMWH solution in a dialysate to provide a dialysed MMWH sample; (f) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin; and (g) purifying the FMMWH. Step (g) may comprise precipitating the FMMWH and isolating the FMMWH.

[0402] The method of synthesis of FMMWH may consist of or consist essentially of the steps: (a) dissolving unfractionated heparin in an aqueous buffer solution adjusted to between about pH 5.0 and about pH 9.0 to provide a first solution; (b) adding an oxidising agent to the first solution to provide a second solution; (c) incubating the second solution at a temperature of from about 0 °C to about 10 °C to form a MMWH solution; (d) inactivating the oxidising agent in the MMWH solution; (e) dialysing the MMWH solution in a dialysate to provide a dialysed MMWH sample; (f) Isolating the MMWH from the dialysed heparin sample; (g) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin; and (h) purifying the FMMWH. Step (h) may comprise precipitating the FMMWH and isolating the FMMWH.

[0403] In the sixteenth and seventeenth aspects, the kit may comprise the FMMWH in a unit dosage form, in a dosage as defined herein. The kit may comprise a pharmaceutical package. The kit may comprise the necessary reagents to synthesise the FMMWH for use according to the invention.

[0404] For the avoidance of doubt, embodiments related to each aspect of the invention apply mutatis mutandis to the other aspects of the invention. Further aspects and embodiments of the present invention will be evident from the discussion herein.

[0405] Every document cited herein, including any cross-referenced or related patent or application, is hereby incorporated by reference in its entirety unless expressly excluded or otherwise limited.

[0406] It will be appreciated that various modifications may be made to the embodiments shown without departing from the spirit and scope of the invention as defined by the accompanying claims.EXAMPLES

[0407] The invention will now be demonstrated by reference to the following non-limiting examples.

[0408] Unless otherwise mentioned, room temperature and pressure are 20 °C (293.15 K, 68 °F) and 1 atm (14.696 psi, 101.325 kPa), respectively.Experimental MethodsSize Exclusion Chromatography (SEC)

[0409] SEC is performed on a GEC Superdex75 (10 / 100) column on an Agilent 1200 HPLC system with variable wavelength UV monitor or GEC AKTA system with variable wavelength UV monitor. The mobile phase is 0.15M NaCI in water, which is passed through the column at 0.4 mL / min. The optical density is measured at 232 nm. SEC is performed with reference to an 11 kD standard.Freeze drying

[0410] Freeze drying (also known as lyophilisation or cryodesiccation) is a drying process carried out at low temperature. Freeze drying generally involves reducing temperature and pressure to below the substance's triple point and removing the frozen solvent (e.g. water ice) by sublimation. For aqueous compositions, such as those disclosed herein, freeze drying may be carried out at temperatures of from about -20 °C to -80 °C, preferably about -40 °C, and pressures of from about 1000 Pa (0.01 bar) to about 10 Pa (0.0001 bar).Synthesis of FMMWH

[0411] 3 grams of unfractionated heparin is dissolved in 50 mL of milli Q water at room temperature. 1.19 grams of solid NaIC is then added under stirring and the reaction is allowed to proceed at room temperature under stirring for 3 hours.

[0412] Then 0.9 grams of solid NaCI is added to the reaction mixture to obtain a NaCI concentration of 1.8% m / v. The solution is cooled to 15 °C. 14 mL methanol is added to the reaction mixture. The solution is cooled to 5 °C and 116 mL of methanol is added to obtain a final methanol concentration of 73% v / v while maintaining the temperature between 5 and 10 °C.

[0413] After completion of the methanol addition, the precipitate is allowed to settle for 2 hours at room temperature. The supernatant is removed by decanting and the precipitate is washed with methanol several times to obtain a water content less than 5%. The precipitate is then isolated by filtration, washed with methanol and dried in vacuo at room temperature overnight.

[0414] The precipitate is then suspended in trifluoroethanol and methylamine (2M in THF) is added and the reaction mixture stirred for 30 minutes. Sodium triacetoxyborohydride is then added and the reaction mixture stirred for 30 minutes. After completion of the reaction, the pH is then adjusted to pH 4 with hydrochloric acid and the reaction mixture stirred for 15 minutes. The pH is subsequently adjusted to pH 7 with sodium hydroxide. 2% m / v NaCI is added followed by MeOH to arrive at 73% v / v MeOH in the reaction mixture. The methanolic reaction mixture is stirred for 10 minutes and the precipitate allowed to settle. The precipitate is then resuspended with methanol and collected by filtration. The product is dried in vacuo at room temperature overnight.Manufacturing Process of Derivatives

[0415] All derivatives were prepared by performing a reaction between medium molecular weight heparin and an oxyamine (2 molar equivalents per oxidized uronic acid residue; Ulrich, Sebastien, et al. Oxidised hep"Oxime ligation : a chemoselective click-type reaction for accessing multifunctional biomolecular constructs." Chemistry-A European Journal 20.1 (2014): 34-4)). Medium molecular weight heparin was prepared by reacting unfractionated heparin with sodium periodate as previously described (see e.g. WO2024105271, incorporated herein by reference). The stoichiometry was determined by using the mass of tri-sulphated disaccharide and 100% oxidation of the uronic acids. The reaction occurred for 1 hour in unbuffered milli Q water. After 1 hour, several product isolation methods to isolate the product were tested :• Precipitation• Dialysis• Ion exchange (IW) followed by elution• (direct) lyophilizationSynthesisPreparation of medium molecular weight heparin3 grams of unfractionated heparin was dissolved in 50 mL of milli Q water at room temperature. 1.19 grams of solid NaIC was then added under stirring and the reaction was allowed to proceed at room temperature under stirring for 3 hours.Then 0.9 grams of solid NaCI was added to the reaction mixture to obtain a NaCI concentration of 1.8% m / v. The solution was cooled to 15 °C. 14 mL methanol was addedto the reaction mixture. The solution was cooled to 5 °C and 116 mL of methanol was added to obtain a final methanol concentration of 73% v / v while the temperature was maintained between 5 and 10 °C.After completion of the methanol addition, the precipitate was allowed to settle for 2 hours at room temperature. The supernatant was removed by decanting and the precipitate was washed with methanol several times to obtain a water content less than 5%. The precipitate was then isolated by filtration, washed with methanol and dried in vacuo at room temperature overnight.Oxidized heparin with benzylhydroxylamine400 mg medium molecular weight heparin (ID24-016-1, 0.675 mmole) was dissolved in 2.5 mL milli Q at RT and 0.157 mL benzylhydroxylamine (1.35 mmole, 2 eq, density = 1.058 g / cm3) was added to the solution. The solution was stirred for 1 hour. After 1 hour, the solution was transferred to a centrifuge tube with a filter membrane of 3kDa (wash these tubes 3x with milli Q before use). The solution was centrifuged at 4000 rpm for 5 minutes at 5°C. The permeate was removed and 5 mL milli Q water was added to the concentrate. The tube was vortexed and centrifuged again. The was repeated at least 3x. 5 mL milli Q water was added to the concentrate and this was lyophilized for 1 night. This resulted in 355 mg ID24-021-5k, corresponding to a yield of 89%.a) Oxidized heparin with o-carboxylhydroxylamine hemihydrochloride400 mg medium molecular weight heparin (ID24-016-1, 0.675 mmole) was dissolved in 2.5 mL milli Q at RT and 0.147 mL o-carboxylhydroxylamine hemihydrochloride (approx. 1.35 mmole, approx..2 eq,) was added to the solution. The solution was stirred for 1 hour. After 1 hour, 2.4 mL MeOH was added to the reaction solution to perform a 50% MeOH precipitation. The MeOH was removed and the remaining solution was lyophilized. This resulted in sample ID24-021-6a.Dialysis was also performed on this reaction resulting in sample ID24-021-6c.b) Oxidized heparin with 2,2,2-trifluoroethoxyamine hydrochloride400 mg medium molecular weight heparin (ID24-016-1, 0.675 mmole) was dissolved in 2.5 mL milli Q water at RT and 0.183 g 2,2,2-trifluoroethoxyamine hydrochloride (1.35 mmole, 2 eq) was added to the solution. He solution was stirred for 1 hour. After 1 hour, the rection solution was lyophilized. This resulted in product ID24-021-7a.NMR of functionalised medium molecular weight heparins

[0416] The present study was focused on the NMR analysis of three FMMWHs, schematically shown above. Mono-dimensional3H NMR spectra were expected to show some structural differences mainly for the well-separated signals, useful for the comparison with the reference oxy-heparin starting material. Due to the high structural microheterogeneity of heparin-based derivatives, two-dimensional *H-13C HSQC NMR technique was expected to resolve peaks superimposed in monodimensional3H NMR spectra (FIGs. 3-6), allowing to obtain more structural information about these derivatives.Materials and methods

[0417] The able below reports the samples characterized in this study, the information related to their structure and amount.NMR analysisReagent and Materials• 5 mm NMR tubes, Bruker• Deuterium oxide (D2O), > 99.8 %, Sigma Aldrich, or equivalent• Ethylenediaminetetraacetic-dl6 acid (EDTA-dl6), 98%, Cambridge Isotope Laboratories• Trimethylsilylpropionic-2,2,3,3-D4 acid sodium salt (TSP), Sigma-Aldrich• Sodium dihydrogen phosphate hydrate (NaH2PO4-H2O), Sigma-Aldrich, or equivalent• Disodium hydrogen phosphate dihydrate (Na2HPO4 2H2O), Sigma-Aldrich, or equivalentDeuterium oxide with 0.002 % TSP

[0418] 4 mg of TSP were dissolved in 2 mL of deuterium oxide; then, 0.1 mL of the obtained solution was diluted to 10.0 mL with deuterium oxide.PBS preparation

[0419] To prepare 150 mM PBS at pH 7.1 containing 0.3 mM EDTA-dl6, 49.7 mg of sodium dihydrogenphosphate hydrate, 202.9 mg of disodium hydrogenphosphate dihydrate and 0.92 mg of EDTA-dl6 were dissolved in 10 mL of water (final pH 7.1). The solution was then lyophilized. The freeze-dried material was dissolved in approximately 2 mL of deuterium oxide and freeze-dried again. The obtained material was further dissolved in lOmL deuterium oxide with 0.002% TSP.Equipment

[0420] XH and HSQC NMR spectra were acquired using a Bruker Avance 500 MHz spectrometer, equipped with a 5 mm cryoprobe.Sample preparation

[0421] The samples G18085 and G18086 were dissolved in 600 pL deuterated water; the pH of the final solutions was neutral. Because the solutions of G18087 and G18088 after dissolving in 600 pL deuterated water had very low pH (<3), these two samples were freeze-dried and then dissolved again in 600 pL of 150 mM PBS with 0.3 mM EDTA-dl6 and 0.002% TSP (pH 7.1).Proton spectra acquisition

[0422] XH NMR spectra were acquired using the following parameters:HSQC spectra acquisition

[0423] *H-13C HSQC-DEPT NMR spectra of the oxy-heparins were acquired applying the following acquisition parameters.NMR spectra processing

[0424] The acquired1H and HSQC-DEPT NMR spectra were processed using a Bruker Topspin 4.3.0 software, spectra were calibrated by setting the SR value, determined in calibration procedure of each instrument.Results

[0425] MonodimentionalXH and bidimensionalHSQC NMR spectra were acquired for all the samples. As mentioned in the Material and methods, once dissolved in D2O, the G18087 and G18088 gave rise to solutions with very low pH (<3). Because heparin signals are susceptible to pH, in particular uronic acid related signals, these two samples were freeze dried and re-dissolved in 150 mM PBS with 0.3 mM EDTA-dl6 and 0.002% TSP (pH 7.1), buffer used in for the quantitative NMR methods of heparins. For all the samples instead of 1H-13C HSQC, 1H-13C HSQC-DEPT NMR spectra were acquired in order todistinguish between the CH and CH2 related cross peaks, both possible markers of the derivatized structures (highlighted with asterisks in FIG. 1).

[0426] The ^-^C-HSQC NMR spectrum of starting material G18085 confirmed the structure of an oxy-heparin by the presence of the cross peaks related to the trisulfated disaccharide I2S-ANS,6S and typical acetal related cross peaks as well as the absence of non-sulfated iduronic and glucuronic acids (FIG. 3), suggesting that periodate oxidation was complete. Together with various signals detectable in the "aldehyde" range (FIG. 2 of the proton spectrum, a signal at 8.46 ppm compatible with formate is detected in all the samples.

[0427] The monodimentional proton NMR spectra of the derivatives G18086, G18087 and G18088 (FIG. 2) show that the N-sulfo-glucosamine (1ANS6X at 5.42 ppm) and 2-O-sulfo- iduronic acid signals (1I2S at 5.22 ppm and 5I2S at 4.83 ppm) related to highly sulfated regions of heparin, expected to be unchanged during periodate oxidation, do not show any significant changes in their intensities (FIG. 2). Of note, the intensity of the signals in the proton spectrum of G18086 was significantly lower than for all the other samples. A high Pl value, measured during spectra acquisition, suggests that this sample may contain high salt amount.

[0428] As for reference G18085, the main signals ofXH-NMR spectra of the derivatives belong to the highly sulfated sequences I2S-ANS,6x, unmodified by periodate and further derivatization reaction. Very broad new signals appeared at 4.6 and 5.2 ppm in G18086 and G18088, respectively. It cannot be ruled out the presence of similar signals in the spectrum of G18087, most likely overlapping with other signals. In fact, further 2D HSQC- DEPT NMR showed the presence of CH2 and CH = N cross peaks in all the derivatives absent in the starting material (FIG. 7-9). For all the derivatives, the acetal related cross peaks disappeared and new cross peaks in the anomeric region of G18087 and G18088 are observed (FIG. 7-9. In the case of G18086, the absence of new cross peaks in the anomeric region can be related to the low amount of the material. The absence of acetals and the appearance of new signals in the anomeric region support the completeness of the reaction. However, the signal compatible with CH = N is much lower than that of the "new" CH2 (compatible with the marker structure) (FIG. 2, 7, 8 and 9, so that the presence of both bound and free derivatizing reagent cannot be excluded.Conclusions

[0429] The performed NMR analyses revealed the presence of new signals in both 1H and 1H-13C HSQC spectra of all the derivatives, compatible with the expected structures.Biological testing of functionalised medium molecular weight heparins

[0430] The functionalised medium molecular weight heparins were tested in a factor Xa assay. Factor Ila (also known as thrombin) acts as a serine protease that converts soluble fibrogen into insoluble strands of fibrin, as well as catalysing other coagulation-related reactions. Factor Xa is the activated from of the coagulation factor X. Factor X is a serine endopeptidase enzyme, which play a key role at several stages of the coagulation system.

[0431] Heparin (unfractionated heparin) and its derivative, e.g. low molecular weight heparin binding to a plasma cofactor, antithrombin (AT), to inactive several coagulation factors Ila, Xa, Xia and Xlla. The inactivation of factor Xa by heparins is termed "indirect" since it relies on the presence of AT and not a direct interaction with Factor Xa.

[0432] As shown in the table below, the FMMWHs exhibit very low activity against Factor Xa, unlike unfractionated heparin and low molecular weight heparin.

[0433] The functionalised medium molecular weight heparins were tested in a ristocetin- induced platelet aggregation (RIPA) assay.

[0434] Protocol according to "Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the platelet physiology subcommittee of SSC / ISTH", Journal of Thrombosis and Haemostasis, 2013, 11 : 1183-1189.

[0435] Blood samples were taken from a non-smoker not on any anti-platelet therapy (for example, aspirin). An anti-platelet therapy may also be known as a platelet agglutination inhibitor or a platelet aggregation inhibitor.

[0436] Blood was drawn, with no venostasis, from a donor into 109 mM sodium citrate solution (VACUETTE, 3.5 mL #454327, lot#A21013FQ). The first 3 to 4 mL of blood drawn was discarded.

[0437] Blood samples were allowed to 'rest' at room temperature for 15 min before centrifugation. Platelet rich plasma (PRP) was prepared by centrifuging blood samples at 200 g for 10 min at 21 °C, without using brake. Platelet poor plasma (PPP) was preparedby centrifuging blood samples, from which PRP was removed, at 1500 g for 15 min at21 °C.

[0438] An assessment of PRP quality was made by carrying out a platelet count of the PRP. Platelet count in PRP was 421 G / L. The platelet count of PRP samples was not (and should not be) adjusted to a standardised value with autologous PPP.

[0439] After centrifugation, PRP samples were allowed to sit at room temperature for 15 min before Light Transmission Aggregometry (LTA) studies were carried out. PRP was used to set 0% light transmission in the aggregometer. Autologous PPP was used to set 100% light transmission in the aggregometer. LTA studies were carried out at 37 °C. Before adding agonist, baseline tracings for LTA were observed for oscillations and stability for at least 1 minute. The volume of agonist added for LTA was consistent, and never more than 10% of the total sample volume.

[0440] The functionalised medium molecular heparins were prepared in water at a concentration of 5.2 mM to provide a sample solution.Ristocetin working solution:- 20 pL of Ristocetin stock (24 mg / mL ristocetin in saline) was combined with 380 pL of saline to provide a working solution of 1.2 mg / mL ristocetin in saline.Sample working solution :-10 pL of 5.2mM sample solution was combined with 163 pL saline to provide a 300 pM working solution of each of the samples.RIPA protocol:

[0441] 360 pL PRP was diluted with Agrastat (5 pg / mL; 1 / 10, antagonist of GPIIIbllla). 20 pL of sample working solution or 20 pL saline (control) was added. The solution was stirred for 2 seconds and then incubated for 10 minutes at 37 °C without stirring. The solution was then incubated for 1 minute at 37 °C with agitation (baseline tracking). 20 pL of ristocetin working solution was added and the platelet agglutination measured.

[0442] Each experiment was carried out in duplicate. The final concentration of the sample was 15 pM. The amplitude, slope and Area under the curve (AUC) calculated by software.

[0443] The results are shown in the table below.Discussion

[0444] Due to medium molecular weight heparin (MMWH) provided by oxidising unfractionated heparin with sodium periodate having potentially reactive aldehyde groups, MMWH analogues with new moieties appended in place of the aldehydes were synthesised. The initial objective of the analogue program was to better understand the structureactivity relationship (SAR) of the aldehydes and whether polarity of the aldehyde or other analogues could vary the RIPA or Factor Xa activity. Since oximes, with the NH2 moiety, is selective and reacts specifically with aldehydes, the initial derivative provided was an 2,2,2-trifluoroethoxyamine derivative, as seen in FIG 1. Upon confirming the oxime pathway was suitable, the next objective was to use the oxime pathway to replace the aldehyde groups of MMWH with O-benzylhydroxylamine, an apolar moiety, and 0- carboxylhydroxylamine, a polar moiety, to assess a highly polar moiety and a highly apolar moiety in place of the two aldehyde moieties. The synthetic procedure is summarized below with the derivates being shown within FIG. 1. All analogues were synthesized on small scale and tested for purity, structural confirmation (NMR), Factor Xa activity, and RIPA activity. The analytical results are summarized within Table 1 above.

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Bongers TN, de Maat MPM, van Goor M-LPJ, Bhagwanbali V, van Vliet HHDM, Gomez Garcia EB, et al. High von Willebrand factor levels increase the risk of first ischemic stroke:influence of ADAMTS13, inflammation, and genetic variability. Stroke. 2006 Nov;37(ll) :2672-7.64. Bongers TN, Emonts M, de Maat MPM, de Groot R, Lisman T, Hazelzet JA, et al. Reduced ADAMTS13 in children with severe meningococcal sepsis is associated with severity and outcome. Thromb Haemost. 2010 Jun; 103(6): 1181-7.65. Bhogal P, Jensen M, Collins G, Spooner O, Makalanda L, Hart D, et al. Letter in response to: Coagulation markers are independent predictors of increased oxygen requirements and thrombosis in COVID-19. J Thromb Haemost. 2020; 18(12):3382-4.66. Sobel M, McNeill PM, Carlson PL, Kermode JC, Adelman B, Conroy R, et al. Heparin inhibition of von Willebrand factor-dependent platelet function in vitro and in vivo. J Clin Invest. 1991 May;87(5) : 1787-93.67. Sobel M, Soler DF, Kermode JC, Harris RB. Localization and characterization of a heparin binding domain peptide of human von Willebrand factor. J Biol Chem. 1992 May 5;267(13) :8857-62.69. van Haren FMP, Page C, Laffey JG, Artigas A, Camprubi-Rimblas M, Nunes Q, et al. Nebulised heparin as a treatment for COVID-19: scientific rationale and a call for randomised evidence. Crit Care Lond Engl. 2020 Jul 22;24(1) :454.70. Dixon B, Campbell DJ, Santamaria JD. Elevated pulmonary dead space and coagulation abnormalities suggest lung microvascular thrombosis in patients undergoing cardiac surgery. Intensive Care Med. 2008 Jul;34(7) : 1216-23.71. Dixon B, Schultz MJ, Smith R, Fink JB, Santamaria JD, Campbell DJ. Nebulized heparin is associated with fewer days of mechanical ventilation in critically ill patients: a randomized controlled trial. Crit Care Lond Engl. 2010;14(5): R180.72. Dixon B, Smith R, Santamaria JD, Orford NR, Wakefield BJ, Ives K, et al. A trial of nebulised heparin to limit lung injury following cardiac surgery. Anaesth Intensive Care. 2016 Jan;44(l) :28-33.73. Dixon B, Schultz MJ, Hofstra JJ, Campbell DJ, Santamaria JD. Nebulized heparin reduces levels of pulmonary coagulation activation in acute lung injury. Crit Care Lond Engl. 2010; 14(5) :445.74. Clausen TM, Sandoval DR, Spliid CB, Pihl J, Perrett HR, Painter CD, et al. SARS- CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2. Cell. 2020 Nov 12; 183(4) : 1043-1057. el5.75. Liu L, Chopra P, Li X, Bouwman KM, Tompkins SM, Wolfert MA, et al. Heparan sulfate proteoglycans as attachment factor for SARS-CoV-2. bioRxiv. 2021 Jan 4;2020.05.10.087288.Embodiments of invention1. A functionalised medium molecular weight heparin comprising one or more of Structure I, wherein structure I comprises:wherein X is selected from the group consisting of -C(=O)H and -CH2-L-R2; wherein L is selected from the group consisting of -N(H)-, N(Ri)- and -O-C(=O)-; R2 is selected from the group consisting of alkyl, fluoroalkyl, carboxylic acid, sulfonate, aryl, heteroaryl, heteroalkyl, and heterocycle; wherein at least one X is -CH2-L-R2.2. The functionalised medium molecular weight heparin according to embodiment 1 wherein the alkyl comprises C1-C5 alkyl.3. The functionalised medium molecular weight heparin according to embodiment 2 wherein the C1-C5 alkyl is a methyl group.4. The functionalised medium molecular weight heparin according to embodiment 1 wherein R2 is fluoroalkyl and the fluoroalkyl comprises C1-C5 fluoroalkyl.5. The functionalised medium molecular weight heparin according to embodiment 4 wherein the C1-C5 fluoroalkyl is trifluoromethyl or fluoromethyl.6. The functionalised medium molecular weight heparin according to embodiment 1 wherein R2 is carboxylic acid and the carboxylic acid is -C(=O)-OH or -C(R3)(H)-C(=O)- OH.7. The functionalised medium molecular weight heparin according to embodiment 1 wherein R2 is sulfonate and the sulfonate is -S(=O)2-OH.8. The functionalised medium molecular weight heparin according to embodiment 1 wherein R2 is aryl and the aryl is phenyl.9. The functionalised medium molecular weight heparin according to embodiment 1 wherein R.2 is aryl and the aryl is phenol.10. The functionalised medium molecular weight heparin according to embodiment 1 wherein R.2 is heteroaryl and the heteroaryl is imidazole.11. The functionalised medium molecular weight heparin according to any preceding embodiment wherein one X is -C(=O)H.12. The functionalised medium molecular weight heparin according to any one of embodiments 1-10 wherein both X are -CH2-L-R2.13. The functionalised medium molecular weight heparin according to any preceding embodiment where L is -N(H)-.14. The functionalised medium molecular weight heparin according to any one of embodiments 1-12 where L is -O-C(=O)-.15. The functionalised medium molecular weight heparin according to any preceding embodiment wherein the functionalised medium molecular weight heparin has an average molecular weight from greater than about 8000 Da (g / mol) to about 13500 Da (g / mol).16. A composition comprising the functionalised medium molecular weight heparin according to any one of embodiments 1-15.17. The functionalised medium molecular weight heparin according to any one of embodiments 1 to 15 or the composition according to embodiment 16 for use in the treatment of endotheliopathy in a patient or for use in the treatment of a disease or condition in a patient.18. The functionalised medium molecular weight heparin or the composition for use according to embodiment 17 wherein the patient has an endotheliopathy characterised by a plasma von Willebrand factor antigen to ADAMTS13 ratio of at least about 2.19. The functionalised medium molecular weight heparin or the composition for use according to embodiment 18 wherein the disease or condition is COVID-19, infection, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, septicaemia, sepsis, cardiovascular disease, diabetes mellitus, trauma, in particular brainor head trauma, burns, inhalational injury, drugs and drug reactions, haematological conditions, subarachnoid haemorrhage, aneurysmal diseases, stroke, brain parenchymal haemorrhage, radiation induced injury, ischemic stroke, pancreatitis, hepatological conditions, renal diseases, or chronic obstruction pulmonary disease (COPD), or combinations thereof.20. The functionalised medium molecular weight heparin or the composition for use according to embodiment 19 wherein the disease or condition is COVID-19.21. The functionalised medium molecular weight heparin or the composition for use according to embodiment 18 wherein the treatment inhibits the haematogenous spread of cancer.22. A method of synthesis of functionalised medium molecular weight heparin, the method comprising the steps of:(a) dissolving unfractionated heparin in an aqueous solution to provide a first solution;(b) adding an oxidising agent to the first solution to provide a second solution;(c) incubating the second solution at a temperature of from about 15 °C to about 30 °C to form a medium molecular weight heparin solution; and(d) reacting the medium molecular weight heparin with a functionalising agent to provide functionalised medium molecular weight heparin.23. The method of embodiment 22 wherein the functionalisation agent comprises (i) an amine or (ii) a reducing agent and carboxylic acid or activated carboxylic acid.24. A kit suitable for preparing functionalised medium molecular weight heparin according to any one of embodiments 1 to 15, wherein the kit comprises: (a) unfractionated heparin; (b) an aqueous solution (c) an oxidising agent; (d) a functionalising agent; and (e) optionally, an inactivating agent.

Claims

1. ClaimsWhat is claimed is:

1. A functionalised medium molecular weight heparin, wherein the functionalised medium molecular weight heparin comprises a functional group.

2. The functionalised medium molecular weight heparin according to claim 1 wherein the functionalised medium molecular weight heparin comprises a linker.

3. The functionalised medium molecular weight heparin according to claim 2 wherein the linker comprises an oxime linker or an amine linker.

4. The functionalised medium molecular weight heparin according to any previous claim wherein the functionalised medium molecular weight heparin comprises a sulphated saccharide.

5. The functionalised medium molecular weight heparin according to any previous claim wherein the functional group comprises a polar functional group, an apolar functional group, an acidic functional group, or a basic functional group.

6. The functionalised medium molecular weight heparin according to any previous claim wherein the functional group is selected from the group comprising an aryl, a phenyl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, an alcohol, a thiol, an ether, a sulphide, an amine, a carboxylic acid, and / or a fluoroalkyl.

7. The functionalised medium molecular weight heparin according to any previous claim wherein the functional group comprises a benzyl, a carboxylic acid or a trifluoro alkyl.

8. The functionalised medium molecular weight heparin according to claims 4-7 wherein in the linker comprises an oxime linker.

9. The functionalised medium molecular weight heparin according to any previous claim wherein the functionalised medium molecular weight heparin comprises one or more of structure A, wherein structure A comprises:wherein X is -CHn-L-Ri, wherein L is the linker, Ri is the functional group and n= 1 or 2.

10. The functionalised medium molecular weight heparin according to any previous claim wherein the functionalised medium molecular weight heparin comprises one or more of structure B, wherein structure B comprises:wherein Ri comprises the functional group.

11. The functionalised medium molecular weight heparin according to any previous claim wherein the functionalised medium molecular weight heparin comprises one or more of structure C, wherein structure C comprises:wherein Ri comprises the functional group.

12. The functionalised medium molecular weight heparin according to any previous claim wherein the functionalised medium molecular weight heparin comprises one or more of structure D, wherein structure D comprises:

13. The functionalised medium molecular weight heparin according to claims 1-11 wherein the functionalised medium molecular weight heparin comprises one or more of structure E, wherein structure E comprises:

14. The functionalised medium molecular weight heparin according to claims 1-11 wherein the functionalised medium molecular weight heparin comprises one or more of structure F, wherein structure F comprises:

15. The functionalised medium molecular weight heparin according to any preceding claim wherein the functionalised medium molecular weight heparin has an average molecular weight from greater than about 8000 Da (g / mol) to about 13500 Da (g / mol).

15. The functionalised medium molecular weight heparin according to any previous claim, wherein the functional group is a heterologous functional group17. The functionalised medium molecular weight heparin according to any previous claim wherein the functionalised medium molecular weight heparin comprises at least three units of a GlcNS6S-IdoA2S or IdoA2S-GlcNS6S disaccharide.

18. A composition comprising the functionalised medium molecular weight heparin according to any one of claims 1-17.

19. The functionalised medium molecular weight heparin according to any one of claims 1 to 17 or the composition according to claim 18 for use in the treatment of endotheliopathy in a patient or for use in the treatment of a disease or condition in a patient.

20. The functionalised medium molecular weight heparin or the composition for use according to claim 19 wherein the patient has an endotheliopathy characterised by a plasma von Willebrand factor antigen to ADAMTS13 ratio of at least about 2.

21. The functionalised medium molecular weight heparin or the composition for use according to claim 20 wherein the disease or condition is COVID-19, infection, viral infection, acute respiratory distress syndrome (ARDS), cancer, bacterial infection, septicaemia, sepsis, cardiovascular disease, diabetes mellitus, trauma, in particular brain or head trauma, burns, inhalational injury, drugs and drug reactions, haematological conditions, subarachnoid haemorrhage, aneurysmal diseases, stroke, brain parenchymal haemorrhage, radiation induced injury, ischemic stroke, pancreatitis, hepatological conditions, renal diseases, or chronic obstruction pulmonary disease (COPD), or combinations thereof.

22. The functionalised medium molecular weight heparin or the composition for use according to claim 20 wherein the disease or condition is COVID-19.

23. The functionalised medium molecular weight heparin or the composition for use according to claim 20 wherein the treatment inhibits the haematogenous spread of cancer.

24. A method of synthesising a functionalised medium molecular weight heparin comprising:I. providing a medium molecular weight heparin comprising a carbonyl; and,II. reacting the medium molecular weight heparin with a functionalising agent to provide the functionalised medium molecular weight heparin; wherein the functionalising agent comprises an oxyamine.

25. The method according to claim 24 wherein the oxyamine comprises:wherein Ri comprises a functional group.

25. The method according to claim 24 wherein the oxyamine comprises:wherein R.i comprises a functional group.

27. The method according to any one of claims 24-25 wherein the carbonyl is an aldehyde.

28. The method according to any one of claims 24-27 wherein step I comprises incubating unfractionated heparin with an oxidising agent to produce the medium molecular weight heparin.

29. The method according to any one of claims 24-28 wherein step II is carried out in a solvent wherein the solvent comprises water30. The method according to any one of claims 24-29 wherein step II is carried out at an acidic pH.

31. The method according to any one of claims 28-30 wherein the oxidising agent comprises a periodate.

32. The method according to claim 31 wherein the periodate is sodium periodate.

33. The method according to any one of claims 24 to 32 further comprising purifying the functionalised medium molecular weight heparin.

34. The method according to claim 33 wherein purifying the functionalise medium molecular weight heparin comprises precipitation, dialysis, chromatography, solvent exchange and / or lyophilization.

35. The method according to claims any one of claims 24-34 wherein step II is carried out for about 1 hour.

36. The method according to any one of claims 24-35 wherein the functional group comprises a polar functional group, an apolar functional group, an acidic functional group, or a basic functional group.

37. The method according to any one of claims 24-36 wherein the functional group is selected from the group comprising an aryl, a phenyl, a heteroaryl, an alkyl, a cycloalkyl, an alkenyl, an alkynyl, an alcohol, a thiol, an ether, a sulphide, an amine, a carboxylic acid, or a fluoroalkyl.

38. The method according to any one of claims 24-37 wherein the functional group is selected from the group consisting of an aryl, a carboxylic acid or a trifluoro alkyl.

39. The method according to any one of claims 24-38 wherein the functionalising agent has the structure:

40. The method according to any one of claims 24-38 wherein the functionalization agent has the structure:

41. The method according to any one of claims 24-38 wherein the functionalization agent has the structure:

42. The method according to any one of claims 24-41 wherein the medium molecular weight heparin has an average molecular weight from greater than about 8000 Da (g / mol) to about 13500 Da (g / mol).

43. The method according to any one of claims 24-42 wherein the medium molecular weight heparin comprises at least three units of a GlcNS6S-IdoA2S or IdoA2S-GlcNS6S disaccharide.

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