Reducing interleukin and chemokine using low anticoagulant heparin

Abstract

This invention is in the field of medicine. More particularly the invention is in the field of treating sepsis and related disorders, and disorder wherein certain cytokines are elevated. The invention further relates to treating diseases where cytokines need to be suppressed. Further described is a companion diagnostic test.

Description

[0001] Reducing interleukin and chemokine using low anticoagulant heparin

[0002] Field of the invention

[0003] This invention is in the field of medicine. More particularly the invention is in the field of treating sepsis and related disorders, and disorder wherein certain cytokines are elevated. The invention further relates to treating diseases where cytokines need to be suppressed. Further described is a companion diagnostic test.

[0004] Background of the invention

[0005] Sepsis is defined as a life-threatening organ dysfunction due to a dysregulated host response to infection. Even though patients with sepsis are treated with antibiotics and supportive care, mortality rates remain high around 40% when progressed to septic shock. At this moment, sepsis is the number one cause in hospital death in the USA. Furthermore, patients who survive sepsis often suffer from long-term sequelae with significant health care impact and social implications. The reported incidence of sepsis varies between 100 and 700 per 100,000 persons per year and is rising due to an aging population [Refs.1-5],

[0006] Histones are major protein components of chromatin in eukaryotic cell nuclei, serving as spools around which deoxyribonucleic acid (DNA) are wound and thereby playing a role in gene regulation. Extracellular histones are released upon cell injury and have been suggested to play a pivotal role in the progression of sepsis to septic shock. A physiological role of extracellular histones, that possess intrinsic cytotoxic properties, is likely to contribute to antibacterial defence mechanisms, when neutrophil extracellular traps (NETs) decorated with extracellular histones are formed in response to bacterial exposure. In pathophysiological conditions, however, excessive presence of extracellular histones can also damage host endothelial and epithelial cells, as well as platelets and red blood cells (RBC), and induce inflammation and coagulation. Subsequently, damaged cells on their turn can release histones from their nuclei, when these cells are not properly cleared. This can result in a self-enforcing feedback cascade, accelerating tissue damage and additional release of histones, ultimately resulting in organ damage. Indeed, abundant evidence from preclinical studies indicates neutralization of extracellular histones confers a strong survival benefit during experimental sepsis. In agreement, patients with sepsis and other types of critical illness have elevated circulating histone levels, which correlate with increased mortality and adverse outcome [Refs. 6-12],

[0007] The severity of sepsis is due to an activation cascade that will lead to an auto amplifying cytokine production: the cytokine storm. Cytokines (and chemokines) are a broad category of relatively small proteins (<40 kDa) that are produced and released with the aim of cell signalling. General understanding of the processes that trigger this tremendous amount of cytokine production has made dramatic progress over the last decades, but unfortunately, these findings could not translate yet into effective treatments [Ref. 20],

[0008] Despite the manifold of different therapies, there exists a need in the art for reliable, safe, affordable and easily applicable therapies for sepsis and related disorders as presented above, in particular by reducing or suppressing cytokine or chemokine production. In addition, therapies are needed for other diseases where cytokines and chemokines play a role and need to be suppressed.

[0009] US 2014 / 162978 A1 describes compositions for treating sepsis based on animal models; the disclosure remains silent on appropriate dosage for human patients.

[0010] The present invention addresses the above shortcomings, problems and concerns by the uses and methods as defined by the appended claims.

[0011] Summary of the invention

[0012] In a first aspect the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, or acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, and wherein the use comprises reducing the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject.

[0013] In a second aspect the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease in a human subject by reducing IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from: sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), COVID-19, Idiopatic Pulmonary Fibrosis (IPF), inflammatory bowel disease (IBD), Chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, asthma, multiple sclerosis (MS), ankylosing spondylitis, pancreatitis, abdominal aortic aneurism, atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, chronic kidney disease, diabetic nephropathy, HIV, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, Parkinson’s disease, severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemia-reperfusion in kidney transplantation and cardiothoracic surgery, preferably wherein the cancer is selected from multiple myeloma, breast cancer, lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, and prostate cancer; and wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject and wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours.

[0014] In a third aspect the invention relates to pentasaccharide depleted heparin for use in reducing one or more of IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in a human subject in the treatment of sepsis, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject and wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours.

[0015] Brief description of the figures

[0016] Fig. 1 depicts the variation of IL-6 and M6229 over time in different serum from septic patients. For each patient a separate graph is shown. Serum samples were obtained at time points T=0, 6, 10, and 24 hours. The left axis shows the amount of pentasaccharide depleted heparin (M6229) in the serum in microgram per mL and the right axis depicts the amount of IL-6 in the serum in picogram per mL. IL-6 values for patients AMC003, AMC004, and AMC005 were below detection threshold and omitted.

[0017] Fig. 2 depicts the variation of IL-17AA and M6229 over time in different serum from septic patients. For each patient a separate graph is shown. Serum samples were obtained at time points T=0, 6, 10, and 24 hours. The left axis shows the amount of pentasaccharide depleted heparin (M6229) in the serum in microgram per mL and the right axis depicts the amount of IL-17A in the serum in picogram per mL.

[0018] Fig. 3 depicts the variation of CCL-2 and M6229 over time in different serum from septic patients. For each patient a separate graph is shown. Serum samples were obtained at time points T=0, 6, 10, and 24 hours. The left axis shows the amount of pentasaccharide depleted heparin (M6229) in the serum in microgram per mL and the right axis depicts the amount of CCL-2 in the serum in picogram per mL.

[0019] Fig. 4 depicts the variation of CCL-4 and M6229 over time in different serum from septic patients. For each patient a separate graph is shown. Serum samples were obtained at time points T=0, 6, 10, and 24 hours. The left axis shows the amount of pentasaccharide depleted heparin (M6229) in the serum in microgram per mL and the right axis depicts the amount of CCL-4 in the serum in picogram per mL.

[0020] Fig. 5 depicts the variation of CXCL8 (IL-8) and M6229 over time in different serum from septic patients. For each patient a separate graph is shown. Serum samples were obtained at time points T=0, 6, 10, and 24 hours. The left axis shows the amount of pentasaccharide depleted heparin (M6229) in the serum in microgram per mL and the right axis depicts the amount of CXCL8 in the serum in picogram per mL.

[0021] Fig. 6 plots the results of Figure 1 (IL-6) as a percentage reduction with compared to the value obtained for T=0, so a positive percentage represents a reduction of the respective cytokine with respect to T=0. The top graph plots percentage changes for each individual patient, the middle graph plots average percentage change for all patients, while the bottom graphs plot average percentage change for the highest dosage patient cohort only (AMC006-AMC011).

[0022] Fig. 7 plots the results of Figure 1 (IL-17A) as a percentage reduction with compared to the value obtained for T=0, so a positive percentage represents a reduction of the respective cytokine with respect to T=0. The top graph plots percentage changes for each individual patient, the middle graph plots average percentage change for all patients, while the bottom graphs plot average percentage change for the highest dosage patient cohort only (AMC006-AMC011).

[0023] Fig. 8 plots the results of Figure 1 (CCL2) as a percentage reduction with compared to the value obtained for T=0, so a positive percentage represents a reduction of the respective cytokine with respect to T=0. The top graph plots percentage changes for each individual patient, the middle graph plots average percentage change for all patients, while the bottom graphs plot average percentage change for the highest dosage patient cohort only (AMC006-AMC011).

[0024] Fig. 9 plots the results of Figure 1 (CCL4) as a percentage reduction with compared to the value obtained for T=0, so a positive percentage represents a reduction of the respective cytokine with respect to T=0. The top graph plots percentage changes for each individual patient, the middle graph plots average percentage change for all patients, while the bottom graphs plot average percentage change for the highest dosage patient cohort only (AMC006-AMC011).

[0025] Fig. 10 plots the results of Figure 1 (CXCL8 I IL-8) as a percentage reduction with compared to the value obtained for T=0, so a positive percentage represents a reduction of the respective cytokine with respect to T=0. The top graph plots percentage changes for each individual patient, the middle graph plots average percentage change for all patients, while the bottom graphs plot average percentage change for the highest dosage patient cohort only (AMC006-AMC011).

[0026] Figures 11-16 plot the percentage change of each of IL-6, IL-17, IL-8, MCP-1 (CCL2) and MIP-ip (CCL4) per cohort, wherein cohort 1 comprises patients administered 0.15 mg / (kg / hr) body weight of pentasaccharide depleted heparin, cohort 2 comprises patients administered 0.45 mg / (kg / hr) body weight of pentasaccharide depleted heparin, and cohort 3 comprises patients administered 0.90 mg / (kg / hr) body weight of pentasaccharide depleted heparin. Figure 11 combines all three cohorts, Figure 12 cohorts 2 and 3, Figures 13-15 each individual cohort 1, 2, and 3 respectively, and Figure 16 shows only patients diagnosed with Acute respiratory distress syndrome (ARDS) within cohort 3.

[0027] Definitions

[0028] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever. Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. For purposes of the present invention, the following terms are defined below.

[0029] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, a method for administrating a pharmaceutical agent includes the administrating of a plurality of molecules (e.g., 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).

[0030] As used herein, “about” and “approximately", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed invention. Unless otherwise clear from context, all numerical values provided herein include numerical values modified by the term “about.” As used herein, “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0031] As used herein, "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... , etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less" i.e., 5, 4, 3, ....-10, -11, etc.

[0032] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. It also encompasses the more limiting “to consist of”. As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[0033] As used herein, "exemplary" or “for example” means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations, including those disclosed herein.

[0034] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and should not be construed as a limitation on the scope of the invention. The description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range including both integers and non-integers. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, 6 etc. This applies regardless of the breadth of the range.

[0035] As used herein, the term “pharmaceutical composition” refers to a composition formulated in pharmaceutically acceptable or physiologically-acceptable compositions for administration to a cell or subject. The compositions of the invention may be administered in combination with other agents as well, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy. The pharmaceutical composition often comprise, in addition to a pharmaceutical active agent, one or more pharmaceutical acceptable carriers (or excipients).

[0036] As used herein, "treatment", "treating", "palliating", “alleviating” and "ameliorating" in the context of a subject to be treated, all refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder. As used herein, “prevention" and "preventing" refers to an approach for reducing in part or in full the change of developing adverse effects, for example normally associated with the use of a particular drug or agent. Within the context of the current invention, for example, the terms may refer to preventing, treating or reducing the effects of a neurological disorder.

[0037] As used herein the term cytokines refers to a broad range of small proteins involved in cell signaling by binding to a cytokine receptor. The broad term cytokines includes chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but not hormones or growth factors.

[0038] Detailed description of the invention

[0039] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0040] It is contemplated that any method, use or composition described herein can be implemented with respect to any other method, use or composition described herein. Embodiments or preferences discussed in the context of methods, use and / or compositions of the invention may likewise be employed with respect to any other method, use or composition described herein. Thus, an embodiment or preference pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[0041] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

[0042] The present invention broadly relates to the inventors’ finding that the administration of pentasaccharide depleted heparin results in the abrupt reduction of certain cytokines, for example in septic patients. This finding opens the possibility for pentasaccharide depleted heparin mediated treatment of diseases, such as sepsis, where these cytokines are relevant. More specifically the inventors found that IL6, IL-17A, IL8, CCL2, and CCL4 were reduced quickly in serum of septic patients upon administration of pentasaccharide depleted heparin. It is therefore deemed plausible that pentasaccharide depleted heparin may benefit patients suffering from other diseases where these cytokines play a role or have increased expression.

[0043] Therefore, in a first aspect the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, or acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr), preferably at least 0.45 mg / (kg*hr), more preferably at least 0.90 mg / (kg*hr), body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 12, 18, 24, 30, or at least 48 hours, and wherein the use comprises reducing the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject. Alternatively, the invention relates to a method of treating or preventing sepsis, SIRS, septic shock, or acute respiratory distress syndrome (ARDS) in a human subject, wherein the method comprises reducing the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject by administering pentasaccharide depleted heparin to the subject in a dose of at least 0.15 mg / (kg*hr), preferably at least 0.45 mg / (kg*hr), more preferably at least 0.90 mg / (kg*hr), body weight of the subject wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours. Further disclosed herein is pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, or acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr), preferably at least 0.45 mg / (kg*hr), more preferably at least 0.90 mg / (kg*hr), body weight of the subject and wherein the use comprises reducing the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject. Also disclosed herein is pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, or acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr), preferably at least 0.45 mg / (kg*hr), more preferably at least 0.90 mg / (kg*hr), body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours. The response of the immune system during sepsis is a complex dynamic and time-dependent process. Cytokines and chemokines play pivotal roles in the immune response during sepsis, modulating both the pro-inflammatory and anti-inflammatory phases of this severe condition. Cells of the innate immune system release high levels of pro-inflammatory cytokines triggering a “cytokine storm”, but at the same time in sepsis can occur a compensatory mechanism at the initial stages of the septic episode by releasing anti-inflammatory mediators.

[0044] Pentasaccharide depleted heparin has been suggested for the treatment of sepsis, for example in WO 2013 / 007771 A1 and US 2014 / 162978 A1. Therein is described an example where mice are challenged with a bolus injection of Lipopolysaccharide (LPS) (360 mg / kg of D-galactosamine (Gal) and 5 microgram / kg Escherichia coli LPS) and received after one hour a bolus injection of saline, 10 units of pentasaccharide depleted heparin or 20 units of pentasaccharide depleted heparin. The results demonstrate that control mice died after 12 hours, while the 10 unit group this was delayed to 48 hours. The 20 unit group was still alive after 96 hours. Wildhagen 2014 also describes a mouse cecal ligation and puncture (CLP) model resulting in a microbial peritonitis where 4 h after CLP 570 pg of pentasaccharide depleted heparin is injected intraperitoneally.

[0045] Although these results are promising they are no indication that pentasaccharide depleted heparin can actually be used as a treatment for sepsis for several reasons. First, in the described LPS experiment a bolus injection of LPS is administered, while in an ongoing infection bacteria are present and replicating introducing a constant stream of LPS and simultaneously introduction of new bacteria through reproduction, putting a continuous and escalating burden in the immune system. Second, the experiments are conducted in mice and human patients may response differently. Third both experiments describe a single bolus intraperitoneal injection of pentasaccharide depleted heparin only. Fourthly for the CLP mouse model it is known that in this model no elevated CRP levels (an important indicator of sepsis in humans) can be expected in contrast to human sepsis (Howosaka et al., journal of surgical research, March 2023, (283) 572 - 580), making it questionable whether both the LPS (no microbial infection) and CLP model (no CRP response) are representative for human sepsis. Herein the inventors surprisingly demonstrate that pentasaccharide depleted heparin is also effective in humans, in actual sepsis patients, and surprisingly found continuous intravenous administration to be highly effective. These results are reflected by measuring of cytokine plasma levels in the patients, which show a sharp decline after 24 hours in pentasaccharide depleted heparin administered subjects, and provided below in Examples 1-5 and Figures 1-10. Further it shown that surprisingly the subset of patients with Acute respiratory distress syndrome (ARDS) in cohort 3 demonstrate particular good response to treatment with pentasaccharide depleted heparin. In addition Example 7 shows safety data supporting that pentasaccharide depleted heparin can be safely administered continuously intravenously over a period of 120 hours in two tested dosages, further supporting the inventors’ findings that pentasaccharide depleted heparin can be safely administered for longer periods of time (e.g. up to 120 hours or even longer) and is likely to benefit septic patients even more.

[0046] IL-6, also known as interleukin 6 or IL6, is one of the primary hyperinflammatory cytokines secreted by macrophages in response to specific microbial molecules. IL-6 is produced in the early phase of sepsis and it is produced by various immune cells, including macrophages, monocytes, and endothelial cells, in response to microbial components like lipopolysaccharide (LPS). Due to IL-6 being one of the earlier cytokines, it stimulates hepatocytes (liver cells) to produce acute-phase proteins such as C-reactive protein (CRP), fibrinogen, and serum amyloid A. This interleukin is responsible for stimulating acute phase protein synthesis and the production of neutrophils in the bone marrow, as well as to produce its recruiting to the site of infection. IL-6 is also essential to produce lymphocyte activation. In fact, IL-6 promotes the proliferation of B cells and increases the responses mediated by the pro-inflammatory T-helper 17 cells (Th17) and suppresses the function of antiinflammatory regulatory T cells (Tregs). Importantly, IL-6 have been linked to endothelial dysfunction and in disseminated intravascular coagulation.

[0047] When used herein the term IL-6 refers to a protein encoded by the gene annotated as ENSG00000136244, or when the subject is not human, the corresponding animal ortholog.

[0048] IL-17A, herein interchangeably referred to as IL-17 and also known as interleukin 17A, IL17A or CTLA8, is a pro-inflammatory cytokine produced by Th17 cells which induces the release of many cytokines (such as IL-6, G-CSF, GM-CSF, IL-1 p, TGF-p, TNF-a) and chemokines (including IL-8, GRO-a and MCP-1), with the final goal of increasing inflammation, the generation of granulocytes by bone marrow and attracting leukocytes to the site of infection. Furthermore, IL-17A also induces TNF-a expression in macrophages, which is a pro- inflammatory cytokine involved in the recruitment and stimulation of neutrophils and monocytes.

[0049] When used herein the term IL-17A refers to a protein encoded by the gene annotated as ENSG00000112115, or when the subject is not human, the corresponding animal ortholog.

[0050] IL-8, also known as interleukin 8 or CXCL8 and used herein interchangably, is a pro-inflammatory chemokine that plays a central role in the immune response, particularly during sepsis. It functions primarily as a chemoattractant for neutrophils, guiding them to the site of infection or injury. In sepsis, the overproduction of IL-8 leads to excessive recruitment and activation of neutrophils, contributing to the inflammatory cascade and the development of systemic inflammation. Elevated IL-8 levels in septic patients are correlated with the severity of the disease, including the development of organ dysfunction and increased mortality. IL-8 not only promotes inflammation but also induces endothelial cell activation, leading to increased vascular permeability, which is a hallmark of septic shock.

[0051] When used herein the term IL-8 or CXCL8 refers to a protein encoded by the gene annotated as ENSG00000169429, or when the subject is not human, the corresponding animal ortholog.

[0052] CCL2, also known as monocyte chemoattractant protein-1 (MCP-1), chemokine ligand 2 or CCL-2, plays a crucial role in sepsis by recruiting monocytes and macrophages to infection sites, amplifying the inflammatory response, and contributing to organ dysfunction, so CCL2 can predict the severity of a septic episode. While CCL2 is essential for immune defense, excessive levels can lead to tissue damage and worse outcomes in septic patients as consequence of the excessive inflammatory response. In fact, CCL2 is involved in the amplification of this response by recruiting immune cells that produce more pro-inflammatory cytokines, such as TNF-a, IL-1, and IL-6, oxidative stress and proteolytic enzymes. CCL2 is involved in the pathophysiology of sepsis contributing to Acute Lung Injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) as well as to acute kidney injury (AKI). When used herein the term CCL2 refers to a protein encoded by the gene annotated as ENSG00000108691 , or when the subject is not human, the corresponding animal ortholog.

[0053] CCL4, also known as Macrophage Inflammatory Protein-1 p, Ml P-1 p, chemokine ligand 4 or CCL-4 or CCL4L1 is a chemokine that plays a significant role in the immune response during sepsis. It is involved in recruiting and activating immune cells, particularly monocytes, macrophages, and lymphocytes, at sites of infection or injury. Among the wide array of functions of CCL4, it can act as chemotactic agent by binding to CCR5 receptor of immune cells, which is crucial for the immune surveillance and response to pathogens in sepsis. However, it acts as a mediator in the pro-inflammatory response by attracting immune cells and provoking the release of cytokines and chemokines to amplify the immune response. Particularly, CCL4 helps to activate both cytotoxic T cells and NK cells, which are crucial for clearing intracellular pathogens such as viruses or bacteria that have invaded host cells. In sepsis, the activation of these cells is part of the broader immune response aimed at eradicating the infection. It is noteworthy to mention that excessive or dysregulated CCL4 production during sepsis can contribute to hyperinflammation, tissue damage, and multi-organ failure.

[0054] When used herein the term CCL4 refers to a protein encoded by the gene annotated as ENSG00000275824, or when the subject is not human, the corresponding animal ortholog.

[0055] In view of the above described effects of IL-6, IL-8, IL-17A, CCL2 and CCL4, in an embodiment the pentasaccharide depleted heparin is used to achieve one or more of the below:

[0056] - production of one or more of acute-phase proteins selected from C-reactive protein (CRP), fibrinogen, and serum amyloid A by hepatocytes is reduced;

[0057] - production and / or recruitment of neutrophils is reduced;

[0058] - lymphocyte activation is reduced;

[0059] - B cell proliferation is reduced;

[0060] - release of one or more cytokines selected from IL-6, G-CSF, GM-CSF, I L-1 p, TGF-p, TNF-a, IL-8, GRO-a and MCP-1 is reduced;

[0061] - inflammation is decreased;

[0062] - production and / or recruitment of granulocytes is reduced; and - production and / or recruitment of monocytes, macrophages and / or lymphocytes is reduced.

[0063] For example, by reducing IL-6, production of one or more of acute-phase proteins selected from C-reactive protein (CRP), fibrinogen, and serum amyloid A by hepatocytes is reduced; and / or production and / or recruitment of neutrophils is reduced; and / or lymphocyte activation is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, or acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, and wherein the use comprises one or more of: reducing production of one or more of acute-phase proteins selected from C-reactive protein (CRP), fibrinogen, and serum amyloid A by hepatocytes; and / or reducing recruitment of neutrophils; and / or reducing lymphocyte activation. In an embodiment this is achieved by reducing IL-6 levels in the blood of the patient.

[0064] In a further example, by reducing IL-17A, release of one or more cytokines selected from IL-6, G-CSF, GM-CSF, IL-1 (3, TGF- , TNF-a, IL-8, GRO-a and MCP-1 is reduced; and or inflammation is decreased; and / or production and / or recruitment of granulocytes is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, or acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, and wherein the use comprises one or more of: reducing release cytokines selected from IL-6, G-CSF, GM-CSF, I L-1 p, TGF-p, TNF-a, IL-8, GRO-a and MCP-1; and / or decreasing inflammation; and / or reducing production and / or recruitment of granulocytes. In an embodiment this is achieved by reducing IL-17A levels in the blood of the patient.

[0065] In a further example, by reducing CCL2, production and / or recruitment of monocytes and / or macrophages is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, and wherein the use comprises reducing production and / or recruitment of monocytes and / or macrophages. In an embodiment this is achieved by reducing CCL2 levels in the blood of the patient.

[0066] In a further example, by reducing CCL4, production and / or recruitment of monocytes, macrophages, and / or lymphocytes is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, and wherein the use comprises reducing production and / or recruitment of monocytes, macrophages, and / or lymphocytes. In an embodiment this is achieved by reducing CCL4 levels in the blood of the patient.

[0067] In a further example, by reducing IL-8, production and / or recruitment of neutrophils is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, or acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, and wherein the use comprises reducing production and / or recruitment of neutrophils. In an embodiment this is achieved by reducing IL-8 levels in the blood of the patient.

[0068] Histones are highly cationic proteins which can be neutralized by polyanions, such as heparin. In non-clinical animal models of sepsis, heparin treatment increases survival independent of its anticoagulant activity. A randomized, blinded clinical trial in septic shock patients receiving an infusion of unfractionated heparin (UFH) showed a decrease in histone levels 24 hours after treatment start. Several meta-analyses of clinical trials in which septic patients were treated with UFH or low molecular weight heparins (LMWH) showed a statistically significant reduction in 28-day mortality as the result of heparin treatment. Nevertheless, it is known that sepsis patients, in particular septic shock patients, are at an increased risk for bleeding complications, frequently requiring coagulation factor supplementation due to a dysregulated hemostatic balance. In such cases administration of LMWH or heparin is not desirable due to the associated increased bleeding risk [13-16], For this reason alternative treatment options are desirable which do not increase the risk of bleeding complications. Heparin is a first-line anticoagulant and has been used for over a century. It is a heterogeneous, linear, highly sulfated, anionic glycosaminoglycan with a broad distribution in relative molecular weight and charge density. These structural properties allow heparin to selectively interact with multiple proteins, leading to heparin's various pharmacological functions, such as anticoagulant, anti-viral, antitumor and anti-inflammatory activities (Wang et al.).

[0069] Heparin is a mixture of polysaccharide chains (Casu et al. 1989). "Structure of heparin and heparin fragments." Ann N Y Acad Sci 556: 1-17). The composition of the polysaccharide chains and their length varies. Chains with a so-called pentasaccharide domain bind strongly to anti-thrombin (AT), which is one of the major circulating anticoagulant proteins (Casu et al. 1981).

[0070] Pentasaccharide depleted heparin is known in the art as a form of heparin with a decreased anti-coagulant activity. Several methods are known for its preparation. For example, heparin may be depleted from its pentasaccharides by affinity chromatography, thereby obtaining non-anticoagulant heparin or heparin with a decreased anti-coagulant activity. Heparin may also be chemically treated in order to obtain heparin with a decreased anti-coagulant activity included but not limited to regioselectively desulfated heparins, and ‘glycol-split’ heparins like for example heparin obtained by periodate oxidation followed by borohydride reduction. Thus when used herein the term low anticoagulant heparin intends to refer to any heparin that is modified to reduce its anticoagulant properties. Non limiting examples are pentasaccharides depleted heparin, regioselectively desulfated heparins and glycol-split heparins like heparin obtained by periodate oxidation followed by borohydride reduction.

[0071] Pentasaccharide depleted heparin may be obtained from unfractionated heparin (UFH) by methods known in the art, for example described in W02013007771A1 or WO2016198578A1 (each herein incorporated by reference in its entirety). In a preferred method, the pentasaccharide depleted heparin is obtained by affinity chromatography. Therein, UFH is passed through a column that contains immobilized AT (antithrombin III). The molecules that contain the pentasaccharide sequence bind to the column, whereas other material passes. Unbound material is called Low Affinity Materal (LAM), whereas material that does bind is called High Affinity Material (HAM). LAM is substantially reduced in pentasaccharides and subsequently in its anticoagulant activity, whereas HAM has an elevated anticoagulant activity.

[0072] LAM may also be described as the pentasaccharide-depleted fraction of unfractionated heparin. The terms pentasaccharide depleted heparin, LAM and M6229 are used interchangeably herein.

[0073] The term pentasaccharide-depleted heparin in this context is used to refer to a fraction of heparin wherein the content of pentasaccharides is substantially reduced in comparison to commercially available heparin.

[0074] The term substantially reduced or decreased as used herein means reduced with at least 10%, such as 20% or 30%, more preferably 40% or 50%, even more preferred, more than 60% or 70% or 80% such as 90% or more than 98% such as more than 99% or even 100%. It is most preferred when the pentasaccharide depleted fraction does not contain any detectable pentasaccharides when tested for thrombin generation as described by Hemker et al., (2003) infra. Conversely, heparin with a decreased anti-coagulant activity means heparin with a reduced anti-coagulant activity, such as reduced with at least 10%, such as 20% or 30%, more preferably 40 or 50%, even more preferred, more than 60% or 70% or 80% such as 90% or more than 98% such as more than 99% or even 100%. It is most preferred when the heparin with a decreased anti-coagulant activity does not contain any detectable anticoagulant activity when tested for thrombin generation as described by Hemker et al., (2003) infra. In the experimental section it is described in detail how a pentasaccharide-depleted heparin may be obtained. It is called LAM therein, abbreviation of Low Affinity Material.

[0075] Thus in an embodiment the heparin has been depleted of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 98%, 99% or even 100% of the pentasaccharide. When referring to pentasaccharide depleted heparin, the terms depleted or reduced when referring to pentasaccharide are to be read with respect to the unfractionated heparin (UFH) from which the pentasaccharide depleted heparin is obtained. Thus heparin depleted of at least 50% of the pentasaccharide means that the pentasaccharide depleted heparin, when compared to the UHF from which it was obtained has 50% or less pentasaccharides based on number of molecules (mol).

[0076] Sepsis is an extreme immune response to infection that can lead to tissue damage, organ failure, or death if not treated right away. Sepsis occurs when chemicals released by the body into the bloodstream to fight an infection trigger inflammation, blood clots, and leaky blood vessels throughout the body. This reduces blood flow and the amount of oxygen and nutrients to the body’s tissues and organs. Bacterial infections are the most common cause of sepsis, but it can also be caused by other types of infections, for example but not limited to viral infections. Signs and symptoms may include fever or very low body temperature, chills, fast heart rate, rapid or trouble breathing, warm or sweaty skin, confusion, and severe pain. Sepsis can occur in anyone with an infection, but is more common in older adults, infants, pregnant women, or people who are in the hospital or who have a weakened immune system, chronic medical condition, or severe injury or illness.

[0077] Systemic inflammatory response syndrome (SIRS) is serious condition in which there is inflammation throughout the whole body. SIRS may be caused by an infection, trauma, surgery, ischemia (lack of blood supply to a part of the body), or certain conditions, such as an autoimmune disorder or pancreatitis. To be diagnosed with SIRS, a person must have two or more of the following: low or high body temperature, increased heart rate, increased breathing rate, and an abnormal white blood cell count. When SIRS occurs as a result of a known infection, it may be called sepsis. Sepsis can be severe or life threatening and lead to multiple organ failure and shock. Also called systemic inflammatory response syndrome.

[0078] Septic shock is a serious condition that occurs when a body-wide infection leads to dangerously low blood pressure.

[0079] Acute respiratory distress syndrome (ARDS) is a sudden and severe lung condition in which fluid accumulates in the small air sacs (alveoli) of the lungs, leading to rapid onset of breathing difficulties and dangerously low blood oxygen levels. This syndrome usually occurs in people who are severely ill, often following a major injury, infection, or trauma. ARDS often develops within hours to days of a critical illness or injury, such as severe infection (sepsis), pneumonia, trauma, aspiration of foreign material, or exposure to toxic fumes. Cytokines play a central role in acute respiratory distress syndrome (ARDS) by driving and modulating the inflammation that characterizes the disease. For example, Key cytokines like TNF-a, I L-1 p, IL-6, and IL-8 are rapidly elevated in the lungs and blood following the initial insult. These molecules trigger an inflammatory cascade, increasing vascular permeability and leading to fluid leakage into alveoli, causing pulmonary edema and impaired gas exchange.

[0080] When used herein preventing sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS) may for example refer to treatment of a subject at risk of developing sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS). For example a subject may be at risk for developing sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS) when the subject has an infection or a chronic disease.

[0081] In a further preferred embodiment the methods, uses and products described herein are for preventing, treating or ameliorating Acute respiratory distress syndrome (ARDS) in a subject, particularly for preventing, treating or ameliorating ARDS in a subject with sepsis.

[0082] The inventors herein show in vivo data of septic patients using three different dosages of low anticoagulant heparin (pentasaccharide depleted heparin): 0.15 mg / (kg*hr) body weight, 0.45 mg / (kg*hr) body weight and 0.9 mg / (kg*hr) body weight; each administered for 6 hours. In particular the examples below describe in detail the setup and outcome of a first in human trial conducted in 10 human patients with sepsis. Particularly Example 4 and the appended Figures 1-16 describe reduced levels of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the patients. This effect was observed for all three tested dosages regimes, but a more pronounced efficacy was observed at the higher dosages 0.45 mg / (kg*hr) body weight and particularly 0.9 mg / (kg*hr) body weight. In addition the effect was observed surprisingly fast with significant reductions of the mentioned cytokines in plasma at 6 hours post onset of the treatment.

[0083] Thus in an embodiment the low anticoagulant heparin is administered to the subject in a dose of at least 0.45 mg / (kg*hr) body weight of the subject. In an embodiment the low anticoagulant heparin is administered to the subject in a dose of at least 0.9 mg / (kg*hr) body weight of the subject. Thus in an embodiment the low anticoagulant heparin administration is provided in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, such as for example 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.0 mg / (kg*hr) body weight of the subject or more. In an embodiment the low anticoagulant heparin administration is provided in a dose of 2.0 mg / (kg*hr) body weight of the subject or less, such as for example 2.0, 1.95, 1.90, 1.85, 1.80, 1.75, 1.70, 1.65, 1.60, 1.55, 1.50, 1.45, 1.40, 1.35, 1.30, 1.25, 1.20, 1.15, 1.10, 1.05, or 1.0 mg / (kg*hr) body weight of the subject or less. Thus in a further embodiment the low anticoagulant heparin administration is provided in a dose of between 0.15 and 2.0 mg / (kg*hr) body weight of the subject, preferably between 0.20 and 1.5, more preferably between 0.25 and 1.0 mg / (kg*hr) body weight of the subject. The dosage unit mg / (kg*hr) refers to the total amount of compound in mg administered in one hour per kg body weight of the subject. The administration may be continuous (e.g. intravenously) or by one or more single administrations (e.g. injections). In the latter case it is understood that the compound may be provided less than once per hour and the unit mg / (kg*hr) refers to the average administered dose in mg per kg body weight of the subject per hour over the total time of treatment. For example this may be the case when the low anticoagulant heparin is administered subcutaneously, e.g. by daily subcutaneous injection.

[0084] Thus in an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.20 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.25 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.30 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11 , 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.35 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.40 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.45 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.50 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.60 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.75 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours. In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.90 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, preferably at least 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even at least 72 hours.

[0085] The inventors show herein that for shorter administration times (e.g. 6 hours) a higher dose of 0.9 can be used and shows good results in septic patients, and particularly in septic patients with ARDS. Therefore in a further preferred embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at between 0.15 and 0.90 mg / (kg*hr) body weight of the subject, preferably between 0.30 and 0.90 mg / (kg*hr) body weight of the subject, more preferably between 0.45 and 0.90 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of 5 to 24, preferably 5 to 12, more preferably 5 to 10, e.g. 5, 6, 7, 8, 9, 10 hours. In an embodiment these dosage regimes are for the treatment, prevention or amelioration of ARDS. In an embodiment these dosage regimes are for the treatment, prevention or amelioration of ARDS in a septic patient.

[0086] The inventors further show that lower dosages (e.g. 0.15, 0.30, 0.45 mg / (kg*hr) body weight of the subject) can be safely used for longer time periods such as 120 hours. Therefore in a further preferred embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at between 0.15 and 0.45 mg / (kg*hr) body weight of the subject, preferably between 0.15 and 0.30 mg / (kg*hr) body weight of the subject, or between 0.30 and 0.45 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of 24 hours or more, preferably 48 hours or more, such as 48, 72, 96, or 120 hours or more.

[0087] It is further anticipated the patients may benefit from an initial high dosage followed by long term administration of lower dosages. For example a dosage of 0.45 - 0.9 mg / (kg*hr) body weight of the subject may be administered for the first 1 to 8, preferably 2 to 6 hours followed by a dosage of 0.15 and 0.45 mg / (kg*hr) body weight of the subject for at least 24, preferably at least 48, 72, 96, or even at least 120 hours or alternatively up to a total administration time of 120 hours.

[0088] In an embodiment the dosage may be based on an assumed average body weight for an adult, e.g. 70 kg. This may for example be useful in case urgent administration is needed and there is no time to determine the patient’s weight. In such case thus dosage of 0.15 mg / (kg*hr) body weight of the subject corresponds to an administration rate of 10.5 mg / hr, 0.45 mg / (kg*hr) body weight of the subject corresponds to an administration rate of 31.5 mg / hr, and 0.90 mg / (kg*hr) body weight of the subject corresponds to an administration rate of 63 mg / hr of pentasaccharide depleted heparin.

[0089] In an alternative embodiment the pentasaccharide depleted heparin is not continuously administered (e.g. by IV) but administered for a specified period by repeated subcutaneous injections. For example to reach a comparable dosage regime a subcutaneous injection can be administered every 12 or 24 hours for a period of 24, 28, 72, 96, or 120 hours or even longer. In such case for an injection every 12 hours for example 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or even 1200 mg can be injected each time period, assuming an average adult body weight of approximately 60 kg. For an injection every 24 hours for example 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, or even 2400 mg can be injected each time period, assuming an average adult body weight of approximately 70 kg.

[0090] Although heparin has been suggested as a potential treatment for sepsis, so far no successful treatment has been reported, as for example evidenced by Jaimes et al. who suggested that unfractionated heparin (UFH) may be a feasible and safe intervention in sepsis, however their study was not able to demonstrate a beneficial effect on the chosen primary outcomes or in the 28-day mortality rate. Without wishing to be bound by theory, it is postulated that heparin based therapies are not successful due to anticoagulant properties of heparin. The maximum dose of heparin that can be used is typically around 0.1 mg / (kg*hr) body weight of the patient. Due to these anticoagulant properties higher doses of heparin cannot be used in septic patients, among others, as the anticoagulant properties cause bleeding and may be fatal. Because the herein described low anticoagulant heparin has low anticoagulant properties, a higher dose can be administered which surprisingly results in quick reduction of IL-6, IL-8, IL-17A, CCL2, and / or CCL4 levels in the patient when exceeding the safe level of unfractionated heparin (e.g. dose levels of 0.15, 0.45 or even 0.9 mg / (kg*hr)). W02013007771A1 suggest the use of pentasaccharide depleted heparin in the treatment of sepsis, however does not disclose any dosages, let alone which dosages can be safely used or above which dosage IL-6, IL-8, IL-17A, CCL2, and / or CCL4 is effectively reduced in patients. The present invention provides the surprising finding that contrary to unfractionated heparin, low anticoagulant heparin can be safely administered at higher doses and more importantly results in the quick reduction of serum IL-6, IL-8, IL-17A, CCL2, and / or CCL4 levels at such high doses.

[0091] In the clinical trial presented herein pentasaccharide-depleted heparin was administered continuously for six hours, however it is understood that shorter or longer administration times may be used. It is further envisioned that the low anticoagulant heparin administration is provided for a much longer period as currently tested., e.g.

[0092] 72 hours, 120 hours or even longer. Example 7 provides safety data in healthy subjects that such data are feasible, Example 8 provides the synopsis of a clinical trial to test this hypothesis in humans suffering from sepsis. Therefore in an embodiment the low anticoagulant heparin is administered over a period of at least 2 hours, preferably at least 4 hours more preferably at least 5 hours most preferably about 6 hours or longer, for example 8, 10, 12, 18, 24, 30, 36, 42, 48, 54, 60, or even 72 hours or longer. Thus for example the low anticoagulant heparin administration is provided for about 4 to 12 or even for about 4 to 48 hours, preferably 5 to 8 hours, or 5 to 48 hours, such as for example 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 42, or 48 hours. Alternatively the low anticoagulant heparin administration is provided for 48 hours or longer, for example at least 60, preferably at least 72, more preferably at least 96 most preferably at least 120 hours or even longer. In a further preferred embodiment the low anticoagulant heparin administration is provided for 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140 hours or longer.

[0093] It is further envisioned that the same absolute amount of low anticoagulant heparin is administered over a shorter or longer period of time, for example by one or more bolus subcutaneous injections or by continuous intravenous administration or one or more bolus intravenous injections. Therefore in an embodiment the low anticoagulant heparin is administered to the subject in a dose of at least 0.90 mg / kg body weight of the subject, preferably at least 2.7 mg / kg body weight of the subject, more preferably at least 5.4 mg / kg body weight of the subject. In an embodiment the low anticoagulant heparin is administered to the subject in a dose of between 0.9 and 10 mg / kg body weight of the subject, preferably between 1.0 and 8 mg / kg body weight of the subject, more preferably between 1.2 and 6 mg / kg body weight of the subject. In an embodiment the low anticoagulant heparin is provided in a dose of 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.5, 5.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10 mg / kg body weight of the subject.

[0094] The present data is obtained with intravenous infusion in patients, however the invention should not be construed as limited to such mode of administration. It is anticipated that any form of parenteral administration is suitable, including but not limited to intravenous, subcutaneous, intramuscular or intradermal administration. In an embodiment the low anticoagulant heparin is administered parenterally. In an embodiment the low anticoagulant heparin is administered intravenously or subcutaneously. The term intravenous administration encompasses bolus administration which may be administered as an IV push, administration through an IV infusion and secondary administration. When used herein secondary administration refers to medication to be administered intravenously at the same time as an infusion may be connected to the primary tubing, and is also referred to as a secondary IV, or IV piggyback.

[0095] Although bolus and intermittent administration modes are also contemplated as part of the invention, a continuous administration mode, such as but not limited to intravenous administration, is preferred. Without wishing to be bound by theory, it is contemplated that this allows for maintaining a near-steady therapeutic concentration that minimizes peak trough fluctuations. This could improve efficacy for timedependent drugs and reduce peak-related toxicity compared with bolus or intermittent dosing, and also may allow faster attainment and tighter maintenance of target exposure when combined with a loading dose and therapeutic monitoring. It is further contemplated that subcutaneous administration may result in almost stable plasma values of pentasaccharide depleted heparin.

[0096] Thus for example in case the low anticoagulant heparin is administered by subcutaneous injection, a dose of 0.15 mg / (kg*hr) body weight of the subject may for example correspond to a single subcutaneous injection of 0.15 mg / kg body weight of the subject each hour, or an injection of 0.30 mg / kg body weight of the subject each two hours, or an injection of 0.45 mg / kg body weight of the subject each three hours, etc. The dose can be scaled accordingly in case a higher dosage regime is used, e.g.

[0097] 0.45 or 0.9 mg / (kg*hr). Thus, in case the low anticoagulant heparin is administered by subcutaneous injection, a dose of 0.45 mg / (kg*hr) body weight of the subject may for example correspond to a single subcutaneous injection of 0.45 mg / kg body weight of the subject each hour, or an injection of 0.90 mg / kg body weight of the subject each two hours, or an injection of 1.35 mg / kg body weight of the subject each three hours, etc. In case the low anticoagulant heparin is administered by subcutaneous injection, a dose of 0.90 mg / (kg*hr) body weight of the subject may for example correspond to a single subcutaneous injection of 0.90 mg / kg body weight of the subject each hour, or an injection of 1.8 mg / kg body weight of the subject each two hours, or an injection of 2.7 mg / kg body weight of the subject each three hours, etc.

[0098] Cytokine and chemokines such as IL-6, IL-17A, CCL2, CCL4, and IL-8 (CXCL8) are known to be present in increased amounts in septic patients. Therefore the present invention aims to reduce the level of one or more level of one or more of IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject. Thus in an embodiment the level of one or more of IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced to 80% or less, preferably 70%, 60%, or even 50% or less compared to just before the start of the administration of the pentasaccharide depleted heparin. In an embodiment the reduction is a significant reduction. In an embodiment the reduction is observed in 2 or more hours, preferably 4 or more or even 6 or more hours after onset of pentasaccharide depleted heparin administration. For example the reduction may be observed after 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 30, 36, 42, 48, 54, 60, 70, 80, 90, or 100 hours after onset of the administration of the pentasaccharide depleted heparin. In a preferred embodiment the reduction of one or more of IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is observed between 2 and 72 hours, preferably between 2 and 48 hours, 2 and 36 hours, 2 and 24 hours, 2 and 18 hours or even 2 and 12 hours, for example between 3 and 10 hours or between 4 and 8 hours.

[0099] The term reduced or lower, when referring to an amount of cytokine or chemokine intends to compare the amount of cytokine or chemokine in the serum of a patient with the amount present just prior to the start of administering treatment (pentasaccharide depleted heparin). It is understood that the levels of cytokine or chemokine in the plasma of the subject may initially reduce and later increase, therefore the time point to assess reduction of cytokine or chemokine is preferably chosen to be a timepoint when administration of pentasaccharide depleted heparin is ongoing, or alternatively, when pentasaccharide depleted heparin is still detectable in the serum of the patient. Thus for example when the pentasaccharide depleted heparin is administered over a period of 6 hours, the time point to assess reduction oof chemokine or cytokine is preferably within 6 hours, e.g. between 4 and 6 hours after administration, but may alternatively be chosen up to 10 hours after start of administration (e.g. 7, 8, 9, or 10 hours after start of administration of pentasaccharide depleted heparin) when pentasaccharide depleted heparin are still detectable. When a longer period of administration of pentasaccharide depleted heparin is chosen, for example 120 hours, the time point to determine reduction of cytokine or chemokine levels is ideally between 4 and 120 hours after start of administration, for example between 10 and 120 hours, more preferably between 24 and 120, between 48 and 120 hours or even between 60 and 120 hours. It is further envisioned that also when the pentasaccharide depleted heparin is provided for a longer period of time (such as up to 120 hours or longer as described in this paragraph, the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, such as for example 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.0 mg / (kg*hr) body weight of the subject, or more.

[0100] When used herein the term “in the serum of the subject” refers to serum isolated from a patient at a certain time point. Serum is the clear liquid part of the blood that remains after blood cells and clotting proteins have been removed. Thus when a cytokine has a certain level in serum it is implied that corresponding levels of the cytokine were present in the blood of the patient. The terms serum and plasma are used interchangeably herein.

[0101] In an embodiment the level of two or more, preferably three, four, five, or all five cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced. For example, the level of IL-6 and IL-8, IL-6 and IL-17A, IL-6 and CCL2, or IL-6 and CCL4 are reduced, or the level of IL-8 and IL-17A, IL-8 and CCL2, or IL-8 and CCL4 are reduced, or the level of IL-17A and CCL2, or IL-17A and CCL4 are reduced or the level of CCL2 and CCL4 are reduced. Thus in an embodiment the level of IL-6 and one or more, preferably two, three, or all four of cytokines, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced. In a further embodiment the level of IL-8 and one or more, preferably two, three, or all four of cytokines, IL-6, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced. In a further embodiment the level of IL-17A and one or more, preferably two, three, or all four of cytokines, IL-6, IL-8, CCL2, and CCL4 in the serum of the subject is reduced. In a further embodiment the level of CCL2 and one or more, preferably two, three, or all four of cytokines, IL-6, IL-17A, IL-8, and CCL4 in the serum of the subject is reduced. In a further embodiment the level of CCL4 and one or more, preferably two, three, or all four of cytokines, IL-6, IL-17A, CCL2, and IL-8 in the serum of the subject is reduced.

[0102] In an embodiment the pentasaccharide depleted heparin is administered to the subject when one or more, preferably least two, three, four, or all five, of the following conditions is / are met:

[0103] the amount of IL-6 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL; the amount of IL-8 in the serum exceeds 10 pg / mL, preferably exceeds 12, 14, 16, 18, 20, 25, 30, 35, 40, or 50 pg / mL;

[0104] the amount of IL-17A in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL;

[0105] the amount of CCL2 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL; or

[0106] the amount of CCL4 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL.

[0107] In an embodiment the amount of one or more of IL-6, IL-17A, CCL2, CCL4, and IL-8 are detected using the companion diagnostic test described herein below.

[0108] Because of the beneficial effect of pentasaccharide depleted heparin on reducing IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in the subject it is further envisioned that pentasaccharide depleted heparin is used in the treatment of diseases where one or more of IL-6, IL-8, IL-17A, CCL2, and / or CCL4 play a role and need to be reduced. Thus in a second aspect the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof. Alternatively the invention relates to a method of treating or preventing a disease by reducing IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in a subject in need thereof, the use comprising administering pentasaccharide depleted heparin to the subject.

[0109] In view of the above described effects of IL-6, IL-8, IL-17A, CCL2 and CCL4, in an embodiment the pentasaccharide depleted heparin is used to achieve one or more of the below:

[0110] - production of one or more of acute-phase proteins selected from C-reactive protein (CRP), fibrinogen, and serum amyloid A by hepatocytes is reduced;

[0111] - production and / or recruitment of neutrophils is reduced;

[0112] - lymphocyte activation is reduced;

[0113] - B cell proliferation is reduced;

[0114] - release of one or more cytokines selected from IL-6, G-CSF, GM-CSF, I L-1 p, TGF-p, TNF-a, IL-8, GRO-a and MCP-1 is reduced;

[0115] - inflammation is decreased;

[0116] - production and / or recruitment of granulocytes is reduced; and - production and / or recruitment of monocytes, macrophages and / or lymphocytes is reduced.

[0117] For example, by reducing IL-6, production of one or more of acute-phase proteins selected from C-reactive protein (CRP), fibrinogen, and serum amyloid A by hepatocytes is reduced; and / or production and / or recruitment of neutrophils is reduced; and / or lymphocyte activation is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease, wherein the use comprises one or more of: reducing production of one or more of acute-phase proteins selected from C-reactive protein (CRP), fibrinogen, and serum amyloid A by hepatocytes; and / or reducing recruitment of neutrophils; and / or reducing lymphocyte activation. In an embodiment this is achieved by reducing IL-6 levels in the blood of the patient.

[0118] In a further example, by reducing IL-17A, release of one or more cytokines selected from IL-6, G-CSF, GM-CSF, IL-1 (3, TGF- , TNF-a, IL-8, GRO-a and MCP-1 is reduced; and or inflammation is decreased; and / or production and / or recruitment of granulocytes is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease wherein the use comprises one or more of: reducing release cytokines selected from IL-6, G-CSF, GM-CSF, IL-1 , TGF-0, TNF-a, IL-8, GRO-a and MCP-1; and / or decreasing inflammation; and / or reducing production and / or recruitment of granulocytes. In an embodiment this is achieved by reducing IL-17A levels in the blood of the patient.

[0119] In a further example, by reducing CCL2, production and / or recruitment of monocytes and / or macrophages is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease wherein the use comprises reducing production and / or recruitment of monocytes and / or macrophages. In an embodiment this is achieved by reducing CCL2 levels in the blood of the patient.

[0120] In a further example, by reducing CCL4, production and / or recruitment of monocytes, macrophages, and / or lymphocytes is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease wherein the use comprises reducing production and / or recruitment of monocytes, macrophages, and / or lymphocytes. In an embodiment this is achieved by reducing CCL4 levels in the blood of the patient.

[0121] In a further example, by reducing IL-8, production and / or recruitment of neutrophils is reduced. Therefore, in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease wherein the use comprises reducing production and / or recruitment of neutrophils. In an embodiment this is achieved by reducing IL-8 levels in the blood of the patient.

[0122] Thus in an aspect the invention relates to Pentasaccharide depleted heparin for use in the treatment or prevention of a disease in a human subject by reducing IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof.

[0123] In an embodiment the disease is selected from sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, inflammatory bowel disease (IBD), Chronic obstructive pulmonary disease (COPD), pancreatitis, abdominal aortic aneurism, atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, and Parkinson’s disease.

[0124] In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing IL-6 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), COVID-19 (cytokine storm in severe cases), Idiopatic Pulmonary Fibrosis (IL-6 contributes in epithelial to mesenchymal transition), Sepsis, Cancer, such as a cancer selected from multiple myeloma, breast cancer, or lung cancer, Inflammatory Bowel Disease (IBD), including Crohn’s disease and ulcerative colitis.

[0125] In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing IL-8 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from Chronic Obstructive Pulmonary Disease (COPD), Cancer for example cancer selected from gastric cancer, pancreatic cancer, or colorectal cancer, Psoriasis, and Asthma. In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing IL-17A serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from Psoriasis and Psoriatic Arthritis, Multiple Sclerosis (MS), Ankylosing Spondylitis, Rheumatoid Arthritis (RA), and Inflammatory Bowel Disease (IBD), such as Crohn’s disease.

[0126] In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing CCL2 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from type 2 Diabetes, Obesity, Cancer such as breast cancer, prostate cancer, or pancreatic cancer, Chronic Kidney Disease, such as diabetic nephropathy, and Asthma.

[0127] In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing CCL4 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from HIV Infection and Progression, Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), and Systemic Lupus Erythematosus (SLE).

[0128] In an embodiment the disease is selected from: sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), COVID-19, Idiopatic Pulmonary Fibrosis (IPF), inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, Chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, asthma, multiple sclerosis (MS), ankylosing spondylitis, pancreatitis, abdominal aortic aneurism, atherosclerosis, cardiovascular disease, type 2 diabetes mellitus, obesity, chronic kidney disease, diabetic nephropathy, HIV, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, and Parkinson’s disease, preferably wherein the cancer is selected from multiple myeloma, breast cancer, lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, prostate cancer, severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemia-reperfusion in kidney transplantation and cardiothoracic surgery. In a preferred embodiment the disease is selected from: severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemiareperfusion in kidney transplantation and cardiothoracic surgery.

[0129] Severe ANCA-associated vasculitis (AAV) is a serious autoimmune condition where the immune system attacks and inflames small and medium-sized blood vessels. It is characterized by the presence of anti-neutrophil cytoplasmic antibodies (ANCA) and can affect multiple organ systems, with kidney and lung damage being particularly significant. The condition is potentially fatal if untreated. Cytokines play a critical pathogenic and regulatory role in Severe AAV by modulating immune responses, inflammation, and tissue injury. High cytokine levels are notably associated with severe phenotypes, such as renal involvement in AAV. Therefore the inventors anticipate that reducing levels of IL-6, IL-8, IL-17A, CCL2, and CCL4 could help to ameliorate or treat severe AAV, or assist in reducing symptoms associated with severe AAV.

[0130] Lupus nephritis is a serious complication of lupus, an autoimmune disease, where the immune system attacks and inflames the kidneys. Cytokines are critically involved in the pathogenesis of lupus nephritis (LN), influencing immune cell infiltration, inflammation, and tissue injury within the kidneys. Emerging research indicates that specific cytokine profiles correlate with disease activity, histopathological features, and response to therapy in LN. Targeting cytokine pathways (e.g., IL-17) offers promising therapeutic avenues to modulate immune responses and prevent renal damage.

[0131] Thrombotic microangiopathy (TMA) is a group of disorders characterized by the formation of microvascular clots that cause damage to small blood vessels, leading to hemolytic anemia, thrombocytopenia, and organ damage, particularly affecting the kidneys and brain. Cytokines are crucial mediators in the development and progression of TMA, as they modulate endothelial cell activation, immune responses, and coagulation pathways. Pro-inflammatory cytokines such as IL-6, IL-1, and TNF-a are often elevated, contributing to endothelial injury, increased vascular permeability, and further promotion of thrombosis. Therefore the inventors anticipate that reducing levels of IL-6, IL-8, IL-17A, CCL2, and CCL4 could help to ameliorate or treat TMA, or assist in reducing symptoms associated with TMA. Pre-eclampsia is a pregnancy-specific syndrome characterized by new-onset hypertension and organ dysfunction, typically emerging after 20 weeks of gestation. It is primarily driven by abnormal placentation resulting in poor remodelling of uterine spiral arteries, leading to placental ischemia and systemic endothelial dysfunction. The placental ischemia and oxidative stress induce the release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-a), interleukins (e.g., IL-6), and others into the maternal circulation. These cytokines promote systemic inflammation and endothelial activation / damage, exacerbating hypertension and organ dysfunction. Therefore the inventors anticipate that reducing levels of IL-6, IL-8, IL-17A, CCL2, and CCL4 could help to ameliorate or treat pre-eclampsia, or assist in reducing symptoms associated with pre-eclampsia.

[0132] Ischemia-reperfusion injury (IRI) in kidney transplantation is a pathophysiological process that occurs when the blood supply to the transplanted kidney is temporarily interrupted (ischemia) and then restored (reperfusion). This injury is especially common in kidneys from deceased donors due to warm and cold ischemia during harvesting and storage. IRI leads to cell damage, inflammation, and microvascular dysfunction, resulting in delayed graft function, acute rejection, and long-term chronic graft dysfunction. Cytokines are key mediators in the inflammatory response to IRI, with pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-a), interleukins (IL-1, IL-6), and chemokines being rapidly upregulated. Therefore the inventors anticipate that reducing levels of IL-6, IL-8, IL-17A, CCL2, and CCL4 could help to ameliorate or treat IRI, or assist in reducing symptoms associated with IRI.

[0133] Ischemia-reperfusion injury (IRI) in cardiothoracic surgery refers to tissue damage that occurs when blood supply to the heart or lungs is temporarily interrupted and then restored during procedures like coronary artery bypass grafting (CABG) or heart transplantation. Although restoring blood flow is essential to salvage ischemic tissue, reperfusion paradoxically causes further injury by triggering oxidative stress, inflammation, and cellular dysfunction. Pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-a), interleukin-1 (IL-1), and interleukin-6 (IL-6) are rapidly released from injured endothelial and immune cells following reperfusion. Therefore the inventors anticipate that reducing levels of IL-6, IL-8, IL-17A, CCL2, and CCL4 could help to ameliorate or treat IRI, or assist in reducing symptoms associated with IRI.

[0134] It is understood that the dosages, dosage regimens, and uses described above can also be applied to the uses, treatments, method or products of other aspects or embodiments of the invention or disclosure, such as but not limited to use non sepsis related applications as broadly described herein. Some non-limiting details are provided below.

[0135] In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, preferably in a dose of at least 0.45 mg / (kg*hr) body weight of the subject, more preferably in a dose of at least 0.9 mg / (kg*hr) body weight of the subject. Thus in an embodiment the low anticoagulant heparin administration is provided in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, such as for example 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.0 mg / (kg*hr) body weight of the subject or more. In an embodiment the low anticoagulant heparin administration is provided in a dose of 2.0 mg / (kg*hr) body weight of the subject or less, such as for example 2.0, 1.95, 1.90, 1.85, 1.80, 1.75, 1.70, 1.65, 1.60, 1.55, 1.50, 1.45, 1.40, 1.35, 1.30, 1.25, 1.20, 1.15, 1.10, 1.05, or 1.0 mg / (kg*hr) body weight of the subject or less. Thus in a further embodiment the low anticoagulant heparin administration is provided in a dose of between 0.15 and 2.0 mg / (kg*hr) body weight of the subject, preferably between 0.20 and 1.5, more preferably between 0.25 and 1.0 mg / (kg*hr) body weight of the subject. The dosage unit mg / (kg*hr) refers to the total amount of compound in mg administered in one hour per kg body weight of the subject. The administration may be continuous (e.g. intravenously) or by one or more single administrations (e.g. injections). In the latter case it is understood that the compound may be provided less than once per hour and the unit mg / (kg*hr) refers to the average administered dose in mg per kg body weight of the subject per hour over the total time of treatment. For example this may be the case when the low anticoagulant heparin is administered subcutaneously, e.g. by daily subcutaneous injection.

[0136] In an embodiment the pentasaccharide depleted heparin is administered over a period of at least 2 hours, preferably at least 4 hours more preferably at least 5 hours, or wherein the pentasaccharide depleted heparin is administered over a period of at least 48 hours, preferably at least 60, at least 72, at least 96, more preferably at least 120 hours or longer. Thus for example when the pentasaccharide depleted heparin is administered over a period of 6 hours, the time point to assess reduction oof chemokine or cytokine is preferably within 6 hours, e.g. between 4 and 6 hours after administration, but may alternatively be chosen up to 10 hours after start of administration (e.g. 7, 8, 9, or 10 hours after start of administration of pentasaccharide depleted heparin) when pentasaccharide depleted heparin are still detectable. When a longer period of administration of pentasaccharide depleted heparin is chosen, for example 120 hours, the time point to determine reduction of cytokine or chemokine levels is ideally between 4 and 120 hours after start of administration, for example between 10 and 120 hours, more preferably between 24 and 120, between 48 and 120 hours or even between 60 and 120 hours. It is further envisioned that also when the pentasaccharide depleted heparin is provided for a longer period of time (such as up to 120 hours or longer as described in this paragraph, the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, such as for example 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.0 mg / (kg*hr) body weight of the subject, or more.

[0137] In an embodiment the pentasaccharide depleted heparin is administered parenterally preferably wherein the pentasaccharide depleted heparin is administered intravenously or subcutaneously.

[0138] In an embodiment the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced to 80% or less, preferably 70%, 60%, or even 50% or less, in 6 hours when compared to just prior to administering the pentasaccharide depleted heparin, preferably wherein the level of two or more, preferably three, four, five, or all five of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced, more preferably wherein the level of IL-6 and one or more, preferably two, three, four, or all four of the cytokines IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced.

[0139] In an embodiment the pentasaccharide depleted heparin is administered to the subject when one or more, preferably least two, three, four, or all five, of the following conditions is / are met: the amount of IL-6 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL;

[0140] the amount of IL-8 in the serum exceeds 10 pg / mL, preferably exceeds 12, 14, 16, 18, 20, 25, 30, 35, 40, or 50 pg / mL;

[0141] the amount of IL-17A in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL;

[0142] the amount of CCL2 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL; or

[0143] the amount of CCL4 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL.

[0144] In an embodiment the amount of one or more of IL-6, IL-17A, CCL2, CCL4, and IL-8 are detected using the companion diagnostic test described herein below.

[0145] In a further aspect the invention relates to pentasaccharide depleted heparin for use in the treatment, prevention or amelioration of a diseases or disorder associated with an elevated level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject, the use comprising administering the pentasaccharide depleted heparin to the subject. Alternatively the invention relates to a method of treating, preventing or ameliorating a diseases or disorder associated with an elevated level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject, the method comprising administering pentasaccharide depleted heparin to the subject in need thereof.

[0146] In an embodiment the pentasaccharide depleted heparin is administered to the subject when one or more, preferably least two, three, four, or all five, of the following conditions is / are met:

[0147] the amount of IL-6 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL;

[0148] the amount of IL-8 in the serum exceeds 10 pg / mL, preferably exceeds 12, 14, 16, 18, 20, 25, 30, 35, 40, or 50 pg / mL;

[0149] the amount of IL-17A in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL;

[0150] the amount of CCL2 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL; or the amount of CCL4 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL.

[0151] In an embodiment the amount of one or more of IL-6, IL-17A, CCL2, CCL4, and IL-8 are detected using the companion diagnostic test described herein below.

[0152] In an embodiment the disease is selected from sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, inflammatory bowel disease (IBD), Chronic obstructive pulmonary disease (COPD), pancreatitis, abdominal aortic aneurism, atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, and Parkinson’s disease.

[0153] In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing IL-6 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), COVID-19 (cytokine storm in severe cases), Idiopatic Pulmonary Fibrosis (IPF), Sepsis, Cancer, such as a cancer selected from multiple myeloma, breast cancer, or lung cancer, Inflammatory Bowel Disease (IBD), including Crohn’s disease and ulcerative colitis.

[0154] In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing IL-8 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from Chronic Obstructive Pulmonary Disease (COPD), Cancer for example cancer selected from gastric cancer, pancreatic cancer, or colorectal cancer, Psoriasis, and Asthma.

[0155] In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing IL-17A serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from Psoriasis and Psoriatic Arthritis, Multiple Sclerosis (MS), Ankylosing Spondylitis, Rheumatoid Arthritis (RA), and Inflammatory Bowel Disease (IBD), such as Crohn’s disease.

[0156] In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing CCL2 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from type 2 Diabetes, Obesity, Cancer such as breast cancer, prostate cancer, or pancreatic cancer, Chronic Kidney Disease, such as diabetic nephropathy, and Asthma.

[0157] In an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment or prevention of a disease by reducing CCL4 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof, wherein the disease is selected from HIV Infection and Progression, Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), and Systemic Lupus Erythematosus (SLE).

[0158] In an embodiment the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, preferably in a dose of at least 0.45 mg / (kg*hr) body weight of the subject, more preferably in a dose of at least 0.9 mg / (kg*hr) body weight of the subject. Thus in an embodiment the low anticoagulant heparin administration is provided in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, such as for example 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.0 mg / (kg*hr) body weight of the subject or more. In an embodiment the low anticoagulant heparin administration is provided in a dose of 2.0 mg / (kg*hr) body weight of the subject or less, such as for example 2.0, 1.95, 1.90, 1.85, 1.80, 1.75, 1.70, 1.65, 1.60, 1.55, 1.50, 1.45, 1.40, 1.35, 1.30, 1.25, 1.20, 1.15, 1.10, 1.05, or 1.0 mg / (kg*hr) body weight of the subject or less. Thus in a further embodiment the low anticoagulant heparin administration is provided in a dose of between 0.15 and 2.0 mg / (kg*hr) body weight of the subject, preferably between 0.20 and 1.5, more preferably between 0.25 and 1.0 mg / (kg*hr) body weight of the subject. The dosage unit mg / (kg*hr) refers to the total amount of compound in mg administered in one hour per kg body weight of the subject. The administration may be continuous (e.g. intravenously) or by one or more single administrations (e.g. injections). In the latter case it is understood that the compound may be provided less than once per hour and the unit mg / (kg*hr) refers to the average administered dose in mg per kg body weight of the subject per hour over the total time of treatment. For example this may be the case when the low anticoagulant heparin is administered subcutaneously, e.g. by daily subcutaneous injection. In an embodiment the pentasaccharide depleted heparin is administered over a period of at least 2 hours, preferably at least 4 hours more preferably at least 5 hours, or wherein the pentasaccharide depleted heparin is administered over a period of at least 48 hours, preferably at least 60, at least 72, at least 96, more preferably at least 120 hours or longer. Thus for example when the pentasaccharide depleted heparin is administered over a period of 6 hours, the time point to assess reduction oof chemokine or cytokine is preferably within 6 hours, e.g. between 4 and 6 hours after administration, but may alternatively be chosen up to 10 hours after start of administration (e.g. 7, 8, 9, or 10 hours after start of administration of pentasaccharide depleted heparin) when pentasaccharide depleted heparin are still detectable. When a longer period of administration of pentasaccharide depleted heparin is chosen, for example 120 hours, the time point to determine reduction of cytokine or chemokine levels is ideally between 4 and 120 hours after start of administration, for example between 10 and 120 hours, more preferably between 24 and 120, between 48 and 120 hours or even between 60 and 120 hours. It is further envisioned that also when the pentasaccharide depleted heparin is provided for a longer period of time (such as up to 120 hours or longer as described in this paragraph, the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, such as for example 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, or 1.0 mg / (kg*hr) body weight of the subject, or more.

[0159] In an embodiment the pentasaccharide depleted heparin is administered parenterally preferably wherein the pentasaccharide depleted heparin is administered intravenously or subcutaneously.

[0160] In an embodiment the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced to 80% or less, preferably 70%, 60%, or even 50% or less, in 6 hours when compared to just prior to administering the pentasaccharide depleted heparin, preferably wherein the level of two or more, preferably three, four, five, or all five of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced, more preferably wherein the level of IL-6 and one or more, preferably two, three, four, or all four of the cytokines IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced. In an embodiment the invention relates to pentasaccharide depleted heparin for use in a method of treating, ameliorating or preventing sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), COVID-19, Idiopatic Pulmonary Fibrosis (IPF), inflammatory bowel disease (IBD), Chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, asthma, multiple sclerosis (MS), ankylosing spondylitis, pancreatitis, abdominal aortic aneurism, atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, chronic kidney disease, diabetic nephropathy, HIV, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, Parkinson’s disease, severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemia-reperfusion in kidney transplantation and cardiothoracic surgery, preferably wherein the cancer is selected from multiple myeloma, breast cancer, lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, and prostate cancer, in a subject in need thereof, the use comprising administering the pentasaccharide depleted heparin when the amount of IL-6 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL. Alternatively, the invention relates to pentasaccharide depleted heparin for use in a method of treating, ameliorating or preventing sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), COVID-19, Idiopatic Pulmonary Fibrosis (IPF), inflammatory bowel disease (IBD), Chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, asthma, multiple sclerosis (MS), ankylosing spondylitis, pancreatitis, abdominal aortic aneurism, atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, chronic kidney disease, diabetic nephropathy, HIV, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, Parkinson’s disease, severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemia-reperfusion in kidney transplantation and cardiothoracic surgery, preferably wherein the cancer is selected from multiple myeloma, breast cancer, lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, and prostate cancer, in a subject in need thereof, the use comprising administering the pentasaccharide depleted heparin when the amount of IL-8 in the serum exceeds 10 pg / mL, preferably exceeds 12, 14, 16, 18, 20, 25, 30, 35, 40, or 50 pg / mL. Alternatively the invention relates to pentasaccharide depleted heparin for use in a method of treating, ameliorating or preventing sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), COVID-19, Idiopatic Pulmonary Fibrosis (IPF), inflammatory bowel disease (IBD), Chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, asthma, multiple sclerosis (MS), ankylosing spondylitis, pancreatitis, abdominal aortic aneurism, atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, chronic kidney disease, diabetic nephropathy, HIV, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, Parkinson’s disease, severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemia-reperfusion in kidney transplantation and cardiothoracic surgery, preferably wherein the cancer is selected from multiple myeloma, breast cancer, lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, and prostate cancer, in a subject in need thereof, the use comprising administering the pentasaccharide depleted heparin when the amount of IL-17A in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL. Alternatively the invention relates to pentasaccharide depleted heparin for use in a method of treating, ameliorating or preventing sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), COVID-19, Idiopatic Pulmonary Fibrosis (IPF), inflammatory bowel disease (IBD), Chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, asthma, multiple sclerosis (MS), ankylosing spondylitis, pancreatitis, abdominal aortic aneurism, atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, chronic kidney disease, diabetic nephropathy, HIV, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, Parkinson’s disease, severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemia-reperfusion in kidney transplantation and cardiothoracic surgery, preferably wherein the cancer is selected from multiple myeloma, breast cancer, lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, and prostate cancer, in a subject in need thereof, the use comprising administering the pentasaccharide depleted heparin when the amount of CCL2 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL. Alternatively the invention relates to pentasaccharide depleted heparin for use in a method of treating, ameliorating or preventing sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), COVID-19, Idiopatic Pulmonary Fibrosis (IPF), inflammatory bowel disease (IBD), Chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, asthma, multiple sclerosis (MS), ankylosing spondylitis, pancreatitis, abdominal aortic aneurism, atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, chronic kidney disease, diabetic nephropathy, HIV, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, Parkinson’s disease, severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemia-reperfusion in kidney transplantation and cardiothoracic surgery, preferably wherein the cancer is selected from multiple myeloma, breast cancer, lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, and prostate cancer, in a subject in need thereof, the use comprising administering the pentasaccharide depleted heparin when the amount of CCL4 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL. Dosages and administration regimens as broadly described herein can be used for the aforementioned applications as well.

[0161] In a further aspect the invention relates to pentasaccharide depleted heparin for use in reducing one or more of IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in a human subject in the treatment of sepsis, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject and wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours. In an embodiment the method comprises monitoring the levels of one or more of IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject, and administering the pentasaccharide depleted heparin when one or more, preferably least two, three, four, or all five, of the following conditions is / are met:

[0162] the amount of IL-6 in the serum exceeds 5 pg / mL; the amount of IL-8 in the serum exceeds 10 pg / mL;

[0163] the amount of IL-17A in the serum exceeds 5 pg / mL;

[0164] the amount of CCL2 in the serum exceeds 5 pg / mL; or

[0165] the amount of CCL4 in the serum exceeds 5 pg / mL.

[0166] Alternatively the invention describes a method reducing one or more of IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in a human subject in the treatment of sepsis, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject and wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours. In an embodiment the method comprises monitoring the levels of one or more of IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject, and administering the pentasaccharide depleted heparin when one or more, preferably least two, three, four, or all five, of the following conditions is / are met:

[0167] the amount of IL-6 in the serum exceeds 5 pg / mL;

[0168] the amount of IL-8 in the serum exceeds 10 pg / mL;

[0169] the amount of IL-17A in the serum exceeds 5 pg / mL;

[0170] the amount of CCL2 in the serum exceeds 5 pg / mL; or

[0171] the amount of CCL4 in the serum exceeds 5 pg / mL.

[0172] In a further aspect the invention relates to pentasaccharide depleted heparin for use in reducing one or more of IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in a human subject in the treatment of sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), COVID-19, Idiopatic Pulmonary Fibrosis (IPF), inflammatory bowel disease (IBD), Chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, asthma, multiple sclerosis (MS), ankylosing spondylitis, pancreatitis, abdominal aortic aneurism, atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, chronic kidney disease, diabetic nephropathy, HIV, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, Parkinson’s disease, severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemia-reperfusion in kidney transplantation and cardiothoracic surgery, preferably wherein the cancer is selected from multiple myeloma, breast cancer, lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, and prostate cancer, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject and wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours. In an embodiment the method comprises monitoring the levels of one or more of IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject, and administering the pentasaccharide depleted heparin when one or more, preferably least two, three, four, or all five, of the following conditions is / are met:

[0173] the amount of IL-6 in the serum exceeds 5 pg / mL;

[0174] the amount of IL-8 in the serum exceeds 10 pg / mL;

[0175] the amount of IL-17A in the serum exceeds 5 pg / mL;

[0176] the amount of CCL2 in the serum exceeds 5 pg / mL; or

[0177] the amount of CCL4 in the serum exceeds 5 pg / mL.

[0178] Further disclosed herein is a companion diagnostic test for quantifying one or more, for example, two, three, four or all five cytokines selected from IL-6, IL-17A, CCL2, CCL4, and IL-8. In an embodiment the test is provided in the form of a kit. In an embodiment the test is ELISA based and includes an antibody or antibodies specific for one or more, for example, two, three, four or all five cytokines selected from IL-6, IL-17A, CCL2, CCL4, and IL-8. In an embodiment the test further includes a standard for one or more, for example, two, three, four or all five cytokines selected from IL-6, IL-17A, CCL2, CCL4, and IL-8 to allow quantification of the detected cytokines. The standard may for example be a sample or series of samples with known concentrations of one or more, for example, two, three, four or all five cytokines selected from IL-6, IL-17A, CCL2, CCL4, and IL-8. In an embodiment the test is suitable for detecting the cytokines in a plasma sample, for example a plasma sample obtained from a patient.

[0179] Thus in an embodiment the invention relates to pentasaccharide depleted heparin for use in the treatment, prevention or amelioration of a diseases or disorder associated with an elevated level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject, the use comprising detecting the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject using a companion diagnostic test, and the pentasaccharide depleted heparin is administered to the subject when one or more, preferably least two, three, four, or all five, of the following conditions is / are met: the amount of IL-6 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL;

[0180] the amount of IL-8 in the serum exceeds 10 pg / mL, preferably exceeds 12, 14, 16, 18, 20, 25, 30, 35, 40, or 50 pg / mL;

[0181] the amount of IL-17A in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL;

[0182] the amount of CCL2 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL; or

[0183] the amount of CCL4 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / m;

[0184] wherein the companion diagnostic test is a test for quantifying one or more, for example, two, three, four or all five cytokines selected from IL-6, IL-17A, CCL2, CCL4, and IL-8. In an embodiment the test is the companion diagnostic test as broadly described herein above.

[0185] Alternatively the invention relates to a method of treating, preventing or ameliorating a diseases or disorder associated with an elevated level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject, the method comprising detecting the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject using a companion diagnostic test, and the pentasaccharide depleted heparin is administered to the subject when one or more, preferably least two, three, four, or all five, of the following conditions is / are met:

[0186] the amount of IL-6 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL;

[0187] the amount of IL-8 in the serum exceeds 10 pg / mL, preferably exceeds 12, 14, 16, 18, 20, 25, 30, 35, 40, or 50 pg / mL;

[0188] the amount of IL-17A in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL;

[0189] the amount of CCL2 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / mL; or

[0190] the amount of CCL4 in the serum exceeds 5 pg / mL, preferably exceeds 6, 7, 8, 9, 10, 15, 20, or 25 pg / m;

[0191] wherein the companion diagnostic test is a test for quantifying one or more, for example, two, three, four or all five cytokines selected from IL-6, IL-17A, CCL2, CCL4, and IL-8. In an embodiment the test is the companion diagnostic test as broadly described herein above.

[0192] EXAMPLES

[0193] Example 1 - Preparation of an A T-column.

[0194] The AT-column was prepared according to the package insert of a 5 ml HiTrap column (GE Healthcare®). After washing the isopropanol from the column ~2.5 mg AT in 5 ml coupling buffer was applied to the column. Then, the described procedure to immobilize the protein to the column material and to wash the column was employed (according to the package insert). Finally the column was equilibrated with 140 mM NaCI, 20 mM Tris (pH 7.4).

[0195] Example 2: Separation of UFH into LAM and HAM.

[0196] To the column was applied 2 mg unfractionated heparin. LAM was eluted with 140 mM NaCI, 20 mM Tris (pH 7.4) and HAM with 2 M NaCI, 20 mM Tris (pH 7.4). The last buffer was applied in a block gradient. In Fig. 1 an example of the elution pattern is shown.

[0197] To obtain a larger amount of LAM, the procedure described in Figure 1 was repeated several times.

[0198] To determine whether the LAM was free of HAM two tests were used. Firstly, collected HAM was reapplied to the AT-column and run as described above. No HAM-peak was found. Secondly the effect of LAM on thrombin generation was measured. The reaction mixture (120 pl) contained normal pooled plasma in a 1.5 x dilution, 3 pl LAM or buffer, 4 pM DOPL (60% DOPC, 20% DOPC and 20% DOPE), 5 pM tissue factor (Innovin), 100 mM CaCI2 and 417 pM ZGGR-AMC. The reaction was started with CaCI2 + ZGGR-AMC. Thrombin generation was measured as described by Hemker, H. C., P. Giesen, et al. (2003). Thrombin generation was not inhibited by the added 3 pl HAM. The column fractions containing LAM were collected. The buffer was switched to ammonium bicarbonate (pH 7.8) with Sephadex G-25 medium and the fractions were lyophilized. Dried LAM was weighed and dissolved in phosphate buffered saline to reach the desired concentration.

[0199]

[0200] Study Overview

[0201] Brief Summary

[0202] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. Mortality is high and survivors frequently suffer from long-term sequelae. Extracellular histones have been identified as essential mediators in the pathogenesis of sepsis and septic shock. These toxic molecules are released by damaged cells in response to infection and high extracellular levels can induce tissue injury and multiple organ dysfunction syndrome. Extracellular histones can be neutralized by complexation with the new candidate drug called M6229, a non-anticoagulant heparin, allowing the use of elevated dose levels relative to regular unfractionated heparin. This project aims at the roll-out of a first-in-man clinical study in sepsis patients evaluating the safety, tolerability, pharmacokinetics and pharmacodynamic effects of intravenously administered M6229 in subjects suffering from sepsis.

[0203] Inclusion Criteria:

[0204] 1. Male or female patients aged > 18 years old.

[0205] 2. Signed informed consent by patient or legal representative.

[0206] 3. Diagnosed with sepsis, defined by the Sepsis-3 criteria as a life-threatening organ dysfunction caused by a dysregulated host response to an infection. Organ dysfunction is defined by 1 of the following:

[0207] a. Increase in SOFA score of >2. i. The baseline SOFA score can be assumed to be zero in patients not known to have pre-existing organ dysfunction.

[0208] b. Acute kidney injury i. Defined as eGFR < 15 mL / min. c. Acute respiratory distress syndrome i. Defined by the Berlin criteria, d. The need of mechanical ventilation, e. Alteration in mental status.

[0209] 4. The patients have to be included in the study within 72 hours of ICU admission due to sepsis or within 72 hours after sepsis diagnosis on the ICU. M6229 has to be administered within 84 hours after ICU admission due to sepsis or within 84 hours after sepsis diagnosis on the ICU.

[0210] Exclusion Criteria:

[0211] 1. Subject has an advance directive to withhold life-sustaining treatments. 2. Subject is breastfeeding or intents to get pregnant within 30 days of enrolling into the study.

[0212] 3. Subject is of childbearing potential and has a positive pregnancy test.

[0213] a. A woman is considered to be of childbearing potential under the age of 60 years, unless surgically sterile.

[0214] 4. Clinical suspicion or confirmation of a viral hemorrhagic shock syndrome including, but not limited to, dengue fever.

[0215] 5. Bleeding risk:

[0216] a. Clinical: i. Active bleeding; ii. Head trauma; iii. Intracranial surgery or stroke in the past 3 months; iv. History of intracerebral arteriovenous malformation, cerebral aneurysm or mass lesions of the central nervous system; v. Cerebral haemorrhage; vi. History of a bleeding diatheses; vii. Gastrointestinal bleeding in the past 6 weeks; viii. Presence of an epidural or spinal catheter; ix. Contraindication for IV therapeutic UFH. b. Laboratory: i. Platelet count <50 x109 / L; ii. INR >2.0; iii. Baseline aPTT >45 seconds prior to enrolment, 1 ,5x upper limit of normal (ULN).

[0217] 6. Use of any of the following treatments:

[0218] 1. UFH to treat a thrombotic event within 12 hours before infusion;

[0219] 2. LMWH within 24 hours before infusion;

[0220] 3. Warfarin (if used within 7 days before study entry AND if the INR exceeds 2.0 at enrolment);

[0221] 4. Direct oral anticoagulant (DOAC) use 3 days prior to enrollment.

[0222] 5. Thrombolytic therapy within 3 previous days;

[0223] 6. Use of llb / llla inhibitors within the previous 7 days.

[0224] 7. Confirmed antiphospholipid syndrome.

[0225] 8. Known allergy to fish.

[0226] 9. Cardiopulmonary resuscitation in the previous 7 days.

[0227] 10. Liver failure defined as Child-Pugh Score Class C.

[0228] 11. Abnormal liver function (ASAT and / or ALAT > 5 times upper limit of normal (ULN)).

[0229] 12. Extracorporeal membrane oxygenation (ECMO) support dependent.

[0230] 13. Pulmonary embolism or clinical suspicion of deep venous thrombosis (DVT).

[0231] 14. Life expectancy of less than 24 hours.

[0232] 15. Treating physician refusal. 16. Known adverse reaction to UFH, including heparin induced thrombocytopenia (HIT).

[0233] 17. Participation in any other investigational drug study or other interventional study with interfering endpoints.

[0234] 18. Any other clinical condition which, in the opinion of the investigator, would not allow safe completion of the protocol.

[0235] Study plan:

[0236] Continuous intravenous infusion of M6229, a low-anticoagulant fraction of heparin. Dose-escalation is based on a modified continual reassessment method (mCRM) including escalation with overdose control (EWOC).

[0237] Outcome Measures

[0238] aPTT changes before, during and after infusion of M6229 [Safety and tolerability]; Anti-coagulation effects of M6229 determined by a change in aPTT at different time points during and after infusion of M6229; Up to 72 hours after start infusion

[0239] Peak plasma concentration (Cmax) [Pharmacokinetics]; Peak plasma concentration of M6229 in plasma; Up to 72 hours after start infusion.

[0240] Steady state concentration (Css) [Pharmacokinetics]; Steady state concentration of M6229 in plasma; Up to 72 hours after start infusion.

[0241] Time to peak concentration (Tmax) [Pharmacokinetics]; Time to peak concentration of M6229 in plasma; Up to 72 hours after start infusion.

[0242] Area under the plasma concentration versus time curve (AUC) [Pharmacokinetics]; Area under the plasma concentration versus time curve of M6229; Up to 72 hours after start infusion.

[0243] Clearance [Pharmacokinetics]; Clearance of M6229; Up to 72 hours after start infusion.

[0244] Terminal half-life (t1 / 2) [Pharmacokinetics]; Terminal half-life is the time required for the plasma concentration of M6229 to fall by 50% during the terminal phase; Up to 72 hours after start infusion.

[0245] Volume of distribution (Vd) [Pharmacokinetics]; Volume of distribution of M6229; Up to 72 hours after start infusion. Histone plasma level changes before, during and after infusion of M6229 [Efficacy]; Change in histone plasma levels before and at different time-points after M6229 administration; Up to 72 hours after start infusion.

[0246] Secondary Outcome Measures

[0247] Incidence of excessive anti-coagulation effects [Safety and tolerability];

[0248] Excessive anti-coagulation effects are:

[0249] Clinical evidence or suspicion of severe non-surgical bleeding, defined as the administration of > 2 units of blood products in 24 hours from start of infusion; aPTT > 90 seconds; Up to 72 hours after start infusion.

[0250] Incidence of adverse reactions [Safety and tolerability]; Adverse reactions that are considered definitely and probably related to M6229 as specified in the protocol; Up to 72 hours after start infusion.

[0251] Changes in ECG corrected QT interval (QTc) [Safety and tolerability]; Changes in ECGs QTc that are considered definitely and probably related to M6229; Up to 24 hours after start infusion.

[0252] Amount of M6229 excreted in urine [Pharmacokinetics]; Urine pharmacokinetic parameters of M6229 (amount of M6229 excreted in urine); Up to 24 hours after start infusion.

[0253] Change in plasma levels of D-Dimer before, during and after M6229 administration [Efficacy]; Change in plasma levels of biomarkers of inflammation, coagulation and fibrinolysis (e.g. D-dimer, IL-6, IL-8) before and at different time-points after M6229 administration; Up to 72 hours after start infusion.

[0254] Change in plasma levels of interleukins before, during and after M6229 administration [Efficacy]; Change in plasma levels of biomarkers of inflammation, coagulation and fibrinolysis (e.g. D-dimer, IL-6, IL-8) before and at different time-points after M6229 administration; Up to 72 hours after start infusion

[0255] Correlation of histone plasma levels and abovementioned biomarkers with M6229 plasma levels (PK / PD) [Efficacy]; Besides histone plasma levels, the investigators will also measure other biomarkers of inflammation, coagulation and fibrinolysis (e.g. D-dimer, IL-6, IL-8); Up to 72 hours after start infusion Severity of organ dysfunction based on Sequential Organ Failure Assessment (SOFA) score [Efficacy]; SOFA scores will be reported. Moreover, the investigators will compare these data with historic controls. For this, data will be used from a subset of patients included in a previously conducted study conducted in two tertiary teaching hospitals in the Netherlands named "Molecular Diagnosis and Risk Stratification of Sepsis" (MARS) study. The MARS study was a prospective observational study performed between January 2011 and January 2014 in the ICUs of the Amsterdam UMC, location AMC and UMC Utrecht; 30 days.

[0256] Time on mechanical ventilation [Efficacy]; Ventilator free-days and time on mechanical ventilation. Data will be compared with historic controls from the MARS cohort; 30 days.

[0257] Time on renal replacement therapy [Efficacy]; Renal replacement therapy free-days and time on renal replacement therapy. Data will be compared with historic controls from the MARS cohort; 30 days.

[0258] Time on vasopression therapy [Efficacy]; Vasopressor free-days and time on vasopressors. Data will be compared with historic controls from the MARS cohort; 30 days.

[0259] Length of stay [Efficacy]; ICU and hospital length of stays. Data will be compared with historic controls from the MARS cohort; 30 days

[0260] Mortality rate [Efficacy]; ICU and hospital mortality. Data will be compared with historic controls from the MARS cohort; 30 days.

[0261] Pentasaccharide depleted heparin was prepared as indicated above. Patients diagnosed with sepsis were administered pentasaccharide depleted heparin in a dosage as indicated below:

[0262] Dose level 1 - patients 2 and 3: 0.15 mg pentasaccharide depleted heparin per kilogram of body weight per hour;

[0263] Dose level 2 - patients 4 and 5: 0.45 mg pentasaccharide depleted heparin per kilogram of body weight per hour;

[0264] Dose level 3 - patients 6-11 : 0.9 mg pentasaccharide depleted heparin per kilogram of body weight per hour;

[0265] dosages were assigned to patients arbitrarily. Administration was performed intravenously by infusion for a duration of 6 hours after diagnosis. Blood samples were taken at the following time points in hours: T=0 (start of pentasaccharide depleted heparin administration), T=3, T=6 (end of pentasaccharide depleted heparin administration), T=24, T=48, T=72. Serum was isolated form the sample for further analysis

[0266] Example 4 - Measurement of the Cytokine Human Magnetic 25-plex Panel for Lu mi nex Platform

[0267] This example describes the results obtained for the first in human trail described above. 10 patients were administered pentasaccharide depleted heparin as described herein in the following dosage regimes: Cohort 1 - patients 2 and 3: 0.15 mg pentasaccharide depleted heparin per kilogram of body weight per hour; Cohort 2 -patients 4 and 5: 0.45 mg pentasaccharide depleted heparin per kilogram of body weight per hour; and Cohort 3 - patients 6-11: 0.9 mg pentasaccharide depleted heparin per kilogram of body weight per hour; All patients were administered for 6 hours.

[0268] Blood samples were collected at different time points to evaluate plasma cytokine values.

[0269] The method employed for measure the cytokines is Cytokine Human Magnetic 25-plex Panel for Luminex Platform. The Human Cytokine Magnetic 25-Plex Panel is designed for the quantitative determination of IL-1p, IL-1RA, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40 / p70), IL-13, IL-15, IL-17A, TNF-a, IFN-a, IFN-y, GM-CSF, MIP-1a, Ml P-1 p, IP-10, MIG, Eotaxin, RANTES, and MCP-1 in human serum, plasma, and tissue culture supernatant.

[0270] The procedure involves the following steps: (1) Add 50 pL of Assay Diluent followed by 50 pL of sample in sample well of the 96-well plate. (2) Cover and incubate the plate for 2 hours at room temperature under agitation. (3) To detect analytes, incubate the plate for 1 hour at room temperature with 100 pL 1X Biotinylated Detector Antibody. (4) After decanting liquid and washing the wells, incubate the plate for 30 minutes at room temperature with 100 pL 1X Streptavidin-RPE solution. (5) To read assay results decant liquid and wash assay well for 2-3 minutes. (6) Uncover the plate and insert the plate into the Luminex Platform instrument and analyze.

[0271] Results

[0272] Plasma samples are obtained from a total of 10 patients (AMC002, AMC003, AMC004, AMC005, AMC006, AMC007, AMC008, AMC009, AMC010, AMC011). Most of patients included samples taken at Oh, 6h, 10h and 24h, except for AMC005 (no sample at 24h) and AMC006 (no sample at 6h).

[0273] In this study, the levels of key cytokines in plasma samples from septic patients over different time points (TO, T6, T10, T24) were evaluated. In the current experiment measured the levels of different cytokines from plasma samples taken at different times in septic patients who were treated with M6229 were measured. Results for individual time points are presented in Figures 1-5 for each of IL-6, IL-17A, CCL2, CCL4, and CXCL8 (IL-8). These results are then combined in Figures 6-10 and presented as percentage change of the respective cytokine with respect to T=0 for each individual patient (top graphs), average change for all patients (middle graphs) and average change for patients receiving the highest dose (bottom graphs).

[0274] IL-6 is one of the most relevant hyperinflammatory cytokines participating in sepsis. Results showed that in most patients IL-6 decreased in all patients from T10, and in some patients (AMC007, AMC008, AMC009, AMC009, AMC10) even atT6. In patients AMC003, AM004, and AMC005 the detected levels of IL-6 are below the detection limit, these results have been disregarded. The results are depicted in Figure 1.

[0275] IL-17A is a pro-inflammatory cytokine produced by Th17 cells which induces the release of many cytokines that we also have been measured such as IL-6 and chemokines (including IL-8 and MCP-1). It was found that generally the levels of IL-17a (CTLA-8) and induced cytokines and chemokines (IL-6 and MCP-1 (CCL2) showed a similar decrease in the levels at 6 and 10 h and recovered to similar levels at longer times, except for IL-6 which decreased from 6h. The results are depicted in Figure 2.

[0276] CCL2 (MCP-1), plays a crucial role in sepsis by recruiting monocytes and macrophages to infection sites, amplifying the inflammatory response, producing more pro-inflammatory cytokines (TNF-a, IL-1, and IL-6), and contributing to organ dysfunction as consequence of its excessive levels. Results indicate the levels of CCL2 decreased in AMC005, AMC007, AMC008, AMC009, and ACM11. The results are depicted in Figure 3.

[0277] CCL4 (MIP-1P) is a chemokine that plays a significant role in the immune response during sepsis. It acts as a mediator in the pro-inflammatory response by attracting immune cells and provoking the release of cytokines and chemokines to amplify the immune response. Results indicate the levels of CCL4 decreased in AMC005, AMC008, AMC009, AMC010, and ACM11. The results are depicted in Figure 4.

[0278] IL-8 (CXCL8) is a pro-inflammatory chemokine that plays a central role in the immune response, particularly during sepsis, where it contributes to the inflammatory cascade and the development of systemic inflammation, endothelial cell activation, which in turn leads to increased vascular permeability and septic shock. The results show how the levels of the pro-inflammatory IL-8 decreased in all patients from T6, except for patient AM C002. The results are depicted in Figure 5.

[0279] Last the datapoints are combined for the different cohorts (Cohort 1 - patients 2 and 3: 0.15 mg pentasaccharide depleted heparin per kilogram of body weight per hour; Cohort 2 - patients 4 and 5: 0.45 mg pentasaccharide depleted heparin per kilogram of body weight per hour; and Cohort 3 - patients 6-11: 0.9 mg pentasaccharide depleted heparin per kilogram of body weight per hour) and plotted in Figures 11-16 for the percentage change of each of IL-6, IL-17, IL-8, MCP-1 (CCL2) and MIP-ip (CCL4) per cohort. Figure 11 combines all three cohorts, Figure 12 cohorts 2 and 3, Figures 13-15 each individual cohort 1, 2, and 3 respectively, and Figure 16 shows only patients diagnosed with Acute respiratory distress syndrome (ARDS) within cohort 3. From these figures it can be concluded that a higher dosage regime, such as 0.45 mg pentasaccharide depleted heparin per kilogram of body weight per hour or even 0.90 mg pentasaccharide depleted heparin per kilogram of body weight per hour is even more beneficial for septic patients, and particularly patients diagnosed with ARDS benefit from treatment with pentasaccharide depleted heparin.

[0280] Acute respiratory distress syndrome was diagnosed in the trail by a physician based on a physical exam (assessment for signs of rapid breathing, fast heart rate, and cyanosis), preferably combined with imaging (X-ray or CT scan of the chest to identify bilateral lung infiltrates and rule out other causes) and / or measurement of blood oxygen levels to assess severity of hypoxia. Example 5 - Clinical Trial Protocol

[0281] A randomized, single-blind, placebo-controlled, study to examine the safety, tolerability, and pharmacokinetics of M6229 administered as a 120-hour continuous infusion at two dose levels versus placebo in healthy subjects.

[0282] OVERVIEW OF STUDY DESIGN

[0283] This is a randomized, single-blind, placebo-controlled study with M6229 and will be conducted in healthy subjects at a single study center. This study will consist of singleblind, randomized, placebo-controlled, IV infusion cohorts, in which the safety, tolerability and PK of M6229 will be assessed when administered continuously for 120 hours. Treatment cohorts will be studied sequentially.

[0284] Subjects

[0285] In this randomized, single-blind, placebo-controlled, ascending IV dose study, up to 2 cohorts of healthy subjects will be studied sequentially. Within each cohort 6 subjects will be randomized to M6229 (N=4) or to matching placebo (N=2). Accordingly, approximately 2 subjects will be enrolled in this study. Each cohort will consist of 3 male and 3 female subjects (two male and two female subjects on M6229 and one male and one female placebo subject).

[0286] Treatments

[0287] Subjects will receive the following fixed treatments (body weight of 70 kg taken as a reference) in a randomized order (Treatment cohorts will be studied sequentially as listed below): Treatment A: a 120-hour continuous infusion with M6229 at 17.5 mg / h or placebo, i.e. saline; Treatment B: a 120-hour continuous infusion with M6229 at 52.5 mg / h or placebo, i.e. saline; Initiation to the high dose cohort (Treatment B) can only occur after review of the data from the low dose cohort (T reatment A) by the Dose Escalation committee (DEC). The dose-escalation decision will be based on preliminary safety and tolerability data, especially focusing on coagulation, collected in at least four subjects (including three subjects on active treatment) of a given dose group for at least 48 hours postdose.

[0288] Screening will take place from Days -28 to -2, prior to the start day of assigned treatment. For each treatment, subjects will be admitted to the clinical site on Day -1. Starting on Day 1, subjects will receive the treatment they are assigned to. Subjects will be discharged from the clinical site on Day 9 provided that all required assessments have been performed, and there are no medical reasons for a prolonged stay. The clinical study will be completed with an end-of-study visit (ESV), which will take place between 5 to 9 days after the last treatment-defined assessment (or after early withdrawal).

[0289] The study duration for each subject is approximately 10 days (Day -1 included, screening and ESV not included). Safety and tolerability will be assessed throughout the study from signing of the informed consent form (IGF) onwards until the subject's last study-related activity. Specific evaluations and their timing can be found in the Time and Events Schedule.

[0290] STUDY DRUGS, FORMULATION, DOSE, AND MODE OF ADMINISTRATION Product: Active ingredient is M6229 which consists of unfractionated heparin depleted of pentasaccharide containing anticoagulant heparin fraction. (anti-Xa < 2 lU / mL). Formulation: Solution for injection of the active ingredient M6229 in a saline citrate buffer, pH 7. Strength: 70 mg / mL. Exact strength may differ per Investigational Medicinal Product (IMP) batch and will be displayed on the label. Vial content: 10 mL in a 25R glass type I vial. Dose: Administration by continuous intravenous (IV) infusion over 120 hours at 17.5 and 52.5 mg / h (i.e. 0.25 and 0.75 mg / kg / h considering a subject with a body weight of 70 kg as a reference).

[0291] Placebo: Saline

[0292] STUDY EVALUATIONS

[0293] Pharmacokinetics

[0294] Serial pharmacokinetic venous blood samples will be collected each period from predose until discharge from the clinical site at selected time points as specified in the Time and Events Schedule and processed, handled and identified following to the laboratory manual. Plasma concentrations of M6229 will be determined during each treatment at pre-dose, during infusion and until 72 hours after stopping the 120h infusion. A full 24-hour continuous urine collection during the last day of infusion (96h-120h across Day 5-6) at steady-state as specified in the Time and Events Schedule. Urine sample will be processed, handled and identified till analysis following the laboratory manual. Citrate plasma samples and urine samples will be analyzed to determine concentrations of M6229 using a validated, specific, and sensitive fluorescence assay. The following key plasma parameters of M6229 will be calculated using non-compartmental methods and actual sampling times on the dosing day: Cmax, tmax, AUCIast, tlast, AUC°°, t1 / 2, Cl and Vd. The following key urine parameters of M6229 will be calculated using non-compartmental methods and actual collection period. For urine samples, the following PK parameters of M6229 will be derived: Ae96-120h, Ae96h-120h %dose and CLR. Other PK parameters may be estimated as appropriate for exploration of the data. Additional parameters may be included if deemed appropriate.

[0295] Safety evaluations

[0296] Safety and tolerability will be evaluated throughout the study from signing of the ICF onwards until the last study-related activity and will include: adverse events (AEs), ECGs, vital signs, clinical laboratory results, and physical examinations (see the Time and Events Schedule for details).

[0297] STATISTICAL METHODS

[0298] Sample Size Determination

[0299] No formal statistical hypothesis testing is planned for this study due to the exploratory and descriptive nature of the study. All subject data will be listed. All analyses will be descriptive, providing summary statistics (number of observations, arithmetic mean, standard deviation, minimum, median and maximum) for quantitative data, and frequency tables (absolute and relative frequencies) for qualitative / ordinal data. All placebo subjects will be pooled across dose cohorts. A more technical and detailed elaboration of the statistical analysis will be included in a separate Statistical Analysis Plan (SAP). The sample size of 4 subjects receiving M6229 and 2 receiving placebo per dose cohort is a customary sample size employed in early development studies of similar design and objectives, and it is expected to allow clinical judgment of safety and tolerability and assessment of the PK profile. Two subjects receiving placebo per dose cohort should be sufficient to allow judgment of safety and tolerability at the two M6229 dose levels. If a subject withdraws prior to dosing, an additional subject will be enrolled as a replacement. Pharmacokinetics

[0300] Descriptive statistics will be calculated for the plasma and urine concentrations of M6229 and for the derived PK parameters, as applicable. Statistics will include sample size, mean, median, minimum, maximum, standard deviation (SD), percent coefficient of variation, and geometric mean. For each subject, plasma concentration-time data will be graphically presented. Similarly, graphs of the mean plasma concentration-time profiles and overlay graphs with combined individual plasma concentration-time profiles will be produced. Plasma and urine pharmacokinetic parameters will be subjected to an exploratory graphical analysis including various transformations in order to get a general overview. Additional analyses, may be performed if deemed necessary.

[0301] Safety

[0302] All subjects who received a dose, independent of the duration of dosing, will be included in the safety and tolerability analysis. Safety will be evaluated by examining the incidence and type of adverse events, and by evaluating the clinical laboratory test values, physical examination results, 12-lead ECGs, and vital signs measurements, (see the Time and Events Schedule for details).

[0303]

[0304] Upon completion of the trial on healthy volunteers without adverse effects M6229 will be administered to patients with sepsis by continuous intravenous (IV) infusion over 120 hours at 17.5 and 52.5 mg / h (i.e. 0.25 and 0.75 mg / kg / h considering a subject with a body weight of 70 kg as a reference).

[0305]

[0306] A clinical trails was conducted in healthy individuals to test different dosages of M6229 in healthy individuals by continuous intravenous (IV) infusion over 120 hours. Dose levels are based on the assumption of an average body weight of 70 kg.

[0307] Investigational medicinal product: M6229

[0308] Formulation and route of administration: Solution for injection of the active ingredient M6229 in a saline citrate buffer, pH 7; continuous IV infusion over 120 hours - Dose:

[0309] Treatment A: 17.5 mg / h or placebo

[0310] Treatment B: 52.5 mg / h or placebo

[0311] Treatment C: 25 mg / h or placebo

[0312] - Batch number: 21F005F01; 24B005F01

[0313] Reference therapy, dose and mode of administration, batch number:

[0314] Comparator: Saline (Placebo)

[0315] - Formulation and route of administration: 0.9% w / v Sodium Chloride; continuous IV infusion over 120 hours

[0316] - Dose: not applicable

[0317] - Batch number: 24027453

[0318] Duration of Treatment:

[0319] Treatment A: All 6 subjects received a 120-hour continuous IV infusion with M6229 or placebo.

[0320] Treatment B: 3 subjects received a continuous IV infusion with M6229 or placebo for less than 24 h.

[0321] Treatment C: All 6 subjects received a 120-hour continuous IV infusion with M6229 or placebo.

[0322] Criteria for Evaluation:

[0323] Safety

[0324] All standard safety assessments including physical examination, vital signs, adverse events (AEs), hematology, chemistry, coagulation, urinalysis, immunogenicity and ECG.

[0325] Pharmacokinetics

[0326] Plasma parameters of M6229: Cmax, tmax, AUCIast, tlast, AUC , t1 / 2, Cl and Vd. Urine parameters of M6229: Ae96-i20h, Ae96h-i20h %dOse, and CLR.

[0327] Summary Conclusions:

[0328] Safety Results:

[0329] When M6229 was administered at low dose (17.5 mg / h) 3 AEs reported in 3 subjects were judged as related to the study drug. Two of them (abdominal distension and piercing associated complication) were of common terminology criteria for adverse event (CTCAE) grade 1 and 1 (increase in alanine aminotransferase [ALT]) was of CTCAE grade 2. The increase in ALT (below 5 x upper limit of normal [ULN]) was reversible and not accompanied by any other indicators of hepatocellular toxicity and most likely due to its glycosaminoglycan structure typical for unfractionated heparin. Treatment with unfractionated heparin or low molecular weight heparins is associated with frequent increases in serum aminotransferase levels, which are usually transient and not associated with clinical symptoms or significant liver injury. With intermediate dose (25 mg / h), 3 AEs in 3 subjects were reported and judged as related to the study drug. Two AEs were CTCAE grade 1 (intermenstrual bleeding, epistaxis) and the other AE was CTCAE grade 2 (aPTT prolongation), which met a pre-defined AESI criterion (aPTT increase above 2.5 times baseline value). No stopping criteria were met and all subjects in the intermediate dose group completed the study. In the high dose (52.5 mg / h) cohort 2 AEs were reported in 2 subjects, which constituted adverse events of special interest (AESIs) and subsequently met the individual withdrawal criterion (aPTT prolongation > 85 sec). Both subjects were prematurely withdrawn from treatment. Both aPTT prolongations were judged as CTCAE grade 2 (moderate) in intensity and related to M6229. aPTT increase was not accompanied by any sign of spontaneous bleeding. After stop of infusion, aPTT turned back to the normal range within 6 to 8 h.

[0330] Pharmacodynamic Results:

[0331] Plasma anti-factor Xa activity remained below the limit of quantification (<0,10 U / rnL) by the end of the 120- hour M6229 infusion at any of the dose levels. No clinically relevant increases in prothrombin time (I NR) were observed in any of the 3 treatment cohorts.

[0332] Immunogenicity results:

[0333] Three out of 4 subjects in both, the 17.5 mg / h and the 25 mg / h cohort showed positive results for anti-PF4-heparin complex, IgG, IgM and IgA at the ESV. However, this finding was not accompanied by thrombocytopenia or signs or symptoms of thrombosis, confirmed by negative heparin induced platelet activation (HIPA) and PF4-induced platelet activation assay (PIPA) testing.

[0334] No serious adverse events (SAEs) and no deaths occurred in the study. There were no apparent trends or doserelated changes in hematology, biochemistry, urinalysis, physical examinations, vital signs or ECG intervals in any of the subjects.

[0335] Key Pharmacokinetic Results: Full PK profiles were obtained after administration of M6229 as a 120-hour IV infusion at dose levels of 17.5 mg / h and 25.0 mg / h, whereas for the 52.5 mg / h infusion, the aPTT exceeding the maximal acceptable value (85 sec) resulted in a premature termination of the infusion.

[0336] M6229 showed predictable PK behavior with close to dose-proportional systemic exposure expressed as Cmax and AUC. During the 120-h infusion, steady state conditions were virtually reached within 24 h. Under steady state conditions, mean M6229 plasma concentrations were reported between 5.26 pg / mL and 5.91 pg / mL at the low dose and between 8.08 pg / mL and 9.51 pg / mL at the intermediate dose level. M6229 levels were on average quantifiable until 4 h to 8 h after the end of the infusion. Less than 5% of the M6229 dose was excreted unchanged into urine, at steady state. Combining data from all three cohorts, a positive correlation was observed for the aPTT value versus M6229 plasma concentration, both during and after infusion. Conclusions:

[0337] Clinical findings observed during and after a continuous IV infusion for 120 h of M6229 at dose levels of 17.5 mg / h, 25 mg / h, and 52.5 mg / h include mainly asymptomatic transaminase increases which is in line with what has been described for heparin. The observed elevations in ALT did not qualify as drug-induced liver injury (DILI) and regressed after discontinuation of M6229 infusion. A time-and dose-dependent aPTT prolongation was observed under M6229 infusion over a period of 120 h. Increases of aPTT between 1.5 to 2.5x fold above baseline values may be expected during infusion of M6229 at low (17.5 mg / h) and intermediate (25 mg / h) dose levels. Significant increases of aPTT (>85 sec) were seen during infusion of M6229 at high dose level at exposure times beyond 6 h. However, all observed changes in aPTT spontaneously returned to within normal limits in less than 24 h after stopping the infusion. Although aPTT was affected, no effect of M6229 administration on plasma anti-factor Xa activity, and no clinically relevant increases in prothrombin time (INR) were observed in any of the 3 treatment cohorts, excluding significant bleeding risk at this M6229 exposure level. At therapeutic doses of heparin, its major anticoagulant effect is binding to ATI 11 and inactivation of factors Ila, Xa, IXa, and XI la. At high heparin concentrations, it binds to heparin cofactor II and catalyzes inactivation of factor Ila. It remains to be elucidated whether the high dose of M6229 (52.5 mg / h) may still promote binding to heparin cofactor II and catalysis of factor Ila, which could potentially explain the observed increases in aPTT. The clinical relevance of these aPTT increases is not clear since they were not associated with spontaneous bleeding signs and M6629 does not show any anti-factor Xa activity. Since antifactor Xa activity is responsible for the anticoagulant effect of low molecular weight heparins (LMWH) and also significantly contributes to the anticoagulant effect of UFH, it is difficult to adequately assess to what extent coagulation is impacted by this residual effect of M6229 on aPTT. Therefore, careful monitoring of aPTT and bleeding signs is recommended during future M6229 treatment. At the dose-ranges and time-windows used, M6229 did not lead to immunogenic safety problems like platelet activation, thrombocytopenia or thrombosis. Furthermore, no spontaneous bleeding or thrombosis were reported in any participant.

[0338] No SAEs and no deaths occurred in the study. There were no apparent trends or dose-related changes in hematology, biochemistry, urinalysis, physical examinations, vital signs or ECG intervals in any of the subjects.

[0339] Example 8

[0340] The following clinical study will be conducted in septic patients and is expected to result in significant improvement of patient outcomes, as well as significant reduction of cytokine levels as well as other parameters as defined in the primary and secondary endpoints. Dose levels are based on the assumption of an average body weight of 70 kg.

[0341] SYNOPSIS

[0342]

[0343]

[0344]

[0345] > >

[0346]

[0347]

[0348] REFERENCES

[0349] 1. Singer et al: The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). Jama 2016, 315(8):801-810.

[0350] 2. Cecconi et al.: Sepsis and septic shock. Lancet 2018, 392(10141):75-87.

[0351] 3. Gotts et al.: Sepsis: pathophysiology and clinical management. Bmj 2016, 353:i1585.

[0352] 4. Rudd et al: Global, regional, and national sepsis incidence and mortality, 1990-2017: analysis for the Global Burden of Disease Study. Lancet 2020, 395(10219):200-211.

[0353] 5. Prescott et al.: Enhancing Recovery From Sepsis: A Review. Jama 2018, 319(1):62-75.

[0354] 6. Marino-Ramirez et al.: Histone structure and nucleosome stability. Expert Rev Proteomics 2005, 2(5):719-729.

[0355] 7. Chaput et al.: Sepsis: the dark side of histones. Nat Med 2009, 15(11):1245-1246.

[0356] 8. Cheng et al.: Circulating Histones Are Major Mediators of Multiple Organ Dysfunction Syndrome in Acute Critical Illnesses. Crit Care Med 2019, 47(8):e677-e684.

[0357] 9. Wildhagen et al.: Extracellular histone H3 levels are inversely correlated with antithrombin levels and platelet counts and are associated with mortality in sepsis patients. Thromb Res 2015, 136(3):542-547.

[0358] 10. Huckriede et al.: Histone H3 Cleavage in Severe COVID-19 ICU Patients. Frontiers in Cellular and Infection Microbiology 2021 , 11(846).

[0359] 11. Cavalier et al.: Circulating Nucleosomes as Potential Markers to Monitor COVID-19 Disease Progression. Front Mol Biosci 2021, 8:600881-600881.

[0360] 12. Shaw et al: Circulating histones play a central role in COVID-19-associated coagulopathy and mortality. Haematologica 2021, 106(9):2493-2498.

[0361] 13. Wang et al.: Heparin therapy reduces 28-day mortality in adult severe sepsis patients: a systematic review and meta-analysis. Crit Care 2014, 18(5):563.

[0362] 14. Gould et al.: Extracellular Histones Increase Tissue Factor Activity and Enhance Thrombin Generation by Human Blood Monocytes. Shock 2016, 46(6):655-662.

[0363] 15. Zarychanski et al: The efficacy and safety of heparin in patients with sepsis: a systematic review and metaanalysis. Crit Care Med 2015, 43(3):511-518. 16. Fan et al.: Efficacy and safety of low-molecular-weight heparin in patients with sepsis: a meta-analysis of randomized controlled trials. Sci Rep 2016, 6:25984. 17. Investigator’s Brochure M6229, Version 3.0, 2024

[0364] 18. Wildhagen et al.: Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis. Blood 2014, 123(7):1098-1101.

[0365] 19. Korninger et al.: Studies on the specific fibrinolytic effect of human extrinsic (tissue-type) plasminogen activator in human blood and in various animal species in vitro. Thrombosis and haemostasis 1981, 46(2):561-565.

[0366] 20. Chousterman et al., Semin Immunopathol. 2017 Jul;39(5):517-528.

Claims

69CLAIMS1. Pentasaccharide depleted heparin for use in the treatment or prevention of sepsis, SIRS, septic shock, or acute respiratory distress syndrome (ARDS) in a human subject, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject, wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours, andwherein the use comprises reducing the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject.

2. Pentasaccharide depleted heparin for use according to claim 1, wherein the level of one or more of IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced to 80% or less, preferably 70%, 60%, or even 50% or less, when compared to just before the start of the administration of the pentasaccharide depleted heparin, preferably wherein the reduction is observed in 2 or more hours, preferably 4 or more or even 6 or more hours after onset of pentasaccharide depleted heparin administration.

3. Pentasaccharide depleted heparin for use according to any claim 1 or 2, wherein one or more of the following is / are achieved:- production of one or more of acute-phase proteins selected from C-reactive protein (CRP), fibrinogen, and serum amyloid A by hepatocytes is reduced;- production and / or recruitment of neutrophils is reduced;- lymphocyte activation is reduced;- B cell proliferation is reduced;- release of one or more cytokines selected from IL-6, G-CSF, GM-CSF, I L-1 p, TGF-p, TNF-a, IL-8, GRO-a and MCP-1 is reduced;- inflammation is decreased;- production and / or recruitment of granulocytes is reduced; and- production and / or recruitment of monocytes, macrophages and / or lymphocytes is reduced.

704. Pentasaccharide depleted heparin for use according to any one of the preceding claims, wherein the level of two or more, preferably three, four, or all five cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced, preferably wherein the level of IL-6 and one or more, preferably two, three, or all four of cytokines, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced.

5. Pentasaccharide depleted heparin for use according to any one of the preceding claims, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.45 mg / (kg*hr) body weight of the subject, preferably wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.9 mg / (kg*hr) body weight of the subject, and / or wherein the pentasaccharide depleted heparin is administered parenterally, preferably wherein the pentasaccharide depleted heparin is administered intravenously or subcutaneously.

6. Pentasaccharide depleted heparin for use according to any one of the preceding claims, wherein the pentasaccharide depleted heparin is administered over a period of at least 48 hours, preferably at least 60, at least 72, at least 96, more preferably at least 120 hours or longer.

7. Pentasaccharide depleted heparin for use according to any one of the preceding claims, wherein the heparin has been depleted of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 98%, 99% or even 100% of the pentasaccharide.

8. Pentasaccharide depleted heparin for use in the treatment or prevention of a disease in a human subject by reducing IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in the subject, the use comprising administering the pentasaccharide depleted heparin to the subject in need thereof,wherein the disease is selected from: sepsis, SIRS, septic shock, acute respiratory distress syndrome (ARDS), cancer, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), COVID-19, Idiopatic Pulmonary Fibrosis (IPF), inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, Chronic obstructive pulmonary disease (COPD), psoriasis, psoriatic arthritis, asthma, multiple sclerosis (MS), ankylosing spondylitis, pancreatitis, abdominal aortic aneurism,71atherosclerosis, (rheumatoid) arthritis, cardiovascular disease, type 2 diabetes mellitus, obesity, chronic kidney disease, diabetic nephropathy, HIV, age related macular degeneration, haemorrhagic stroke, Alzheimer’s disease, Parkinson’s disease, severe antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, severe systemic lupus erythematosus with kidney involvement (Lupus Nephritis), thrombotic microangiopathy (TMA), pre-eclampsia, and ischemia-reperfusion in kidney transplantation and cardiothoracic surgery, preferably wherein the cancer is selected from multiple myeloma, breast cancer, lung cancer, gastric cancer, pancreatic cancer, colorectal cancer, and prostate cancer; andwherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject and wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours.

9. Pentasaccharide depleted heparin for use according to claim 8, wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.45 mg / (kg*hr) body weight of the subject, preferably in a dose of at least 0.9 mg / (kg*hr) body weight of the subject.

10. Pentasaccharide depleted heparin for use according to claim 8 or 9, wherein the pentasaccharide depleted heparin is administered over a period of at least 48 hours, preferably at least 60, at least 72, at least 96, more preferably at least 120 hours or longer.

11. Pentasaccharide depleted heparin for use according to any one of claims 8 to 10, wherein the pentasaccharide depleted heparin is administered parenterally preferably wherein the pentasaccharide depleted heparin is administered intravenously or subcutaneously.

12. Pentasaccharide depleted heparin for use according to any one of claims 8 to 11, wherein the level of one or more of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced to 80% or less, preferably 70%, 60%, or even 50% or less, when compared to just before the start of the administration of the pentasaccharide depleted heparin,72preferably wherein the reduction is observed in 2 or more hours, preferably 4 or more or even 6 or more hours after onset of pentasaccharide depleted heparin administration,preferably wherein the level of two or more, preferably three, four, or all five of the cytokines IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced,more preferably wherein the level of IL-6 and one or more, preferably two, three, or all four of the cytokines IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject is reduced.

13. Pentasaccharide depleted heparin for use according to any one of claims 8 to 12, wherein the pentasaccharide depleted heparin is administered to the subject when one or more, preferably least two, three, four, five, or all six, of the following conditions is / are met:the amount of IL-6 in the serum exceeds 5 pg / mL;the amount of IL-8 in the serum exceeds 10 pg / mL;the amount of IL-17A in the serum exceeds 5 pg / mL;the amount of CCL2 in the serum exceeds 5 pg / mL; orthe amount of CCL4 in the serum exceeds 5 pg / mL.

14. Pentasaccharide depleted heparin for use in reducing one or more of IL-6, IL-8, IL-17A, CCL2, and / or CCL4 serum levels in a human subject in the treatment of sepsis,wherein the pentasaccharide depleted heparin is administered to the subject in a dose of at least 0.15 mg / (kg*hr) body weight of the subject and wherein the pentasaccharide depleted heparin is administered over a period of at least 5 hours.

15. Pentasaccharide depleted heparin for use according to claim 14, wherein the method comprises monitoring the levels of one or more of IL-6, IL-8, IL-17A, CCL2, and CCL4 in the serum of the subject, and administering the pentasaccharide depleted heparin when one or more, preferably least two, three, four, or all five, of the following conditions is / are met:the amount of IL-6 in the serum exceeds 5 pg / mL;73the amount of IL-8 in the serum exceeds 10 pg / mL; the amount of IL-17A in the serum exceeds 5 pg / mL; the amount of CCL2 in the serum exceeds 5 pg / mL; or the amount of CCL4 in the serum exceeds 5 pg / mL.

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