Drugs for treating sepsis and hypercytokinemia

Abstract

The present invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, in the treatment of sepsis and hypercytokinemia.

Description

[Technical Field]

[0001] The present invention relates generally to therapeutic agents for treating sepsis and hypercytokinemia, and more particularly to the use of dextran sulfate, or a pharmaceutically acceptable salt thereof, in the treatment of sepsis and hypercytokinemia. [Background technology]

[0002] Sepsis is a life-threatening condition that occurs when the body's response to infection causes injury to its own tissues and organs. Sepsis is an inflammatory immune response triggered by infection. Bacterial infection is the most common cause, but fungal, viral, and protozoal infections can also lead to sepsis. Common sites of primary infection include the lungs, brain, urinary tract, skin, and abdominal organs.

[0003] Hypercytokinemia, also known as cytokine storm, is a physiological response in which the innate immune system causes an uncontrolled and excessive release of proinflammatory cytokines. Hypercytokinemia can be caused by many infectious and non-infectious causes, especially viral infections.

[0004] Cytokines are regulators of the immune response to infection and play an important role in controlling inflammation and injury. Proinflammatory cytokines stimulate systemic inflammation, while anti-inflammatory cytokines inhibit inflammation and enhance healing. Key inflammatory cytokines controlling the early response in sepsis include interleukin-1α (IL-1α), IL-1β, IL-6, and tumor necrosis factor-α (TNFα). Proinflammatory cytokines act as endogenous pyrogens, upregulate the synthesis of secondary mediators and other inflammatory cytokines by both macrophages and mesenchymal cells, such as fibroblasts, epithelial and endothelial cells, stimulate the production of acute-phase proteins, or attract inflammatory cells.

[0005] Sepsis is characterized by the excessive production of cytokines in the circulation, leading to a cytokine storm (hypercytokinemia) and a systemic inflammatory response. Therefore, it has been suggested that inhibiting excessive cytokine production or removing cytokines and other inflammatory mediators from the blood may suppress systemic inflammation during sepsis and hypercytokinemia and improve patient outcomes.

[0006] Corticosteroids have been used to treat sepsis by suppressing cytokine production. The main disadvantage of corticosteroids is their broad effects, generally suppressing cytokine production, both pro- and anti-inflammatory cytokines. In addition, the suppression can be difficult to control, sometimes leading to cytokine levels that are too low, thereby preventing the patient's immune system from fighting the infection that is the underlying cause of sepsis or hypercytokinemia.

[0007] Another group of drugs suggested for treating sepsis are neutralizing antibodies. The drawback of this group of drugs is their specificity in targeting a single cytokine. Thus, the activity of other cytokines may remain untouched or may actually increase due to compensatory mechanisms. Another disadvantage of antibodies is their relatively long half-life in the body. As a result, their cytokine-blocking effect may last for a relatively long period of time, which may actually be too long for the patient's immune system to effectively combat the infection that causes sepsis or hypercytokinemia.

[0008] Thus, there remains a need for effective treatments for sepsis and hypercytokinemia. Summary of the Invention

[0009] The aim is to provide a drug for treating sepsis.

[0010] Another objective is to provide a therapeutic agent for hypercytokinemia.

[0011] These and other objects are achieved by the embodiments disclosed herein.

[0012] Aspects of the present invention relate to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the treatment of sepsis.

[0013] Another aspect of the present invention relates to dextran sulfate, or a pharmaceutically acceptable salt thereof, for use in the treatment of hypercytokinemia.

[0014] Dextran sulfate or a pharmaceutically acceptable salt thereof can suppress inflammatory cytokines in selected immune cells. This means that dextran sulfate or a pharmaceutically acceptable salt thereof prevents or at least significantly inhibits the increased levels of these cytokines in the circulating blood, which causes a cytokine storm and a systemic inflammatory response in patients. Cytokine suppression achieved by dextran sulfate or a pharmaceutically acceptable salt thereof still allows the patient's immune system to remain active and fight infection, the underlying cause of sepsis or hypercytokinemia.

[0015] Some embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0016] [Figure 1]PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: (1A) LPS (0.01 ng / ml), (1B) peptidoglycan (30 ng / ml), (1C) pokeweed mitogen (1.0 μg / ml), (1D) PHA-L (1.0 μg / ml), (1E, 1F) CpG (0.2 μM or 1.0 μM) + IL-15 (15 ng / ml), or (1G, 1H) cytostim (10 μl / ml or 30 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at either 60 μg / ml, 200 μg / ml, or 600 μg / ml). IL-6 levels in the supernatants were quantified by ELISA. Data are presented as the mean ± SEM obtained from 6–12 (LPS) donors. Data are plotted as percentage stimulation + vehicle. (-) indicates at least one donor was below the limit of detection. **Mann-Whitney U test comparison between vehicle + LPS and 600 μg / ml ILB™ + LPS, p=0.005. *Mann-Whitney U test comparison between vehicle + PHA-L and 600 μg / ml ILB™ + PHA-L, p=0.048. [Figure 2] Monocytes purified from PBMCs were stimulated with either LPS (0.01 ng / ml) or peptidoglycan (30 ng / ml) in the absence of stimuli (medium) or with LPS (0.01 ng / ml) or peptidoglycan (30 ng / ml). Then, they were cultured for 24 hours in the presence or absence (vehicle) of LMW-DS (ILB™; 60 μg / ml, 200 μg / ml, or 600 μg / ml) (2A, 2D), dexamethasone (3.0 μM) (2B, 2E), or heparin (2.0 μg / ml, 6.0 μg / ml, or 20 μg / ml) (2C, 2F). IL-6 levels in cell culture supernatants were quantified by ELISA. Data are presented as mean ± SEM, n = 10. * indicates values ​​below the detection limit (5 pg / ml). + P < 0.05, +++ P < 0.001 for significant differences relative to stimulation (Mann-Whitney U test). [Figure 3]PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60 μg / ml, 200 μg / ml, or 600 μg / ml). Interferon-gamma (IFNγ) levels in the supernatants were quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, and (*) indicates data from 6 donors. Compared to vehicle stimulation: #P<0.05, ##P<0.01, ###P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 4]PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60 μg / ml, 200 μg / ml, or 600 μg / ml). Interleukin-8 / chemokine (C-X-C motif) ligand 8 (IL-8 / CXCL8) in the supernatants was quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, and (*) indicates data from 6 donors. Compared to vehicle stimulation: #P<0.05, ##P<0.01, ###P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 5]PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60 μg / ml, 200 μg / ml, or 600 μg / ml). Tumor necrosis factor alpha (TNFα) levels in the supernatants were quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, and (*) indicates data from 6 donors. Compared to vehicle stimulation: #P<0.05, ##P<0.01, ###P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 6] PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60 μg / ml, 200 μg / ml, or 600 μg / ml). IL-1β levels in the supernatants were quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, and (*) indicates data from 6 donors. Compared to vehicle stimulation: #P<0.05, ##P<0.01, ###P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 7] PBMCs were cultured for 24 hours in the absence (medium, unstimulated) or presence of stimuli: LPS (0.01 ng / ml), peptidoglycan (30 ng / ml), PHA-L (1.0 μg / ml), CpG (0.2 μM) + IL-15 (15 ng / ml), pokeweed mitogen (1.0 μg / ml), or Cytostim (10 μl / ml) + vehicle (0.027% saline) or LMW-DS (ILB™ at 60 μg / ml, 200 μg / ml, or 600 μg / ml). IL-10 levels in the supernatants were quantified by Luminex. Unless otherwise indicated, data are presented as percentage stimulation + vehicle and mean ± SEM from 12 donors. (-) indicates at least one replicate was below the limit of quantification, (+) indicates at least one replicate was above the limit of quantification, (^) indicates data from 11 donors, and (*) indicates data from 6 donors. Compared to vehicle stimulation: #P<0.05, ##P<0.01, ###P<0.001; NS indicates no significant difference (Mann-Whitney test, two-tailed). [Figure 8] Serum levels of IL-6 after administration of LMW-DS (5, 10, and 24 weeks) as a percentage of pre-LMW-DS levels. Data presented as a percentage of cytokine levels from pre-LMW-DS treatment. Data presented as mean ± SEM of 8 patients. +P<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention relates generally to therapeutic agents for treating sepsis or hypercytokinemia, and in particular to the use of dextran sulfate, or a pharmaceutical salt thereof, in the treatment of sepsis or hypercytokinemia.

[0018] Traditionally, sepsis is defined as a systemic inflammatory response syndrome (SIRS) in response to an infectious process. SIRS is the presence of two or more of the following: abnormal body temperature (<36°C or >38°C), abnormal heart rate (>90 beats / min), abnormal respiratory rate (>20 breaths / min), or abnormal blood gases (PaCO2 <32 mmgH), and white blood cell (WBC) count (<4000 / mm 3 or >12,000 / mm 3 Severe sepsis is defined as sepsis accompanied by sepsis-induced organ failure or tissue hypoperfusion (manifested as hypotension, elevated lactate, or decreased urine output). Severe sepsis is an infectious disease state associated with multiple organ dysfunction syndrome (MODS). Septic shock is severe sepsis plus persistent hypotension despite the administration of intravenous fluids.

[0019] Sepsis is characterized by the excessive production of cytokines in the circulating blood, which causes a cytokine storm (hypercytokinemia) and leads to a systemic inflammatory response. Therefore, it has been suggested that inhibiting excessive cytokine production or removing cytokines and other inflammatory mediators from the blood may suppress the cytokine storm and systemic inflammation during sepsis and improve patient outcomes. However, one challenge in sepsis and hypercytokinemia is how to target components of the response without causing long-term or complete immunosuppression.

[0020] Hypercytokinemia, or cytokine storm, is a physiological response in humans and other animals in which the innate immune system triggers an uncontrolled, excessive release of proinflammatory cytokines. Cytokine storms can be triggered by many infectious and noninfectious etiologies, particularly viral respiratory infections, such as influenza A virus subtype H5N1, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), and SARS-CoV-2. Other causative pathogens include Epstein-Barr virus, cytomegalovirus, and group A streptococcus, as well as noninfectious conditions such as graft-versus-host disease. Viruses can invade lung epithelial cells and alveolar macrophages, producing viral nucleic acids that stimulate infected cells to release cytokines and chemokines, activating macrophages and dendritic cells, among others, and releasing additional cytokines, resulting in a cytokine storm.

[0021] Dextran sulfate, or a pharmaceutically acceptable salt thereof, can selectively reduce levels of inflammatory cytokines, particularly interleukin 6 (IL-6), but may also reduce tumor necrosis factor alpha (TNFα), IL-1β, IL-8, and interferon gamma (IFNγ). This suppression of cytokine levels is particularly induced in Toll-like receptor (TLR) 4-activated monocytes, macrophages, and microglia (myeloid cells). Such TLR4-based activation of myeloid cells is important in bacterial infections, including those caused by gram-negative bacteria, because TLR4 constitutes the sensing receptor for gram-negative lipopolysaccharide (LPS). Other TLR4 ligands include the F protein of syncytial virus, mannuronic acid from Gram-negative bacteria, teichuronic acid from Gram-positive bacteria, Chlamydia pneumoniae HSP60, flavolipin from Flavobacterium meningosepticum, mannan from Saccharomyces cerevisiae and Candida albicans, and the dengue virus NS1 protein. Activation of TLR4 in myeloid cells is seen in patients with cytokine storm and sepsis.

[0022] Thus, reducing inflammatory cytokines with dextran sulfate, or a pharmaceutically acceptable salt thereof, in patients suffering from infectious disease, hypercytokinemia and / or sepsis leads to suppression of the cytokine storm and systemic inflammation during sepsis, improving patient outcomes.

[0023] Dextran sulfate, or a pharmaceutically acceptable salt thereof, offers advantages over current drugs used to treat sepsis and hypercytokinemia, such as corticosteroids and neutralizing anti-cytokine antibodies.

[0024] First, corticosteroids generally have a broad effect of suppressing cytokine production, both pro- and anti-inflammatory cytokines. Furthermore, the suppression is nearly complete, as shown in Figures 2B and 2E. However, such comprehensive immunosuppression may actually be harmful to patients with sepsis, as it significantly inhibits the immune system, thereby reducing its effectiveness in combating the infectious disease that is the underlying cause of hypercytokinemia or sepsis.

[0025] The experimental data presented herein demonstrate that dextran sulfate, or a pharmaceutically acceptable salt thereof, does not completely block cytokine activity and, moreover, does not suppress cytokine levels from all cells of the immune system (Figures 1A-1H, 3-7). This means that dextran sulfate, or a pharmaceutically acceptable salt thereof, may be useful in treating hypercytokinemia or sepsis by reducing inflammatory cytokine levels and, at the same time, enabling activation of the immune system to fight infection, the underlying cause of hypercytokinemia or sepsis.

[0026] For example, experimental data show that dextran sulfate can suppress IL-6 cytokine production by activated cells in the innate immune system, particularly myeloid cells, such as monocytes and macrophages (Figure 1A), while not significantly affecting IL-6 cytokine production by activated cells in the adaptive immune system, such as B lymphocytes (Figures 1B, 1C, 1F, 1H).

[0027] Furthermore, the IL-6 suppressive effect of dextran sulfate or its pharmaceutically acceptable salts is not a general effect among sulfated polysaccharides. Experimental data presented herein showed that heparin, another sulfated polysaccharide, did not significantly suppress IL-6 levels in LPS-activated monocytes but rather increased the levels of this proinflammatory cytokine (Figure 2C).

[0028] Dextran sulfate, or a pharmaceutically acceptable salt thereof, also has an advantage over neutralizing antibodies that have been suggested as therapeutic agents for treating sepsis. Such neutralizing antibodies have the disadvantage that they target only a single cytokine, thereby preventing the activity of other cytokines. , meaning that the cytokine activity of antibodies may remain untouched or may actually increase due to compensatory mechanisms. Another disadvantage of antibodies is their relatively long half-life in the body (up to 2 weeks). As a result, their cytokine activity blocking effect may last for a relatively long period of time, which may in fact be too long for the patient's immune system to successfully combat infections that induce hypercytokinemia or sepsis. Dextran sulfate, or its pharmaceutically acceptable salts, has a relatively much shorter half-life (C max is approximately 2-3 hours in humans), thus simplifying dosing and administration to achieve cytokine suppression for a well-defined period, if required, while allowing the immune system to fight the infection inducing hypercytokinemia or sepsis as soon as the acute septic phase has passed.

[0029] IL-1β, also known as leukocyte pyrogen, leukocyte-intrinsic mediator, monocyte factor, lymphocyte-activating factor, or catabolin, is produced by activated macrophages as a proprotein, which is proteolytically processed by caspase-1 to its active form. IL-1β is a key mediator of the inflammatory response and is involved in various cellular activities, such as cell proliferation, differentiation, and apoptosis. IL-1β has been reported to play a role in sepsis, and it is persistently elevated in patients who die from sepsis (Mera et al., Multiplex cytokine profiling in patients with sepsis, APMIS, 119(2): 155-163 (2011)).

[0030] Dextran sulfate, or a pharmaceutically acceptable salt thereof, induced a reduction in LPS-stimulated secretion of IL-1β (FIG. 6).

[0031] IL-6, also known as interferon-β2 and B-cell stimulating factor-2 (BSF-2), is a pleiotropic interleukin that functions as both a pro- and anti-inflammatory cytokine. IL-6 is secreted by T cells and macrophages and stimulates the immune response to tissue injury, leading to inflammation. IL-6 is also secreted by macrophages in response to specific microbial molecules called pathogen-associated molecular patterns (PAMPs) that bind to pattern recognition receptors (PRRs), such as toll-like receptors (TLRs). IL-6 production is elevated in patients with sepsis (Mera et al., (2011); Gouel-Cheron et al., Early interleukin-6 and slope of monocyte human leukocyte antigen-DR: A powerful association to predict the development of sepsis after major trauma, PloS one, 7(3): e33095 (2012)), suggesting that IL-6 is associated with the development of sepsis. Furthermore, IL-6 levels are higher in patients with septic shock than in those without, and are even higher in those who die from severe sepsis (Wu et al., Serial cytokine levels in patients with severe sepsis, Inflammation Research, 58(7): 385-393 (2009)), suggesting that IL-6 is an important cytokine in the pathophysiology of severe sepsis. In addition, increased levels of IL-6 have been found to be associated with the highest risk of death in septic patients (Kellum et al., Understanding the inflammatory cytokine response in pneumonia and sepsis: Results of the Genetic and Inflammatory Markers of Sepsis (GenIMS) Study, Archives of Internal Medicine, 167(15): 1655-1663 (2007)).Among the cytokine milieu induced during sepsis, plasma IL-6 has the best correlation with mortality (Kumar et al., Cytokine profile in elderly patients with sepsis, Indian Journal of Critical Care Medicine, 13(2): 74-78 (2009)).

[0032] Dextran sulfate, or a pharmaceutically acceptable salt thereof, induced a concentration-dependent reduction in LPS-stimulated secretion of IL-6 (Figs. 1A, 2A).

[0033] Interleukin-8 (IL-8), also known as chemokine (C-X-C motif) ligand 8 (CXCL8), is a chemokine produced primarily by macrophages. IL-8 is one of the key mediators of inflammatory responses. Its primary function is to induce chemotaxis in its target cells, e.g., neutrophil granulocytes. IL-8 functions as a chemical signal that attracts neutrophils to the inflammatory site. Serum and plasma levels of IL-8 are elevated in patients with sepsis (Livaditi et al., Neutrophil CD64 expression and serum IL-8: Sensitive early markers of severity and outcome in sepsis, Cytokine, 36(5-6): 283-290 (2006)). Furthermore, early levels of IL-8 are the most predictive factor for mortality in patients with sepsis (Mera et al., (2011)), indicating that IL-8 plays a role in sepsis.

[0034] Dextran sulfate, or a pharmaceutically acceptable salt thereof, induced a reduction in LPS- and Cytostim-stimulated secretion of IL-8 (FIG. 4).

[0035] IFNγ, also known as type II interferon, is a cytokine important for innate and adaptive immunity against viral and intracellular bacterial infections. CD4 and CD8 T cells primarily produce IFNγ upon antigen stimulation, and NK cells also produce IFNγ in the innate immune response. IFNγ is the most important cytokine used to define Th1 cells. Several studies have shown that IFNγ promoted inflammatory responses during septic shock (Romero et al., The role of interferon-gamma in the pathogenesis of acute intraabdominal sepsis, Journal of Leukocyte Biology, 88(4): 725-735 (2010)). IFNγ expression was persistently elevated in patients who died from sepsis (Mera et al., (2011)).

[0036] Dextran sulfate, or a pharmaceutically acceptable salt thereof, induced a reduction in LPS-, PHA-L-, and Cytostim-stimulated secretion of IFNγ (FIG. 3).

[0037] TNFα, also known as cachexin or cachectin, stimulates the acute phase response involved in systemic inflammation. Plasma levels of TNFα have been demonstrated to be significantly increased in patients and animal models of sepsis (Mera et al., (2011)). TNFα has become the most well-studied inflammatory cytokine in sepsis.

[0038] Dextran sulfate, or a pharmaceutically acceptable salt thereof, induced a reduction in LPS-, PHA-L-, and Cytostim-stimulated secretion of TNFα (FIG. 5).

[0039] IL-10, also known as human cytokine synthesis inhibitory factor (CSIF), is a key cytokine in anti-inflammatory responses. CD4+ Th2 cells, monocytes, and B-cells produce IL-10. IL-10 potently inhibits the expression of Th1 cytokines, including both IL-2 and IFNγ. After binding to its high-affinity IL-10 receptor, IL-10 also suppresses the production of TNFα, IL-1, IL-6, IL-8, IL-12, GM-CSF, MIP-1α, and MIP-2α in monocytes, macrophages, neutrophils, and NK cells. IL-10 is one of the key cytokines in the pathophysiology of sepsis. Measurement of serum cytokines in patients with severe sepsis showed that IL-10 levels were significantly enhanced (Rau et al., Clinical manifestations but not cytokine profiles differentiate adult-onset still's disease and sepsis, The Journal of Rheumatology, 37(11): 2369-237641 (2010); Surbatovic et al., Immune cytokine response in combat casualties: Blast or explosive trauma with or without secondary sepsis, Military Medicine, 172(2): 190-195 (2007)). Increased serum IL-10 levels correlated with sepsis scores and mortality. A high IL-10-to-TNFα ratio was associated with mortality.Furthermore, persistent overproduction of IL-10 is a major risk factor for sepsis severity and fatal outcome (Gogos et al., Pro-versus anti-inflammatory cytokine profile in patients with severe sepsis: A marker for prognosis and future therapeutic options, The Journal of Infectious Diseases, 181(1):176-180 (2000)), suggesting that septic patients are severely immunosuppressed.

[0040] Dextran sulfate, or a pharmaceutically acceptable salt thereof, induced a reduction in LPS-, PHA-L pokeweed-, and Cytostim-stimulated secretion of IL-10 (FIG. 7).

[0041] In one embodiment, dextran sulfate, or a pharmaceutically acceptable salt thereof, is used in the treatment of sepsis.

[0042] In certain embodiments, dextran sulfate, or a pharmaceutically acceptable salt thereof, is used in the treatment of severe sepsis.

[0043] In another specific embodiment, dextran sulfate, or a pharmaceutically acceptable salt thereof, is used in the treatment of septic shock.

[0044] In another embodiment, dextran sulfate, or a pharmaceutically acceptable salt thereof, is used in the treatment of hypercytokinemia.

[0045] In the following, the (average) molecular weight and sulfur content of dextran sulfate mentioned also apply to any pharmaceutically acceptable salt of dextran sulfate. Thus, a pharmaceutically acceptable salt of dextran sulfate preferably has the average molecular weight and sulfur content discussed in the following embodiments.

[0046] Dextran sulfate outside the preferred range of embodiments is believed to result in less efficacy and / or negative side effects on cells or subjects.

[0047] For example, dextran sulfates with molecular weights greater than 10,000 Da (10 kDa) typically have a lower efficacy-to-side effect profile than dextran sulfates with lower molecular weights. This means that the maximum dose of dextran sulfate that can be safely administered to a subject is lower for larger dextran sulfate molecules (>10,000 Da) than for dextran sulfate molecules with average molecular weights within the preferred range. As a result, when dextran sulfate is to be administered to a subject in vivo, such larger dextran sulfate molecules are less suitable for clinical use.

[0048] Dextran sulfate is a sulfated polysaccharide, particularly a sulfated glucan, i.e., a polysaccharide made from many glucose molecules. The average molecular weight defined herein indicates that individual sulfated polysaccharides may have molecular weights different from this average molecular weight, but the average molecular weight represents the average molecular weight of the sulfated polysaccharides. This further means that there is likely to be a natural distribution of molecular weights of dextran sulfate samples around this average molecular weight.

[0049] The average molecular weight of dextran sulfate, or more precisely, the weight average molecular weight (M w ) is usually determined using indirect methods such as gel exclusion / permeation chromatography, light scattering, or viscosity. Determination of average molecular weight using such indirect methods depends on many factors, including the choice of column and eluent, flow rate, calibration procedure, etc.

[0050] Weight average molecular weight (M w ):

number

[0051] In one embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M of 10,000 Da or less. w In certain embodiments, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 2,000 Da to 10,000 Da. w It has.

[0052] In another embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 2,500 Da to 10,000 Da, preferably in the range of 3,000 Da to 10,000 Da. w In certain embodiments, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 3,500 Da to 9,500 Da, for example, in the range of 3,500 Da to 8,000 Da. w It has.

[0053] In another specific embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 4,500 Da to 7,500 Da, for example, in the range of 4,500 Da to 6,500 Da or in the range of 4,500 Da to 5,500 Da. w It has.

[0054] Thus, in some embodiments, dextran sulfate or a pharmaceutically acceptable salt thereof has an M of 10,000 Da or less, 9,500 Da or less, 9,000 Da or less, 8,500 Da or less, 8,000 Da or less, 7,500 Da or less, 7,000 Da or less, 6,500 Da or less, 6,000 Da or less, or 5,500 Da or less. w It has.

[0055] In some embodiments, the dextran sulfate or pharmaceutically acceptable salt thereof has an M of 1,000 Da or more, 1,500 Da or more, 2,000 Da or more, 2,500 Da or more, 3,000 Da or more, 3,500 Da or more, 4,000 Da or more, or 4,500 Da or more. w Any of these embodiments may be used in combination with any of the above-presented M w and these may be combined with an upper limit of 10,000 Da or less.

[0056] In certain embodiments, the M of dextran sulfate or a pharmaceutically acceptable salt thereof as set forth above is w is the average M w and is preferably determined by gel exclusion / permeation chromatography, size exclusion chromatography, light scattering or viscosity-based methods.

[0057] Number average molecular weight (M n ):

number

[0058] In one embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an M as measured by NMR spectroscopy in the range of 1,850 to 3,500 Da. n It has.

[0059] In certain embodiments, dextran sulfate or a pharmaceutically acceptable salt thereof has an M as measured by NMR spectroscopy in the range of 1,850 Da to 2,500 Da, preferably in the range of 1,850 Da to 2,300 Da, for example in the range of 1,850 Da to 2,000 Da. n It has.

[0060] Thus, in some embodiments, the dextran sulfate or pharmaceutically acceptable salt thereof has an M of 3,500 Da or less, 3,250 Da or less, 3,000 Da or less, 2,750 Da or less, 2,500 Da or less, 2,250 Da or less, or 2,000 Da or less. n Furthermore, dextran sulfate or a pharmaceutically acceptable salt thereof has an M of 1,850 Da or more. n It has.

[0061] In one embodiment, the dextran sulfate or pharmaceutically acceptable salt thereof has an average number of sulfates per glucose unit in the range of 2.5 to 3.0.

[0062] In certain embodiments, dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of sulfates per glucose unit in the range of 2.5 to 2.8, preferably in the range of 2.6 to 2.7.

[0063] In one embodiment, the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of glucose units in the range of 4.0 to 6.0.

[0064] In a particular embodiment, dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of glucose units in the range of 4.5 to 5.5, preferably in the range of 5.0 to 5.2.

[0065] In one embodiment, dextran sulfate or a pharmaceutically acceptable salt thereof has an Mn as measured by NMR spectroscopy in the range of 1,850 to 3,500 Da, an average number of sulfates per glucose unit in the range of 2.5 to 3.0, and an average sulfation at the C2 position in the glucose units of the dextran sulfate of at least 90%.

[0066] In one embodiment, the dextran sulfate has an average glucose unit number of about 5.1, an average sulfate number per glucose unit in the range of 2.6 to 2.7, and an M in the range of 1,850 Da to 2,000 Da. n It has.

[0067] In one embodiment, the pharmaceutically acceptable salt of dextran sulfate is sodium dextran sulfate. In a specific embodiment, the sodium dextran sulfate has an average glucose unit count of about 5.1, an average sulfate count per glucose unit in the range of 2.6 to 2.7, and a Na concentration in the range of 2,100 Da to 2,300 Da. + M containing counter ions n It has.

[0068] In one embodiment, the dextran sulfate has an average glucose unit count of 5.1, an average sulfate count per glucose unit of 2.7, and a Na concentration of about 1,900-1,950 Da as measured by NMR spectroscopy. + Excluded average M n and Na by NMR spectroscopy measurement of approximately 2,200-2,250 Da. + Content average M n It has.

[0069] Dextran sulfate according to embodiments can be provided as a pharmaceutically acceptable salt of dextran sulfate, for example, the sodium salt or potassium salt.

[0070] In one embodiment, dextran sulfate, or a pharmaceutically acceptable salt thereof, as disclosed in WO2016 / 076780 is used.

[0071] The subject is preferably a mammalian subject, more preferably a primate, and especially a human subject. However, dextran sulfate or a pharmaceutically acceptable salt thereof can also be used for veterinary purposes. Non-limiting examples of animal subjects include primates, cats, dogs, pigs, horses, mice, and rats.

[0072] Dextran sulfate or a pharmaceutically acceptable salt thereof is preferably administered to a subject by injection, particularly intravenous (iv), subcutaneous (sc), or intraperitoneal (ip) injection, preferably by iv or sc injection. Other possible parenteral administration routes include intramuscular and intraarticular injection. Alternatively, or in addition, dextran sulfate or a pharmaceutically acceptable derivative thereof may be injected, for example, directly into a tissue or organ or other site in the subject's body where the desired effect is to occur.

[0073] The dextran sulfate or a pharmaceutically acceptable salt thereof of the embodiment is preferably formulated as an aqueous injection solution using a selected solvent or excipient. The solvent is conveniently an aqueous solvent, particularly a buffer solution. Non-limiting examples of such buffer solutions include citrate buffers, such as citrate monohydrate (CAM) buffer, or phosphate buffers. For example, the dextran sulfate of the embodiment can be dissolved in saline, such as 0.9% NaCl saline, and then optionally buffered with 75 mM CAM and adjusted to a pH of about 5.9 using sodium hydroxide. Non-buffered solutions are also possible, including aqueous injection solutions such as saline, i.e., NaCl (aqueous). Furthermore, if a buffer solution is desired, buffer systems other than CAM can be used.

[0074] Embodiments are not limited to injection, and other routes of administration can alternatively be used, including oral, nasal, buccal, rectal, transdermal, transbronchial, or topical. The active compound dextran sulfate is then formulated with suitable excipients or carriers, which are selected based on the particular route of administration.

[0075] Suitable dosage ranges for dextran sulfate or a pharmaceutically acceptable salt thereof can vary depending on the use, such as in vitro or in vivo, the size and weight of the subject, the severity of the septic condition the subject is being treated for, and other considerations. For human subjects in particular, possible dosage ranges would be 1 μg / kg to 100 mg / kg body weight, preferably 10 μg / kg to 50 mg / kg body weight.

[0076] In a preferred embodiment, dextran sulfate or a pharmaceutically acceptable salt thereof is formulated to be administered at a dose ranging from 0.05 to 50 mg / kg body weight of a subject, preferably 0.05 or 0.1 to 40 mg / kg body weight of a subject, and more preferably 0.05 or 0.1 to 30 mg / kg body weight of a subject, or 0.1 to 25 mg / kg body weight of a subject, or 0.1 to 15 mg / kg body weight of a subject, or 0.1 to 10 mg / kg body weight of a subject. Preferred doses are selected from the range of 0.25 to 5 mg / kg body weight, preferably 0.5 to 2.5 mg / kg body weight, and more preferably 0.75 to 2 mg / kg body weight of a subject.

[0077] Dextran sulfate or a pharmaceutically acceptable salt thereof can be administered in a single dose, such as in the form of a single bolus injection, which can be infused into the subject very quickly, but is conveniently infused over a period of time, such that the dextran sulfate solution is infused into the patient over several minutes, such as 5-10 minutes.

[0078] Alternatively, dextran sulfate or a pharmaceutically acceptable salt thereof can be administered multiple times, ie, at least twice, during the treatment period.

[0079] Dextran sulfate or a pharmaceutically acceptable salt thereof can be administered sequentially, simultaneously, or in the form of a composition comprising dextran sulfate or a pharmaceutically acceptable salt thereof and at least one other active agent, wherein the at least one active agent can be selected from any agent useful in the treatment of sepsis.

[0080] The present invention also relates to the use of dextran sulfate, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of sepsis and / or hypercytokinemia.

[0081] The present invention also provides a method for treating sepsis and / or hypercytokinemia. The method comprises administering dextran sulfate, or a pharmaceutically acceptable salt thereof, to a subject suffering from sepsis and / or hypercytokinemia. In certain embodiments, the dextran sulfate, or a pharmaceutically acceptable salt thereof, is administered to a subject suffering from an infection or infectious disease, particularly an infection or infectious disease that may cause sepsis and / or hypercytokinemia in the subject.

[0082] The terms "treatment" and "treating" as used herein refer to the management and care of a patient for the purpose of combating sepsis and / or hypercytokinemia. The terms are intended to include the full range of treatments for sepsis and / or hypercytokinemia, such as the administration of dextran sulfate or a pharmaceutically acceptable salt thereof, to alleviate symptoms or complications, delay the progression of the disease, reduce or alleviate symptoms and complications, and / or cure or eliminate the disease, as well as to prevent sepsis and / or hypercytokinemia. Prevention should be understood as the management and care of a patient for the purpose of combating sepsis and / or hypercytokinemia, including the administration of dextran sulfate or a pharmaceutically acceptable salt thereof to prevent the onset of symptoms or complications. Treatment can be carried out in an acute or chronic manner. As used herein, treatment also encompasses the prophylaxis or prevention of sepsis and / or hypercytokinemia, as well as the prevention of sepsis and / or hypercytokinemia, including the prevention of sepsis and / or hypercytokinemia symptoms.

[0083] The term "therapeutically effective amount" of dextran sulfate, or a pharmaceutical salt thereof, as used herein means an amount sufficient to cure, prevent, reduce, or partially arrest the clinical symptoms of sepsis and / or hypercytokinemia and its complications. An amount sufficient to accomplish this is defined as a "therapeutically effective dose." Effective amounts for each purpose will depend on the severity of the disease and the patient's weight and general condition. It will be understood that determining appropriate dosages can be accomplished using routine experimentation, constructing a matrix of values, and testing different points within the matrix, all of which is within the ordinary skill of a skilled physician or veterinarian.

[0084] Example Example 1 This example investigated the ability of low molecular weight dextran sulfate (LMW-DS) to affect the stimulated release of IL-6 from human peripheral blood mononuclear cells (PBMCs) in vitro.

[0085] Human PBMCs can be stimulated in vitro with a variety of agents that directly and indirectly activate various cell subsets. Monitoring cytokine release allows for the investigation of the potential effects of drugs and the prediction of their effects in patients. IL-6 is a proto-inflammatory cytokine that has been shown to be associated with numerous disease states, including sepsis.

[0086] material and method PBMCs were isolated from healthy donors by Ficoll-Paque PLUS (GE Healthcare; 11778538) density gradient centrifugation. 2 × 10 PBMCs were collected. 5Cells per well were cultured for 24 hours at 37°C under 5% CO2 in the presence or absence (vehicle) of three concentrations of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780)—60 μg / ml, 200 μg / ml, and 600 μg / ml—with or without stimulation (unstimulated phosphate-buffered saline (PBS) vehicle control) or stimulation (lipopolysaccharide (LPS), peptidoglycan, pokeweed mitogen, phytohemagglutinin-L (PHA-L), CpG + IL-15, or Cytostim). After centrifugation, cell culture supernatants were removed and stored at -20°C until analysis of IL-6 by ELISA. IL-6 levels in the supernatants were quantified by ELISA (R&D Systems; DY206) according to the manufacturer's instructions.

[0087] result Previously collected internal data guided the selection of stimulatory concentrations, using submaximal concentrations of LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15, and Cytostim. In PBMC mixtures, all stimuli increased IL-6 release into the cell culture supernatant. Due to the encouraging results generated from cells from the first six donors regarding the effect of LMW-DS on LPS stimulation, we decided to extend the LPS testing to six additional donors. Furthermore, compared to unstimulated PBMCs, the original selected concentrations of CpG + IL-15 and Cytostim gave relatively small increases in IL-6 release. Therefore, higher concentrations of CpG + IL-15 and Cytostim were investigated in cells from the additional six donors. These higher concentrations induced greater increases in IL-6 release compared to unstimulated cells.

[0088] LPS LPS is a Toll-like receptor (TLR) 4 agonist. In human PBMC mixtures, the primary cell type directly activated by LPS is monocytes, which express TLR4. Monocytes are part of the innate immune system. These myeloid cells can also be used to model responses to other myeloid cells, such as macrophages and microglia. In this example, LPS caused a substantial increase in IL-6 release into the cell culture supernatant (Figure 1A), and LMW-DS produced a concentration-dependent and statistically significant decrease in IL-6 (Figure 1A). This indicates that LMW-DS has the potential to reduce the pro-inflammatory consequences of IL-6 after TLR4 activation in monocytes.

[0089] peptidoglycan Peptidoglycan is a TLR2 agonist and is expressed primarily by monocytes and B lymphocytes in PBMC mixtures. The latter is a component of the acquired immune system well known for its important role in the generation of specific antibodies to antigens. Although peptidoglycan resulted in a substantial increase in IL-6 release into cell culture supernatants, experiments with cells from six donors showed little evidence that LMW-DS caused an overall reduction in IL-6 release, even at the highest concentration tested (Figure 1B).

[0090] pokeweed mitogen Phytolacca mitogen is a lectin derived from pokeweed (Phytolacca americana). It induces T-lymphocyte-dependent activation of B lymphocytes. In this study, pokeweed mitogen significantly increased IL-6 release by PBMC mixtures, but this release was largely unaffected by LMW-DS (Figure 1C).

[0091] PHA-L PHA-L is an L-type subunit lectin derived from kidney beans (kintoki beans) that crosslinks T lymphocyte surface receptors upon their activation. PHA-L induced a relatively small increase in IL-6 release from PBMC mixtures, which was significantly inhibited by LMW-DS in a concentration-dependent manner (Fig. 1D).

[0092] CpG+IL-15 CpG-oligodeoxynucleotides are short, single-stranded deoxyribonucleic acid (DNA) molecules that activate TLR9, which is expressed primarily by monocytes and B cells in PBMC mixtures. IL-15 synergizes with CpG in stimulating B lymphocytes. Unlike pokeweed mitogen, CpG + IL-15 directly activates B lymphocytes, i.e., in a T lymphocyte-independent manner. In a first-round experiment with PBMCs from six donors, selected concentrations of CpG-ODN + IL-15 caused only a slight increase in IL-6 release into the cell culture supernatant. While this relatively low level of IL-6 release was overall unaffected by LMW-DS (Figure 1E), in additional experiments performed with additional donors to increase the number of LPS stimulations, these same additional donors were subjected to higher concentrations of CpG + IL-15 to elicit a more potent IL-6 release beyond that evident from unstimulated cells; this was achieved, yet there was no overall effect of LMW-DS (Figure 1F).

[0093] Cytostim Cytostim is an antibody-based T lymphocyte activator. It binds to the T cell receptor (TCR) and crosslinks it with major histocompatibility complex (MHC) molecules on antigen-presenting cells. Thus, Cytostim stimulates both cluster of differentiation 4 (CD4) and CD8 T lymphocytes. In this example, initial rounds of experiments with PBMCs from six donors used concentrations of Cytostim that resulted in only a relatively small increase in IL-6 release into the cell culture supernatant, which was generally unaffected by LMW-DS (Figure 1G). PBMCs from these same additional donors were examined with higher concentrations of Cytostim to produce greater IL-6 release compared to that from unstimulated cells, yet there was no overall effect of LMW-DS (Figure 1H).

[0094] Although all stimuli investigated in this example were able to increase IL-6 release from cells in the PBMC mixture, the targeted effect of LMW-DS (versus LPS and PHA-L) on IL-6 release suggests a refined mode of action rather than a general ability to reduce IL-6 release.

[0095] Example 2 Monocytes are circulating innate immune cells that are a key component of the immune system. In addition, because they are readily available, they can be used to model other less accessible myeloid cells, such as macrophages and microglia.

[0096] Similar to various myeloid cells, monocytes can be activated via Toll-like receptor (TLR) agonists, such as TLR2 and TLR4 receptor agonists peptidoglycan and lipopolysaccharide (LPS), respectively. Activation can be monitored by the expression of activation markers (via flow cytometry) and / or by the secretion of cytokines, such as the pro-inflammatory cytokine interleukin-6 (IL-6). In Example 1, LMW-DS reduced IL-6 secretion in human peripheral blood mononuclear cell (PBMC) preparations in response to LPS stimulation. However, the diverse cell types in PBMC mixtures hinder interpretation of the exact cell type mediating this response. This study investigated the ability of LMW-DS to modulate LPS-stimulated IL-6 release from purified human monocytes to identify its precise intracellular target. IL-6 is a pro-inflammatory cytokine that has been implicated in numerous pathologies, including sepsis.

[0097] material and method Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Paque PLUS (GE Healthcare; 11778538) density gradient centrifugation. Monocytes were purified using the EasySep™ Human Monocyte Enrichment Kit (StemCell), which purifies monocytes "intactly" to preserve their phenotype.

[0098] Monocytes were cultured for 24 hours at 37°C under 5% CO2 in the presence or absence (vehicle) of three concentrations of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780)—600 μg / ml, 200 μg / ml, and 60 μg / ml—with or without stimulation (unstimulated PBS vehicle control) or stimulation (LPS or peptidoglycan)—and in the presence or absence of heparin (2.0, 6.0, or 20 μg / ml; equivalent to 0.406, 1.218, and 4.06 units / ml; Sigma-Aldrich) or dexamethasone (3.0 μM; Sigma-Aldrich). After centrifugation, cell culture supernatants were removed and stored at -20°C until analysis of IL-6 by ELISA. The levels of IL-6 in the supernatants were quantified by ELISA (R&D Systems) according to the manufacturer's instructions.

[0099] result Previously collected internal data guided the selection of stimulatory concentrations and the use of submaximal concentrations of LPS and peptidoglycan, which also correspond to the same concentrations of LPS and peptidoglycan used in Example 1 when a human PBMC mixture was used as the IL-6 source.

[0100] The TLR2 agonist peptidoglycan and the TLR4 agonist LPS resulted in the release of IL-6 into cell culture supernatants from purified human monocytes (Fig. 2).

[0101] Average results from monocytes from 10 healthy donors showed a statistically significant, concentration-dependent inhibition of LPS-stimulated IL-6 release into the cell culture supernatant by LMW-DS (Figure 2A), and heparin produced a statistically significant, concentration-dependent enhancement of LPS-stimulated IL-6 release into the cell culture supernatant (Figure 2C). As expected, the glucocorticoid steroid dexamethasone statistically significantly inhibited LPS-stimulated IL-6 release into the cell culture supernatant (Figure 2B).

[0102] Average results from monocytes from 10 healthy donors showed a statistically significant, concentration-dependent increase in peptidoglycan-stimulated IL-6 release into the cell culture supernatant by LMW-DS (Figure 2D). This enhancement was mirrored to some extent by heparin, although the trend did not reach statistical significance (Figure 2F). As expected, the presence of dexamethasone statistically significantly inhibited peptidoglycan-stimulated IL-6 release into the cell culture supernatant (Figure 2E).

[0103] Monocytes are part of the innate immune system. These myeloid cells can also be used to model responses to other myeloid cells, such as macrophages and microglia. In this example, the clear and substantial effect of LMW-DS in suppressing the LPS-induced increase in IL-6 release from purified monocytes provides strong evidence that these cells are targets of LMW-DS.

[0104] Example 3 Activation of immune responses in diseased or infected tissues is reflected by changes in the phenotypic balance of peripheral blood mononuclear cells (PBMCs). Thus, evaluation of drug effects on components of the adaptive and innate immune systems may reveal mechanistic cellular pathways to better understand clinical changes relevant to experimental therapies and, potentially, identification of cellular and / or molecular biomarkers predictive of therapeutic efficacy for different disease states.

[0105] Human PBMCs can be directly and indirectly stimulated in vitro with a variety of agents that activate different cell subsets and mimic immune responses associated with compromised tissue. Monitoring cytokine release from PBMCs allows for the investigation of potential drug effects and the prediction of drug action in specific patients.

[0106] material and method Example 1 investigated the ability of LMW-DS to modulate IL-6 secretion from human PBMCs using various stimuli. This study performed a more extensive analysis of the cell culture supernatants resulting from Example 1. Therefore, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Paque PLUS density gradient centrifugation. PBMCs were collected at 2 x 10 5 Cells per well were cultured at 37°C in 5% CO2 in the presence or absence (vehicle) of three concentrations of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780) at 600 μg / ml, 200 μg / ml, and 60 μg / ml, either unstimulated (unstimulated PBS vehicle control) or stimulated (LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15, or Cytostim).

[0107] Therefore, treatment was as follows for each PBMC donor: i. Vehicle ii. Stimulation iii. Stimulation + LMW-DS (60μg / ml) iv. Stimulation + LMW-DS (200μg / ml) v. Stimulation + LMW-DS (600μg / ml) Each was repeated three times, resulting in a total number of samples: 1. LPS: 165 samples (from 11 donors) 2. Peptidoglycan from Bacillus subtilis; 90 samples (from 6 donors) 3. PHA-L; 90 samples (from 6 donors) 4. 0.2 μM CpG + IL-15; 90 samples (from 6 donors) 5. 1.0 μM CpG + IL-15; 90 samples (from 6 donors) 6. Phytolacca mitogen; 90 samples (from 6 donors) 7. 10 μl of Cytostim; 90 samples (from 6 donors) 8. 30 μl of Cytostim; 90 samples (from 6 donors) Total number of supernatant samples from all stimulations (all cell types) = 795

[0108] After treatment, cell culture supernatants were removed, centrifuged, and stored at -20°C until thawed for multiplex analysis of various cytokines using a 5-plex human magnetic Luminex assay (R&D Systems; catalog number LXSAHM-05). Luminex analysis was performed exactly according to the manufacturer's protocol.

[0109] result Previously collected internal data guided the selection of stimulatory concentrations to use submaximal concentrations of LPS, peptidoglycan, pokeweed mitogen, PHA-L, CpG + IL-15, and Cytostim; submaximal concentrations of stimuli tend to allow both increased and decreased modulation (if present) to be identified. In PBMC mixtures, all stimuli increased cytokine release into the cell culture supernatant, although some stimuli were more effective than others (Figures 3-7).

[0110] LPS LPS (lipopolysaccharide) is a Toll-like receptor (TLR) 4 agonist. In human PBMC mixtures, the primary cell type directly activated by LPS is monocytes, which express TLR4. Monocytes are part of the innate immune system. These myeloid cells can also be used to model responses to other myeloid cells, such as macrophages and microglia. In this study, when assessing the effects of LPS, there was a slight increase in the secretion of IFNγ, TNFα, IL-1β, and IL-10, but a significant increase in IL-8 secretion (Figures 3-7). LMW-DS caused a concentration-dependent slight decrease in IFNγ and IL-10 (Figures 3 and 7), and the presence of the highest concentration of LMW-DS resulted in a slight decrease in stimulated secretion of IL-1β, IL-8, and TNFα (Figures 4-6).

[0111] peptidoglycan Peptidoglycan is a TLR2 agonist and is expressed primarily by monocytes and B lymphocytes in PBMC mixtures. The latter is a component of the acquired immune system well known for its important role in the generation of specific antibodies to antigens. Peptidoglycan causes a substantial increase in the release of IL-1β, IL-8, and TNFα into cell culture supernatants; however, examination of results from all donors revealed little evidence that LMW-DS, even at the highest concentration tested, resulted in an overall reduction in cytokine release, although there was a corresponding increase in IL-1β and TNFα secretion (Figures 4-6).

[0112] PHA-L PHA-L (phytohemagglutinin-L) is an L-type subunit lectin derived from kidney beans (kintoki beans) that crosslinks T lymphocyte surface receptors upon their activation. In PBMC mixtures, PHA-L induced increases in IFNγ, IL-8, IL-10, and TNFα, with an overall small increase in IL-1β (Figures 4-7). LMW-DS produced a small increase in the stimulated release of IFNγ, IL-8, and TNFα, but not IL-1β (Figures 4-6). In contrast, LMW-DS produced a large, concentration-dependent decrease in IL-10 secretion (Figure 7).

[0113] pokeweed mitogen Phytolacca mitogen is a lectin derived from pokeweed (Phytolacca americana). It induces T-lymphocyte-dependent activation of B lymphocytes. In this study, pokeweed mitogen significantly increased the secretion of IFNγ, IL-1β, IL-8, IL-10, and TNFα by PBMC mixtures (Figures 4-7), but this release was largely unaffected by LMW-DS, except for a concentration-dependent reduction in IL-10 secretion (Figure 7).

[0114] CpG+IL-15 CpG-ODN is a short, single-stranded DNA molecule that activates TLR9, which is expressed primarily by monocytes and B cells in PBMC mixtures. IL-15 synergizes with CpG in stimulating B lymphocytes. Unlike pokeweed mitogen, CpG + IL-15 directly activates B lymphocytes, i.e., in a T lymphocyte-independent manner. In this study, there was only strong evidence for this stimulation to increase IL-8 secretion. Overall, in these experiments, LMW-DS had little effect on this response (Figures 4-7).

[0115] Cytostim Cytostim is an antibody-based T lymphocyte activator. It binds to the T cell receptor (TCR) and crosslinks it with major histocompatibility complex (MHC) molecules on antigen-presenting cells. Thus, Cytostim stimulates both CD4 and CD8 T lymphocytes. Overall, Cytostim led to increased secretion of IFNγ, IL-1β, IL-8, IL-10, and TNFα (Figures 4-7), with only a slight decrease associated with the presence of LMW-DS, except for IL-10 secretion (Figure 7), which showed a significant concentration-dependent decrease in the presence of LMW-DS.

[0116] Overall, the data support the possible action of LMW-DS to benefit patients with sepsis. Post-infectious sepsis is considered a dysregulated immune response that results in organ damage. Sepsis is responsible for a large proportion of morbidity, mortality, and healthcare expenditure. Worldwide, there are an estimated 31.5 million cases of sepsis per year, with an estimated 46,000 deaths per year in the UK, and an estimated cost to the NHS of £1.5-2 billion per year.

[0117] During sepsis in response to infection, excessive production of inflammatory cytokines (cytokine storm) can lead to septic shock. Several cytokines regulated by LMW-DS are elevated and thought to be involved in the pathogenesis of sepsis. For example, IFNγ, IL-1β, IL-6, IL-8, and TNFα continue to increase in non-survivors (Mera et al., (2011)). TNFα and IL-1β play a key role in sepsis, acting on cells such as macrophages, where they amplify the inflammatory cascade and increase the release of other pro-inflammatory cytokines and reactive oxygen and nitrogen species, and on endothelial cells, where they mediate inflammation-induced activation of blood coagulation (Schulte et al., Cytokines in sepsis: potent immunoregulators and potential therapeutic targets - an updated view, Mediators of Inflammation, 2013: 165974 (2013)). Additional roles of TNFα include promoting neutrophil extravasation through its effects on endothelial cells, and it has been demonstrated that blocking TNFα with monoclonal antibodies can also improve survival in patients with severe sepsis. IL-6 can enhance the activation of T cells, B cells, and the coagulation system, and IL-6 levels correlate with the clinical severity of sepsis (Schulte et al., (2013)). IL-6 knockout reduces lung injury in a mouse model of acute lung injury. IL-8 acts to potently attract and activate neutrophils, and levels correlate with the severity of sepsis (Kraft et al., Predictive value of IL-8 for sepsis and severe infections after burn injury, Shock, 43(3): 222-227 (2015)).In addition to their pathological role in sepsis, cytokines can also play host-protective and immunomodulatory roles in host defense, so despite the promise of targeting them mentioned above, the role of cytokines in sepsis remains a "double-edged sword" (Chaudhry et al., Role of cytokines as a double-edged sword in sepsis, In Vivo, 27(6): 669-684 (2013)).

[0118] One challenge in sepsis is how to target components of the response without producing long-term immunosuppression. While the benefits of neutralizing TNFα in sepsis have been demonstrated, monoclonal antibodies with long half-lives, such as infliximab, adalimumab, and certolizumab, have half-lives of approximately 14 days, creating challenges in the timing and route of administration. Drugs that target multiple cytokines, such as LMW-DS, with a relatively short duration of action at specific stages of the disease, could therefore potentially provide additional clinical benefit to patients with sepsis.

[0119] Example 4 material and method Serum samples were collected from motor neuron disease (MND) / amyotrophic lateral sclerosis (ALS) patients in a clinical trial (EudraCT No. 2018-000668-28) before and 5, 10, and 24 weeks after treatment with LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780). Frozen serum samples were thawed and IL-6 was quantified using a Luminex assay system (Bio-Plex 200 system with Bio-Plex Manager software) according to the manufacturer's instructions. After the initial screening visit, patients received a single weekly injection of LMW-DS (ILB®, Tikomed AB, Viken, Sweden, WO 2016 / 076780) at 1.0 mg / kg body weight in saline into the subcutaneous fat of the lower abdomen, with a maximum of 1.5 mL at each injection site.

[0120] Statistical analysis was performed using the non-parametric Mann-Whitney U test, with p<0.05 considered significant.

[0121] result Figure 8 shows various serum levels of IL-6 assayed by the Luminex assay system in serum samples collected from each of the eight ALS patients. To allow for comparison across patient cohorts, the percent change in blood concentrations of IL-6 was assessed after patients began receiving LMW-DS at 5, 10, and 24 weeks. Analysis showed a statistically significant decrease in measured IL-6 levels at 10 weeks of treatment.

[0122] The above-described embodiments should be understood as a few illustrative examples of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, and changes can be made to the embodiments without departing from the scope or spirit of the present invention. In particular, different part measures in different embodiments can be combined into other configurations, where technically feasible.

Claims

1. Use of dextran sulfate or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of sepsis, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has a number average molecular weight (M n ) in the range of 1,850 to 3,500 Da, as measured by nuclear magnetic resonance (NMR) spectroscopy.

2. 2. The use of claim 1, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof is formulated for systemic administration to the subject.

3. 3. The use according to claim 2, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof is formulated for intravenous or subcutaneous administration to the subject, preferably for subcutaneous administration to the subject.

4. The use according to any one of claims 1 to 3, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average sulfur content in the range of 15 to 20%.

5. The use according to any one of claims 1 to 4, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has a Mn in the range of 1,850 to 2,500 Da as measured by NMR spectroscopy.

6. The use according to claim 5, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an M n in the range of 1,850 to 2,300 Da as measured by NMR spectroscopy.

7. The dextran sulfate or a pharmaceutically acceptable salt thereof has an M in the range of 1,850 to 2,000 Da as measured by NMR spectroscopy. ｎ 7. The use according to claim 6, wherein

8. The use according to any one of claims 1 to 7, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of sulfates per glucose unit in the range of 2.5 to 3.

0.

9. The use of claim 8, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of sulfates per glucose unit in the range of 2.5 to 2.

8.

10. The use of claim 9, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of sulfates per glucose unit in the range of 2.6 to 2.

7.

11. The use according to any one of claims 1 to 10, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of glucose units in the range of 4.0 to 6.

0.

12. The use of claim 11, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of glucose units within the range of 4.5 to 5.

5.

13. The use of claim 12, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average number of glucose units in the range of 5.0 to 5.

2.

14. The use according to any one of claims 1 to 13, wherein the dextran sulfate or a pharmaceutically acceptable salt thereof has an average of 5.1 glucose units and an average number of sulfates per glucose unit of 2.6 to 2.

7.

15. The use according to any one of claims 1 to 14, wherein the dextran sulfate or pharmaceutically acceptable salt thereof is formulated as an injectable aqueous solution.

16. The use according to any one of claims 1 to 15, wherein the pharmaceutically acceptable salt thereof is the sodium salt of dextran sulfate.

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