Use of heparan sulfate (HS) oligosaccharides in diseases

Abstract

Disclosed are methods of treating drug-induced liver injury using synthetic heparan sulfate oligosaccharides having 12, 14, or 16 saccharide residues. For example, the drug-induced liver injury can be acetaminophen (APAP)-induced acute liver failure. Also disclosed are methods of treating sepsis using synthetic heparan sulfate oligosaccharides having 12, 14, 16, or 18 saccharide residues, as well as select 12-, 14- and 16-mer oligosaccharides and their pharmaceutical compositions.

Description

[0001] DESCRIPTION

[0002] USE OF HEPARAN SULFATE (HS) OLIGOSACCHARIDES IN DISEASES

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63 / 612,744, filed December 20, 2023; the disclosure of which is incorporated herein by reference in its entirety.

[0005] GOVERNMENT INTEREST

[0006] This invention was made with government support under Grant Number HL094463 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] REFERENCE TO SEQUENCE LISTING XML

[0008] The Sequence Listing XML associated with the instant disclosure has been electronically submitted to the United States Patent and Trademark Office via the Patent Center as a 10,958 byte UTF-8-encoded XML file created on December 17, 2024 and entitled “421-545-PCT.xml”. The Sequence Listing submitted via Patent Center is hereby incorporated by reference in its entirety.

[0009] TECHNICAL FIELD

[0010] The presently disclosed subject matter relates methods of treating acute care indications, e.g., drug-induced liver injury or sepsis using synthetic oligosaccharides related in structure to heparan sulfate. The presently disclosed subject matter further relates to compositions comprising synthetic oligosaccharides.

[0011] BACKGROUND

[0012] Sterile inflammation is a natural process that initiates tissue repair in response to cellular damage. However, exaggerated inflammation after an initial insult often damages surrounding healthy tissues and is a key contributor to many disease processes. High mobility group box 1 (HMGB1) is a DNA binding protein that regulates transcription and is released from the nucleus during necrotic cell death (Zitvogel, 2010; Chen, 2010). Extracellularly, HMGB1 is a damage associated molecular pattern (DAMP) protein that acts as a pro-inflammatory molecule, orchestrating migration and activation of inflammatory cells to the injury site (Bianchi, 2017).

[0013] Acetaminophen (APAP), also known as paracetamol or N-acetyl-para- aminophenol, is a widely used analgesic. Ingestion of a supratherapeutic dose causes acute liver failure (ALF) (Heard, 2008). The misuse of opioids or co-formulations of opioids and APAP, can also cause ALF. In the US, nearly 50% of drug-induced liver injury has been attributed to APAP toxicity (Lee, 2007), which accounts for -80,000 emergency room visits annually (Blieden, 2014). The mechanism for APAP toxicity begins with its metabolic conversion to the reactive chemical species, A-acetyl- / ?- benzoquinone imine (NAPQI), which causes hepatocyte necrosis. (Tacke, 2015). Necrotic hepatocytes release HMGB1 which drives chemotaxis of neutrophils through the receptor for advanced glycation end-products (RAGE), activating sterile inflammation and amplifying liver injury (Huebener 2015).

[0014] Sepsis is a serious medical condition that can be caused by an unregulated inflammatory response to an existing infection. Sepsis can result in organ failure, tissue damage and death. Septic patients are typically treated by antibiotics and supportive care, such as fluid resuscitation, but the dysregulated host response remains unaddressed (4). While the uncontrolled inflammatory response of sepsis is complex, it is believed to include DAMPs, e.g., extracellular histones H3 (H3) and HMGB1 (5- 7).

[0015] Accordingly, additional compositions and methods for treating drug-induced liver injury and sepsis remain an ongoing need.

[0016] SUMMARY

[0017] This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.

[0018] In some embodiments, the presently disclosed subject matter provides a method of treating drug-induced liver injury or sepsis in a subject in need thereof, the method comprising administering to the subject a synthetic heparan sulfate (HS) oligosaccharide having a structure of Formula (I): wherein: m and n are each selected from 0, 4, 5, 6, and 7; Ri is selected from -H and -SO3H; R2 is selected from -SO3H and -COCH3; R3 is selected from -H and -SO3H; and R4 is selected from the group comprising -H, alkyl, aryl, substituted alkyl, substituted aryl, and a functional handle; subject to the proviso that the synthetic HS oligosaccharide is selected from (i)-(vi) or a pharmaceutical composition thereof, wherein: (i) m is 0, n is 4, Ri is -SO3H, R2 is -SO3H, and R3 is -SO3H; (ii) m is 0, n is 5; Ri is -H, R2 is -SO3H, and R3 is -SO3H; (iii) m is 0, n is 6, Ri is -H, R2 is -SO3H, and R3 is -SO3H; (iv) m is 4, n is 0, Ri is -SO3H, and R2 is -SO3H; (v) m is 5, n is 0, Ri is -SO3H, and R2 is -SO3H; and (vi) m is 6, n is 0, Ri is -SO3H, and R2 is -SO3H.

[0019] In some embodiments, the drug-induced liver injury is acetaminophen (APAP)-induced acute liver failure. In some embodiments, the subject in need of treatment is a mammalian subject, optionally a human subject.

[0020] In some embodiments, the synthetic HS oligosaccharide is selected from the group comprising (i), (iv), (v), and (vi). In some embodiments, R4 is substituted aryl, optionally p-nitrophenyl.

[0021] In some embodiments, the presently disclosed subject matter provides the use of a synthetic heparan sulfate (HS) oligosaccharide or a pharmaceutical composition thereof in a method of treating drug-induced liver injury, optionally acetaminophen (APAP)-induced acute liver failure, or sepsis in a subject in need thereof, wherein the synthetic HS oligosaccharide has a structure of Formula (I): wherein: m and n are each selected from 0, 4, 5, 6, and 7; Ri is selected from -H and -SO3H; R2 is selected from -SO3H and -COCH3; R3 is selected from -H and -SO3H; and R4 is selected from the group comprising -H, alkyl, aryl, substituted alkyl, substituted aryl, and a functional handle; subject to the proviso that the synthetic HS oligosaccharide is selected from (i)-(vi) or a pharmaceutical composition thereof, wherein: (i) m is 0, n is 4, Ri is -SO3H, R2 is -SO3H, and R3 is -SO3H; (ii) m is 0, n is 5, Ri is -H, R2 is -SO3H, and R3 is -SO3H; (iii) m is 0, n is 6, Ri is -H, R2 is -SO3H, and R3 is -SO3H; (iv) m is 4, n is 0, Ri is -SO3H, and R2 is -SO3H; (v) m is 5, n is 0, Ri is -SO3H, and R2 is -SO3H; and (vi) m is 6, n is 0, Ri is -SO3H, and R2 is -SO3H.

[0022] In some embodiments, the presently disclosed subject matter provides a synthetic heparan sulfate (HS) oligosaccharide having a structure of Formula (I): wherein: m and n are each selected from 0, 4, 5, 6, and 7; Ri is selected from -H and -SO3H; R2 is selected from -SO3H and -COCH3; R3 is selected from -H and -SO3H; and R4 is selected from the group comprising -H, alkyl, aryl, substituted alkyl, substituted aryl, and a functional handle; subject to the proviso that the synthetic HS oligosaccharide is selected from (ii), (iii), (v), or (vi), wherein: (ii) m is 0, n is 5, Ri is -H, R2 is -SO3H, and R3 is -SO3H; (iii) m is 0, n is 6, Ri is -H, R2 is -SO3H, and R3 is -SO3H; (v) m is 5, n is 0, Ri is -SO3H, and R2 is -SO3H; and (vi) m is 6, n is 0, Ri is -SO3H, and R2 is -SO3H. In some embodiments, the synthetic HS oligosaccharide is (v) or (vi). In some embodiments, the synthetic HS oligosaccharide is (ii) or (iii), optionally wherein the synthetic HS oligosaccharide is (ii). In some embodiments, R4 is substituted aryl, optionally p-nitrophenyl. In some embodiments, the presently disclosed subject matter provides a pharmaceutical composition comprising (a) a synthetic HS oligosaccharide having a structure of Formula (I) wherein the synthetic HS oligosaccharide is selected from (ii), (iii), (v), or (vi); and (b) a pharmaceutically acceptable carrier or adjuvant.

[0023] In some embodiments, the presently disclosed subject matter provides a method of treating sepsis in a subject in need thereof, the method comprising administering to the subject a synthetic heparan sulfate (HS) oligosaccharide having a structure of Formula (I): wherein: m is 0; n is 7; Ri is H; R2 is -SO3H; R3 is -SO3H; and R4 is -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle, optionally wherein R4 is p- nitrophenyl.

[0024] Accordingly, it is an object of the presently disclosed subject matter to provide methods of treating drug-induced liver injury or sepsis in a subject in need thereof and to provide related synthetic heparan sulfate oligosaccharides and pharmaceutical compositions. An object of the presently disclosed subject matter having been stated hereinabove, and which is achieved in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds when taken in connection with the accompanying drawings and examples as best described herein below.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] The presently disclosed subject matter can be better understood by referring to the following figures. The figures are not intended to limit the scope of this presently disclosed subject matter, but merely to clarify and exemplify the presently disclosed subject matter.

[0027] Figures 1A-1K: 18-mer NS2S inhibits inflammation in CLP-induced septic mice. (Figure 1A) Chemical structures of 6-mer NS2S, 12-mer NS2S, and 18-mer NS2S. Short-hand structure for each oligosaccharide is also presented for clarity. Chemoenzymatic synthesis of the HS oligosaccharides is shown in Figure 5A. (Figures 1B-1H) Mice underwent CLP surgery were administered (S.Q.) 20 mg / kg of 18-mer at 0, 6, 12 hours after CLP, and euthanized 24 hours after CLP to collect plasma for the following analysis. Concentrations of the biomarkers are individually presented for male and female groups in Figures 15A-15H and 16A-16V. (Figure IB) Circulating H3 in mouse plasma was evaluated by Western analysis. A representative western analysis image is presented on top. The bar graph represents the H3 band intensities from the individual samples. The full image is presented in Figures 17A- 17D. (Figure 1C) Circulating HMGB1 in mouse plasma was evaluated by ELISA (n = 6 male and 3 female). (Figures 1D-1H) The levels of IL-6, MCP-1, soluble iCAM- 1, creatinine, and BUN were tested in mice plasma. The data is expressed as mean±SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test, n = 6-10 male and 3-5 female. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (Figures 1L1K) Photomicrographs of proximal renal tubules. (Figure II) Normal tubules of control mice. (Figure 1 J) 24 hours post-CLP, proximal tubular epithelial cells have moderate vacuolation (blue arrowheads) and are less tall than in control mice. (Figure IK) 24 hours post-CLP with 18-mer treatment, proximal tubular epithelial cells are similar to control mice.

[0028] Figures 2A-2E: 18-mer NS2S reduces inflammation by targeting extracellular H3 and the LPS / HMGB1 complex. (Figure 2A) The viability of endothelial cells EA.hy926 was analyzed by flow cytometry. Cells were treated with 30 g / mL of H3 and the indicated concentration of HS for an hour before analysis (n =3). The statistical difference in cell viability with the same HS treatment but the different concentrations is shown compared to the untreated group (group 3) (&P<0.05 for 6- mer, #P<0.05 for 12-mer, *P<0.05 for 18-mer)(bars from left to right: 6-mer, light gray; 12-mer, medium gray; 18-mer, dark gray). (Figures 2B-2E) Plasma and peritoneal lavage were collected from mice 2 hours after biotinylated LPS (B*LPS) administration (i.p., 5 mg / kg) with or without the indicated concentration of HS (n = 4-7). (Figures 2B-2C) The protein level of IL-6 was evaluated by ELISA. (Figures 2D-2E) The complex of B*LPS / HMGB1 was isolated by affinity pull-down using streptavidin resin from mouse peritoneal lavage, shown as a representative image of immunoblot with HMGB1 (Figure 2D) and bar graph for the intensity of the band. (Figure 2E) Samples before and after passing streptavidin resin are labeled “preload” and “elution,” respectively. Data expressed as mean±SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05; **p<0.01.

[0029] Figures 3A-3G: 18-mer NS2S enlists ApoA-I to reduce inflammation and exert a protective effect against sepsis. (Figures 3 A-3D) Plasma and peritoneal lavage were collected from mice 2 hours after B*LPS administration (i.p., 5 mg / kg) with or without the indicated concentration of HS (n = 4-7). (Figure 3 A) The SDS-PAGE image of the peritoneal lavage stained with Ponceau S. The membrane is the same as Fig. 2D. The protein migrated at 25 kDa was identified as ApoA-I by proteomic analysis. (Figure 3B) Immunoblot of the level of B*LPS / HMGB1 (top) and B*LPS / ApoA-I (bottom) from peritoneal lavage. Samples before and after affinity purification by streptavidin resin are labeled “preload” and “elution,” respectively. The full images are presented in Figures 19A and 19B. (Figures 3C and 3D) The concentration of LPS in the plasma and peritoneal lavage (n = 4-7). Data expressed as mean±SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. ***p<0.001. (Figure 3E) The protein level of TNF-a from the cell supernatant of LPS-stimulated Raw264.7, with or without HDL pretreatment (n = 3). (Figure 3F) The dissociation of human HDL by HS oligosaccharides. Human HDL (200 mg / mL) was incubated with 6-mer NS2S, 12-mer NS2S, and 18-mer NS2S (5 mg / mL) under mildly acidic conditions and centrifuged. The supernatant and precipitation from each reaction were analyzed by SDS-PAGE followed by Coomassie blue staining. (Figure 3G) The 72-hour survival in CLP mice was administered (S.Q.) with saline, 6-mer, and 18-mer at 0, 6, 12, 24, 36, and 52 hours, (sham, n=8 male; CLP, n=22 male and 6 female; CLP + 18-mer, n=20 male and 6 female, CLP + 6-mer, n=20 male and 6 female). Data analyzed by log-rank test; overall, p = 0.0006; CLP vs. Sham, p=0.0019; CLP vs. CLP + 18-mer, p= 0.0015; CLP vs. CLP + 6-mer, p= 0.2040. Survival data were individually presented for male and female groups in Figures 15A and 15E.

[0030] Figure 4: Proposed the mechanism of action by 18-mer NS2S in sepsis. Sepsis causes systemic inflammation and releases extracellular H3 and HMGB1 and bacterial LPS. 18-mer NS2S displays the protection by directly neutralizing H3 and indirectly targeting HMGB1. 18-mer NS2S binds to H3 and neutralizes the cytotoxicity of H3 (Action 1). 18-merNS2S causes the structural changes of HDL and releases ApoA-I (Action 2). ApoA-I binds to LPS to allow a clearance from the circulation to reduce the plasma concentration of LPS (Action 3). ApoA-I displaces HMGB1 from the LPS-HMGB1 complex, which is a pathway to deliver LPS into the cells to cause cell death (Action 4). [Illustration created with BioRender]

[0031] Figures 5A-5C: Chemoenzymatic synthesis of 6-merNS2S, 12-merNS2S and 18-mer NS2S. (Figure 5 A) The synthesis is initiated from / / ra-nitrophenyl glucuronide (GlcA-pNP) monosaccharide. It took eight enzymatic modification steps to obtain 6-mer NS2S, 18 steps to obtain 12-mer NS2S, and 30 steps to obtain 18-mer NS2S. Abbreviations: pmHS2, heparosan synthase 2 from Pasteurella miillocida: NST, A-sulfotransferase; PAPS, 3 ’-phosphoadenosine 5 ’-phosphosulfate; C5-epi, C5- epimerase; 2-OST, 2-O-sulfotransferase. (Figures 5B and 5C) The anticoagulant activity was evaluated by testing the inhibition effect of 6-mer NS2S, 12-mer NS2S, 18-mer NS2S, unfractionated heparin (UFH), and fondaparinux (FPX) to FXa and Flla. UFH and FPX were used as controls in this experiment. All HS (UFH, FPX, 6- mer, 12-mer, 18-mer) was tested at 5 pg / mL. The data are expressed as mean (n=3) ± SEM.

[0032] Figures 6A-6D: Hematologic analysis of CLP induced septic mice. Plasma collected from mice receiving sham or CLP surgery were used to analyze hematologic changes (Figure 6A, white blood cells (WBC); Figure 6B, neutrophils (Neu); Figure 6C, lymphocytes (Lym); and Figure 6D, platelets (PLT)). Data expressed as mean ± SEM and analyzed by unpaired student’s t-test, n = 7-12 mice in the 24-hour group, n = 3-5 mice in the 72-hour group. *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

[0033] Figures 7A-7E: 18-mer NS2S reduces CLP-induced systemic inflammation independent of infection. Mice were subjected to a sham procedure or CLP and sacrificed 24 hours after the sham or CLP surgery. In the 18-mer NS2S group, mice were administered 18-mer NS2S (S.Q., 20mg / kg) at 0, 6, and 12 hours after CLP. (Figures 7A-7C) The protein level of IL-6, MCP-1, and iCAM-1 were tested from mouse peritoneal lavage. Data expressed as mean±SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test, n = 6-10 male and 3-5 female mice. *p<0.05; **p<0.01; ***P<0.001. (Figure 7D) Colony forming units (CFU) count (Log 10) in the peritoneal lavage harvested from CLP or 18-mer treated CLP mice 24 hours after CLP surgery. (Figure 7E) Heatmap analysis of cytokine and chemokine measurements performed by multiplex analysis for sham, CLP, and 18- mer NS2S groups. Plasma was evaluated with samples taken 24 hours after the sham or CLP procedure. Heat map showing the evaluation of cytokine and chemokine values in LoglO. IL, Interleukin; TNF, tumor necrosis factor; IFNg, Interferon gamma; KC, Keratinocyte-derived chemokines; IP, Interferon gamma-induced protein; GM-CSF, Granulocyte macrophage colony-stimulating factor; KIM-1, kidney injury molecule 1; RANTES, Regulated on activation, normal T cell expressed and secreted; MIP, Macrophage inflammatory protein; G-CSF, Granulocyte-colony stimulating factor; RAGE, Receptor for advanced glycation end products.

[0034] Figures 8A-8F: 18-mer NS2S reduces CLP-induced kidney inflammation and damage. Mice were subjected to a sham procedure or CLP. In the 18-mer NS2S group, mice were administered 18-mer NS2S (S.Q., 20mg / kg) at 0, 6, 12 hours and sacrificed 24 hours after CLP. (Figures 8A-8C) The mRNA levels of IL-6, MCP-1, and iCAM- 1 were tested from mouse kidney homogenates. Data expressed as mean±SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test, n = 6- 10 male and 3-5 female mice. *p<0.05; **p<0.01. (Figures 8D-8F) Represented images of H&E stained inner stripe of the outer medulla (ISOM) from sham (Figure 8D), CLP (Figure 8E), and 18-mer (Figure 8F) mice. Eosinophilic material and epithelial degeneration in the thin segment and thick ascending limb of Henle’s (asterisks).

[0035] Figure 9: 18-mer NS2S, but not NAc 18-mer, protects against histone-induced lethality. Mice are administered with histone (75 mg / kg) alone or with 18-mer NS2S (18-mer) or NAc 18-mer (75 mg / kg) by retro-orbital injection. Both male and female mice were tested (histone, n=8 male and 2 female ; histone + 18-mer, n=5 male and 2 female ; histone + NAc 18-mer, n = 10 male and 1 female). Data analyzed by log-rank test; overall, p = 0.0016; Histone vs. Histone + 18-mer, p=0.0003; Histone vs. Histone + NAc 18-mer, p= 0.1709.

[0036] Figures 10A and 10B: 18-mer NS2S is partially resistant to heparanase digestion as determined by HPLC. (Figure 10A) 18-mer NS2S (upper plot line in Figure 10A) or (Figure 10B) 18-mer NS6S (upper plot line in Figure 10B) (200 pg / mL) were incubated with heparanase (20 pg / mL)(lower plot line in Figures 10A and 10B) at 37 °C in 50 mM MOPS at pH 6. After 24 hours of incubation, the reaction was inactivated by heating at 95°C 5 min and analyzed by HPLC. A dashed line in Figure 10A and Figure 10B indicates the eluted position of undigested 18-mer NS2S and 18-mer NS6S. Under this condition, NAc 18-mer was totally resistant to heparanase digestion.

[0037] Figures HA and 11B: Structural analysis of the products 18-mer NS2S or 18- mer NS6S after heparanase digestion by LC / MS. (Figure 11 A) 18-mer NS2S or (Figure 1 IB) 18-mer NS6S show the degradation sites after heparanase digestion. The digested products were subjected to LC-MS analysis.

[0038] Figures 12A-12H: Evaluation of 18-mer NS2S effects on HMGB1 / LPS induced inflammation. (Figures 12A and 12B) Plasma and peritoneal lavage were collected from mice 2 hours after biotinylated LPS (B*LPS) administration (i.p., 5 mg / kg) with or without HS treatment (n = 4-7). The protein level of TNF-a was evaluated by ELISA. (Figures 12C and 12D) The level of IL-6 in peritoneal lavage and plasma (n = 4) from endotoxemia mice with or without 18-mer NS2S treatment. (Figure 12E) Peritoneal lavage was collected from mice 2 hours after B*LPS administration (i.p., 5 mg / kg) and incubated with LPS, 6-mer NS2S, or 18-mer NS2S (lOmg / mL) for Ih at room temperature. The B*LPS / HMGB1 complex in the lavage. (Figure 12F) Peritoneal lavage was collected from mice 2 hours after B*LPS or LPS administration (i.p., 5 mg / kg). The B*LPS / HMGB1 complex in the lavage was isolated by affinity pull-down using streptavidin resin following immunoblot of HMGB1. Samples are labeled as “Preload”: prior to the affinity-pull down experiment; “Flow-through”: flow-through lavage after incubation with streptavidin; “Wash”: PBS buffer incubated with streptavidin, and “Elution” : elution buffer incubated with streptavidin, after denatured at 95°C. The full images are presented in Figures 18A-18C. (Figures 12G and 12H) The complex of 18-mer NS2S and LPS cannot be captured using an ultracentrifugal approach (Figure 12G). The ultracentrifugal approach was validated by binding to antithrombin (AT III) using 6- mer AXa as positive control and 6-mer as negative control (Figure 12H). Presence of HS was detected by absorbance at O.D. 310nm with triplicates. Figures 13A-13C: Investigation of the apoA-I / LPS complex. (Figure 13A) Peritoneal lavage were collected from mice 2 hours after biotinylated LPS (B*LPS) administration (i.p., 5 mg / kg) with or without 18-mer treatment. The SDS-PAGE profile of the peritoneal lavage was analyzed by Coomassie staining after the pulldown assay using streptavidin resin. A band around 25kDa was sent to proteomic analysis and identified as apoA-I. (Figure 13B) Peritoneal lavage were collected from mice 2 hours after B*LPS administration and incubated with different concentration of recombinant apoA-I. The presence of B*LPS / HMGB1 complex are shown by affinity pull-down assay and immunoblot of HMGB1. (Figure 13C) Peritoneal lavage were collected from mice 2 hours after B*LPS administration (i.p., 5 mg / kg) with 6- mer NS2S, 12-mer NS2S, and 18-mer NS2S treatment. The presence of B*LPS / HMGB1 complex are shown by affinity pull-down assay and immunoblot of HMGB1. Samples before and after passing streptavidin resin are labeled “preload” and “elution”, respectively. The full image is presented in Figure 20B.

[0039] Figures 14A-14H: Concentration of apoA-I and HDL in plasma and peritoneal lavage from Biotinylated LPS (B*LPS) injected mice and CLP mice. (Figures 14A- 14D) Concentration of apoA-I and HDL were measured by ELISA in plasma and peritoneal lavage that obtained at 2 hours after I.P. injection of biotinylated LPS (B*LPS, 5mg / kg) and 18-mer NS2S (50 mg / kg) / B*LPS co-injected mice. (Figures 14E-14H) Concentration of apoA-I and HDL were measured by ELISA in plasma and peritoneal lavage that obtained at 24 hours after sham or CLP surgery with or without 18-mer treatment (S.Q., 0, 6, 12 hours after CLP surgery) (n = 6-9). Data expressed as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05; **p<0.01.

[0040] Figures 15A-15H: Comparison of survival studies and DAMPs levels in plasma between male (Figures 15A-15D) and female (Figures 15E-15H) mice. (Figures 15Aand 15E) The 72-hour survival in CLP mice was administered (s.q.) with saline, 6-mer NS2S, and 18-mer NS2S at 0, 6, 12, 24, 36, and 52 hours. (Figure 15 A) sham, n=8; CLP, n=22; CLP +18-mer NS2S, n=20, CLP + 6-mer NS2S, n=20. Data analyzed by log-rank test; overall, p = 0.0065; CLP vs. Sham, p=0.0053; CLP vs. CLP + 18-mer NS2S, p= 0.0134; CLP vs. CLP + 6-mer NS2S, p= 0.1842. (Figure 15E) n=6. Data analyzed by log-rank test; overall, p = 0.0495; CLP vs. CLP + 18-mer NS2S, p= 0.1353. (Figures 15B and 15F) The 300-min survival in histone (75 mg / kg, r.o.) injected mice were administered (r.o.) with 18-mer NS2S or NAc 18-mer (75 mg / kg, r.o.). (Figure 15B) Histone, n=8; Histone + 18-mer NS2S, n=5, Histone + NAc 18-mer, n=10. Data analyzed by log-rank test; overall, p = 0.0079; Histone vs. Histone + 18-mer NS2S, p=0.0011; Histone vs. Histone + NAc 18-mer, p= 0.5473. (Figure 15F) Histone, n=2; Histone + 18-mer NS2S, n=2, Histone + NAc 18-mer, n=l. Data analyzed by log-rank test; overall, p = 0.0775. (Figures 15C, 15D, 15G, and 15H) Mice were subjected to a sham procedure or CLP and sacrificed 24 hours after the sham or CLP surgery. In the 18-mer group, mice were administered 18-mer (S.Q., 20 mg / kg) at 0, 6, and 12 hours after CLP. The protein level of H3 (Figure 15C, Male; Figure 15G, Female) and HMGB1 (Figure 15D, Male; Figure 15H, Female) were tested from mouse plasma. Data expressed as mean±SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test, n = 6-10 male and 3-5 female mice. *p<0.05.

[0041] Figures 16A-16V: Comparison of inflammatory markers levels and kidney damage markers in plasma, peritoneal lavage, and kidney between male (Figures 16A- 16K) and female (Figures 16L-16V) mice. Mice were subjected to a sham procedure or CLP and sacrificed 24 hours after the sham or CLP surgery. In the 18-mer group, mice were administered 18-mer NS2S (S.Q., 20mg / kg) at 0, 6, and 12 hours after CLP. The protein or mRNA levels of IL-6 (Figures 16A-16C, Male; Figures 16L-16N, Female), MCP-1 (Figures 16D-16F, Male; Figures 16O-16Q, Female), and iCAM-1 (Figures 16G-16I, Male; Figures 16R-16T, Female) were tested from mouse plasma , peritoneal lavage, or kidney homogenates. Creatinine and BUN (Figures 16J-16K, Male; Figures 16U-16V, Female) were analyzed from mouse plasma. Data expressed as mean±SEM and analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test, n = 6-10 male and 3-5 female mice. *p<0.05, **p<0.01; ***p<0.001, ****p<0.0001.

[0042] Figures 17A-17D: Full western blot and Ponceau S images from H3 plasma from CLP mice (see Figures IB, 15C, and 15G). The nitrocellulose membrane was stained by primary antibody anti-H3 (Figure 17A, Male; Figure 17B, Female) following staining by Ponceau S (Figure 17C, Male; Figure 17D, Female). Sample input were plasma collected at 24 hours after: lane 1-3: sham; lane 4-6: CLP; lane 7- 9: CLP + 18-mer.

[0043] Figures 18A-18C: Full western blot of LPS pull-down assay with ex vivo competitive binding (Figures 12E and 12F). (Figure 18A)Pull-down assay with peritoneal lavage from B*LPS injected mice incubated with LPS, NS2S 6-mer or 18- mer NS2S was shown. Lane 1-4: preload from peritoneal lavage incubated with PBS (1), LPS (2), 6-mer(3), 18-mer (4); lane 5-8: elution from peritoneal lavage incubated with PBS (5), LPS (6), 6-mer(7), 18-mer (8). (Figures 18A and 18C) Pull-down assay followed by immunoblot of HMGB1 with LPS I.P injection (Figure 18B) biotinylated LPS (B*LPS) I.P. injection (Figure 18C) . Sample input: lane 1 : preload; land 2: flow- through; lane 3: wash; lane 4: elution. Pull-down assay with peritoneal lavage from B*LPS injected mice incubated with 18-mer NS2S was shown in lane 5-8: lane 5: preload; land 6: flow-through; lane 7: wash; lane 8: elution.

[0044] Figures 19A and 19B: Full western blot of pull-down assay with 18-mer NS2S / B*LPS co-injection (Figure 3B). Pull-down assay with biotinylated LPS (B*LPS) and 18-mer NS2S (5 or 50 mg / kg) co-i.p. injection, immunoblot with HMGB1 (Figure 19A) and apoA-I (Figure 19B). Sample input: lane 1-3: preload from peritoneal lavage from mice injected B*LPS (1), B*LPS + 18-mer (5 mg / kg) (2), B*LPS + 18mer (50 mg / kg) (3). Lane 4-6: elution from peritoneal lavage from mice injected B*LPS (4), B*LPS + 18mer (5 mg / kg) (5), B*LPS + 18mer (50 mg / kg) (6).

[0045] Figures 20A and 20B: Full western blot of pull-down assay with apoAl ex vivo competition and HS / B*LPS co-injection (Figures 13B and 13C). (Figure 20A) Pull-down assay with peritoneal lavage from B*LPS injected mice incubated with recombinant apoAl (10, 50, 100 g / mL) was shown. Lane 2-5: preload from peritoneal lavage incubated with PBS (2), apoA-I ( l O g / mL) (3), apoA-I (50 / zg / mL) (4), apoA-I ( l OO g / mL) (5); lane 6-9: elution from peritoneal lavage incubated with PBS (6), apoA-I (10 / zg / mL) (7), apoA-I (50 / zg / mL) (8), apoA-I (100 / zg / mL) (9). Lane 1 is peritoneal lavage without incubation of other solution. (Figure 20B) Pull-down assay with B*LPS and 6-mer NS2S, 12-mer NS2S or 18-mer NS2S co-i.p. injection, immunoblot with apoAl. Sample input: lane 1-5 were preload from peritoneal lavage from mice injected sterile saline (1), B*LPS (2), B*LPS + 6-mer (3), B*LPS + 12- mer (4), B*LPS + 18-mer (5). Lane 6-10 were elution from peritoneal lavage from mice injected sterile saline (6), B*LPS (7), B*LPS + 6-mer (8), B*LPS + 12-mer (9), B*LPS + 18-mer (10).

[0046] Figures 21A-21F are schematic drawings showing the chemical structures of exemplary HS oligosaccharides of the presently disclosed subject matter. Figure 21 A is a schematic drawing showing the chemical structure of a HS dodecaoligosaccharide of the presently disclosed subject matter, referred to herein as 12-mer NS2S6S or GLY-202. Figure 21B is a schematic drawing showing the chemical structure of a HS tetradecaoligosaccharide of the presently disclosed subject matter, referred to herein as 14-mer NS2S. Figure 21C is a schematic drawing showing the chemical structure of a HS hexadecaoligosaccharide of the presently disclosed subject matter, referred to herein as 16-mer NS2S. Figure 21D is a schematic drawing showing the chemical structure of a HS dodecaoligosaccharide of the presently disclosed subject matter, referred to herein as 12-mer NS6S. Figure 21E is a schematic drawing showing the chemical structure of a HS tetradecaoligosaccharide of the presently disclosed subject matter, referred to herein as 14-mer NS6S. Figure 21F is a schematic drawing showing the chemical structure of a HS hexadecaoligosaccharide of the presently disclosed subject matter, referred to herein as 16-mer NS6S.

[0047] Figure 22 is a schematic diagram showing the chemoenzymatic synthesis of exemplary 12-mer HS oligosaccharides.

[0048] Figure 23 is a graph that shows the in vivo efficacy of HS oligosaccharides (18-mer NS2S, 16-mer NS6S, 14-mer NS6S, 12-mer NS6S, and 12-mer NS2S6S) in reducing alanine aminotransferase (ALT) in a mouse model of acetaminophen (APAP)-induced acute liver failure.

[0049] Figures 24A-24C are graphs showing in vivo effects of exemplary HS oligosaccharides on myeloperoxidase (MPO) activity, neutrophils, and necrotic area in a mouse model of acetaminophen (APAP)-induced acute liver failure. Figure 24A is a graph showing the effect of HS oligosaccharide 12-mers, 12-mer NS6S and 12- mer NS2S6S, in reducing MPO activity in an in vivo mouse model of APAP -induced acute liver failure. Figure 24B is a graph showing the effect of HS oligosaccharide 12-mers, 12-mer NS6S and 12-mer NS2S6S, in reducing neutrophils in liver tissue in an in vivo mouse model of APAP-induced acute liver failure. Figure 24C is a graph showing the effect of HS oligosaccharide 12-mers, 12-mer NS6S and 12-mer NS2S6S, in reducing the percentage of necrotic area in liver tissue in an in vivo mouse model of APAP -induced acute liver failure.*, p < 0.05; **, p < 0.01; ***, p < 0.001. P -values determined from Dunnet’s multiple comparisons following a one-way ANOVA.

[0050] Figures 25A and 25B are graphs showing survival curves in a mouse model of acetaminophen (APAP)-induced acute liver failure after treatment with exemplary HS oligosaccharides of the presently disclosed subject matter. Figure 25 A is a graph showing survival curves in a mouse model of APAP-induced acute liver failure for mice treated with 12-mer NS2S6S (GLY-202) or 18-mer NS2S (18-mer) twice daily beginning 30 minutes (early treatment) after APAP overdose. The lines for the 18- mer and the 12-mer overlap (both groups showed 100% survival). An untreated control group survival curve (APAP) is also shown. N = 5 for each group. Figure 25B is a graph showing survival curves in a mouse model of APAP-induced acute liver failure for mice treated with 12-mer NS2S6S (GLY-202) or 18-mer NS2S (18- mer) twice daily beginning 3 hours (delayed treatment) after APAP overdose. An untreated control group survival curve (APAP) is also shown. N = 9-10 for each group.

[0051] Figure 26 is a western blot images from HMGB1 pulldown using HS oligosaccharides. Sample input: Lane 1 : 18-mer NS2S; Lane 2: 18-mer NS6S; Lane 3: 16-mer NS6S; Lane 4: 14-mer NS6S. Sample Flow Through: Lane 5: 18-mer NS6S; Lane 6: 16-mer NS6S; Lane 7: 14-mer NS6S. Sample Elution: Lane 8: 18- mer NS2S; Lane 9: 18-mer NS6S; Lane 10: 16-mer NS6S; Lane 11 : 14-mer NS6S.

[0052] Figure 27 is a graph showing the effect of the combined administration of GLY-202 and NAC on plasma ALT concentration in a mouse model of APAP overdose. NAC, GLY-202, or both NAC and GLY-202 were administered 3 hours after APAP overdose. For comparison, data is shown for mice where NAC was also administered alone 30 minutes after APAP overdose or where neither NAC nor GLY- 202 were administered. The ALT concentration is expressed in units per liter (U / L).

[0053] DETAILED DESCRIPTION

[0054] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures and Examples, in which representative embodiments are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

[0055] I. DEFINITIONS

[0056] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.

[0057] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0058] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one skilled in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0059] In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.

[0060] Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.

[0061] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to "a cell" includes a plurality of such cells, and so forth. Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0062] As used herein, the term “about,” when referring to a value or to an amount of a composition, mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

[0063] As used herein, the term “and / or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and / or D” includes A, B, C, and D individually, but also includes any and all combinations and sub-combinations of A, B, C, and D.

[0064] The term “comprising”, which is synonymous with “including” “containing” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.

[0065] As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0066] As used herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel_characteristic(s) of the claimed subject matter.

[0067] With respect to the terms “comprising”, “consisting of’, and “consisting essentially of’, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.

[0068] As used herein the term “alkyl” refers to C1-20 inclusive, linear (z.e., "straightchain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (z.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms (z.e., a C1-8 alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" refers, in particular, to C1-8 straight-chain alkyls. In other embodiments, “alkyl” refers, in particular, to C1-8 branched-chain alkyls.

[0069] Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more alkyl group substituents, which can be the same or different. The term "alkyl group substituent" includes but is not limited to alkyl, substituted alkyl, halo, nitro, amino, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyl, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as “alkylaminoalkyl”), or aryl.

[0070] Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0071] The term "aryl" is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine. The term "aryl" specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In particular embodiments, the term “aryl” means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

[0072] The aryl group can be optionally substituted (a “substituted aryl”) with one or more aryl group substituents, which can be the same or different, wherein “aryl group substituent” includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R", wherein R' and R" can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

[0073] Thus, as used herein, the term "substituted aryl" includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

[0074] Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like.

[0075] The term “aralkyl” refers to an -alkyl-aryl group, optionally wherein the alkyl and / or aryl group comprises one or more alkyl or aryl group substituents.

[0076] In some embodiments, the term “bivalent” refers to a group that can bond (e.g., covalently bond) or is bonded to two other groups, such as other alkyl, aralkyl, cycloalkyl, or aryl groups. Typically, two different sites on the bivalent group (e.g., two different atoms) can bond to groups on other molecules. For example, the bivalent group can be an alkylene group.

[0077] "Alkylene" can refer to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and / or substituted with one or more "alkyl group substituents." There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH2-); ethylene (-CH2-CH2-); propylene (-(CH2)3-); cyclohexylene (-CeHio-); -CH=CH— CH=CH-; -CH=CH-CH2-; -(CH2)q-N(R)- (CH2)I— , wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (-O-CH2-O-); and ethylenedioxyl (-O-(CH2)2-O- ). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.

[0078] “Arylene” refers to a bivalent aryl group.

[0079] “Functional handle” is used to refer to chemical groups that facilitate the chemoenzymatic synthesis. In some embodiments, a functional handle is with or without UV absorbance and / or binds or does not bind to a C18-column. In some embodiments, the functional handle can also be referred to as a detectable tag. In some embodiments, the functional handle comprises an alkyl, aryl, substituted alkyl, or substituted aryl group as defined herein, such as p-nitrophenyl.

[0080] “GlcA” as used herein refers to a glucuronic acid residue in an oligo- or polysaccharide.

[0081] “GlcNS” as used herein refers to a glucosamine residue in an oligo- or polysaccharide where the amine moiety is sulfated (i.e., a hydrogen atom of the amine moiety is replaced by a -SO3H group).

[0082] “GlcNS6S” as used herein refers to a glucosamine residue in an oligo- or polysaccharide where both the amine moiety and the hydroxyl group at carbon 6 are sulfated (i.e., a hydrogen atom is replaced by a -SO3H group).

[0083] “IdoA2S” as used herein refers to an iduronic acid residue in an oligo- or polysaccharide wherein the hydroxyl group at carbon 2 is sulfated (i.e., the OH hydrogen atom is replaced by -SO3H). II. HEPARAN SULFATE (HS) AND SYNTHETIC HS OLIGOSACCHARIDES

[0084] Heparan sulfate (HS) is a sulfated polysaccharide comprising disaccharide units which each comprise an uronic acid residue (a glucuronic acid (GlcA) or a iduronic acid (IdoA) residue) linked to a glucosamine (GlcN) residue, where the uronic acid and / or GlcN residues can each carry one or more sulfo groups. The saccharide length and sulfation pattern of HS oligosaccharides can determine their function (15). HS isolated from natural sources is typically a mixture of HS compounds with different sizes and sulfation patterns. However, pure HS oligosaccharides can be prepared using chemoenzomatic routes (16). See also U.S. Patent Application Publication Nos. 2021 / 0169923 and 2022 / 0265699, the disclosures of which are incorporated herein by reference in their entireties.

[0085] In accordance with the presently disclosed subject matter, a family of synthetic HS oligosaccharides are described that can be used to treat acute care indications such as sepsis and drug-induced liver injury, optionally APAP-induced acute liver failure. For example, while a synthetic HS octadecasaccharide (18-mer), referred to herein as 18-mer NS2S, has recently been described for use in treating APAP-induced acute liver failure (17), as described hereinbelow, it has now also be found to protect against sepsis in a mouse model. More particularly, the 18-mer NS2S not only inhibits the pro-inflammatory activity of extracellular histone H3 and HMGB1, but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). As demonstrated herein, the 18-mer NS2S protects against sepsis-related injury and improves survival in cecal ligation and puncture (CLP) mice and reduces inflammation in an endotoxemia mouse model. The 18-mer NS2S neutralizes the cytotoxic H3 through direct interaction with the protein. Furthermore, the 18-mer NS2S enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide (LPS), a toxic complex contributing to cell death and tissue damage in sepsis. Overall, the presently disclosed study provides evidence that the 18-mer NS2S, mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.

[0086] As further described herein, smaller oligosaccharides (e.g., 12-, 14-, and 16- mers) have been found to have similar biological properties to the 18-mer NS2S. For example, smaller HS oligosaccharides have been shown to lack the anti-coagulant activity of some other HS compounds but can have the ability to inhibit HMBG1- induced sterile inflammation and / or to inhibit histone cytotoxicity and / or can inhibit other inflammatory mediators. In contrast to synthetic 18-mers, smaller oligosaccharides can be significantly less expensive to produce. Thus, according to one aspect, the presently disclosed subject matter provides compositions and / or methods for effective treatment of acute care indications with less expensive, more readily synthesized, short chain oligosaccharides that can avoid the purification barriers related to long chain oligosaccharides.

[0087] Accordingly, in some embodiments, the presently disclosed subject matter provides a synthetic HS oligosaccharide and / or the use thereof in treating an acute medical condition (e.g., sepsis and / or drug-induced liver injury (e.g., acetaminophen overdose)). The synthetic HS oligosaccharides can be prepared via chemoenzymatic synthetic routes and can be provided as single compounds of a particular oligosaccharide length and sulfation pattern.

[0088] In some embodiments, the synthetic HS oligosaccharide has a structure of

[0089] Formula (I): wherein: m and n are each selected from 0, 3, 4, 5, 6, and 7, subject to the proviso that the sum of m and n is 3, 4, 5, 6, or 7; Ri is selected from -H and -SO3H; R2 is selected from -SO3H and -COCH3; R3 is selected from -H and -SO3H; and R4 is selected from the group comprising -H, alkyl, aryl, substituted alkyl, substituted aryl, and a functional handle.

[0090] In some embodiments, one of m and n is 0. Thus, in some embodiments, m is 0 and n is 3, 4, 5, 6, or 7. In some embodiments, m is 0 and n is 4, 5, or 6. In some embodiments, m is 0 and n is 7. In some embodiments, n is 0 and m is 3, 4, 5, 6, or 7. In some embodiments, n is 0 and m is 4, 5, or 6. In some embodiments, R2 is - SO3H. In some embodiments; the synthetic HS oligosaccharide is a compound of Formula (I) above, selected from (i)-(vi), wherein:

[0091] (i) is a compound having the structure: where m is 0, n is 4, Ri is -SO3H, R2 is -SO3H, and R3 is -SO3H;

[0092] (ii) is a compound having the structure: where m is 0, n is 5, Ri is -H, R2 is -SO3H, and R3 is -SO3H;

[0093] (iii) is a compound having the structure: where m is 0, n is 6, Ri is -H, R2 is -SO3H, and R3 is -SO3H;

[0094] (iv) is a compound having the structure: where m is 4, n is 0, Ri is -SO3H, and R2 is -SO3H;

[0095] (v) is a compound having the structure: where m is 5; n is 0; Ri is -SO3H, and R2 is -SO3H; and

[0096] (vi) is a compound having the structure: where m is 6; n is 0; Ri is -SO3H; and R2 is -SO3H.

[0097] In some embodiments, R4 is H. In some embodiments, R s alkyl (e.g., Cl- C6 alkyl, such as methyl or ethyl). In some embodiments, R4 is a functional handle. In some embodiments, R4 is substituted aryl. In some embodiments, R4 is p- nitrophenyl (pNP or p-NP).

[0098] More particularly, an exemplary compound (i) has the structure shown in Figure 21A. This compound can also be referred to as GlcNS6S-GlcA-GlcNS6S- IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA- pNP; 12-mer NS2S6S; or GLY-202.

[0099] An exemplary compound (ii) has the structure shown in Figure 2 IB. This compound can also be referred to as GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-IdoA2S- GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-GlcA-pNP or 14-mer NS2S.

[0100] An exemplary compound (iii) has the structure shown in Figure 21C. This compound can also be referred to as GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-IdoA2S- GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-GlcA-pNP or 16-mer NS2S.

[0101] An exemplary compound (iv) has the structure shown in Figure 2 ID. This compound can also be referred to as GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S- GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-pNP or 12-mer NS6S.

[0102] An exemplary compound (v) has the structure shown in Figure 2 IE. This compound can also be referred to as GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S- GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-pNP or 14- mer NS6S.

[0103] An exemplary compound (vi) has the structure shown in Figure 2 IF. This compound can also be referred to as GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S- G1CA-G1CNS6S-G1CA-G1CNS6S-G1CA-G1CNS6S-G1CA-G1CNS6S-G1CA-G1CNS6S- GlcA-pNP or 16-mer NS6S. In some embodiments, the compound of Formula (I) is an 18-mer. In some embodiments, m is 0, n is 7, Ri is -H, R2 is -SO3H; and R3 is -SO3H. In some embodiments, R4 is p-nitrophenyl. In some embodiments, the compound is GlcNS- GlcA-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS- IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-GlcA-pNP (18-mer NS2S).

[0104] In some embodiments, the synthetic HS oligosaccharide is a compound of wherein: m and n are each selected from 0, 4, 5, 6, and 7 (e.g., 0, 4, 5, and 6); Ri is selected from -H and -SO3H; R2 is selected from -SO3H and -COCH3; R3 is selected from -H and -SO3H; and R4 is selected from the group comprising -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle; subject to the proviso that the synthetic HS oligosaccharide is selected from (ii), (iii), (v), and (vi), wherein: (ii) m is 0; n is 5; Ri is -H, R2 is -SO3H; and R3 is -SO3H; (iii) m is 0; n is 6; Ri is -H, R2 is -SO3H; and R3 is -SO3H; (v) m is 5; n is 0; Ri is -SO3H, and R2 is -SO3H; and (vi) m is 6; n is 0; Ri is -SO3H; and R2 is -SO3H. In some embodiments, the R4 is substituted aryl. In some embodiments, R4 is pNP.

[0105] In some embodiments, the synthetic HS oligosaccharide is (v) or (vi). In some embodiments, the synthetic HS oligosaccharide is 14-mer NS6S or 16-mer NS6S.

[0106] In some embodiments, the synthetic HS oligosaccharide is (ii) or (iii). In some embodiments, the synthetic HS oligosaccharide is 14-mer NS2S or 16-mer NS2S. In some embodiments, the synthetic HS oligosaccharide is (ii), e.g., 14-mer NS2S.

[0107] III. PHARMACEUTICAL COMPOSITIONS

[0108] In some embodiments, the presently disclosed subject matter provides pharmaceutical and / or therapeutic compositions comprising one or more synthetic HS oligosaccharide (i.e., a compound of Formula (I), e.g., (i)-(vi) or 18-mer NS2S), as disclosed herein. In some embodiments, a pharmaceutical composition can comprise (a) a synthetic HS compound (i.e., a compound of Formula (I)) and (b) a pharmaceutically acceptable carrier or adjuvant. In some embodiments, the pharmaceutical and / or therapeutic composition comprises two or more compounds of Formula (I). In some embodiments, the composition comprises one or more or two or more of (i), (ii), (iii), (iv), (v), and (vi). In some embodiments, R4 is substituted aryl, e.g., pNP. In some embodiments, the composition comprises one or more or two or more of 12-mer NS2S6S, 14-mer NS2S, 16-mer NS2S; 12-mer NS6S, 14-mer NS6S, and 16-mer NS6S. In some embodiments, the one or more synthetic HS oligosaccharides comprise at least one of 14-mer or 16-mer (i.e., (ii), (iii), (v) or (vi)). In some embodiments, the one or more synthetic HS oligosaccharides comprise at least one of (ii), (v), and (vi), such as at least one of 14-mer NS2S, 14-mer NS6S, and 16-merNS6S. In some embodiments, the composition comprises 12-merNS2S6S. In some embodiments, the composition comprises an 18-mer of Formula (I), e.g., 18- mer NS2S.

[0109] In some embodiments, the carrier is pharmaceutically acceptable for use in humans. The carrier or adjuvant desirably should not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, ammo acid copolymers and inactive virus particles.

[0110] Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonate and benzoates.

[0111] Pharmaceutically acceptable carriers in therapeutic compositions can additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, can be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated for administration to the patient.

[0112] Suitable formulations of pharmaceutical compositions of the presently disclosed subject matter include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats, bactericidal antibiotics and solutes which render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. The formulations can be presented in unitdose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use. Some exemplary ingredients are SDS in the range of in some embodiments 0.1 to 10 mg / ml, in some embodiments about 2.0 mg / ml; and / or mannitol or another sugar in the range of in some embodiments 10 to 100 mg / ml, in some embodiments about 30 mg / ml; and / or phosphate-buffered saline (PBS). Any other agents conventional in the art having regard to the type of formulation in question can be used. In some embodiments, the carrier is pharmaceutically acceptable. In some embodiments the carrier is pharmaceutically acceptable for use in humans.

[0113] Pharmaceutical compositions of the presently disclosed subject matter can have a pH between 5.5 and 8.5, preferably between 6 and 8, and more preferably about 7. The pH can be maintained by the use of a buffer. The composition can be sterile and / or pyrogen free. The composition can be isotonic with respect to humans. Pharmaceutical compositions of the presently disclosed subject matter can be supplied in hermetically-sealed containers.

[0114] A therapeutic method according to the presently disclosed subject matter comprises administering to a subject in need thereof a HS or related compound as disclosed herein.

[0115] An effective dose of a pharmaceutical composition of the presently disclosed subject matter is administered to a subject in need thereof. The terms “therapeutically effective amount,” “therapeutically effective dose,” “effective amount,” “effective dose,” and variations thereof are used interchangeably herein and refer to an amount of a therapeutic composition or pharmaceutical composition of the presently disclosed subject matter sufficient to produce a measurable response (e.g., reduced plasma alanine aminotransferase (ALT) in liver injury). Actual dosage levels can be varied so as to administer an amount that is effective to achieve the desired therapeutic response for a particular subject.

[0116] In some embodiments, the quantity of a therapeutic composition of the presently disclosed subject matter administered to a subject will depend on a number of factors including but not limited to the subject’s size, weight, age, the target tissue or organ, the route of administration, the condition to be treated, and the severity of the condition to be treated.

[0117] The potency of a therapeutic composition can vary, and therefore a “therapeutically effective” amount can vary. However, using the assay methods described herein below, one skilled in the art can readily assess the potency and efficacy of the pharmaceutical compositions of the presently disclosed subject matter and adjust the therapeutic regimen accordingly.

[0118] In some embodiments, the synthetic HS oligosaccharides and / or their pharmaceutical compositions can be used to treat an acute care condition (e.g., drug- induced liver injury or sepsis) in a subject in need of treatment thereof. The subject treated in the presently disclosed subject matter is desirably a human subject, although it is to be understood that the principles of the disclosed subject matter indicate that the compositions and methods are effective with respect to invertebrate and to all vertebrate species, including mammals, which are intended to be included in the term “subject.” Moreover, a mammal is understood to include any mammalian species in which treatment of sepsis, acute liver failure (e.g., acetaminophen overdose), is desirable, particularly agricultural and domestic mammalian species.

[0119] The methods of the presently disclosed subject matter are particularly useful in the treatment of warm-blooded vertebrates. Thus, the presently disclosed subject matter concerns mammals and birds.

[0120] More particularly, provided herein is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans) and / or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered, kept in zoos, kept as pets (e.g., parrots, finches, etc.) as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical and / or social importance to humans. Thus, provided herein is the treatment of livestock, including, but not limited to, domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

[0121] IV. METHODS OF TREATING ACUTE CARE CONDITIONS

[0122] In some embodiments, the presently disclosed subject matter provides a method of treating an acute care condition, such as an acute care condition involving sterile inflammation and / or an unregulated inflammatory response. In some embodiments, the acute care condition is a drug-induced livery injury or sepsis. In some embodiments, the acute care condition is drug-induced liver injury. In some embodiments, the drug-induced liver injury is APAP-induced acute liver failure. In some embodiments, the acute care condition is sepsis.

[0123] In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human subject. In some embodiments, the subject is a subject who has not experienced ischemia-reperfusion injury.

[0124] In some embodiments, the presently disclosed subject matter provides a method of treating drug-induced liver injury or sepsis in a subject in need thereof. In some embodiments, the method comprises administering to the subject a synthetic HS oligosaccharide having a structure of Formula (I): wherein m and n are each selected from 0, 3, 4, 5, 6, and 7, subject to the proviso that the sum of m and n is 3, 4, 5, 6, or 7; Ri is selected from -H and -SO3H; R2 is selected from -SO3H and -COCH3; R3 is selected from -H and -SO3H; and R4 is selected from the group comprising -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.

[0125] In some embodiments, one of m and n is 0. Thus, in some embodiments, m is 0 and n is 3, 4, 5, 6, or 7. In some embodiments, m is 0 and n is 4, 5, or 6. In some embodiments, n is 0 and m is 3, 4, 5, 6, or 7. In some embodiments, n is 0 and m is 4, 5, or 6. In some embodiments, R2 is -SO3H. In some embodiments, the synthetic HS oligosaccharide has a structure of Formula (I): wherein: m and n are each selected from 0, 4, 5, 6 and 7 (e.g., 0, 4, 5, or 6); Ri is selected from -H and -SO3H; R2 is selected from -SO3H and -COCH3; R3 is selected from -H and -SO3H; and R4 is selected from the group comprising -H, alkyl, aryl, substituted alkyl, substituted aryl, and a functional handle; subject to the proviso that the synthetic HS oligosaccharide is selected from (i)-(vi) or a pharmaceutical composition thereof. In some embodiments, the synthetic HS oligosaccharide has a structure of Formula (I) wherein: (i) m is 0, n is 4, Ri is -SO3H, R2 is -SO3H, and R3 is -SO3H; (ii) m is 0, n is 5, Ri is -H, R2 is -SO3H, and R3 is -SO3H; (iii) m is 0, n is 6, Ri is -H, R2 is -SO3H, and R3 is -SO3H; (iv) m is 4, n is 0, Ri is -SO3H, and R2 is - SO3H; (v) m is 5, n is 0, Ri is -SO3H, and R2 is -SO3H; and (vi) m is 6, n is 0, Ri is - SO3H, and R2 is -SO3H.

[0126] In some embodiments, R4 is H. In some embodiments, R4 is alkyl (e.g., methyl or ethyl). In some embodiments, R4 is a substituted aryl. In some embodiments, R4 is pNP.

[0127] In some embodiments, the synthetic HS oligosaccharide is selected from the group comprising 12-mer NS2S6S, 14-mer NS2S, 16-mer NS2S, 12-mer NS6S, 14- mer NS6S, and 16-mer NS6S.

[0128] In some embodiments, the synthetic HS oligosaccharide is selected from (i), (iv), (v), and (vi). In some embodiments, the synthetic HS oligosaccharide is (i), e.g., 12-mer NS2S6S (GLY-202). In some embodiments, the synthetic HS oligosaccharide is (iv), e.g., 12-mer NS6S. In some embodiments, the synthetic HS oligosaccharide is (v), e.g., 14-mer NS6S. In some embodiments, the synthetic HS oligosaccharide is (vi), e.g., 16-mer NS6S.

[0129] In some embodiments, the synthetic HS oligosaccharide is administered about 3 hours, about 6, hours, about 9 hours, about 12 hours or more than 12 hours after the subject is exposed to a drug (e.g., APAP) that induces liver injury. In some embodiments, the presently disclosed subject matter provides a synthetic HS oligosaccharide or a pharmaceutical composition thereof for use in a method of treating drug-induced liver injury or sepsis in a subject in need thereof. In some embodiments, the drug-induced liver injury is APAP -induced acute liver failure. In some embodiments, the synthetic HS oligosaccharide has a structure of Formula wherein: m and n are each selected from 0, 4, 5, 6, and 7 (e.g., 0, 4, 5, and 6); Ri is selected from -H and -SO3H; R2 is selected from -SO3H and -COCH3; R3 is selected from -H and -SO3H; and R4 is selected from the group comprising -H, alkyl, aryl, substituted alkyl, substituted aryl, and a functional handle; subject to the proviso that the synthetic HS oligosaccharide is selected from (i)-(vi) or a pharmaceutical composition thereof wherein: (i) m is 0, n is 4, Ri is -SO3H, R2 is -SO3H, and R3 is - SO3H; (ii) m is 0, n is 5, Ri is -H, R2 is -SO3H, and R3 is -SO3H; (iii) m is 0, n is 6, Ri is -H, R2 is -SO3H, and R3 is -SO3H; (iv) m is 4, n is 0, Ri is -SO3H, and R2 is - SO3H; (v) m is 5, n is 0, Ri is -SO3H, and R2 is -SO3H; and (vi) m is 6, n is 0, Ri is - SO3H, and R2 is -SO3H.

[0130] In some embodiments, R4 is H. In some embodiments, R4 is alkyl (e.g., methyl or ethyl). In some embodiments, R4 is a substituted aryl. In some embodiments, R4 is pNP.

[0131] In some embodiments, the synthetic HS oligosaccharide is selected from the group comprising 12-mer NS2S6S, 14-mer NS2S, 16-mer NS2S, 12-mer NS6S, 14- mer NS6S, and 16-mer NS6S.

[0132] In some embodiments, the synthetic HS oligosaccharide is selected from (i), (iv), (v), and (vi). In some embodiments, the synthetic HS oligosaccharide is (i), e.g., 12-mer NS2S6S (GLY-202). In some embodiments, the synthetic HS oligosaccharide is (iv), e.g., 12-mer NS6S. In some embodiments, the synthetic HS oligosaccharide is (v), e.g., 14-mer NS6S. In some embodiments, the synthetic HS oligosaccharide is (vi), e.g., 16-mer NS6S. In some embodiments, the presently disclosed subject matter provides a method of treating sepsis in a subject in need thereof, the method comprising administering to the subject a synthetic HS oligosaccharide having a structure of Formula (I): wherein: m is 0; n is 7; Ri is H; R2 is -SO3H; R3 is -SO3H; and R4 is -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle. In some embodiments, R4 is p-nitrophenyl. In some embodiments, the compound is GlcNS-GlcA-GlcNS- IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS- IdoA2S-GlcNS-IdoA2S-GlcNS-GlcA-pNP (i.e., 18-mer NS2S).

[0133] In some embodiments, the presently disclosed subject matter provides a synthetic HS oligosaccharide for use in treating sepsis in a subject in need of treatment thereof, wherein the synthetic HS oligosaccharide has a structure of Formula (I) wherein: m is 0; n is 7; Ri is H; R2 is -SO3H; R3 is -SO3H; and R4 is -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle. In some embodiments, the synthetic HS oligosaccharide is 18-mer NS2S.

[0134] The methods of the presently disclosed subject matter are particularly useful in the treatment of warm-blooded vertebrates. Thus, the presently disclosed subject matter concerns mammals and birds.

[0135] More particularly, provided herein is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans) and / or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered, kept in zoos, kept as pets (e.g., parrots, finches, etc.) as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical and / or social importance to humans. Thus, provided herein is the treatment of livestock, including, but not limited to, domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

[0136] EXAMPLES

[0137] The following examples are included to further illustrate various embodiments of the presently disclosed subject matter. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the presently disclosed subject matter.

[0138] EXAMPLE 1

[0139] MATERIALS AND METHODS FOR HS OLIGOSACCHARIDES

[0140] HS oligosaccharides, including 6-mer NS2S, 12-mer NS2S, and 18-mers NS2S, were chemoenzymatically synthesized based on previous methods (Arnold, 2020b; Xu, 2014). The binding affinity of the oligosaccharides to H3 was determined by surface plasmon resonance (SPR). The evaluation of HS protection was investigated in the cecal ligation and puncture (CLP) model with various biomarker analysis by enzyme-linked immunosorbent assay (ELISA), Luminex multiplex assay, western blot, and quantitative polymerase chain reaction (qPCR). The mechanistic studies were conducted with the LPS-induced endotoxemia model and pull-down assays, followed by proteomic analysis for identifying apoA-I. Additional method details are as follows:

[0141] Synthesis ofNS2S Heparan Sulfates

[0142] The syntheses of 6-mer NS2S, 12-mer NS2S, 18-mer NS2S were completed according to the method published previously (Arnold, 2020b). Briefly, PmHS2 (heparan synthase 2 from P. multocidd) was used with UDP-sugars to elongate the monosaccharide, GlcA-pNP (p-nitrophenyl glucuronide, from Carbosyn), to the desired size of backbones. The backbone was then subjected to modification with N- sulfotransferase (NST), Cs-epimerase (Cs-epi), and 2-O-sulfotransferase (2-OST). Four primary steps were involved in the synthesis, including step elongation- 1 to add GlcNTFA, step elongation-2 to add GlcA, step N-sulfation, and step of epimerization / 2-O-sulfation. See Figure 5A. These steps were repeated to prepare the final products. Finally, the products were purified using a Q-Sepharose column (GE Health) by anion-exchange chromatography with an overall yield of ~4%, ~17% and ~48% for 18-mer NS2S, 12-mer NS2S and 6-mer NS2S, respectively. Using the chemoenzymatic method, both NAc 18-mer and 18-mer NS6S were also synthesized. For the synthesis of NAcl 8-mer, only elongation steps were involved; for the synthesis of 18-mer NS6S, elongation, N-sulfation and 6-O-sulfation steps were involved. Electrospray ionization mass spectrometry (ESI-MS) was used to measure the molecular weight and confirm the structures of the products.

[0143] Step elongation- 1 adds a GlcNTFA residue. In an exemplary synthesis of 8- mer (GlcNTFA-GlcA-GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP) intermediate from a 7-mer (GlcA-GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP), the reaction mixture included NaAcO (50 mM, pH 6), MnCh (5 mM), 13 g of 7-mer, pmHS2 (90 pg / ml) and UDP-GlcNTFA (24 mM) in a total volume of 500 mL, then incubated at 37°C overnight. A Q-Sepharose column (0.4 L) was used for purification with a gradient elution from 0.2 M to 0.8 M NaCl in 20 mM sodium acetate pH 5 in 240 min; the flow rate was 4 mL / min.

[0144] Step elongation-2 adds a GlcA residue. In an exemplary conversion of the 8- mer (GlcNTFA-GlcA-GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP) intermediate to a 9-mer intermediate (GlcA-GlcNTFA-GlcA-GlcNS-GlcA-GlcNS-IdoA2S- GlcNS-GlcA-pNP), the reaction mixture included NaAcO (50 mM, pH 6), MnCh (5 mM), 14 g of 8-mer intermediate, pmHS2 (135 pg / ml) and UDP-GlcA (10 mM) in a total volume of 1.3 L, then incubated at 37°C overnight. A Q-Sepharose column (0.4 L) was used for purification with gradient elution from 0.2 M to 0.8 M NaCl in 20 mM sodium acetate pH 5 in 240 min; the flow rate was 4 mL / min.

[0145] Step N-sulfation converts a GlcNH? residue to a GlcNS residue using N- sulfotransferase (NST). In an exemplary N-sulfation reaction, 13 g of G1CNH2-G1CA- GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP was incubated with NST (45 pg / ml) and PAPS (2 equivalence of free amino groups) in a solution containing 2-(N- morpholino) ethanesulfonic acid (MES, 50 mM, pH 7.0) and at 37°C overnight in a reaction volume of 0.7 L. A Q-Sepharose column (0.4 L) was used for purification with gradient elution from 0.2 M to 0.8 M NaCl in 20 mM sodium acetate pH 5 in 240 min; the flow rate was 4 mL / min.

[0146] Step epimerization / 2-O-sulfation converts an internal GlcA residue to an IdoA2S residue using both C5-epi and 2-OST. In an exemplary epimerization / 2-O- sulfation, 13 g of GlcNS-GlcA-GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP (8- mer) was incubated in a solution containing Tris (50 mM) buffer (pH 7.5) and semipurified C5-epi (6 pg / ml), 2-OST (13 pg / ml) and PAPS (2 equivalence of start 8-mer material amount) at 37°C overnight in a total volume of 1 L. A Q-Sepharose column (0.4 L) was used for purification with a gradient elution from 0.4 M to 0.8 M NaCl in 20 mM sodium acetate pH 5 in 320 min; the flow rate was 4 mL / min.

[0147] The saccharide backbones were purified using a C18-column (3 x 15Cm, or 120 g, Biotage) with a gradient elution method (0-80% methanol in H2O, 0.1% trifluoroacetic acid, 5 mL / min). All sulfated saccharide products were purified using a Q-Sepharose column and were eluted with a linear gradient of 0.2 -1 M NaCl in 20 mM NaOAc-HAcO, pH 5.0 in 3-6 hours. Different sizes of Q-Sepharose columns were chosen by the binding affinity of the product and reaction scale. At the end of the synthesis, we obtained 6-mer (13 g, purity 99.5%), 12-mer (12 g, purity 99.2 %), and 18-mer (2.4 g, purity 90.2%). Here are the ESLMS results: 6-mer measured MW = 1455.56 Da (theoretical MW=1455.18 Da): 12-mer measured MW = 2925.53 Da (theoretical MW = 2925.35 Da); 18-mer measured MW = 4456.90 Da (theoretical MW = 4455.55 Da); NAc 18-mer measured MW = 3555.5 Da (theoretical MW = 3553.97) and NS6S 18-mer measured MW = 4616.0 Da (theoretical MW = 4615.7).

[0148] At every synthesis step, the products were monitored by a Shimadzu HPLC equipped with a Propac column (Propac PAI, 10 pm, 9 mm x 250 mm, Thermo Fisher). Buffer A was 20 mM NaAcO (pH 5.0), and buffer B was 2 M NaCl with 20 mM NaAcO (pH 5.0). A linear gradient was used to separate the start material and product. Different gradients were chosen by the sulfation degree of compounds. The UV detector was set at 310 nm to monitor the oligosaccharides. The structures of the intermediates from each step were characterized by ESLMS.

[0149] ESLanalysis were performed at a Thermo LCQ-Deca in negative ionization mode. A syringe pump (Harvard Apparatus) introduces the sample by direct infusion (50 pL / min). The sample (2-5 pg) was diluted in 200 pL of 10 mM ammonium bicarbonate with the electrospray source set to 3 kV and 150 °C. The automatic gain control was 1 x 107for full scan MS. The MS data were acquired and processed using Xcalibur 1.3.

[0150] Endotoxin removal for HS

[0151] Endotoxin-free oligosaccharides(6-mer NS2S, 12-mer NS2S, 18-mer NS2S and NAc 18-mer) were prepared for the animal studies based on a previously published method (Xu, 2017). Briefly, a sterile 50 mL centrifuge tube (Amicon Ultra- 15, Ultracel-lOOk; Merck Millipore) was used to remove endotoxin in 6-mer, 12-mer, and 18-mers by centrifugation of the oligosaccharide solution at 4,000 rpm for 10 min with four repeated washes to collect all HS oligosaccharides. The filtered solutions containing oligosaccharides were collected and dried. The level of endotoxin in the purified oligosaccharides was confirmed by the Limulus Amebocyte Lysate (LAL) kit (sensitivity = 0.125 EU / mL, Associates of Cape Cod Inc.) according to manufacturer’s protocol.

[0152] Determination of the in vitro and ex vivo anti-FXa and anti-IIa activity

[0153] An anti-Xa activity assay was performed on a previously published method (Xu, 2017). Briefly, the dilution was prepared as follows: human FXa (Enzyme Research Laboratories) to 25 U / mL with PBS; chromogenic substrate S-2765 (Diapharma) to 1 mg / mL in water; unfractionated heparin (UFH), fondaparinux (FPX), 6-mer, 12-mer, 18-mer to 5 pg / mL in PBS. The reaction mixture with 60 pL of human plasma (Sigma-Aldrich) and 60 pL of solution containing saccharides at different concentrations was incubated for 2 min at room temperature. FXa (100 pL) was added for another 4 min incubation at room temperature. S-2765 substrate (30 pL) was added, and the reaction was measured at 405 nm continuously for 3 min. The absorbance values were plotted against the reaction time.

[0154] An anti-IIa activity assay was performed based on a previously published method (Xu, 2017). Briefly, the dilution was prepared as follows: thrombin from bovine plasma (Sigma-Aldrich) to 20 U / mL in PBS with bovine serum albumin (1 mg / mL); chromogenic substrate S-2238 (Diapharma) to 1 mg / mL in water; UFH, FPX, 6-mer NS2S, 12-mer NS2S, 18-mer NS2S to 5 pg / mL in PBS. The reaction mixture with 60 pL of antithrombin (35 pg / mL, Cutter biologies) and the saccharide solution (60 pL) was incubated for 2 min at room temperature. Thrombin was added and incubated for 4 min at room temperature before adding S-2238 substrate (30 pL) and measured at 405 nm continuously for 5 min. The absorbance values were plotted against the reaction time.

[0155] Heparanase digestion of 18-mers

[0156] Recombinant heparanase was expressed in insect cells using the baculovirus expression system and purified by a heparin-Sepharose column, as previously described (Peterson, 2012). 18-mer NS2S or 18-mer NS6S (200 pg / mL) was incubated with heparanase (20 pg / mL) at 37 °C in 50 mM MOPS at pH 6. After 24 hours of incubation, the reaction was inactivated by heating at 95°C for 5 min. LC-MS and HPLC analysis of heparanase digested 18-mer and NS6S 18-mer

[0157] LC-MS / MS analysis of heparanase digested oligosaccharides was implemented on a Vanquish Flex UHPLC system (Thermo Fisher, Waltham, MA) coupled with a TSQ Fortis triple-quadrupole mass spectrometer. The ACQUITY Glycan BEH Amide column (1.7 pm, 2.1 x 150 mm; Waters) was used at 60°C. The mobile phase A was 50 mM ammonium formate in water (pH 4.4) and mobile phase B was acetonitrile. The elution gradient and flow rate consisted of 0-6 min 83% B, flow rate 0.3 mL / min; 6.1-45 min 83-5% B, flow rate 0.25 mL / min; 45-55 min 5% B, 0.25 mL / min; 55.1-65 min 83% B, flow rate 0.3 mL / min. On-line triple-quadrupole mass spectrometry was used as the detector. ESLMS analysis was operated in the negative-ion mode using the following parameters: Negative ion spray voltage at 3.0 kV, sheath gas at 55 Arb, aux gas 25 arb, ion transfer tube temperature at 250°C and vaporizer temperature at 400°C. TraceFinder software was applied for data processing. A total of 0.5-1 pg of heparanase digested oligosaccharides in 2 pL was injected per run.

[0158] The reaction of heparanase digestion was also monitored using an ion exchange HPLC column (ProPac PAI) with a gradient elution of buffer B (2M NaCl, 20 mM sodium acetate, pH 5.0) from Buffer A (20 mM sodium acetate, pH 5.0) at a flow rate of 1 ml / min. A linear gradient of 40-50% B in 40 minutes was used for the analysis of 18-mer NS2S, and a linear gradient of 70-85% B in 40 minutes was used for the analysis of 18-mer NS6S. The eluent was detected by an on-line UV detector at 310 nm. Cecal ligation and puncture (CLP) mouse model

[0159] CLP was performed using a previously published method (Rittirsch, 2009) with male 10 to 12 weeks old C57 / BL6 mice (the Jackson Laboratory) with body weights of around 25 to 30 grams to induce polymicrobial sepsis. Mice were housed in the animal facility for at least two weeks before the experiment to ensure the fecal flora stability. Mice were anesthetized with 3% isoflurane for hair removal and the following surgery to perform CLP injury. The mouse abdomen was sterilized with betadine and ethanol, and a 1-cm midline incision was made. The mouse cecum was externalized, ligated 1-cm from the distal end of the cecum with a 4:0 silk suture, punctured through-and-through with a 21 -gauge needle, and gently squeezed to extrude a small amount of feces from the perforation sites. The cecum was reinternalized before the double-layer closure of the peritoneum and skin with 5:0 nylon sutures and the AutoClip system, respectively. Sham mice received a similar process as CLP mice described above, without the cecum externalized and reinternalized but not ligated or punctured. After the surgery, each mouse was given 500 pL of fluid in total after the CLP surgery to prevent dehydration. A 500 pL of fluid included saline, mel oxicam (2mg / kg), with or without the 18-mer (20 mg / kg). Mice were recovered on a warm blanket until they could raise themselves. In the biomarker analysis study, each mouse was given 10 pL / g at 0, 6, and 12 hours, with or without the 18-mer. For example, the treated mice at 25 g were administered 250 pL of 18-mer NS2S (2 mg / mL) or saline. At 24 hours after CLP, mice were anesthetized with 3% isoflurane for blood, peritoneal lavage, and organ collection. Blood was collected through inferior vena cava with 50 pL of 3.8% sodium citrate; peritoneal lavage was collected by flushing 1 mL sterile PBS in the peritoneal space. After sample collection, mice were sacrificed with cervical dislocation. In the survival study, mice were administered subcutaneously with 20 mg / kg of 18-mer NS2S (or 6- mer NS2S) at 0, 6, 12, 24, 36, and 52 hours after CLP injury. Mice were frequently checked at least every 6 hours until sacrificed at 72 hours after CLP. Mice were promptly euthanized during the experiment if they met the humane endpoint. Hematologic analysis

[0160] After blood collection from mice in the CLP model, complete blood count was determined in whole blood by Element HT5 veterinary complete blood count analyzer (Heska).

[0161] Counts of colony forming units (CFU)

[0162] After peritoneal lavage collection from mice in the CLP model, the diluted peritoneal lavage (1 : 100, 1 : 1000, 1 : 10000) in sterile PBS were plated on agar plates (1.5% agarose, Fisher; 2% Difco LB broth, BD) and incubated under aerobic conditions at 37 °C. Bacterial colonies were counted after 24 hours. Results were specified as CFU / mL in loglO.

[0163] Pull-down assay / Immunoblot

[0164] Biotinylated LPS (Invivogen, USA) (5 mg / kg) with or without HS (6-mer NS2S, 12-mer NS2S, 18-mer NS2S at 5 mg / kg or 50 mg / kg) was injected into male 10 to 12 weeks old C57 / BL6 mice intraperitoneally (i.p.). After 2 hours, the mouse was sacrificed, and the mouse peritoneum was lavaged with 1 mL of sterile PBS. Peritoneal lavage was collected and frozen immediately for pull-down assay. In each pull-down reaction, peritoneal lavage (100 pL) was incubated with 50 pL of Pierce high-capacity streptavidin agarose (Thermo Fisher) at room temperature for 1 hour to isolate biotinylated LPS bound complexes. After washing with PBS five times, samples were eluted with lithium dodecyl sulfate (LDS) sample buffer and 50 mM of tris[2-carboxy ethyl] phosphine-HCl (TCEP) solution (Thermo Fisher). Preload (15 pL of peritoneal lavage without streptavidin agarose incubation) and eluted samples were separated using NuPAGE 4-12% Bis-Tris protein gels (Invitrogen), transferred to nitrocellulose membranes (Bio-Rad), and detected by Ponceau S solution (Sigma). Membranes were rinsed and then cut for Western blot staining with anti-HMGBl (1 : 10000, Abeam) and ApoA-I (1 :4000, Novus Biologicals), followed by anti-rabbit HRP (1 : 10000, Cell Signaling Technology).

[0165] In a competitive binding assay, 1-10 pg / mL of recombinant ApoA-I (Sino Biological) or 100 pg / mL of HS oligosaccharide was incubated with collected peritoneal lavage overnight at 4°C on a rotator before incubating with streptavidin agarose. Proteomic analysis

[0166] Briefly, the peritoneal lavage from biotinylated LPS injected mice was eluted by streptavidin agarose for electrophoresis as described above. Using Coomassie staining, the band around 25 kDa was identified and cut for proteomic analysis. See Figures 13 A. To prepare for LC / MS / MS analysis, half of the sample volume (25 pL) was denatured with 8 M urea, followed by reduction with dithiothreitol (DTT) and alkylation with iodoacetic acid (IAA). The sample was digested with trypsin overnight at 37°C before acidification with 0.5% trifluoroacetic acid (TFA) and cleaned by a Cl 8 ziptip. The LC / MS / MS analysis was performed on a liquid chromatography system sold under the tradename THERMO EASY NLC™1200-PHCO QEHF system, 45 min method. The data was searched against reviewed contaminants and Uniport mouse databased using Sequest within Proteome Discoverer 2.4. Only targets with high confidence peptides and proteins (false discovery rate (FDR) < 1%) and proteins with >1 peptide are shown in the result. See Table 1, below, ranked by peptide spectrum match (PSM). As ApoA-I was demonstrated with the most abundant PSM, the LPS-bound protein around 25 kDa was confirmed as ApoA-I.

[0167] Table 1. Potential match of the protein (~M.W. 25kda) bound to LPS.

[0168]

[0169]

[0170]

[0171] After Coomassie staining, the band at ~M.W. 25kda in Figure 13 A was sent to proteomic analysis. The protein was determined as apolipoprotein A-I (apoAl) based on PSM. PSMs: Peptide spectrum match; AAs: amino acids.

[0172] Histone induced lethality in vivo

[0173] According to the previous report (Xu, 2009; Li, 2020), calf thymus histones (75 mg / kg, Sigma) was injected retro-orbitally into the anesthetized mice by 3% inhaled isoflurane with or without 18-merNS2S orNAc 18-mer (75 mg / kg). The mice were monitored for 300 minutes (5 hours) after injection for the survival experiment. After 300-minute monitoring, mice were euthanized by cervical dislocation under isoflurane (3%).

[0174] Histone induced cytotoxicity in vitro

[0175] EA.hy926 cells were obtained from Dr. Rafal Pawlinski's lab (Li, 2020). Cells cultured in less than 20 passages were seeded in 12-well plates (3 x 105cell / well) in DMEM medium (Gibco) with 10% fetal bovine serum (FBS) and 1% Penicillin- Streptomycin. When reaching 90% confluence, cells fasted for 18 hours of incubation in a serum-free DMEM medium. Cells were next incubated in serum-free DMEM containing 30 pg / mL histone H3 (Roche, Germany) with varying concentrations of HS (0 - 50 pg / mL of 6-mer NS2S, 12-mer NS2S, 18-mer NS2S). After 1 hour of incubation, PBS was used to wash cells twice before adding 0.05% trypsin-EDTA (Invitrogen) for detachment. Next, cells were collected by centrifugation (300 x g for 3 min) and resuspended in 1 mL PBS buffer containing 10 pg / mL propidium iodide (Sigma). After 10 min incubation, protected from light, cells were subjected to flow cytometry on Becton Dickinson LSRFortessa to evaluate cell viability. Appropriated unstained and untreated samples were used to calibrate gating and sorting conditions. LPS induced inflammation in macrophages

[0176] Raw264.7 cells were purchased from ATCC (American Type Culture Collection). Cells cultured in less than 20 passages were seeded in 48-well plates (1 X 105cell / well) in DMEM medium (Gibco) with 10% FBS and 1% Penicillin- Streptomycin (Invitrogen). When reaching 90% confluence, cells were fasted by overnight incubation in a serum-free DMEM medium. To test HMGB 1 / LPS mediated inflammation, recombinant HMGB1 isolated from HEK 293 cells was incubated with LPS (Escherichia coli serotype O111 :B4, Sigma, USA) at 37°C for 30 minutes in serum-free DMEM before adding to the cells. Cells were then treated with HMGB 1 / LPS complex with or without 18-mer NS2S for 16 hours, and the supernatants were harvested for analysis by ELISA. To test the effect of HDL on LPS, varying concentrations of human HDL (5 - 500 pg / mL, purchased from Athens Research and Technology, USA) were used to treat the cells for 6 hours. Then, the HDL-treated cells were incubated with 10 ng / mL of LPS for 16 hours in serum-free DMEM before harvesting cell supernatants for analysis by ELISA.

[0177] Biomarker analysis

[0178] Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was isolated from different mice organs, including lung, kidney, liver, and heart, with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. 1-5 pg of total RNA was reverse transcribed into cDNA using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, Inc.). mRNA expression of pro-inflammatory cytokines (IL-6), neutrophil markers (MCP-1), adhesion molecules (iCAM-1), and internal control glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) was evaluated by qPCR using the PowerUp SYBR Green Master Mix (Applied Biosystems). Primer pair sequences are summarized in Table 2, below, designed based on literature and ordered from Integrated DNA Technologies, Inc or Eton Bioscience. The amplification condition was as follows: 50°C (2 min), 95°C (2 min), followed by 40 cycles at 95°C (15 s), 55°C (15 s), 72°C (1 min). The melting curve was tested after the amplification, with the condition as follows: 95°C (15 s), 60°C (1 min), 95°C (15 s). The experiment was done using the PCR system sold under the tradename QUANTSTUDIO™ 6 Flex Real-Time PCR System (Thermo Fisher). mRNA levels were quantified with the 2-AACt method and normalized to the internal control gene GAPDH. The results are shown relative to the expression level of the sham group.

[0179] Table 2. Primer sequence used in the qPCR analysis for kidney samples.

[0180] Western blotting. Mouse plasma was collected 24 hours after CLP or sham surgery. Plasma containing 40 pg total protein (measured by Bradford assay) was denatured at 80°C 10 min before electrophoresis at 130 V with NuPAGE 4-12% BisTris protein gels (Invitrogen) for one hour. The gel was transferred at 30 V for 40 min to the nitrocellulose membrane (Bio-Rad). After blocking with 5% BSA in PBS for 2 hours at room temperature, the membrane was incubated overnight with an antihistone H3 antibody (1 : 1000, Abeam). Anti-rabbit HRP (1 : 10000, Abeam) was incubated for one hour at room temperature before being detected by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific). Enzyme-linked immunosorbent assay (ELISA). Mouse plasma and peritoneal lavage collected from CLP or LPS model or cell supernatant from LPS treated Raw264.7 were diluted and analyzed for inflammation markers IL-6, MCP-1, iCAM- 1 (kits obtained from Thermo Fisher Scientific, Inc.), HMGB1 (kit obtained from Tecan Group Ltd.), and ApoA-I (kits obtained from Abeam, USA). The measurement was performed according to the manufacturer's protocols.

[0181] Multiplex analysis. Mouse plasma collected from CLP model were diluted in 1 :2 and / or 1 :50 and analyzed for sepsis related inflammation and immunological markers by Luminex Discovery Assay, Mouse premixed multi-analyte kit (R&D Systems) and Bio-Plex 200 system. 21 markers were selected based on literature and study interest (Tsuchida, 2022), including G-CSF, GM-GSF, IFNg, IL-loc, IL-5, IL- 6, IL- 10, IL- 12 p70, IL-13, IL- 17, KC (CXCL1), MIP-2 (CXCL2), IP- 10 (CXCL10), MCP-1 (CCL2), RANTES (CCL5), TNFa, P-selectin, KIM-1, iCAM-1, RAGE, Syndecan-1. The measurement was performed by UNC advanced analytics core according to the manufacturer’s protocols.

[0182] Kidney injury markers. Creatinine and BUN assay was performed with mice plasma collected 24 hours after CLP surgery (kits obtained from Crystal Chem and Invitrogen, respectively). The measurement was performed according to the manufacturer's protocols.

[0183] HDL measurement. FIDE was measured in plasma or peritoneal lavage collected from mice that underwent CLP surgery or LPS injection (cholesterol assay kit from Abeam, USA). The measurement was performed according to the manufacturer's protocols. In short, mice plasma or peritoneal lavage was mixed with an equal volume of 2 x precipitation buffer and centrifuged at 5,000 rpm for 10 min to separate HDL from LDL / VLDL fraction. Then, according to the manufacturer's protocols, the diluted peritoneal lavage (2 x) and plasma (50 x) were incubated with cholesterol assay buffer, cholesterol probe, and enzyme mix. The Cholesterol reaction mix was incubated at 37°C for 60 min in the dark and measured at OD 570 nm.

[0184] LPS quantification. Mouse plasma or peritoneal lavage was collected from mice receiving 5 mg / kg i.p. of biotinylated LPS with or without 50 mg / kg of 18-mer for 2 hours. LPS levels in the samples were evaluated by using Pierce LAL chromogenic endotoxin quant kit (Thermo Fisher Scientific) based on the manufacturer's instruction. Briefly, 50 L of plasma (diluted lOO-lOOOx) and peritoneal lavage (diluted 500-5000 x) were added to a sterile 96-well plate along with endotoxin standard in serial dilution. Amebocyte lysates (50 pL) were freshly prepared and added to each well. After 8-min incubation, 100 pL of the chromogenic substrate was pre-warmed and added to each well for 6-min incubation. The readout was measured at OD 405nm after adding 50 uL of 25% acetic acid solution to stop the reaction. All reactions were maintained sterile at 37°C.

[0185] Determination of 18-mer NS2S binding to LPS

[0186] 18-mer NS2S (60 pM) was incubated with various doses of LPS (0 - 100 pM) in 500 pL PBS buffer for 30 min at room temperature. The mixtures were transferred to the 100 kDa centrifugal filter (Millipore) and centrifuged for 10 min at 4,000 pm. The binding ability of LPS determines by decreasing absorbance value of 18-mer NS2S after passing through the filter at UV 310 nm. LPS was measured by the Limulus Amebocyte Lysate (LAL) kit (Associated of Cape Cod Inc.) following the manufacturer's protocol. LPS did not pass through a 100 kDa centrifugal filter under this condition.

[0187] The method was validated in the previous study (Li, 2020). Briefly, the positive and negative control experiments were performed with antithrombin (AT) to HS hexasaccharide: GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S-GlcA-pNP (6- mer AXa) and GlcNS2S-GlcA-GlcNS2S-GlcA-GlcNS2S-GlcA-pNP (NS 6-mer). 6- mer AXa binds to AT but not NS 6-mer. To validate the method, different concentrations of AT (0 - 0.2 mM) were incubated with 0.2 mM of NS 6-mer or 6- mer AXa in ImL PBS buffer for 30 min at room temperature. The binding mixtures were transferred in a 30 kDa centrifugal filter (Millipore) and centrifuged at 4,000 rpm for 10 min. The binding ability of AT determines by decreasing the concentration of 6mer or 6-mer AXa after passing through the filter at UV 310 nm.

[0188] Determination of the binding of HS oligosaccharides to H3 by SPR

[0189] SPR measurements were performed on a T200 SPR (Cytiva). Research grade sensor CM5 chip and HBS-EP+ buffer were from Cytiva. To obtain kinetic data for interactions of H3 and HS oligosaccharides, H3 was immobilized on a CM5 chip according to the standard amine coupling protocol. Briefly, carboxymethyl groups on the CM5 chip surface were first activated using an injection pulse of 35 pL (flow rate, 5 pL / min) of an equimolar mix of N-ethyl-N-(dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (final concentration 0.05 M, mixed immediately prior to injection). Following activation, histone H3 was diluted to 200 pg / mL in 100 mM sodium acetate (pH 5.5) buffer and injected over the activated biosensor surface for immobilization. Excess unreacted sites on the sensor surface were deactivated with a 35 pL injection of IM ethanolamine. Next, a reference flow cell was prepared using an injection pulse of 35 pL (flow rate, 5 pL / min) of an equimolar mix of EDC and NSH, followed by a 35 pL injection of IM ethanolamine.

[0190] Oligosaccharide samples were diluted in HBS-EP+ buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4). Different dilutions of oligo samples were injected at a flow rate of 30 pL / min. At the end of the sample injection, the same buffer flowed over the sensor surface to facilitate dissociation. After a 3 min dissociation time, the sensor surface was regenerated by injecting 30 pL of 2M NaCl to get the fully regenerated surface. The binding response was monitored as time (sensorgram) function at 25°C. The sensorgrams were fit globally to obtain apparent on (ka) and off (kd) rates and binding equilibrium dissociation constant: KD (KD = kd / ka) using the T200 Evaluation software and assuming a 1 : 1 Langmuir model. Dissociation ofHDL by HS

[0191] Human HDL (Athens Research and Technology) (4 pg) was incubated with 1 pg of 6-mer NS2S, 12-mer NS2S, or 18-mer NS2S in 50 mM sodium acetate buffer (pH 5.0) for 1 h at 37°C. Samples were centrifuged at 13,000 rpm at 4°C for 30 min after incubation. The supernatant and pellet were separated, mixed with LDS sample buffer and TCEP solution (Thermo Fisher), and denatured at 95°C for 5 min. In Western blot analysis, samples were separated by electrophoresis with a similar setting described above. The gel was stained in Coomassie blue to visualize ApoA-I (protein band migrated to around 25 kDa).

[0192] Histological Analysis

[0193] Animals were humanely euthanized with 3% isoflurane using CO2 followed by cervical dislocation. Kidneys were collected from all animals and fixed in 10% Neutral Buffered Formalin (Thermo Fisher Scientific). Kidneys were then routinely processed to paraffin, sectioned to 5 pm, and stained with hematoxylin and eosin. Tissues were evaluated microscopically in a masked fashion by a board-certified veterinary pathologist (RSS).

[0194] Statistical analysis

[0195] Data are expressed as the mean ± standard error of the mean (SEM). Statistical significance between experimental and control groups was analyzed by two-tailed unpaired Student t-test and between multiple groups by one-way ANOVA followed by Dunnetf s multiple comparison tests. In addition, Kaplan-Meier survival curves in the CLP study were analyzed by log-rank (Mantel-Cox) test. All statistical analysis was performed using GraphPad Prism software (ver. 8.3.1; GraphPad Software, Inc ).

[0196] EXAMPLE 2

[0197] STRUCTURALLY DEFINED HS OLIGOSACCHARIDES Synthesis of structurally defined HS oligosaccharides

[0198] Three structurally defined oligosaccharides were used to test the relationship between the length of HS oligosaccharides and the anti-inflammatory effect. The 6- mer NS2S, 12-merNS2S, and 18-mer NS2S were synthesized using a chemoenzymatic method involving multiple enzymatic modification steps. See Figure 5A. All three HS have a disaccharide unit of GlcNS-GlcA at the non-reducing end, -IdoA2S-GlcNS- repeating units in the middle, and GlcA-pNP at the reducing end (Arnold, 2020b; Xu, 2014). See Figure 1A. The structures and purities of these oligosaccharides were confirmed by mass spectrometry and anion exchange HPLC. None of the oligosaccharides exhibits anticoagulant activity as determined by antifactor Xa and anti-factor Ila assays. See Figures 5B and 5C. The lack of anticoagulant activity of these oligosaccharides allows us to probe the anti-inflammatory mechanism independent of the anti coagulation effect in a sepsis animal model. 18-mer reduces local and systemic inflammatory responses as well as kidney damage in CLP-injured mice

[0199] A CLP mouse model was used based on literature reports (Rittirsch, 2009). The CLP model was validated using hematological analysis. See Figures 6A-6D. It was hypothesized that the longest synthetic HS, the 18-mer NS2S, could reduce inflammation in CLP-injured mice. See Figures 1B-1F. Based on previous studies with synthetic chondroitin sulfates and other studies with nonanticoagulant HS in sepsis (Wildhagen, 2014; Meara, 2020; Li, 2020), 18-mer NS2S (20 mg / kg) was subcutaneously administered after CLP surgery to mice at 0, 6, and 12 hours, and euthanized 24 hours after CLP to collect plasma, peritoneal lavage, and the kidney for analysis. The 18-mer NS2S treatment decreased the plasma concentration of H3 and HMGB1. See Figures IB and 1C. It also reduced the protein levels of inflammatory cytokine (interleukin-6, IL-6), chemokine (monocyte chemoattractant protein- 1, MCP-1), and adhesion molecule (intracellular adhesion molecule- 1, iCAM-1) in plasma (see Figures 1D-1F) and peritoneal lavage (see Figures 7A-7C), suggesting that 18-mer NS2S mitigated systemic and local inflammation in the peritoneal space. See also Figures 15A-15H, 16A-16V, and 17A-17D. Peritoneal lavage bacterial counts were performed for CLP mice and 18-mer NS2S-treated mice. Although 18- mer NS2S treatment appeared to reduce the bacteria counts, it was not statistically significant. See Figure 7D. Without being bound to any one theory, the result suggested that 18-mer NS2S does not display a direct anti-microbial effect in polymicrobial sepsis in vivo. A multiplex assay covering 21 biomarkers related to sepsis (Tsuchida, 2022) was conducted on plasma collected 24 hours after sham or CLP procedure in the absence or presence of 18-mer administration, showing that 18- mer NS2S had an overall protective effect against sepsis-induced inflammation. See Figure 7E. Compared to the sham group, the CLP group exhibited elevations in the range of several log fold for G-CSF, IL-6, IL-10, KC, MIP-2, IP-10, MCP-1, and RANTES. Notably, 18-mer administration after the CLP procedure resulted in decreased levels compared to the CLP group in all markers mentioned above. Levels of P-selectin, iCAM-1 and syndecan-1 were elevated in all groups: sham, CLP, and 18-mer treated. There is a slight decrease in syndecan-1 levels in 18-mer treated mice compared to CLP but the background level in the sham group was also elevated. Levels of GM-GSF, IFNg, IL-la, IL-5, IL-12p70, IL-13, IL-17, TNFa, KIM-1 and RAGE were consistently low in all groups. See also Tables 3 A and 3B, below, which provide the biomarker levels (in picogram (pg / ml)) for sham (untreated), CLP, and 18-mer-treated mice (3 each). Table 3 A. Biomarker values of multiplex assay. Table 3B. Biomarker values of multiplex assay.

[0200] The protection from 18-mer NS2S treatment against kidney damage was examined. A statistically significant reduction of creatinine and blood urea nitrogen (BUN) was observed in the 18-mer NS2S-treated group. See Figures 1G and 1H. Moreover, the mRNA levels of IL-6, MCP-1, and iCAM-1 also significantly decreased in the kidney after 18-mer NS2S treatment. See Figures 8A-8C. Histological evaluation of the kidneys in CLP animals revealed that the proximal tubule epithelial cells were shortened in height compared to control animals and had notable cytoplasmic vacuolation. This finding in sepsis models had been previously reported (Doi, 2009). Animals administered 18-mer NS2S did not have this tubular vacuolation, and kidneys were comparable microscopically to control kidneys. See Figures 11- IK. Additionally, CLP animals had microscopic findings in the tubules at the margin of the inner and outer stripe of the outer medulla in CLP animals (interpreted to involve the thin and thick segment of the loop of Henle). The findings were characterized by intratubular eosinophilic material and cellular debris, which reflected acute tubular degeneration. This region of the kidney is sensitive to ischemic injury, and the findings are consistent with acute hypoperfusion to the kidney which can occur with septicemia. This tubular degeneration was not observed in 2 of 3 animals administered the 18-mer, similar to control animals. See Figures 8D-8F. See also Figures 18A-18C. Taken together, these findings suggest that the treatment of 18-mer NS2S exerts renoprotection in CLP-injured mice.

[0201] 18-mer NS2S neutralizes the cytotoxicity of histone 3 (H 3)

[0202] An elevated plasma concentration of extracellular histone has been associated with septic damage (Xu, 2009). Abundant H3 was released to plasma at 24 hours in CLP-injured mice; a significant decrease was found in the group treated with 18-mer NS2S. See Figure IB. The effect of size of different HS oligosaccharides on the cytotoxicity of H3 was carried out using the human endothelial cell line EA.hy926. Three oligosaccharides were employed, including 6-mer NS2S, 12-mer NS2S, and 18-mer NS2S. See Figure 2A. 18-mer NS2S treatment improved the cell viability in a dose-dependent manner. While the 12-mer improved the cell viability overall, the efficacy is consistently lower than of 18-mer NS2S under different concentrations, and 6-mer NS2S was much less effective than that of the 12-mer NS2S and 18-mer NS2S. See Figure 2A. The binding affinities (KD) to H3 for 18-mer NS2S and two shorter oligosaccharides, 6-mer NS2S and 12-mer NS2S, were determined by surface plasmon resonance (SPR). See Table 4, below. The association rate constant (ka) and dissociation constant (kd) were also determined. 18-mer displayed the highest binding affinity (KD = 66 nM) to H3. This data demonstrates that the binding affinity of HS oligosaccharide to H3 correlates with the efficacy of neutralizing extracellular H3’s cytotoxicity. Next, 18-mer NS2S’s protection against histone-induced lethality in a mouse model (Xu, 2009; Li, 2020) was examined. 18-mer NS2S can fully protect against the intravenous injection of histones at a lethal dose (see Figure 9), suggesting that the saccharide directly affects extracellular histones in vivo.

[0203] Table 4. Association Rate Constants, Dissociation Constants, and Binding Affinities of NS2S Oligosaccharides.

[0204] The rationales for choosing the structure of an 18-mer that has a repeating IdoA2S-GlcNS disaccharide are primarily based on two factors: 1) neutralization of the toxicity of histone; and 2) resistance to the digestion by heparanase. To this end, two other 18-mers, NAc 18-mer and 18-mer NS6S, with different disaccharide repeating units were tested. Unlike the 18-mer NS2S, NAc 18-mer did not bind to H3. See Table 4, above. The NAc 18-mer also did not protect against the toxicity of histones in mice. See Figure 9. NAc 18-mer includes a disaccharide repeating unit of GlcA-GlcNAc, containing no sulfo groups, suggesting that sulfation is also important for the protection in addition to the size. Heparanase is reportedly upregulated under sepsis conditions (Schmidt, 2012). The enzyme degrades HS oligosaccharides to diminish the protective effect against sepsis. Therefore, an oligosaccharide that is resistant to heparanase digestion is desirable. As shown in Figures 10A and 11 A, the 18-mer NS2S is largely resistant to digestion. See also, Table 5, below. In a control experiment, 18-mer NS6S was shown to be susceptible to the digestion of heparinase. See Figures 10B andl lB. See also, Table 6, below. 18-mer NS6S has a repeating disaccharide unit of GlcA-GlcNS6S that has a sulfation pattern distinct from that of 18-mer NS2S. See Figures 11A and 1 IB.

[0205] Table 5. Products Detected in Digestion of 18-mer NS2S Oligosaccharide.

[0206] Table 6. Products Detected in Digestion of 18-mer NS6S Oligosaccharide. 18-mer NS2S attenuates inflammatory responses mediated by LPS / HMGB1 complex in endotoxemia mice but does not disrupt LPS / HMGB 1 complex in vitro

[0207] HMGB1 is also released under systemic inflammation conditions, including sepsis, and displays pro-inflammatory activity. Elevated plasma concentration of HMGB1 was observed in CLP-injured mice, and treatment with 18-mer NS2S significantly reduced the plasma concentration of HMGB1. See Figure 1C. HMGB1 forms complexes with bacterial LPS to deliver LPS into the cells, and the intracellular LPS augments pyroptosis through the caspase- 11 mediated pathway (Deng, 2018). 18-mer NS2S can interact with HMGB1 with a binding affinity of 186 nM (Arnold, 2020b). Herein, it was tested whether 18-mer NS2S reduces the HMGB1 -mediated inflammation in endotoxemia mice. In this model, the injection of LPS induces the release of HMGB1 to peritoneal space and subsequent elevation of IL-6 and TNF-a (Tang, 2021). Co-injection of the 18-mer NS2S with LPS significantly reduced the concentration of IL-6 in peritoneal lavage and plasma (see Figures 2B and 2C) and the concentration of TNF-a in peritoneal lavage and plasma. See Figures 12A and 12B. However, co-injection of 6-mer NS2S or 12-mer NS2S with LPS showed no reduction in IL-6 and TNF-a. See Figures 2B, 2C, 12A, and 12B. The results confirmed that 18-mer NS2S, but not 6-mer NS2S or 12-mer NS2S, attenuates the inflammatory effect induced by LPS and HMGB1.

[0208] Next, 18-mer NS2S’s ability to prevent the formation of the complex of LPS / HMGB1 in vivo was tested. See Figures 2D and 2E. In this experiment, an endotoxemia mouse model was used in which the release of HMGB1 was induced via peritoneally administering biotinylated LPS or co-injecting 18-mer NS2S and biotinylated LPS. The complex of LPS / HMGB1 was then isolated from peritoneal lavage using an avidin-agarose affinity column followed by immunoblotting for HMGB1. A reduction in LPS / HMGB1 was observed in the co-injection group. See Figures 2D and 2E. Coinciding with the LPS / HMGB1 complex reduction, injection with 18-mer NS2S reduced concentrations of IL-6 in peritoneal lavage and plasma in a dose-dependent manner. See Figures 12C and 12D. The data suggest that a lower concentration of LPS / HMGB 1 complex leads to weaker inflammatory responses from the host.

[0209] Without being bound to any one theory, one explanation for the reduction of the LPS / HMGB1 complex by the 18-mer NS2S injection is that the 18-mer NS2S disassociates the complex. To test the hypothesis, an experiment was performed to determine whether 18-mer NS2S disrupts the complex of LPS / HMGB 1 in an in vitro experiment. This study was carried out using the peritoneal lavage harvested from endotoxemia mice that contained the LPS / HMGB 1 complex. However, the addition of 18-merNS2S to the lavage failed to decrease the amount of HMGB1 bound to LPS. See Figure 12E. Like 18-mer NS2S, 6-mer NS2S did not decrease the complex of LPS / HMGB1. As a positive control, unlabeled LPS effectively displaced HMGB1 from the complex of biotinylated LPS / HMGB1. See Figure 12F. This data suggests that the presence of 18-mer NS2S does not cause the disassociation of the LPS / HMGB1 complex. In a separate experiment, no interaction was observed between 18-mer NS2S and LPS using a centrifugal ultrafiltration approach (Li, 2020) (see Figures 12G and 12H), ruling out the possibility that 18-mer NS2S reduces the complex of LPS / HMGB1 via interacting with LPS.

[0210] 18-mer NS2S induces the formation of ApoA-I / LPS complex in vivo

[0211] The inability to disrupt the LPS / HMGB1 complex in the in vitro experiment led to a hypothesis that reduction of the LPS / HMGB1 complex by 18-mer NS2S involves other factors. To this end, the proteins bound to biotinylated LPS from the endotoxemia mice with and without co-injection of 18-mer NS2S were analyzed. SDS-PAGE analysis with Ponceau S staining clearly revealed an extra protein band (~25 kDa) from the co-injection group. See Figure 3 A. Furthermore, the proteomic analysis confirmed that the protein band was ApoA-I. See Table 1 and Figure 13 A.

[0212] Three lines of evidence demonstrated that the presence of ApoA-I ameliorates the damage associated with LPS / HMGB1 and LPS alone. First, ApoA-I disrupted the LPS / HMGB1 complex in the in vitro experiment. Incubation of recombinant ApoA-I with peritoneal lavage containing biotinylated LPS / HMGB1 complex reduced the binding of HMGB1 to LPS in a dose-dependent manner. See Figure 13B. See also Figures 20A and 20B. Second, there was a substantial increase in ApoA-I / LPS complex in the 18-mer NS2S co-injected group. In contrast, a decrease in the LPS / HMGB1 complex concentration was observed in this group. See Figure 3B. See also Figures 19A and 19B. An inverse correlation between the concentrations of ApoA-I / LPS and LPS / HMGB1 suggests that ApoA-I is used to sequester LPS and to lower the level of LPS / HMGB1 complex. Additionally, the increases in ApoA-I / LPS complex in peritoneal lavage appeared to depend on the size of HS oligosaccharide co-injected with LPS. For the oligosaccharides tested, only the LPS / 18-mer NS2S co- injection group or, to a lesser extent, the LPS / 12-mer NS2S, but not the LPS / 6-mer co-injection group, showed an increase in ApoA-I / LPS complex. See Figure 13C. It should be noted that co-injection of 6-mer NS2S or 12-mer NS2S did not display an anti-inflammatory effect in animals, as demonstrated by comparable levels of IL-6 and TNF-a as in the LPS control group. See Figures 2B, 2C, 12A and 12B. The data suggests that the ability to elevate the complex of ApoA-I / LPS appears to correlate with anti-inflammatory potency. Third, the plasma concentration of LPS was reduced by 7.8- fold in the 18-mer co-injection group. See Figure 3C. Lower plasma LPS concentrations reduced organ damage. It is known that LPS is cleared in 3 min from blood circulation through the liver when it complexes with HDL (Yao, 2016). Without being bound to any one theory, it is possible that 18-mer NS2S increases the formation of the ApoA-I / LPS complex and accelerates the clearance of LPS when ApoA-I / LPS enters the circulation. Although there was an approximately 50% reduction in the concentration of lavage LPS from the 18-mer NS2S co-injection group, this difference was not statistically significant (p = 0.15). See Figure 3D. Further, the concentrations of ApoA-I in peritoneal lavage and plasma from endotoxemia mice and 18-mer NS2S co-injection mice were measured. No differences in the concentrations of ApoA-I were observed between the two groups. See Figures 14A and 14B. Taken together, this data suggests that the 18-mer NS2S decreases the concentration of the LPS / HMGB1 complex by eliciting the action of ApoA-I.

[0213] 18-mer NS2S induces the dissociation of ApoA-I from high-density lipoprotein (HDL) As a primary carrier for ApoA-I, HDL reportedly directly interacts with LPS (De Nardo, 2014), and thereby HDL should also neutralize the cytotoxicity from LPS. Herein it was found that HDL inhibited the release of TNF-a by LPS from raw246.7 cells dose-dependently. See Figure 3E. Notably, high concentrations of HDL (500 mg / mL) completely inhibited the release of TNF-a to background levels, suggesting that HDL effectively neutralizes the cytotoxicity of LPS. Furthermore, HDL was detected in plasma and peritoneal lavage from both endotoxemia and 18-mer NS2S co-injection groups. See Figures 14C and 14D. The co-localization of HDL and ApoA-I in the peritoneal space suggests that both HDL and ApoA-I contribute to reducing the toxic effect of LPS / HMGB1.

[0214] The impact of 18-mer NS2S on the structure of HDL was next investigated. Heparin reportedly caused the dissociation of ApoA-I from HDL particles (Tam, 2008; Digre, 2016). To this end, a study was conducted to determine whether 18-mer NS2S displays a similar effect on HDL using purified human HDL. 18-mer NS2S, 12-mer NS2S, and 6-mer NS2S were each incubated with HDL for one hour before centrifugation. Both supernatants and pellets were subjected to SDS-PAGE analysis. See Figure 3F. SDS-PAGE analysis showed that the addition of 18-mer NS2S to HDL moved ApoA-I from the supernatant fraction to the pellet fraction, indicating that 18- mer NS2S dissociated ApoA-I from HDL particles. The 18-mer NS2S was the most effective at dissociating ApoA-I from HDL. Without being bound to any one theory, the data suggests that HDL undergoes structural changes in the presence of 18-mer NS2S.

[0215] 18-mer NS2S treatment reduces lethality in CLP-injured mice

[0216] A 72-hour survival study with 6-mer NS2S and 18-mer NS2S treatment was conducted. Compared to CLP-injured mice receiving saline, 18-mer NS2S significantly improved survival, while 6-mer NS2S treatment had no significant effect on lethality reduction. See Figure 3G. The concentrations of ApoA-I and HDL were also measured in CLP-injured mice with or without the treatment of 18-mer NS2S. A statistically significant reduction in the concentrations of ApoA-I and HDL in peritoneal lavage was observed in the 18-mer NS2S treated group (see Figures 14E and 14G), possibly attributed to less injury. However, the plasma concentrations of ApoA-I and HDL showed no difference between the two groups. See Figures 14F and 14H.

[0217] Discussion

[0218] During sepsis, histones and HMGB1 are released from damaged cells and immune cells, fueling inflammation by increasing pro-inflammatory cytokines, chemokines, and adhesion molecules, resulting in a dysregulated host response, organ damage, and death (Denning, 2019). Curbing these excessive and dysregulated inflammatory responses represents a potential strategy for treating sepsis. Extracellular histone and HMGB1 are attractive targets as they control the upstream inflammatory response pathways (van der Poll, 2017; Xu, 2009; Stevens, 2017). Several reports emerged in recent years using sulfated glycans, including heparin, chondroitin sulfate E, and synthetic per-(9-sul fated maltotrioses and cellobiose to block the pro-inflammatory activities of histone or HMGB 1 and thereby to treat sepsis in animal models (Wildhagen, 2014; Meara, 2020; Li, 2020; Tang, 2021). According to one aspect of the presently disclosed subject matter it was demonstrated that a synthetic non-anticoagulant heparin-based molecule, an 18-mer NS2S, protected against sepsis-induced inflammation and resulting lethality by simultaneously targeting different pathways. The presently disclosed subject matter revealed that 18- mer NS2S engaged at least two targets to protect the animals from damage in sepsis, as illustrated in Figure 4. First, 18-mer NS2S binds to histone H3 and neutralizes its cytotoxicity. Second, 18-mer NS2S enlists the actions of ApoA-I to disrupt the LPS- HMGB1 complex and reduce the plasma concentration of LPS.

[0219] The cytotoxicity of extracellular histones is attributed to their interaction with lipid bilayer within the cell membrane leading to its damage (Silvestre-Roig, 2019). Among all the histone isoforms, the cytotoxicity and the correlation to sepsis severity of H3 are the most established (Garcia-Gimenez, 2017; Xu, 2009). The neutralization of the cytotoxic effect of histones by sulfated glycans has been achieved through direct interactions between histones and glycans (Wildhagen, 2014; Meara, 2020; Li, 2020). Herein it is demonstrated that 18-mer NS2S directly interacts with histone H3 with the binding affinity of 66 nM and improves cell viability after the H3 challenge (action 1 shown in Figure 4). It has been reported that HS oligosaccharides inhibit the activation of toll-like receptor 4 (TLR4)-mediated endothelial cell activations in response to the exposure to a low dose of histone (Zhang, 2017). The present data does not rule out the possibility that 18-mer NS2S also protects the host from low- concentration histone damage by inhibiting the activation of the TLR4 receptor.

[0220] HMGB 1 is known to contribute to the pathophysiology of sepsis as established in the endotoxemia and the CLP models. Materials from infectious microbes, such as bacterial LPS, trigger the release of extracellular HMGB1, contributing to cell damage. Extracellular HMGB1 and LPS form a complex, and the LPS-HMGB1 complex facilitates the intracellular delivery of LPS (Deng, 2018). Intracellular LPS activates caspase -11 to cleave gasdermin D into peptides that form gasdermin D pores in the membrane, leading to pyroptosis (Ding, 2016). It has been suggested that blocking the formation of the complex of LPS-HMGB1 could reduce the damage associated with sepsis (Tang, 2021). As a synthetic heparin mimetic, 18-mer NS2S reduces the concentration of the LPS-HMGB 1 complex in the peritoneal lavage of the endotoxemia mice; however, 18-mer NS2S did not appear to directly disrupt the LPS / HMGB1 complex in vitro. Thus, the present findings suggest that reducing the complex of LPS / HMGB1 is indirectly achieved.

[0221] The present studies show that 18-mer NS2S’s protection is mediated through enlisting the ApoA-I action to attenuate the functions of HMGB1 (actions 3 and 4 in Figure 4). Without being bound to any one theory, this conclusion points to a new mechanism to explain the anti-inflammatory effect of heparin and heparin-like compounds in sepsis. Known for transporting cholesterol from peripheral tissues to the liver and as a vehicle carrying ApoA-I, HDL displays cardioprotection. HDL’s beneficial functions against sepsis have been recently noted (Morin, 2015; Tanaka, 2020). The effects of HDL in sepsis are attributed to down-regulating macrophage response (De Nardo, 2014), strengthening endothelial cell barriers (Galvani, 2015), and neutralizing LPS (Yao, 2016; Petropoulou, 2015). In septic patients, dysfunctional HDL concerning decreased concentration, size, and cholesterol efflux capacity has been reported (Tanaka, 2019; Guirgis, 2018). Increasing HDL concentrations or improving its functionality have been suggested as new approaches for managing sepsis (Tanaka, 2020; Graziella, 2017). The presently disclosed model indicates that 18-mer NS2S causes the dissociation of ApoA-I from HDL (action 2 in Figure 4). ApoA-I binds to LPS to form a non-toxic ApoA-I / LPS complex that benefits the host in two aspects: it accelerates the clearance of LPS from the circulation (action 3 in Figure 4), and it decreases the formation of the complex of LPS / HMGB1.

[0222] A unique structural feature of the 18-mer NS2S used in this study is that it is less susceptible to the degradation by heparanase. Heparanase is released in sepsis and is involved in the degradation of endogenous HS to disrupt the glycocalyx on the surface of the endothelium (Schmidt, 2012). Therefore, it is conceivable that protection in sepsis by an HS oligosaccharide can be diminished after heparanase degradation. 18-mer NS2S used in the current study does not contain a heparanase cleavable saccharide sequence (Peterson, 2012). Minor cleavage at the non-reducing end of the 18-mer NS2S by heparinase unlikely affects the anti-inflammatory effect.

[0223] HS is a negatively charged polysaccharide that interacts with many proteins. The synthetic homogenous HS 6-mer NS2S, 12-mer NS2S, and 18-mer NS2S used in this study provide new insights into the mechanism of HS oligosaccharides' protection against damage associated with sepsis. 12-mer NS2S exhibited inhibition of the cytotoxicity of H3 and dissociated ApoA-I from HDL; however, the potencies in both assays were lower than those of 18-mer NS2S. The lower potency in both assays explained that 12-mer NS2S did not decrease the concentrations of IL-6 and TNF-a in the peritoneal lavage of endotoxemia mice.

[0224] In summary, the presently disclosed subject matter uses homogenous HS oligosaccharides to investigate the relationship between HS structure and the multifaceted functions, including the neutralization of the pro-inflammatory activities of H3 and HMGB1 and the promotion of the anti-inflammatory activities associated with ApoA-I and HDL.

[0225] EXAMPLE 3

[0226] MATERIALS AND METHODS FOR ADDITIONAL STUDIES WITH HS 12- MERS- 16-MERS

[0227] Chemoenzymatic synthesis of oligosaccharides

[0228] The chemoenzymatic synthesis of HS oligosaccharides has been previously described (Xu, 2016). The chemoenzymatic synthesis of exemplary HS oligosaccharides of the presently disclosed subject matter is shown in Figure 22. Briefly for the synthesis of compound 1 (12-mer NS6S), glucuronic acid-pNP was elongated with UDP-GlcNFTA (step a) and UDP-GlcA (step b) using pmHS2 to reach 12-mer. Next, GlcNFTA was deprotected using LiOH then TV-sulfated with NST (step c) to yield 12-mer A-sulfo glucosamine residues. Next, 6-O-sulfation was installed using 6-OST1 and 6-OST3 (step e) to yield compound 12-mer NS6S, i.e., GlcNS6S- GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S- GlcA-pNP. 12-mer NS2S and 12-mer NS2S6S were synthesized by elongating the monosaccharide to a pentasaccharide intermediate. Next, GlcNFTA was deprotected using LiOH then A -sulfated with NST to yield 5-mer with two A -sulfo glucosamine residues. 5-mer NS was elongated with UDP-GlcNFTA to yield a 6-mer intermediate. The glucuronic acid residue in between the two A -sulfo glucosamine residues undergoes epimerization by C5-epimerize and 2-O-sulfation by 2-OST (step d) to yield the 6-mer intermediate GlcNFTA-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP. Next steps b, c, a, and d were repeated three times to generate compound 2 (i.e., 12- mer NS2S; GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS- IdoA2S-GlcNS-GlcA-pNP). This compound was converted to compound 3 (12-mer NS2S6S) by 6-O-sulfation of GlcNS residues by 6-OST1 and 6-OST-3 (step e) to give the structure GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S- IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP.

[0229] The synthetic HS oligosaccharides can be converted to biotinylated versions using the same method as described in U.S. Patent Application Publication No. 2021 / 0169923, incorporated herein by reference in its entirety.

[0230] Affinity purification ofHMGBl from liver lysate

[0231] Biotinylated oligosaccharides were using to affinity purify HMGB1 from liver lysate following the method described in U.S. Patent Application Publication No. 2021 / 0169923.

[0232] Evaluation of liver injury

[0233] Plasma ALT was measured using the ALT Infinity reagent (Thermo Fisher) following the manufacturer’s instructions. Plasma TNF-a was measured using Mouse TNF-a DuoSet Kit (R&D Systems) according to the manufacturer’s instructions. Plasma HMGB1 levels were determined using HMGB1 ELISA Kit (Tecan US) according to the manufacturer’s instructions.

[0234] Histology / Immunohistochemistry

[0235] Liver tissues were fixed in 10% neutral buffered formalin for 24 hours at room temperature, paraffin-embedded, and sectioned. Liver sections (4 pm) were stained with hematoxylin-eosin (H&E) or immunostained with monoclonal antibodies antineutrophil (Abeam, Ab 2557, NIMP-R14) followed by goat anti-rabbit biotinylated secondary antibodies (Abeam). Embedding, sectioning and H&E staining were performed at the Animal Histopathology and Laboratory Medicine Core Facility at UNC Chapel Hill. H&E analyses were performed by the Translational Pathology Laboratory Core Facility at UNC Chapel Hill using Aperio ImageScope Software (Leica Biosystems, Concord, Canada). IHC images were captured using an HD camera attached to a bright field microscope (Leica DM 1000 LED, Leica Microsystems Inc., IL, USA) and were processed using Imaged. For neutrophil quantitation, five lOOx images were randomly selected for each sample and the average neutrophils / field were reported. Myeloperoxidase (MPO) Activity

[0236] Liver tissue was mechanically homogenized in 50 mM CTAB 50 mM potassium phosphate pH 6 at a ratio of 100 pl buffer per 10 mg tissue. Samples were centrifuged at 15,000 x g for 20 min 4 °C and the supernatant was collected and stored at -20 °C. Total protein concentration was measured by Bradford assay. 10 pl of liver lysate was incubated with 80 pl of 0.75 mM H2O2 and 110 pl of TMB (TMB liquid reagent, ready -to-use, Sigma) for 10 minutes at 37°C with gentle agitation. The reaction was stopped by addition of 2.5 M H2SO4 and read at 450 nm. Activity (U / g protein) was calculated as absorbance of sample minus the absorbance of the blank divided by incubation time. This value was normalized by the protein concentration. Statistical Analysis

[0237] All data are expressed as mean ± SEM. Statistical significance between experimental and control groups were analyzed by two-tailed unpaired Student t test, between multiple groups by one-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison’s test, and Kaplan-Meier survival curves by log-rank test using GraphPad Prism software (version 7.03; GraphPad Software, Inc.).

[0238] EXAMPLE 4

[0239] HS 12-MERS- 16-MERS

[0240] Details on a mouse model of acetaminophen-induced acute liver failure has been previously described (Arnold, 2020a). Alanine aminotransferase (ALT) is a biomarker for liver injury measured from plasma. Figure 23 shows the ability of synthetic HS oligosaccharides to reduce ALT in mice in the model of APAP overdose, as an indicator of their ability to reduce liver injury. Compounds were administered 30 minutes and 12 hours after APAP overdose. Each point on the graph represents 1 mouse. 18-mer NS2S was used as a positive control for the reduction of ALT. All compounds tested (i.e., 18-mer-NS2S, 16-mer NS6S, 14-mer NS6S, 12-mer NS6S, and 12-mer NS2S6S) showed similar in vivo efficacy.

[0241] MPO activity (measured in liver lysate) was studied as a measure of neutrophil activity. See Figure 24A. Neutrophils were also measured by IHC in FFP liver tissue (see Figure 24B) and necrotic area (%) was determined by H&E staining in FFP liver tissue. See Figure 24C. Each point in the graphs of Figures 24A-24C represents 1 mouse. Compounds were administered 30 minutes and 12 hours after APAP overdose.

[0242] Survival of mice after lethal overdose of APAP was monitored. See Figures 25A and 25B. Compounds were administered twice daily beginning at 30 minutes (early treatment) or 3 hours (delayed treatment) after APAP overdose. 18-mer NS2S and 12-mer NS2S6S had similar efficacy.

[0243] Details on HMGB1 binding assay were previously described (17). Binding to HMGB1 serves as a proxy for anti-inflammatory activity. 14-mer and 16-mer NS6S and 12-mer NS2S6S showed similar binding to HMGB1 as positive control 18-mer NS2S. See Figure 26. 12-mer NS6S weakly binds HMGB1.

[0244] EXAMPLE 5

[0245] COMBINED TREATMENT OF NAC AND GLY-202 AT A DELAYED

[0246] ADMINISTRATION TIMEPOINT AFTER APAP OVERDOSE

[0247] Methods:

[0248] Groups of 8-20 male, 10-week-old C57BL6 / J mice were used in to study the effect of GLY-202 and N-acetyl cysteine (NAC) on APAP overdose. Mice were fasted overnight prior to a sub-lethal APAP overdose caused by an intraperitoneal injection of 400 mg / kg of APAP. In one cohort, 50 mg / kg of NAC (pH 7) was intraperitoneally administered 30 minutes after APAP overdose. In another cohort, 50 mg / kg NAC was administered 3 hours after APAP overdose. In a third cohort, 5 mg / kg GLY-202, in sterile saline, was subcutaneously administered 3 hours and 12 hours after APAP overdose. In a fourth cohort, 50 mg / kg NAC and 5 mg / kg GLY-202 were administered 3 hours after APAP overdose followed by a second GLY-202 injection at 12 hours.

[0249] Mice were sacrificed at 24 hours after APAP overdose for plasma collection and ALT measurement. Data was graphed using GraphPad Prism. One-way ANOVA with multiple comparison was performed using GraphPad Prism.

[0250] Results:

[0251] In human patients, NAC is typically administered either orally for up to 72 hours or, more commonly, by IV infusion over 21 hours (Licata, 2022). NAC therapy in the rodent model only consists of a single intraperitoneal injection. Based on the present study, it was determined that administering both NAC and GLY-202 3 hours after APAP overdose results in decreased liver injury as measured by plasma ALT (bar on the right in Figure 27) compared to the APAP control group. NAC alone at 3 hours does not result in a significant decrease in plasma ALT (middle bar in Figure 27) compared to the APAP control group, however the cohorts treated with GLY-202 either alone or in combination with NAC at 3 hours did have a significant decrease in plasma ALT (bar on the right and second from the right in Figure 27) compared to APAP control group. The reduction in plasma ALT in both GLY-202 treated groups is similar to the level of ALT in the NAC cohort treated at 30 minutes after APAP overdose (bar second from left in Figure 27), which is a time point during APAP metabolism in mice where NAC is known to be effective. In summary, the results indicate that GLY-202 (12-mer NS2S6S) given 3 hours after APAP overdose decreases ALT, while NAC loses protective effect at this same time point.

[0252] REFERENCES

[0253] All references listed herein including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (e.g., GENBANK® database entries and all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and / or compositions employed herein.

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[0291] Xu, Y., et al., Homogeneous low-molecular- weight heparins with reversible anticoagulant activity. Nat Chem Biol 10, 248-250 (2014).

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[0295] Zitvogel, L., Kepp, O., Kroemer, G., Decoding cell death signals in inflammation and immunity. Cell 140, 798-804 (2010). It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

CLAIMSWhat is claimed is:

1. A method of treating drug-induced liver injury or sepsis in a subject in need thereof, the method comprising administering to the subject a synthetic heparan sulfate (HS) oligosaccharide having a structure of Formula (I):wherein: m and n are each selected from 0, 4, 5, 6, and 7;Ri is selected from -H and -SO3H;R2 is selected from -SO3H and -COCH3;R3 is selected from -H and -SO3H; andR4 is selected from the group consisting of -H, alkyl, aryl, substituted alkyl, substituted aryl, and a functional handle; subject to the proviso that the synthetic HS oligosaccharide is selected from (i)-(vi) or a pharmaceutical composition thereof, wherein:(i) m is 0, n is 4, Ri is -SO3H, R2 is -SO3H, and R3 is -SO3H;(ii) m is 0, n is 5; Ri is -H, R2 is -SO3H, and R3 is -SO3H;(iii) m is 0, n is 6, Ri is -H, R2 is -SO3H, and R3 is -SO3H;(iv) m is 4, n is 0, Ri is -SO3H, and R2 is -SO3H;(v) m is 5, n is 0, Ri is -SO3H, and R2 is -SO3H; and(vi) m is 6, n is 0, Ri is -SO3H, and R2 is -SO3H.

2. The method of claim 1, wherein the drug-induced liver injury is acetaminophen (APAP)-induced acute liver failure.

3. The method of claim 1 or claim 2, wherein the subject in need of treatment is a mammalian subject, optionally a human subject.

4. The method of any one of claims 1-3, wherein the synthetic HS oligosaccharide is selected from the group consisting of (1), (iv), (v), and (vi).

5. The method of any one of claims 1-4, wherein R4 is substituted aryl, optionally p-nitrophenyl.

6. Use of a synthetic heparan sulfate (HS) oligosaccharide or a pharmaceutical composition thereof in a method of treating drug-induced liver injury, optionally acetaminophen (APAP)-induced acute liver failure, or sepsis in a subject in need thereof, wherein the synthetic HS oligosaccharide has a structure of Formula (I):wherein: m and n are each selected from 0, 4, 5, 6, and 7;Ri is selected from -H and -SO3H;R2 is selected from -SO3H and -COCH3;R3 is selected from -H and -SO3H; andR4 is selected from the group consisting of -H, alkyl, aryl, substituted alkyl, substituted aryl, and a functional handle; subject to the proviso that the synthetic HS oligosaccharide is selected from (i)-(vi) or a pharmaceutical composition thereof, wherein:(i) m is 0, n is 4, Ri is -SO3H, R2 is -SO3H, and R3 is -SO3H;(ii) m is 0, n is 5, Ri is -H, R2 is -SO3H, and R3 is -SO3H;(iii) m is 0, n is 6, Ri is -H, R2 is -SO3H, and R3 is -SO3H;7. A synthetic heparan sulfate (HS) oligosaccharide having a structure of the Formula (I):m and n are each selected from 0, 4, 5, 6, and 7;Ri is selected from -H and -SO3H;R2 is selected from -SO3H and -COCH3;R3 is selected from -H and -SO3H; andR4 is selected from the group consisting of -H, alkyl, aryl, substituted alkyl, substituted aryl, and a functional handle; subject to the proviso that the synthetic HS oligosaccharide is selected from (ii), (iii), (v), or (vi), wherein:(ii) m is 0, n is 5, Ri is -H, R2 is -SO3H, and R3 is -SO3H;(iii) m is 0, n is 6, Ri is -H, R2 is -SO3H, and R3 is -SO3H;(v) m is 5, n is 0, Ri is -SO3H, and R2 is -SO3H; or(vi) m is 6, n is 0, Ri is -SO3H, and R2 is -SO3H.

8. The synthetic HS oligosaccharide of claim 7, wherein the synthetic HS oligosaccharide is (v) or (vi).

9. The synthetic HS oligosaccharide of claim 7, wherein the synthetic HS oligosaccharide is (ii) or (iii), optionally wherein the synthetic HS oligosaccharide is (ii).

10. The synthetic HS oligosaccharide of any one of claims 7-9, wherein R4 is substituted aryl, optionally p-nitrophenyl.

11. A pharmaceutical composition comprising (a) a synthetic HS oligosaccharide of any one of claims 7-10; and (b) a pharmaceutically acceptable carrier or adjuvant.

12. A method of treating sepsis in a subject in need thereof, the method comprising administering to the subject a synthetic heparan sulfate (HS) oligosaccharide having a structure of Formula (I):wherein: m is 0; n is 7; Ri is H;R.2 is -SO3H;R3 is -SO3H; andR4 is -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle, optionally wherein R4 is p-nitrophenyl.

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