Effects of heparan sulfate (HS) oligosaccharides on hepatic ischemia-reperfusion injury

Abstract

Methods for treating hepatic ischemia-reperfusion (I / R) injury in a subject are disclosed. In some embodiments, the methods include providing a subject suffering from or at risk of suffering from hepatic I / R injury; and administering to the subject one or more heparan sulfate (HS) compounds. In some embodiments, the one or more HS compounds comprise about 5 to about 18 sugar units, optionally about 12 to about 18 sugar units. In some embodiments, the one or more HS compounds comprise about 12 sugar units.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 934,845, filed November 13, 2019, which is incorporated by reference herein in its entirety. This invention was made with government support under Grant Nos. HL094463, HL144970, GM128484, and HL142604 awarded by the National Institutes of Health. The United States government therefore has certain rights in this invention. [Technical Field]

[0002] The present invention generally relates to methods and compositions for treating hepatic ischemia-reperfusion injury (herein, ischemia-reperfusion is referred to as I / R or IR). More specifically, the present invention relates to heparan sulfate oligosaccharide compounds for treating hepatic ischemia-reperfusion (I / R) injury and methods of using the same for treating hepatic ischemia-reperfusion (I / R) injury. [Background technology]

[0003] Hepatic ischemia-reperfusion (I / R) injury is a major surgical complication during liver transplantation and liver tumor resection [1]. Liver surgery often requires the use of the Pringle maneuver to reduce blood loss, at the expense of potential I / R injury [2]. Initial injury begins during the ischemic phase, when blood flow to tissues is interrupted, resulting in a lack of oxygen and nutrient flow. When blood flow is restored to these tissues, oxygen and nutrients are reestablished. However, this actually intensifies the initial ischemic injury by inducing thrombo-inflammation, which is characterized by impaired hemostasis and inflammation [1]. Currently, there are no approved drugs to protect against liver injury caused by I / R injury. Thrombosis and inflammation have traditionally been considered separate processes. However, increasing evidence supports a relationship between thrombosis and inflammation, which stimulate and reinforce each other, collectively referred to as thromboinflammation [3]. Thromboinflammation is evident in I / R injury [3], sepsis [4], and trauma [5]. Injury to the endothelium is central to the pathogenesis of thromboinflammation. The endothelium functions as an antiadhesive barrier for the circulatory system by presenting proteoglycans on its cell surface. Heparan sulfate (HS) chains on these proteoglycans can bind antithrombin III and inhibit the coagulation factors FXa and thrombin. In thromboinflammatory conditions, the endothelium loses this antiadhesive and anticoagulant barrier. Furthermore, tissue factor lies beneath the endothelium, which becomes exposed upon vessel wall injury and can function as a potent activator of the extrinsic coagulation pathway and subsequent thrombin generation [3]. Furthermore, I / R injury leads to hypoxic cells releasing high-mobility group box 1 (HMGB1) [6]. HMGB1 has been shown to recruit neutrophils via receptors for advanced glycation end products (RAGE) activation after hepatic I / R [7]. Neutrophil recruitment and infiltration induces further cell death by releasing proteases, including myeloperoxidase (MPO) [8]. Severe thromboinflammation can spread beyond the initially affected tissue, leading to multi-organ system damage [3]. Summary of the Invention [Problem to be solved by the invention]

[0004] To improve patient outcomes, treatments that reduce the degree of thromboinflammation are highly desirable [9]. [Means for solving the problem]

[0005] Several embodiments of the present invention are presented herein, and in many cases, variations and permutations of these embodiments are presented. These are merely exemplary of the many different embodiments. Reference to one or more representative features of an embodiment is likewise exemplary. Such embodiments can typically exist with or without the referenced feature(s); similarly, these features may be applied to other embodiments of the present invention disclosed herein, whether or not they are presented herein. To avoid excessive repetition, not all possible combinations of these features are presented herein.

[0006] Provided according to the present invention are methods for treating hepatic ischemia-reperfusion (I / R) injury in a subject. In some embodiments, the method comprises: (i) providing a subject suffering from or at risk of suffering from hepatic I / R injury; and (ii) administering one or more heparan sulfate (HS) compounds to the subject. In some embodiments, the administering step provides anti-inflammatory and / or anticoagulant activity to the subject. In some embodiments, the one or more HS compounds comprise about 5 to about 18 sugar units, optionally about 12 to about 18 sugar units. In some embodiments, the one or more HS compounds comprise about 12 sugar units. In some embodiments, at least one of the one or more HS compounds binds to HMGB1.

[0007] In some embodiments, the one or more HS compounds are of the following formula: [ka] (In the formula, R1 represents -NHSO3H or -NHCOCH3, R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle, and n represents an integer of 0 to 6.)

[0008] In some embodiments, the one or more HS compounds comprise the following structure: [ka] (wherein R1 represents -SO3H or -COCH3, and R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.)

[0009] In some embodiments, the one or more HS compounds comprise the following structure: [ka] (wherein R1 represents -SO3H or -COCH3, and R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.)

[0010] In some embodiments, the one or more HS compounds are of the following formula: [ka] wherein R represents —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.

[0011] In some embodiments, the one or more HS compounds are of the following formula: [ka] (wherein R1 represents -SO3H or -H, and R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.)

[0012] In some embodiments, the one or more HS compounds are present in non-anticoagulant heparins and low molecular weight heparins and include one of the following structural formulas: [ka] [ka]

[0013] In some embodiments, the one or more HS compounds are of the following formula: It becomes. [ka] (wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or functional group) R stands for dollar 1 and n are defined as follows: In embodiment 1, R 1 is a hydrogen atom, n is 1; In embodiment 2, R 1 is a hydrogen atom, n is 2; In embodiment 3 (OSOH) and embodiment 4 (OH), [ka] In embodiment 5 (OSOH) and embodiment 6 (OH), [ka] )

[0014] In some embodiments, the one or more HS compounds are of the following formula: [ka] wherein R represents —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.

[0015] In some embodiments, the subject in need of this treatment is a mammalian subject. In some embodiments, the one or more HS compounds are administered as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises an HS compound and a pharmaceutically acceptable carrier or adjuvant for administration of the HS compound. In some embodiments, the administering step comprises administering two or more HS compounds, optionally wherein the two or more HS compounds are administered separately but simultaneously, optionally wherein the two or more HS compounds are administered at different times, and optionally wherein the two or more HS compounds are administered in a single composition.

[0016] Provided according to the present invention are heparan sulfate (HS) compounds. In some embodiments, one or more HS compounds are provided as a composition for use in treating hepatic ischemia-reperfusion (I / R) injury in a subject. In some embodiments, the composition comprises one or more heparan sulfate (HS) compounds, optionally, the one or more HS compounds comprise from about 5 to about 18 sugar units, optionally from about 12 to about 18 sugar units, and further optionally, the one or more HS compounds comprise about 12 sugar units. In some embodiments, administering the composition to a subject confers anti-inflammatory and / or anticoagulant activity to the subject.

[0017] In some embodiments, the one or more HS compounds are of the following formula: [ka] (In the formula, R1 represents -NHSO3H or -NHCOCH3, R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle, and n represents an integer of 0 to 6.)

[0018] In some embodiments, the one or more HS compounds comprise the following structure: [ka] (wherein R1 represents -SO3H or -COCH3, and R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.)

[0019] In some embodiments, the one or more HS compounds comprise the following structure: [ka] (wherein R1 represents -SO3H or -COCH3, and R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.)

[0020] In some embodiments, the one or more HS compounds are of the following formula: [ka] wherein R represents —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.

[0021] In some embodiments, the one or more HS compounds are of the following formula: [ka] (wherein R1 represents -SO3H or -H, and R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.)

[0022] In some embodiments, the one or more HS compounds are present in non-anticoagulant heparins and low molecular weight heparins and include one of the following structural formulas: [ka] [ka]

[0023] In some embodiments, the one or more HS compounds are of the following formula: It becomes. [ka] (wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or functional group) R stands for dollar1 and n are defined as follows: In embodiment 1, R 1 is a hydrogen atom, n is 1; In embodiment 2, R 1 is a hydrogen atom, n is 2; In embodiment 3 (OSOH) and embodiment 4 (OH), [ka] In embodiment 5 (OSOH) and embodiment 6 (OH), [ka] )

[0024] In some embodiments, the one or more HS compounds are of the following formula: [ka] wherein R represents —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.

[0025] In some embodiments, the one or more HS compounds bind to HMGB1. In some embodiments, the subject in need of this treatment is a mammalian subject. In some embodiments, the composition comprises a pharmaceutically acceptable carrier or adjuvant for administration of the one or more HS compounds. In some embodiments, the composition comprises two or more HS compounds. Accordingly, it is an object of the present invention to provide methods and compositions for treating hepatic ischemia-reperfusion (I / R) injury. This and other objects are achieved in whole or in part by the present invention. Furthermore, the above-mentioned objects, other objects, and advantages of the present invention will become apparent to those skilled in the art after reviewing the following description, drawings, and examples. [Brief explanation of the drawings]

[0026] The present invention can be better understood by reference to the following figures, which are not intended to limit the scope of the invention, but are merely intended to clarify and illustrate the invention. [Figure 1] Figures 1A-F show that a mouse model of hepatic I / R increases liver injury markers. Figure 1A shows the timeline of the hepatic I / R model. Figure 1B is a bar graph showing plasma ALT concentrations. P=0.0083. Figure 1C is a bar graph showing the percentage (%) of necrotic liver area stained with H&E, quantified using the original 100x magnification. P=0.0077. Figure 1D is a bar graph showing neutrophil infiltration into the ischemic lobe, quantified by immunohistochemical staining from 200x magnification images. P=0.0018. Figure 1E is a bar graph showing plasma HMGB1 concentrations. P=0.0129. Figure 1F is a bar graph showing plasma syndecan-1 concentrations. P=0.0170. Data represent the mean ± SEM. Sham n=4-5, I / R n=4-8. *P<0.05 and **P<0.01 (by Student's t-test). [Figure 2] Figures 2A-C show that HMGB1 binds to a highly sulfated 12-mer. Figure 2A shows the structure of a 12-mer prepared by chemoenzymatic synthesis. Figure 2B is a bar graph showing in vitro FXa activity measurements of the 12-mer, with fondaparinux as a positive control. Figure 2C is a series of images showing Western analysis of HMGB1 pulldown from liver lysates using a biotinylated 12-mer. [Figure 3]Figures 3A and 3B show in vivo measurements of anticoagulation and liver injury using 12-mer-1 and 12-mer-3 after IR. Plasma FXa activity (Figure 3A) and ALT (Figure 3B) were measured from mice treated with sham or IR with 12-mer-1 or 12-mer-3. 12-mer-1 significantly reduced FXa activity (12-mer-1 vs. IR, P = 0.0057) and ALT compared with IR (sham vs. IR, P = 0.0233; 12-mer-1 vs. IR, P = 0.0480; 12-mer-3 vs. IR, P = 0.6200). Data represent mean ± SEM. N = 5-6 in all groups. *P < 0.05 and **P < 0.01 (one-way ANOVA followed by Dunnett's test). [Figure 4] Figures 4A–E show that 12-mer-1 reduces the area of ​​hepatic necrosis in the ischemic lobe after hepatic I / R. The area of ​​hepatic necrosis (%) in Figure 4A was quantified using H&E stained, 100x original magnification images. Sham vs. IR, P = 0.0078; 12-mer-1 vs. IR, P = 0.0287; 12-mer-3 vs. IR, P = 0.1059. Data represent mean ± SEM. Sham n = 3, IR n = 4, 12-mer-1 n = 6, and 12-mer-3 n = 5. *P < 0.05 and **P < 0.01 vs. IR (by one-way ANOVA followed by Dunnett's test). Figures 4B–E are representative images of H&E-stained liver tissue at 100x magnification. Scale bar is 200 µm. [Figure 5]Figures 5A-F show that 12-mer-1 reduces neutrophil accumulation in ischemic livers. Figure 5A shows myeloperoxidase (MPO) activity measured in sham or ischemic liver lysates. Sham vs. I / R, P = 0.0121; 12-mer-1 vs. IR, P = 0.0229. Figure 5B shows quantification of average neutrophils per 100x field. Sham vs. IR, P = 0.0248; 12-mer-1 vs. IR, P = 0.0142; 12-mer-3 vs. IR, P = 0.0705. Data represent mean ± SEM. Sham n = 6, IR n = 3-4, 12-mer-1 n = 4-6, 12-mer-3 n = 6. *P < 0.05 by one-way ANOVA followed by Dunnett's test. Figures 5C-F show representative images of liver tissue immunohistochemically stained for neutrophils. Magnification: 200x, scale bar: 200 µm. Arrows (pink) indicate stained neutrophils. [Figure 6] Figures 6A-D show that hepatoprotection requires HMGB1 binding and anticoagulation. Figure 6A illustrates the 6-mer-AXa structure; the legend for Figure 2A applies. Figure 6B shows Western analysis of HMGB1 pulldown from liver lysates using biotinylated 6-mer-AXa containing 12-mer-3 and 12-mer-1 as a positive control. Figure 6C shows plasma FXa activity measured from mice receiving IR treatment with sham or 12-mer-3 + 6-mer-AXa or 6-mer-AXa alone. IR vs. 12-mer-3 + 6-mer-AXa, P = 0.0089; IR vs. 6-mer-AXa, P = 0.0252. Figure 6D shows plasma ALT. IR vs. sham, P = 0.0026; IR vs. 12-mer-3 + 6-mer-AXa, P = 0.0086. Data represent mean ± SEM. Sham n=4, IR n=3-5, 12-mer-3 + 6-mer-AXa n=6, 6-mer-AXa n=6. [Figure 7]Figures 7A and B show full Western blot images from HMGB1 pulldown using oligosaccharides. Figure 7A shows the input sample: Lane 1: 12-mer-4; Lane 2: 12-mer-2; Lane 3: 12-mer-3; Lane 4: 12-mer-1. Figure 7B shows the eluted sample: Lane 5: 12-mer-4; Lane 6: 12-mer-2; Lane 7: 12-mer-3; Lane 8: 12-mer-1. [Figure 8] Figures 8A and 8B show full Western blot images from HMGB1 pulldowns using 12-mer-1 and 6-mer-AXa oligosaccharides. Figure 8A shows the input sample: Lane 1: 12-mer-3; Lane 2: 12-mer-1; Lane 3: 6-mer-AXa. Figure 8B shows the eluted sample: Lane 4: 12-mer-3; Lane 5: 12-mer-1; Lane 6: 6-mer-AXa. [Figure 9] Figure 9 shows the structural formulas of representative HS anticoagulant (AXa) 8-18-mer structures of the present invention. 12-mer-1 (also referred to as 12-mer AXa (n=3)) is protective in a mouse model of hepatic ischemia-reperfusion. R in the formula is defined as indicated in the figure and can also be defined as -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle. [Figure 10] Figure 10 shows the structural formulas of representative HS non-anticoagulant 16- to 18-mer constructs of the present invention. These constructs are not anticoagulants. The 18-mer (n = 6) construct used in hepatic IR contains R1 = H and R2 = phenyl, X = nitro (para-nitrophenyl), e.g., a functional handle. R2 can also be defined as -H, alkyl, aryl, substituted alkyl, or substituted aryl. [Figure 11]Figures 11A-F show that the 18-mer in Figure 10 reduces injury after hepatic ischemia-reperfusion. Figure 11A shows the experimental design of the IR procedure. Figure 11B is a graph showing plasma ALT levels measured in sham, IR, and IR mice treated with the 18-mer. The 18-mer significantly reduces ALT levels compared to IR. Data represent mean ± SEM. n = 5 for sham, n = 7 for IR and 18-mer. *P < 0.05, **P < 0.01 (one-way ANOVA with Dunnett's post-hoc test). Figure 11C is a graph showing quantification of necrotic areas from H&E-stained ischemic liver lobes. Figures 11D-F show representative images of H&E-stained liver tissue for necrosis quantification. Magnification 100x, scale bar 200 μm.

[0027] [Figure 12] Figures 12A-D are a series of graphs showing that the 18mers of Figure 10 reduce inflammatory markers. Figure 12A shows plasma HMGB1 levels measured by ELISA, which are reduced in the 18mer-treated group compared to IR. Figure 12D shows neutrophil infiltration measured by immunohistochemistry, which is reduced in the 18mer-treated group. Figure 12C shows plasma IL-6 levels measured by ELISA, which are reduced in the 18mer-treated group. Figure 12B shows that plasma TNF-α levels tend to be reduced in the 18mer-treated group, although this is not statistically significant. [Figure 13] FIG. 1 is a schematic showing the chemoenzymatic synthesis of a 12-mer. BEST MODE FOR CARRYING OUT THE INVENTION

[0028] The present invention will now be described more fully, with reference to some, but not all, embodiments of the invention. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will satisfy applicable legal requirements. Heparan sulfate (HS) is structurally similar to the anticoagulant heparin. HS and heparin are composed of repeating disaccharide units of glucuronic acid (GlcA) or iduronic acid (IdoA) linked to a sulfo-bearing glucosamine residue

[10] . Heparin is more highly sulfated than HS and contains more IdoA residues. Clinical studies using heparin and low-molecular-weight heparins (LMWHs) to treat thromboinflammatory diseases such as sepsis and irritable bowel syndrome (IR) have been inconclusive [11,12]. Heparin and its derivatives are complex, structurally uncharacterized mixtures of oligosaccharides containing various chain lengths and chemical modifications. This structural heterogeneity makes it difficult to define the relationship between oligosaccharide structure and its biological function. Furthermore, the lack of structurally homogeneous HS oligosaccharides has hindered efforts to harness the properties of HS for use as a therapeutic agent

[13] . To address this issue, chemoenzymatic methods have been developed to synthesize structurally specific HS oligosaccharides with high efficiency [14-16]. According to a specific embodiment of the present invention, certain oligosaccharides with anticoagulant and anti-inflammatory activities (e.g., HMGB1 binding) are shown to be more effective in reducing IR-mediated liver injury than oligosaccharides that simply bind to HMGB1 or oligosaccharides with only anticoagulant activity. The hepatoprotective effects of the oligosaccharides are enhanced by using synthetic HS oligosaccharides rather than heparin or LMWH.

[0029] Heparan sulfate (HS) is a sulfated glycosaminoglycan abundantly present on cell surfaces and in the extracellular matrix, possessing several biological activities, including anticoagulant and anti-inflammatory properties. Hepatic ischemia-reperfusion injury is associated with coagulation and inflammatory responses. According to the present invention, HS oligosaccharides with defined sulfation patterns were synthesized, and the synthetic anticoagulant HS oligosaccharides were shown to limit hepatic ischemia-reperfusion injury in a mouse model. According to a specific aspect of the present invention, using a small targeted HS library, we demonstrated that oligosaccharides with both anticoagulant activity and binding affinity for the inflammatory target HMGB1 reduced this injury more than oligosaccharides that bind only to HMGB1 or oligosaccharides with only anticoagulant activity. The HS oligosaccharides of the present invention provide a novel therapeutic option for reducing liver damage caused by ischemia-reperfusion injury.

[0030] For clarity and not limitation, in some embodiments of the present invention, the hepatoprotective effects of the HS compounds disclosed herein are a function of their size and / or sulfation pattern. In one non-limiting example, 12-mer oligosaccharides with sulfation patterns that confer anticoagulant and anti-inflammatory activity confer hepatoprotective effects. In another non-limiting example, 12-mer oligosaccharides with sulfation patterns that confer anti-inflammatory activity confer hepatoprotective effects when administered with 6-mers with sulfation patterns that confer anticoagulant activity. In another non-limiting example, 18-mer HS oligosaccharides with sulfation patterns that confer anti-inflammatory activity confer hepatoprotective effects, but are not anticoagulant. In some embodiments of the present invention, heparan sulfate (HS) 12-mers have been shown to alleviate liver damage caused by ischemia-reperfusion injury. The present invention reduces the inflammatory response that damages hepatocytes following liver transplantation or surgery. In some embodiments, the HS compound contains about 5 to about 18 sugar units, including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 sugar units. Representative HS compounds are also disclosed in the drawings. In some embodiments, the HS compound is substituted with -H, alkyl, aryl, substituted alkyl, or substituted aryl. In some embodiments, the HS compound is substituted with a functional handle.

[0031] I. Definition The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Although 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 description of the invention. All technical and scientific terms used herein, unless otherwise defined, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of these techniques or equivalent technical substitutions that would be apparent to one of ordinary skill 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 provided to facilitate the description of the present invention. It will be understood that several techniques and steps are disclosed in describing the present invention. Each of these has distinct advantages, and each can also be used in combination with one or more, or in some cases all, of the other disclosed techniques. Thus, for the sake of clarity, this description refrains from repeating in an unnecessary manner every possible combination of the individual steps, but the specification and claims should nevertheless be read with the understanding that such combinations are fully within the scope of the invention and claims.

[0032] Following long-standing patent law convention, the terms a, an, and the, when used in this application, including the claims, refer to "one or more." Thus, for example, a reference to "a cell" includes a plurality of such cells, and the like. Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the preferred properties discovered for the subject matter disclosed herein. As used herein, the term "about," when referring to a value or composition amount, mass, weight, temperature, time, volume, concentration, percentage, and the like, is meant to encompass variations from the particular value of ±20% in some embodiments, ±10%, ±5%, ±1%, ±0.5%, and in some embodiments, ±0.1%, where such variations are appropriate for practicing the disclosed methods or employing the disclosed compositions.

[0033] The term "comprising," like "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 that means that the named elements are present, but that other elements could be added and still form a composition within the scope of the claim. The term "consisting of" excludes any element, step, or subject matter not expressly recited in the claim. When the phrase "consist of" appears in the body of a claim rather than immediately following a preamble, it is limited to only the recited elements, but does not exclude other elements from the claim as a whole. The term "consisting essentially of" limits a claim to the specified materials or steps and further limits materials or steps that do not materially affect the basic and novel characteristics of the claimed subject matter. With respect to the terms "comprising," "consisting of," and "consisting essentially of," when one of these three terms is used herein, the invention can include the use of either of the other two terms.

[0034] As used herein, the term "alkyl group" refers to a C1-20 hydrocarbon chain that is linear (i.e., "straight-chain"), branched, or cyclic, saturated or at least partially, and in some cases fully unsaturated (i.e., alkenyl and alkynyl groups), including, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertbutyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, methylpropenyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl. "Branched" refers to an alkyl group in which a lower alkyl group is attached to a linear alkyl chain, such as a methyl, ethyl, or propyl group. A "lower alkyl group" refers to an alkyl group having from 1 to about 8 carbon atoms (i.e., a C alkyl group), such as, for example, 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. A "higher alkyl group" refers to an alkyl group having from about 10 to about 20 carbon atoms, such as, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In some embodiments, "alkyl group" refers, in particular, to a C straight chain alkyl group. In other embodiments, "alkyl group" refers, in particular, to a C branched chain alkyl group.

[0035] An alkyl group can be optionally substituted with one or more alkyl group substituents, which can be the same or different ("substituted alkyl groups"). "Alkyl group substituents" include, but are not limited to, alkyl groups, substituted alkyl groups, halo groups, arylamino groups, acyl groups, hydroxyl groups, aryloxyl groups, alkoxyl groups, alkylthio groups, arylthio groups, aralkyloxyl groups, aralkylthio groups, carboxyl groups, alkoxycarbonyl groups, oxo groups, and cycloalkyl groups. Optionally, one or more oxygen atoms, sulfur atoms, or substituted or unsubstituted nitrogen atoms can be inserted along the alkyl chain, where the nitrogen substituent is a hydrogen atom, a lower alkyl group (hereinafter referred to as an "alkylaminoalkyl group"), or an aryl group. Thus, as used herein, the term "substituted alkyl group" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group have been replaced with other atoms or functional groups, such as alkyl groups, substituted alkyl groups, halogen atoms, aryl groups, substituted aryl groups, alkoxyl groups, hydroxyl groups, nitro groups, amino groups, alkylamino groups, dialkylamino groups, sulfate groups, and mercapto groups.

[0036] As used herein, the term "aryl group" refers to an aromatic substituent that can be a single aromatic ring or multiple aromatic rings fused together, covalently linked, or bonded to a common functional group, such as, but not limited to, a methylene or ethylene moiety. The common linking group can also be a carbonyl group, as in benzophenone, or an oxygen atom, as in diphenyl ether, or a nitrogen atom, as in diphenylamine. The term "aryl group" specifically includes heteroaromatic compounds. Aromatic rings include, for example, phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, and benzophenone. In particular embodiments, the term "aryl group" refers to a cyclic aromatic ring of about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and includes 5- and 6-membered hydrocarbon and heteroaromatic rings.

[0037] An aryl group can be optionally substituted with one or more aryl group substituents, which may be the same or different (a "substituted aryl group"). "Aryl group substituents" include alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, aralkyl groups, hydroxyl groups, alkoxyl groups, aryloxyl groups, aralkyloxyl groups, carboxyl groups, acyl groups, halo groups, nitro groups, alkoxycarbonyl groups, aryloxycarbonyl groups, aralkoxycarbonyl groups, acyloxyl groups, acylamino groups, aroylamino groups, carbamoyl groups, alkylcarbamoyl groups, dialkylcarbamoyl groups, arylthio groups, alkylthio groups, alkylene groups, and -NR'R" groups, where R' and R" can each independently be a hydrogen atom, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, or an aralkyl group.

[0038] Thus, as used herein, the term "substituted aryl group" includes an aryl group, as defined herein, in which one or more atoms or functional groups of the aryl group may be replaced with another atom or functional group, including, for example, alkyl groups, substituted alkyl groups, halogen atoms, aryl groups, substituted aryl groups, alkoxyl groups, hydroxyl groups, nitro groups, amino groups, alkylamino groups, dialkylamino groups, sulfate groups, and mercapto groups. Specific examples of aryl groups include, but are not limited to, cyclopentadienyl groups, phenyl groups, furan groups, thiophene groups, pyrrole groups, pyran groups, pyridine groups, imidazole groups, benzimidazole groups, isothiazole groups, isoxazole groups, pyrazole groups, pyrazine groups, triazine groups, pyrimidine groups, quinoline groups, isoquinoline groups, indole groups, carbazole groups, and the like.

[0039] The term "aralkyl group" refers to an -alkyl-aryl group, where optionally the alkyl and / or aryl groups contain one or more alkyl or aryl group substituents. In some embodiments, the term "divalent" refers to a group that can be bonded (e.g., covalently bonded) or bonded to two other groups, such as other alkyl, aralkyl, cycloalkyl, or aryl groups. Typically, two different sites (e.g., two different atoms) of the divalent group can be bonded to groups on other molecules. For example, the divalent group can be an alkylene group.

[0040] "Alkylene" refers to a straight or branched chain divalent aliphatic hydrocarbon group having 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 may be linear, branched, or cyclic. The alkylene group may also be optionally unsaturated and / or substituted with one or more "alkyl group substituents." Optionally, inserted along the alkylene group are one or more oxygen, sulfur, or substituted or unsubstituted nitrogen atoms (wherein the nitrogen substituent is alkyl, as defined above, also referred to herein as "alkylaminoalkyl"). Exemplary alkylene groups include methylene (-CH2-); ethylene (-CH2-CH2-); propylene (-(CH2)3-); cyclohexene (-CH6-); 10 -);-CH=CH-CH=CH-;-CH=CH-CH2-;-(CH2) q -N(R)-(CH2) r - (wherein q and r are each 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-CH-O-); and ethylenedioxyl (-O-(CH)-O-).) An alkylene group may have about 2 to about 3 carbon atoms, or even 6 to 20 carbon atoms.

[0041] "Arylene" refers to a divalent aryl group. "Functional handle" is used to refer to a chemical group that facilitates chemoenzymatic synthesis. In some embodiments, a functional handle may or may not be UV absorbing and / or may or may not bind to a -C18 column. In some embodiments, the functional handle may 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. As used herein, the term "and / or," when used in listing items, refers to items present either singly or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes not only A, B, C, and D individually, but also any and all combinations and subcombinations of A, B, C, and D.

[0042] II. Compounds, Compositions, and Methods The present invention provides heparan sulfate (HS) compounds and compositions comprising same, including pharmaceutical and / or therapeutic compositions comprising HS compounds as disclosed herein. In some embodiments, pharmaceutical compositions may comprise one or more HS compounds as disclosed herein. Methods of treating subjects with the HS compounds are also disclosed. In some embodiments, the present invention provides heparan sulfate (HS) compounds. In some embodiments, the present invention provides compositions, such as pharmaceutical compositions, comprising one or more HS compounds as disclosed herein. Accordingly, in some embodiments, the HS compounds are administered as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises one or more HS compounds and a pharmaceutically acceptable carrier or adjuvant for administration of the one or more HS compounds. In some embodiments, a method of treating hepatic ischemia-reperfusion (I / R) injury in a subject is provided. In some embodiments, the method comprises providing a subject suffering from or at risk of suffering from hepatic I / R injury; and administering to the subject one or more HS compounds disclosed herein. In some embodiments, the one or more HS compounds have a sulfation pattern and / or size based on sugar units that confers anti-inflammatory and / or anticoagulant activity, and administering the composition to the subject confers anti-inflammatory and / or anticoagulant activity in the subject.

[0043] In some embodiments, the HS compound contains about 5 to about 18 sugar units, optionally about 12 to about 18 sugar units. In some embodiments, the HS compound contains about 12 sugar units. In some embodiments, the HS compound binds to HMGB1. In some embodiments, the HS compound is a 12-mer-1 (12-mer-1), 12-mer-2 (12-mer-2), 12-mer-3 (12-mer-3), or 12-mer-4 (12-mer-4), as shown schematically in Figure 2A. In some embodiments, the HS compound is a 6-mer-AXa (schematically shown in Figure 6A). In some embodiments, the HS compound is an 18-mer (18-mer), as shown in Figure 10 and discussed in the Examples. Representative synthetic routes for HS compounds with various sulfation patterns and sizes based on the sugar units are shown in the Examples. Screening approaches for anti-inflammatory and anticoagulant activity are also shown in the Examples.

[0044] For clarity and not limitation, in some embodiments of the present invention, the hepatoprotective effects of the HS compounds disclosed herein are a function of their size and / or sulfation pattern. In one non-limiting example, a 12-mer oligosaccharide with a sulfation pattern that confers anticoagulant and anti-inflammatory activity provides hepatoprotective effects. In some embodiments, an 11-mer or 13-mer with the same or similar sulfation pattern should behave similarly. In another non-limiting example, a 12-mer oligosaccharide with a sulfation pattern that confers anti-inflammatory activity provides hepatoprotective effects when administered with a 6-mer with a sulfation pattern that confers anticoagulant activity. Again, an 11-mer or 13-mer with the same or similar sulfation pattern should behave similarly. In another non-limiting example, HS compounds at the higher end of the size range disclosed herein, such as 18-mer HS oligosaccharides with sulfation patterns that are non-anticoagulant and confer anti-inflammatory activity, provide hepatoprotective effects.

[0045] The present invention reduces the inflammatory response that damages hepatocytes following liver transplantation and surgery. In some embodiments, the HS compound contains about 5 to about 18 sugar units, including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 sugar units. Representative HS compounds are also disclosed in the accompanying drawings. In some embodiments, the HS compound is substituted with -H, alkyl, aryl, substituted alkyl, or substituted aryl. In some embodiments, the HS compound is substituted with a functional handle.

[0046] In some embodiments, both anti-inflammatory activity (such as via HMGB1 inhibition) and anticoagulant activity have been shown to confer protection in IR models. These activities can result from the administration of a single molecule (e.g., the HS compounds referred to herein as 12-mer-1 (12-mer-1) or 12-mer AXa (12-mer-AXa)) or the combined administration of two molecules (e.g., the HS compounds referred to herein as 6-mer-AXa (6-mer-AXa, shown schematically in Figure 6A) + 12-mer NS2S6S (12-mer NS2S6S, also referred to herein as 12-mer-3)). Thus, in some embodiments, a combination therapy is provided that comprises administering one or more HS compounds having a sulfation pattern and size based on the number of sugar units, which provides both anti-inflammatory activity (such as via HMGB1 inhibition) and anticoagulant activity to a subject in need thereof, such as a subject suffering from or at risk of suffering from hepatic I / R injury. Approaches for assessing anti-inflammatory and anticoagulant activity and their associated HS compound structures (including sulfation patterns and sizes based on the number of sugar units) are disclosed herein, including in the Examples. In some embodiments, the combination therapy comprises administering 6-mer-AXa (6-mer-AXa) + 12-mer NS2S6S (12-mer NS2S6S, also called 12-mer-3). In some embodiments, when two or more HS compounds are administered, the two or more HS compounds are administered separately but simultaneously. In some embodiments, the two or more HS compounds are administered at different times, but sufficiently close to each other to have the desired therapeutic effect. In some embodiments, the two or more HS compounds are administered in a single composition or formulation.

[0047] In some embodiments, the HS compound comprises the following formula: [ka] (In the formula, R1 represents -NHSO3H or -NHCOCH3, R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle, and n represents an integer of 0 to 6.) In some embodiments, the HS compound comprises the following structure: [ka] (wherein R1 represents -SO3H or -COCH3, and R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.) In some embodiments, the HS compound comprises the following structure: [ka] (wherein R1 represents -SO3H or -COCH3, and R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.)

[0048] In some embodiments, the HS compound comprises the following formula: [ka] wherein R represents —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle. In some embodiments, the HS compound comprises the following formula: [ka] (wherein R1 represents -SO3H or -H, and R2 represents -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.)

[0049] In some embodiments, the HS compounds are non-anticoagulant heparins and low molecular weight heparins and include one of the following structural formulas: [ka] [ka]

[0050] In some embodiments, the present invention provides a 5-mer, a 6-mer, or a 7-mer. In one embodiment, the 6-mer is a HS compound comprising the 6-mer of the present invention. The HS compound is referred to in the literature as 6-mer-AXa (schematically shown in Figure 6A). In some embodiments, the HS compound has the following formula: [ka] (wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or functional group) R stands for dollar 1 and n are defined as follows: In embodiment 1, R 1 is a hydrogen atom, n is 1; In embodiment 2, R 1 is a hydrogen atom, n is 2; In embodiment 3 (OSOH) and embodiment 4 (OH), [ka] In embodiment 5 (OSOH) and embodiment 6 (OH), [ka] )

[0051] In some embodiments, the functional handle comprises an alkyl, aryl, substituted alkyl, or substituted aryl group as defined herein, such as p-nitrophenyl. In one example, the 6-mer is an HS compound referred to herein as 6-mer-AXa (schematically shown in Figure 6A). In some embodiments, the HS compound is of the following formula: [ka] wherein R represents —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.

[0052] Figure 9 shows the structural formulas of representative HS anticoagulant (AXa) 8-18-mers (8-18 mers) of the present invention. The 12-mer AXa (12-mer-AXa) (n=3) is protective in a mouse model of hepatic ischemia and reperfusion. R in the formula is defined as indicated therein and can also be defined as -H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle. In some embodiments, such HS compounds have a sulfation pattern and / or size based on the sugar units that confers anti-inflammatory and / or anticoagulant activity, and administration of the composition to a subject confers anti-inflammatory and / or anticoagulant activity. Figure 10 shows the structural formula of a representative HS non-anticoagulant 16-18-mer of the present invention. This structure is not anticoagulant. The structure of an 18-mer (n=6) used in liver I / R includes R1=H, R2=phenyl, and X=nitro (para-nitrophenyl), e.g., a functional handle. R2 can also be defined as -H, alkyl, aryl, substituted alkyl, or substituted aryl. Thus, in another non-limiting example, HS compounds at the higher end of the size range of the present invention are non-anticoagulant, have sulfation patterns that confer anti-inflammatory activity, and provide hepatoprotection.

[0053] In some embodiments, the pharmaceutical composition may also include a pharmaceutically acceptable carrier or adjuvant. In some embodiments, the carrier is pharmaceutically acceptable for use in humans. The carrier or adjuvant should desirably not itself induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers may be high-molecular-weight, slowly metabolized macromolecules, such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, ammonium acid copolymers, inactive virus particles, and the like. Pharmaceutically acceptable salts may be used, such as mineral acid salts (such as hydrochloride, hydrobromide, phosphate and sulfate) or organic acid salts (such as acetate, propionate, malonate and benzoate). Pharmaceutically acceptable carriers in therapeutic compositions may further include liquids such as water, saline, glycerol, and ethanol. Such compositions may also contain auxiliary substances, such as wetting or emulsifying agents or pH buffering substances. Such carriers enable the pharmaceutical composition to be formulated for administration to a patient.

[0054] Suitable formulations of the pharmaceutical compositions of the present invention include aqueous or non-aqueous sterile injectable solutions (containing antioxidants, buffers, bactericides, bactericidal antibiotics, and solutes that render the formulation isotonic with the body fluids of the intended recipient), and aqueous or non-aqueous sterile suspensions (containing suspending agents, thickening agents, and the like). The formulations may be presented in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored frozen or lyophilized, requiring only the addition of a sterile liquid carrier (e.g., water for injection) immediately prior to use. Some exemplary ingredients are SDS (in some embodiments, in the range of 0.1-10 mg / ml, in some embodiments, about 2.0 mg / ml); mannitol or other sugars (in some embodiments, in the range of 10-100 mg / ml, in some embodiments, about 30 mg / ml); and / or phosphate-buffered saline (PBS). Any other agent conventional in the art for the type of formulation in question may also be used. In some embodiments, the carrier is pharmaceutically acceptable. In some embodiments, the carrier is pharmaceutically acceptable for use in humans. The pharmaceutical composition of the present invention may have a pH of 5.5 to 8.5, preferably 6 to 8, more preferably about 7. This pH can be maintained by the use of a buffer. The composition may be sterile and / or pyrogen-free. The composition may be isotonic with respect to humans. The pharmaceutical composition of the present invention may be supplied in a sealed container.

[0055] The method of treatment according to the present invention comprises administering to a subject in need thereof an HS compound of the present invention or a related compound thereof. An effective dose of the pharmaceutical composition of the present invention 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 or pharmaceutical composition of the present invention sufficient to produce some effect (e.g., alleviation of symptoms of hepatic ischemia-reperfusion (I / R) injury). Actual dosage levels can be varied to administer an amount effective to achieve the desired therapeutic effect for a particular subject. In some embodiments, the amount of a therapeutic composition of the present invention administered to a subject will depend on many factors, including but not limited to the subject's size, weight, age, target tissue or organ, route of administration, the condition being treated, and the severity of the condition being treated. The potency of therapeutic compositions can vary, and therefore a "therapeutically effective" amount can vary. However, one of skill in the art can readily assess the potency and effectiveness of pharmaceutical compositions of the present invention using the assessment methods described below, and adjust treatment regimens accordingly.

[0056] III. Subjects (patients) While the present invention is preferably directed to humans, it is to be understood that the principles of the present invention demonstrate that the compositions and methods are effective with respect to all vertebrate species, including invertebrates and mammals, which are intended to be encompassed by the term "subject." It is further understood that mammals include any mammalian species in which it is desirable to treat hepatic ischemia-reperfusion (I / R) conditions, particularly agricultural and livestock mammalian species. The method of the present invention is particularly useful for treating warm-blooded vertebrates, and therefore relates to mammals and birds. More specifically, provided herein are treatments for mammals, including mammals important not only to humans but also to those that are endangered or otherwise important (e.g., Siberian tigers), economically important (animals raised on farms for human consumption), and / or socially important to humans (animals kept as pets or in zoos), such as non-human carnivores (e.g., cats and dogs), swine (e.g., pigs, hogs, wild boars), ruminants (e.g., cattle, cows, sheep, giraffes, deer, goats, bison, camels), and horses. Endangered species of birds and poultry kept in zoos, particularly domesticated poultry (i.e., turkeys, chickens, ducks, geese, guinea fowl, and other poultry), are also economically important to humans, and treatments for these species are also provided. Accordingly, provided herein are treatments for livestock, including, but not limited to, livestock (e.g., pigs), ruminants, horses, and poultry.

[0057] Hepatic ischemia-reperfusion (I / R) injury is a major complication of surgery during liver transplantation and liver tumor resection. Liver surgery often requires the use of the Pringle maneuver to reduce blood loss, at the expense of potential I / R injury. Initial injury begins during the ischemic phase, when blood flow to tissues is cut off, resulting in a lack of oxygen and nutrients. Therefore, subjects undergoing surgery during liver transplantation are typical subjects (patients) to be treated. However, any subject suffering from or at risk of suffering from hepatic I / R injury, as would become apparent to one skilled in the art upon review of this disclosure, may also be treated. [Example]

[0058] The following examples are included to provide guidance to those skilled in the art for practicing representative embodiments of the invention. In light of this disclosure and the general level of skill in the art, those skilled in the art will recognize that the following examples are merely illustrative, and that numerous variations, modifications, and variations are possible without departing from the scope of the invention.

[0059] Materials and Methods Chemoenzymatic synthesis of oligosaccharides The synthesis of the 12-mer and 6-mer-AXa has been previously reported

[15] . See also Figure 13. Briefly, to synthesize 12-mer-4, glucuronic acid-pNP was extended with UDP-GlcNTFA (step a) and UDP-GlcA (step b) using pmHS2 to obtain the 12-mer. GlcNTFA was then deprotected using LiOH and then N-sulfated with NST (step c) to generate the N-sulfoglucosamine residue of the 12-mer. Next, 6-O-sulfation using 6-OST1 and 6-OST3 (step d) produced 12-mer-4 (12-mer-4: GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-GlcNS6S-GlcA-pNP). 12-mers-1, -2, and -3 (12-mers-1, -2, and -3) were synthesized by elongating the monosaccharide to a pentasaccharide intermediate. Next, GlcNTFA was deprotected using LiOH and then N-sulfated using NST to produce a 5-mer (pentamer) with two N-sulfoglucosamine residues. The 5-mer (pentamer) NS was extended with UDP-GlcNTFA to produce a 6-mer (hexamer) intermediate. The glucuronic acid residue between the two N-sulfoglucosamine residues underwent epimerization by C5-epimerase and 2-O-sulfation by 2-OST (step e) to generate the 6-mer intermediate GlcNTFA-GlcA-GlcNS-IdoA2S-GlcNS-GlcA-pNP. This 6-mer intermediate was then subjected to steps c, d, and e, and 3-O-sulfation by 3-OST (step f) to generate GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S-GlcA-pNP to generate 6-mer-AXa. To generate 12-mer-2 (12-mer-2), steps b, c, a, and e were repeated three times on the 6-mer (hexamer) intermediate to generate GlcNS-GlcA-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-IdoA2S-GlcNS-GlcA-pNP.12-mer-2 (12-mer-2) was converted to 12-mer-3 (12-mer-3) by 6-O-sulfation of the GlcNS residues with 6-OST1 and 6-OST-3 (step e), yielding the structure GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP. Finally, 12-mer-3 was converted to compound 12-mer-1 (step f) to produce the structure GlcNS6S-GlcA-GlcNS6S3S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP. The purity of these different 12-mers and 6-mer-AXa was >95% as determined by high-resolution DEAE-HPLC. Their chemical structures were confirmed by electrospray ionization mass spectrometry (ESI-MS) and NMR

[15] .

[0060] These oligosaccharides were converted to biotinylated versions using the same method described in PCT International Patent Application (PCT / US2018 / 059152). Briefly, pNP tags (5–10 mg) and 0.5 mg Pd / C-bearing 12-mer and 6-mer-AXa were dissolved in 20 mM NaOAc, pH 5.0, in a total volume of 4 ml. The reaction mixture was evacuated and refilled with H2 three times. The reaction was then incubated at room temperature for 4 h. The charcoal was then removed by filtration. The filtered solution was adjusted to pH 8.5 with 500 mM Na2HPO4. Succinimidyl 6-azidohexanoate (20 molar equivalents of the starting oligosaccharide) was added and incubated overnight at 37 °C. The reaction mixture was purified on a DEAE-HPLC column to produce the azido-tagged oligosaccharides. A sample solution consisting of 0.1 M CuSO4, 0.1 M tris(3-hydroxypropyl-triazolylmethyl)amine (THPTA) (Sigma), 0.15 M sodium ascorbate, 0.01 M azide-tagged oligosaccharides, and 0.02 M biotin-PEG4-alkyne (Sigma) was prepared by bubbling N2 through PBS (pH 7.4) buffer for 5 min. A mixture of 400 μl of THPTA and 80 μl of CuSO4 was vigorously stirred, to which 160 μl of sodium ascorbate, 200 μl of azide-tagged oligomers, and 200 μl of biotin-PEG4-alkyne were added. N2 was bubbled through the mixture for 2 min and incubated overnight at 37 °C. The reaction mixture was purified using a DEAE-HPLC column to yield the biotinylated product. The biotinylated 12-mer and 6-mer-AXa were confirmed by ESI-MS.

[0061] Affinity purification of HMGB1 from liver lysates 6-mer-AXa and 12-mer biotinylated oligosaccharides were prepared for affinity purification of HMGB1 from liver lysates according to the method described in PCT International Patent Application (PCT / US2018 / 059152). Briefly, liver lysates were prepared by rapidly freezing tissue in liquid nitrogen at the time of sacrifice. The tissue was mechanically homogenized in a pH 6 buffer containing 200 mM MES, 500 mM phosphate, and 1 mM EDTA, followed by three freeze-thaw cycles. The lysed samples were centrifuged at 10,000 x g for 15 minutes at 4°C. Biotinylated HS oligosaccharides (final concentration 0.1 mM) were mixed with 20 μl of fresh liver lysate (~0.6 mg) in 100 mM NaCl, 20 mM HEPES, pH 7.2, and incubated overnight at 4°C. The biotinylated HS-bound complex was purified using avidin-Sepharose and increasing concentrations of NaCl washes. The eluate from each sample was separated by gel electrophoresis, transferred to a nitrocellulose membrane, and blotted for HMGB1 using anti-HMGB1 primary antibody (Abcam) followed by anti-rabbit HRP (Abcam).

[0062] Determination of anti-FXa activity of oligosaccharides in vitro and ex vivo The assay was based on a previously reported method

[16] . Briefly, human FXa (Enzyme Research Laboratories) was diluted to 50 μg ml in PBS. The chromogenic substrate S-2765 (Diapharma) was diluted to 1 mg ml in water. -1For in vitro testing, fondaparinux (available under the trade name ARIXTRA) and 12-mer oligosaccharides were dissolved in PBS at various concentrations (0–131 nM). A 16 μl sample was incubated with 60 μl of 35 μl ml-1 antithrombin (Cutter Biologics) for 2 minutes at room temperature. Next, 100 μl of FXa was added and incubated for 4 minutes at room temperature. 30 μl of S-2765 substrate was added, and the absorbance of the reaction mixture was measured at 405 nm continuously for 5 minutes. PBS served as the control sample. The maximum slope of each sample was divided by the maximum slope of the control sample to convert it to % FXa activity. For ex vivo studies, mouse plasma was collected after the 6-hour reperfusion period and evaluated as described above.

[0063] Liver ischemia-reperfusion surgery design Liver ischemia-reperfusion (IR) surgery was performed by the Animal Surgery Core Laboratory at the McAllister Heart Institute at the University of North Carolina at Chapel Hill. This mouse experiment was approved by the UNC Animal Care and Use Committee and conformed to National Institutes of Health guidelines. Male C57BL / 6J mice approximately 8 weeks old were used for IR surgery. Mice were injected subcutaneously (SC) with 1 mg / kg oligosaccharide or an equal volume of saline 30 minutes before surgery. For the 12-mer-3 (12-mer-3) + 6-mer-AXa (6-mer-AXa) combination treatment, equal concentrations of each oligosaccharide were combined into a single solution. Under ketamine / xylazine anesthesia, a midline abdominal incision was made to expose the portal vein. Clamps were placed on the portal vein and bile duct of the three major liver lobes to induce 70% hepatic ischemia. Correct clamp placement was confirmed by visual inspection of blanching of the ischemic liver lobe. Temporary suture closure of the muscle and skin over the clamps was used to prevent dehydration during the ischemic phase. Mice remained on a heating pad under anesthesia for the entire ischemic phase (60 minutes). The clamps were removed after 60 minutes, and as blood began to reperfuse the tissue, the ischemic liver lobes returned to red, indicating proper reperfusion. The incision was closed in two layers with 5-0 silk sutures, and the mice were returned to active status (no anesthesia was used during the reperfusion phase). Six hours later, the mice were re-anesthetized, blood was collected by cardiac puncture, and the ischemic liver lobes were collected for histology (fixed in 10% formalin).

[0064] Assessment of hepatic I / R injury Plasma ALT was measured using the ALT Infinity Reagent (Thermo Fisher) according to the manufacturer's instructions. Plasma TNF-α was measured using the Mouse TNF-α DuoSet Kit (R&D Systems) according to the manufacturer's instructions. Plasma HMGB1 levels were measured using the HMGB1 ELISA Kit (Tecan US) according to the manufacturer's instructions. Plasma syndecan-1 levels were measured using the Mouse Syndecan-1 ELISA (CellSciences) according to the manufacturer's instructions. Plasma IL-6 levels were measured using the Mouse IL-6 ELISA (R&D Systems).

[0065] Histology / Immunohistochemistry Ischemic liver tissue was fixed in 10% neutral-buffered formalin at room temperature for 24 hours, embedded in paraffin, and sectioned. Liver sections (4 μm) were stained with hematoxylin and eosin (H&E) or immunostained with monoclonal antibody anti-neutrophil (Abcam, Ab 2557, NIMP-R14) followed by goat anti-rat or goat anti-rabbit biotinylated secondary antibodies (Abcam). Embedding, sectioning, and H&E staining were performed at the UNC-Chapel Hill Animal Histopathology and Laboratory Medicine Core Facility. H&E analysis was performed at the UNC-Chapel Hill Translational Pathology Laboratory Core Facility using Aperio ImageScope Software (Leica Biosystems, Concord, Canada). IHC images were captured using an HD camera connected to a bright-field microscope (Leica DM 1000 LED, Leica Microsystems Inc., IL, USA) and processed using ImageJ. For neutrophil quantification, five 100x images were randomly selected for each sample, and the mean neutrophils / field was reported.

[0066] Myeloperoxidase (MPO) activity Ischemic liver lobes were mechanically homogenized in 50 mM CTAB, 50 mM potassium phosphate, pH 6, at a ratio of 100 μl of buffer per 10 mg of tissue. The samples were centrifuged at 15,000 × g for 20 min at 4°C, and the supernatant was collected and stored at -20°C. Total protein concentration was measured by Bradford assay. 10 μl of liver lysate was incubated with 80 μl of 0.75 mM H2O2 and 110 μl of TMB (TMB liquid reagent, ready-to-use, Sigma) for 10 min at 37°C with gentle agitation. The reaction was stopped by adding 2.5 M H2SO4 and read at 450 nm. Activity (U / g protein) was calculated as the absorbance of the sample minus the absorbance of the blank, divided by the incubation time. This value was normalized by protein concentration.

[0067] statistical analysis All data are expressed as mean ± SEM. Statistical significance between experimental and control groups was analyzed by two-tailed unpaired Student's t-test, and between multiple groups by one-way analysis of variance followed by Dunnett's or Tukey's multiple comparison test, and Kaplan-Meier survival curves with the log-rank test were plotted using GraphPad Prism software (version 7.03; GraphPad Software, Inc., graphpad.com / scientific-software / prism / ).

[0068] Example 1 Hepatic ischemia-reperfusion (I / R) increases liver injury and inflammation We evaluated the in vivo efficacy of the oligosaccharides using a mouse model of partial hepatic I / R injury. In this model, ischemia was induced in 70% of the liver using a clamp (Figure 1A). After 1 h, the clamp was removed, initiating the reperfusion period. Animals were sacrificed 6 h after reperfusion. Liver injury was measured by elevated plasma alanine aminotransferase (ALT), necrotic cell area, and neutrophil infiltration into the ischemic liver (Figures 1B-1D). Furthermore, ischemia-reperfusion (I / R) resulted in significant increases in plasma HMGB1 (Figure 1E, P = 0.013) and syndecan-1 (Figure 1F, P = 0.017). Elevated plasma HMGB1 and syndecan-1 levels are indicators of cell death and endothelial injury [17, 18].

[0069] Example 2 HMGB1 binds to highly sulfated HS oligosaccharides HMGB1 is involved in impaired inflammatory responses after hepatic I / R [17,19]. A recent report investigated the binding of HMGB1 to HS oligosaccharides with specific repeat lengths

[20] . In this study, we tested a panel of 12-mers with varying degrees of sulfation and 2-O-sulfoiduronic acid residues. Access to these 12-mers allowed us to further analyze the effects of sulfation or 2-O-sulfoiduronic acid residues on HMGB1 binding (Figure 2A). This panel included four 12-mers covering the different sulfation types in this study. 12-mer-1 has the highest degree of sulfation, with 17 sulfo groups and four 2-O-sulfoiduronic acid residues. 12-mer-2 has 10 sulfo groups, the lowest of the four 12-mers, and contains four 2-O-sulfoiduronic acid residues. 12-mer-3 contains 16 sulfo groups and four 2-O-sulfoiduronic acid residues. The structural differences between 12-mer-1 and 12-mer-3 are that 12-mer-1 contains a 3-O-sulfo group on one glucosamine residue, whereas this 3-O-sulfation is absent in 12-mer-3, and 12-mer-4 contains 12 sulfo groups but no 2-O-sulfoiduronic acid residues.

[0070] The anticoagulant activity of 12-mer-1, as measured by inhibiting the activity of factor Xa (anti-FXa), was similar to that of fondaparinux, an FDA-approved anticoagulant available under the trade name ARIXTRA. 50 The values ​​were 63 nM and 18 nM, respectively, but 12-mers-2, -3, and -4 did not exhibit anti-FXa activity and therefore lack anticoagulant activity (FIG. 2B). Next, we used biotin-tagged oligosaccharides to pull down endogenous HMGB1 from liver lysates (Figure 2C). Interestingly, 12-mer-1 and 12-mer-3 successfully pulled down HMGB1, suggesting that the degree of sulfation is an important factor for HMGB1 binding in oligosaccharides of this size.

[0071] Figures 7A and 7B are Western blot images from HMGB1 pulldown using oligosaccharides. Figure 7A shows the input sample: Lane 1: 12-mer-4; Lane 2: 12-mer-2; Lane 3: 12-mer-3; Lane 4: 12-mer-1. Figure 7B shows the eluted sample: Lane 5: 12-mer-4; Lane 6: 12-mer-2; Lane 7: 12-mer-3; Lane 8: 12-mer-1. Figures 8A and 8B show full Western blot images from HMGB1 pulldowns using 12-mer-1 and 6-mer-AXa oligosaccharides. Figure 8A shows the input sample: Lane 1: 12-mer-3; Lane 2: 12-mer-1; Lane 3: 6-mer-AXa. Figure 8B shows the eluted sample: Lane 4: 12-mer-3; Lane 5: 12-mer-1; Lane 6: 6-mer-AXa.

[0072] Example 3 12-mer-1 reduces liver injury after IR In an in vivo model of hepatic IR, 12-mer-1 and 12-mer-3 were used based on their ability to bind to HMGB1. These compounds were administered 30 min before ischemia. The anticoagulant activity of 12-mer-1 was confirmed in plasma (Figure 3A). Although both 12-mer-1 and 12-mer-3 bind to HMGB1, only 12-mer-1 significantly reduced plasma ALT (Figure 3B; 12-mer-1 vs. IR, P = 0.048; 12-mer-3 vs. IR, P = 0.620). Furthermore, 12-mer-1 reduced hepatic necrosis in the ischemic liver lobe compared with the IR group (Figure 4; 12-mer-1 vs. IR, P = 0.0287; 12-mer-3 vs. IR, P = 0.1059). This suggests that the anticoagulant and anti-inflammatory properties of 12-mer-1 confer protection against IR injury to the liver.

[0073] Example 4 12-mer-1 reduces neutrophil accumulation and myeloperoxidase (MPO) in the ischemic liver In hepatic IR, neutrophils are rapidly recruited to postischemic tissues during the reperfusion phase [8]. After migrating to the liver, neutrophils release cytotoxic compounds, such as reactive oxygen species and proteases, to clear damaged tissue

[21] . Neutrophils and their potent cargo are key effectors in sterile inflammation due to their lack of specificity for damaged versus healthy tissue. As a result, neutrophil recruitment continues, perpetuating inflammation. Neutrophil-derived proteases, such as elastase, MMP-9, cathepsin G, proteinase-3, and myeloperoxidase (MPO), have been reported to be involved in IR-induced liver injury [8]. In particular, MPO is highly expressed in neutrophils and serves as a marker of neutrophil accumulation. MPO contributes to tissue oxidative stress by reacting with hydrogen peroxide [8]. MPO activity was measured in ischemic liver lysates (Figure 5A). Treatment with 12-mer-1 (12-mer-1) reduced MPO activity by 60% compared with the IR group. In contrast, MPO activity was nearly identical between the 12-mer-3 and IR groups (96.00 vs. 96.33 U / g protein, respectively). Furthermore, neutrophil accumulation was measured in ischemic tissues by immunohistochemistry (Figure 5B–F). Similar to the trend for MPO, 12-mer-1, but not 12-mer-3, reduced neutrophil infiltration (12-mer-1 vs. IR, P = 0.0142; 12-mer-3 vs. IR, P = 0.0705).

[0074] Example 5 Anticoagulation alone is insufficient for liver protection Next, we investigated whether anticoagulant activity was required for hepatoprotection. To this end, we used 6-mer-AXa (6-mer-AXa) oligosaccharides (Figure 6A). Like 12-mer-1, 6-mer-AXa exhibits anticoagulant activity through FXa inhibition, as previously shown

[15] . Biotinylated 6-mer-AXa does not pull down HMGB1 from liver lysates (Figure 6B), so it serves as a control for anticoagulant activity without HMGB1 binding. To determine whether hepatoprotection after IR requires both anti-inflammatory properties (e.g., HMGB1 binding) and anticoagulant properties from heparan sulfate oligosaccharides, we used 6-mer-AXa oligosaccharides alone or in combination with 12-mer-3 (12-mer-3), based on the size and sulfation pattern of representative HS compounds. Although anticoagulant levels were similar between both treatment groups (Figure 6C), only the combined treatment of oligosaccharides with HMGB1-binding ability (12-mer-3) and anticoagulant activity (6-mer-AXa) statistically significantly reduced plasma ALT after IR. There was no statistical difference in ALT concentrations between the IR-injured and 6-mer-AXa-treated groups. This result indicates that both activities, derived from a single compound with dual activity or from the combination of two compounds with separate functions, play a hepatoprotective role.

[0075] Consideration of Examples 1 to 5 Hepatic ischemia-reperfusion injury (hepatic IR injury), unlike acetaminophen-induced liver injury, has been reported to involve coagulation disorders in addition to inflammation, and is therefore described as thromboinflammatory [3]. Anticoagulant HS oligosaccharides were ineffective against acetaminophen-induced liver injury

[20] . However, because hepatic IR involves thromboinflammatory processes, this study included anticoagulant 12-mer-1 (12-mer-1) to examine how anticoagulant activity and HMGB1 binding affect hepatoprotection in hepatic IR injury. This demonstrated that synthetic HS oligosaccharides are a therapeutic agent in another disease model.

[0076] In Examples 1-5, we investigated the structure-activity relationship of HS oligosaccharides for HMGB1 binding by screening a panel of 12-mer oligosaccharides with various sulfation patterns. Only 12-mer-1 and 12-mer-3, both highly sulfated oligosaccharides, were successful in pulling down HMGB1 from liver lysates. However, 12-mer-1, but not 12-mer-3, was observed to reduce ALT and necrosis in ischemic liver lobes in vivo. The anti-inflammatory effects of 12-mer-1, coupled with its ability to bind HMGB1, reduced tissue MPO levels and reduced neutrophil accumulation in ischemic liver lobes. Interestingly, binding to HMGB1 is not sufficient for the hepatoprotection exhibited by 12-mer-3 in vivo. In an IR model, the use of 6-mer-AXa in combination with 12-mer-3, or 6-mer-AXa alone, demonstrated that both anticoagulant and anti-inflammatory effects play a role in achieving hepatoprotection. Thus, anticoagulant activity was examined with and without HMGB1 binding. Treatment with 6-mer-AXa alone did not statistically significantly reduce ALT levels, whereas combination treatment did. While not wishing to be bound by a particular theory of action, the hepatoprotective effect of 12-mer-1 can be attributed to its dual anticoagulant and anti-inflammatory activities, since both anticoagulant and anti-inflammatory mechanisms are essential to the pathophysiology of hepatic IR.

[0077] Examples 1–5 demonstrate that 2-mer-1 (12-mer-1) is both anticoagulant and an active anti-inflammatory. In addition to the protective mechanism of 12-mer-1 in hepatic IR, 12-mer-1 also possesses several favorable drug-like properties. Because most liver transplant patients also have impaired renal function

[23] , renal clearance of 12-mer-1 was considered. Impaired renal clearance of 12-mer-1 was demonstrated using a renal IR model

[16] . However, 12-mer-1 is a homogenous compound with uniform anticoagulant activity, potentially making it a safe option for patients with renal impairment and therefore amenable to dosage adjustment. Bleeding problems have been reported in liver transplant recipients receiving heparin therapy, with 9% requiring surgical intervention due to bleeding complications

[24] . Low-molecular-weight heparin (LMWH) reduces the risk of bleeding, but protamine does not completely reverse this

[16] . The anticoagulant activity of 12-mer-1 is reversible by protamine, providing the added benefit of ameliorating bleeding complications

[16] . Therefore, the potential for controlled administration and reversibility with protamine are key features of 12-mer-1. Furthermore, 12-mer-1 showed no toxicity in a high-dose rat model

[20] . Therefore, 12-mer-1's antithrombotic and inflammatory properties, reversibility with protamine, lack of toxicity, and the ability to precisely control administration make it an attractive treatment for liver transplant / IR patients. Pharmacokinetic studies are currently investigating the relationship between 12-mer-1 administration and the liver's response to IR injury.

[0078] Heparin and desulfated heparin bind to P-selectin, a tethering molecule for neutrophils in most organs and tissues [J. Wang; Geng, J., Thromb Haemost 90, 7 (2003)]. Interestingly, there are some differences in neutrophil recruitment to the liver compared to the classical model. For example, there is little evidence that selectin- or β2-integrin-mediated adhesion is required for neutrophil migration to the liver [8]. Rather, neutrophils are physically trapped in the hepatic sinusoids, where nearly 80% of leukocyte trafficking occurs [SL Maas, O. Soehnlein, JR Viola, Frontiers in Immunology 9, (2018)]. Therefore, without wishing to be bound by a particular theory of action, we believe that the reduction in neutrophil accumulation after treatment with 12-mer AXa is not due to selectin inhibition. Dalteparin, a low-molecular-weight heparin, reduces liver irritation injury in rats

[22] . Interestingly, in this study, protection was not observed when DX9065a, a selective factor Xa inhibitor, was used. This suggests that dalteparin's protective effect is not solely due to its anticoagulant properties. Dalteparin's reduction in MPO levels suggests an effect on neutrophil recruitment. However, due to incomplete structural characterization of dalteparin, further biochemical analysis is extremely difficult, if not impossible. Chemoenzymatic synthesis techniques have been used to generate heparin oligosaccharides with or without anticoagulant activity. As the anti-inflammatory properties of heparin are increasingly recognized

[25] , this invention helps to overcome the challenge of using heterogeneous mixtures of oligosaccharides for characterization of biological effects. Chemoenzymatic synthesis techniques produce structurally defined HS oligosaccharides. As heparin is increasingly recognized for its anti-inflammatory properties

[25] , this invention contributes to a shift in the therapeutic field from using heterogeneous mixtures of oligosaccharides to a new class of uniform, precise oligosaccharide therapeutics.

[0079] Example 6 Effect of HS oligosaccharides on hepatic ischemia-reperfusion injury - Results of 18-mer background: An 18-mer (18-mer) with repeating units of N-sulfoglucosamine and 2-O-sulfoiduronic acid was used in a mouse model of hepatic ischemia-reperfusion (IR) (Figure 10). This compound is non-anticoagulant because it lacks the specific sulfation required for anticoagulation. As noted above, in some embodiments, when larger HS compounds, such as the 18-mer, are used, the non-anticoagulant 18-mer exhibits a protective effect. Treatment with the 18-mer reduces the severity of IR injury in the liver. IR Procedure: Thirty minutes before the start of the ischemic phase, 1 mg / kg of 18-mer or an equivalent volume of sterile saline was administered by subcutaneous injection. Up to 70% ischemia of the liver was induced by clamping the portal vein and bile duct for 60 minutes. After removing the clamps, the reperfusion phase lasted for 6 hours before the animals were sacrificed for liver tissue and blood collection. As surgical controls, sham surgery was performed so that the animals experienced the same anesthesia, midline abdominal incision, and suture procedure as the IR mice (Figure 11A).

[0080] result: Treatment with the 18-mer reduced liver injury after IR. Plasma alanine aminotransferase (ALT) is a marker of liver injury. Treatment with the 18-mer reduced ALT levels compared to the IR group (Figure 11B). As histological evidence of the protection of the 18-mer, the ischemic liver lobes were stained with H&E for quantification of the necrotic area. The 18-mer significantly reduced the necrotic area compared to IR (Figure 11C-F). In addition to reducing the necrotic area, the 18-mer significantly affected many inflammatory mediators (Figures 12A–D). HMGB1, a damage-associated molecular pattern released after IR, is associated with the spread of sterile inflammation and liver injury [A. Tsung et al., The Journal of Experimental Medicine 204, 2913–2923 (2007); JC Evankovich, SW; Zhang, R; Cardinal, J; et al., J Bio Chem 285, 9 (2010)]. The 18-mer reduced plasma HMGB1 (Figure 12A) and neutrophil infiltration into the ischemic liver lobe (Figure 12D). Furthermore, treatment with the 18-mer reduced other plasma inflammatory markers, including IL-6 and TNF-α.

[0081] Conclusion: The 18-mer potentially alleviates liver IR injury by inhibiting HMGB1-mediated neutrophil infiltration and reducing sterile inflammation. Other compounds with similar structures, including the 16-mer with the same repeating disaccharide units of N-sulfoglucosamine and 2-O-sulfoiduronic acid, are also effective in vivo. Furthermore, these compounds can be modified to contain N-, 6-O-sulfoglucosamine and glucuronic acid residues, which may also be effective in vivo. These compounds can also be functionalized with various chemical handles to study binding in vitro and further elucidate the mechanism of protection from liver IR (Figure 10, position R2).

[0082] References The documents listed below and all documents cited throughout the specification, to the extent that they supplement, explain, provide background to, or teach the methods, techniques, and / or compositions employed herein, are incorporated herein by reference. 1. Konishi, T. & Lentsch, A. B. Hepatic ischemia / reperfusion: mechanisms of tissue injury, repair, and regeneration. Gene Expr 17, 277-287, doi:10.3727 / 105221617X15042750874156 (2017). 2. Man, K. et al. Tolerance of the liver to intermittent pringle maneuver in hepatectomy for liver tumors. JAMA Surgery 134, 533-539, doi:10.1001 / archsurg.134.5.533 (1999). 3. Jackson, S. D., R; Schoenwaelder, SM. Thromboinflammation: challenges of therapeutically targeting coagulation and other host defense mechanisms. Blood 133, 12 (2019). 4. Iba, T., Levy, J. H., Raj, A. & Warkentin, T. E. Advance in the management of sepsis-induced coagulopathy and disseminated intravascular coagulation. J Clin Med 8, 728 (2019). 5. Pierce, A. & Pittet, J.-F. Inflammatory response to trauma: implications for coagulation and resuscitation. Curr Opin Anesthesio 27, 246-252, doi:10.1097 / aco.0000000000000047 (2014). 6. Tsung, A. et al. HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calcium-mediated signaling. J Exp Med 204, 2913-2923, doi:10.1084 / jem.20070247 (2007). 7. Huebener, P. et al. The HMGB1 / RAGE axis triggers neutrophil-mediated injury amplification following necrosis. J Clin Invest 125, 539-550 (2015). 8. Oliveira, T. H. C. d., Marques, P. E., Proost, P. & Teixeira, M. M. M. Neutrophils: a cornerstone of liver ischemia and reperfusion injury. Lab Invest 98, 51-62, doi:10.1038 / labinvest.2017.90 (2018). 9. Lu, L. et al. Innate Immune Regulations and Liver Ischemia-Reperfusion Injury. Transplantation 100, 2601-2610 (2016). 10. Gama, C. et al. Sulfation patterns of glycosaminoglycans encode molecular recognition and activity. Nat Chem Biol 2, 467-473 (2006). 11. Feltracco, P. et al. Perioperative thrombotic complications in liver transplantation. World J Gastroenterol 21, 8004-8013, doi:10.3748 / wjg.v21.i26.8004 (2015). 12. Jaimes, F. et al. Unfractioned heparin for treatment of sepsis: A randomized clinical trial (The HETRASE Study)*. Crit Care Med 37, 1185-1196, doi:10.1097 / CCM.0b013e31819c06bc (2009). 13. Liu, J. & Linhardt , R. J. Chemoenzymatic synthesis of heparan sulfate and heparin. Nat Prod Rep 31, 1676-1685 (2014). 14. Xu, Y. et al. Chemoenzymatic synthesis of homogeneous ultra-low molecular weight heparin. Science 334, 498-501 (2011). 15. Xu, Y. et al. Homogeneous low-molecular-weight heparins with reversible anticoagulant activity. Nat Chem Biol 10, 248-250, doi:10.1038 / nchembio.1459 (2014). 16. Xu, Y. et al. Synthetic oligosaccharides can replace animal-sourced low-molecular weight heparins. Sci Transl Med 9, eaan5954, doi:10.1126 / scitranslmed.aan5954 (2017). 17. Tsung, A. et al. The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion. J Exp Med 201, 1135-1143, doi:10.1084 / jem.20042614 (2005). 18. Teng, Y. H.-F., Aquino, R. S. & Park, P. W. Molecular functions of syndecan-1 in disease. Matrix Biol 31, 3-16, doi:10.1016 / j.matbio.2011.10.001 (2012). 19. Yamamoto, T. & Tajima, Y. HMGB1 is a promising therapeutic target for acute liver failure. Expert Rev Gatroenterol Hepatol 11, 673-682 (2017). 20. Arnold, K. et al. Design of anti-inflammatory heparan sulfate to protect against acetaminophen-induced acute liver failure. Sci Transl Med 12, eaav8075, doi:10.1126 / scitranslmed.aav8075 (2020). 21. Kubes, P. & Mehal, W. Z. Sterile inflammation in the liver. Gastroenterology 143, 1158-1172, doi:https: / / doi.org / 10.1053 / j.gastro.2012.09.008 (2012). 22. Naoaki Harada, M. K. O., MD; Mitsuhiro Uchiba, MD. Dalteparin, a low molecular weight heparin, attenuates inflammatory responses and reduces ischemia-reperfusion-induced liver injury in rats. Crit Care Med 34, 8 (2006). 23. Weber, M. L., Ibrahim, H. N. & Lake, J. R. Renal dysfunction in liver transplant recipients: Evaluation of the critical issues. Liver Transplant 18, 1290-1301, doi:10.1002 / lt.23522 (2012). 24. Kaneko, J. et al. Coagulation and fibrinolytic profiles and appropriate use of heparin after living-donor liver transplantation. Clin Transplant 19, 804-809, doi:10.1111 / j.1399-0012.2005.00425.x (2005). 25. Oduah, E. I., Linhardt, R. J. & Sharfstein, S. T. Heparin: Past, present, and future. Pharmaceuticals (Basel) 9, 38, doi:10.3390 / ph9030038 (2016). 26. U.S. Patent No. 9,951,149, issued April 24, 2018. 27. PCT International Application PCT / US2018 / 059152, International Publication Number WO 2019 / 090203, International Publication Date May 19, 2019 It will be understood that various details of the invention disclosed herein can be changed without departing from the scope of the invention. Furthermore, the above description is intended to be illustrative of the invention and not to be limiting thereof.

Claims

1. 1. A composition for use in treating hepatic ischemia-reperfusion (I / R) injury in a subject, the composition comprising one or more synthetic heparan sulfate (HS) compounds; (i) One or more synthetic HS compounds have the following structure: (In the formula, R １ Ha-SO ３ H and R ２ is —H, alkyl, aryl, substituted alkyl, or substituted aryl) or a synthetic HS compound having (ii) the one or more synthetic HS compounds include one synthetic HS compound having the structure GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP, and (In the formula, R is —H, alkyl, aryl, substituted alkyl, or substituted aryl; R １ = GlcNS6S- and n = 1; R １ = GlcA-GlcNS6S- and n = 1) or (iii) The one or more synthetic HS compounds have the structure: (In the formula, R １ is -H, and R ２ is —H, alkyl, aryl, substituted alkyl, or substituted aryl) A composition comprising a synthetic HS compound having:

2. The one or more synthetic HS compounds have the following structure: (In the formula, R １ Ha-SO ３ H and R ２ is —H, alkyl, aryl, substituted alkyl, or substituted aryl) 10. The composition of claim 1, comprising a synthetic HS compound having the formula:

3. The one or more synthetic HS compounds have the following structure: (In the formula, R １ is -H, and R ２ is —H, alkyl, aryl, substituted alkyl, or substituted aryl) 10. The composition of claim 1, comprising a synthetic HS compound having the formula:

4. wherein the one or more synthetic HS compounds include one synthetic HS compound having the structure GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP and a synthetic HS compound having the following structure: (In the formula, R is —H, alkyl, aryl, substituted alkyl, or substituted aryl; R １ = GlcNS6S- and n = 1; R １ = GlcA-GlcNS6S- and n = 1) 10. The composition of claim 1, comprising a synthetic HS compound having the formula:

5. wherein the one or more synthetic HS compounds include one synthetic HS compound having the structure GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA-pNP and a synthetic HS compound having the following structure: wherein R is —H, alkyl, aryl, substituted alkyl, or substituted aryl.

10. The composition of claim 1, comprising a synthetic HS compound having the formula:

6. The composition of any one of claims 1 to 5, wherein one of the one or more synthetic HS compounds binds to HMGB1.

7. The composition of any one of claims 1 to 6, wherein the subject in need of treatment is a mammalian subject.

8. The composition of any one of claims 1 to 7, comprising a pharmaceutically acceptable carrier or adjuvant for administration of the one or more synthetic HS compounds.

9. A composition according to any one of claims 1 to 8, comprising two or more synthetic HS compounds according to claim 1.

Images