Patent Application
20220265699
The subject matter disclosed herein relates generally to methods and compositions for treating liver ischemia reperfusion (I/R) injury. More specifically, disclosed herein are heparan sulfate oligosaccharide compounds, and methods of using the same, to treat liver ischemia reperfusion (I/R) injury.
Liver ischemia reperfusion (IR) injury is a major complication of surgery during liver transplantation and hepatic tumor resection [1]. Liver surgery often requires the use of the Pringle maneuver to reduce blood loss at the expense of potential IR injury [2]. The initial injury starts with the ischemia phase where blood flow is disrupted to the tissue resulting in a lack of oxygen and flow of nutrients. When blood flow is restored to the tissue, it reestablishes oxygen and nutrients to the ischemic tissue. However, this actually enhances the initial ischemic injury by inducing thromboinflammation which is characterized by disturbances in hemostasis and inflammation [1]. Currently, there are no approved drugs to protect against the liver damage caused by IR injury.
Thrombosis and inflammation are traditionally viewed as separate processes. However, growing evidence supports the relationship between thrombosis and inflammation stimulating and reinforcing one another which is collectively described as thromboinflammation [3]. Thromboinflammation is evident in IR injury [3], sepsis [4], and trauma [5]. Damage to the endothelium is central to thromboinflammation pathogenesis. The endothelium acts as an anti-adhesive barrier for the circulatory system by presenting proteoglycans on the cell surface. The heparan sulfate (HS) chains on these proteoglycans can bind to antithrombin III and inhibit coagulation factors FXa and thrombin. The endothelium loses this anti-adhesive and anti-coagulant barrier in thromboinflammatory conditions. Furthermore, tissue factor lies beneath the endothelium and is exposed during vessel wall injury, where it can serve as a potent activator of extrinsic coagulation pathway and subsequent thrombin generation [3]. Additionally, IR injury causes hypoxic cells to release high mobility group box 1 (HMGB1) [6]. HMGB1 has been shown to recruit neutrophils through receptor for advanced glycation end products (RAGE) activation after liver IR [7]. Neutrophil recruitment and infiltration cause further cell death by releasing proteases including myeloperoxidase (MPO) [8]. Severe thromboinflammation can extend beyond the primary affected tissue and lead to multi-organ system failure [3]. Therapeutics that lessen the degree of thromboinflammation are highly desirable to improve patient outcomes [9].
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.
Provided in accordance with the presently disclosed subject matter are methods of treating liver ischemia reperfusion (I/R) injury in a subject. In some embodiments, the method comprises providing a subject suffering from liver I/R injury or at risk of suffering liver I/R injury; and administering to the subject one or more heparan sulfate (HS) compounds. In some embodiments, the administering provides anti-inflammation and/or anti-coagulant activity in the subject. In some embodiments, the one or more HS compounds comprises about 5 to about 18 saccharide units, optionally about 12 to about 18 saccharide units. In some embodiments, the one or more HS compounds comprises about 12 saccharide units. In some embodiments, at least one of the one or more HS compounds binds HMGB1.
In some embodiments, the one or more HS compounds comprises the following formula:
wherein R1 is —NHSO3H or —NHCOCH3, R2 is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle and n is an integer of 0-6.
In some embodiments, the one or more HS compounds comprises the following structure:
wherein R1 is —SO3H or —COCH3 and R2 is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the one or more HS compounds comprises the following structure:
wherein R1 is —SO3H or —COCH3 and R2 —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the one or more HS compounds comprises the following formula
wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the one or more HS compounds comprises the following formula:
wherein R1 is —SO3H or —H and R2 is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the one or more HS compounds exists in non-anticoagulant heparin and low-molecular weight heparin and comprises one of the following structural formulas:
In some embodiments, the one or more HS compounds comprises the following formula:
wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle; and
in embodiment 1, R1=H, n=1;
in embodiment 2, R1=H, n=2;
in embodiments 3 (OSO3H) and 4 (OH),
in embodiments 5 (OSO3H) and 6 (OH),
In some embodiments, the one or more HS compounds comprises the following formula:
wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the subject in need of treatment is a mammalian subject. In some embodiments, the one or more HS compounds is administered as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a HS compound and a pharmaceutically acceptable carrier or adjuvant for administration of the HS compound.
In some embodiments, the administering comprises administering two or more HS compounds, optionally wherein the two or more HS compounds are administered separately but at the same time, optionally wherein the two or more HS compounds are administered at different times, optionally wherein the two or more HS compounds are administered in a single composition.
Provided in accordance with the presently disclosed subject matter heparan sulfate (HS) compounds. In some embodiments, one or more HS compounds are provided as compositions for use in treating liver ischemia reperfusion (I/R) injury in a subject. In some embodiments, the composition comprises: one or more heparan sulfate (HS) compound, optionally wherein the one or more HS compounds comprises about 5 to about 18 saccharide units, optionally about 12 to about 18 saccharide units, further optionally wherein the one or more HS compounds comprises about 12 saccharide units. In some embodiments, administering the composition to the subject provides anti-inflammation and/or anti-coagulant activity in the subject.
In some embodiments, the one or more HS compounds comprises the following formula:
wherein R1 is —NHSO3H or —NHCOCH3, R2 is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle and n is an integer of 0-6.
In some embodiments, the one or more HS compounds comprises the following structure:
wherein R1 is —SO3H or —COCH3 and R2 is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the one or more HS compounds comprises the following structure:
wherein R1 is —SO3H or —COCH3 and R2—H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the one or more HS compounds comprises the following formula:
wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the one or more HS compounds comprises the following formula:
wherein R1 is —SO3H or —H and R2 is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the one or more HS compounds exists in non-anticoagulant heparin and low-molecular weight heparin and comprises one of the following structural formulas:
In some embodiments, the one or more HS compounds comprises the following formula:
wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle; and
in embodiment 1, R1=H, n=1;
in embodiment 2, R1=H, n=2;
in embodiments 3 (OSO3H) and 4 (OH),
in embodiments 5 (OSO3H) and 6 (OH),
In some embodiments, the one or more HS compounds comprises the following formula:
wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, wherein the one or more HS compounds binds HMGB1. In some embodiments, the subject in need of 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 presently disclosed subject matter to provide methods and compositions for treating liver ischemia reperfusion (I/R) injury. This and other objects are achieved in whole or in part by the presently disclosed subject matter. Further, objects of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, drawings and examples.
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.
Representative images of neutrophil immunohistochemically stained liver tissue. 200× magnification. 200 μm scale bar. Pink arrows indicate stained neutrophils.
The presently disclosed subject matter now will be described more fully hereinafter, in which some, but not all embodiments of the presently disclosed subject matter are described. Indeed, the presently disclosed subject matter can be embodied in many 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 satisfy applicable legal requirements.
Heparan sulfate (HS) is structurally similar to the anticoagulant drug heparin. HS and heparin are comprise disaccharide repeating units of glucuronic acid (GlcA) or iduronic acid (IdoA) linked to glucosamine residues that carry sulfo groups [10]. Heparin has higher sulfation and more IdoA residues than HS. Clinical studies using heparin and low molecular weight heparin (LMWH) to treat thromboinflammatory diseases like sepsis and IR are inconclusive on its effect [11,12]. Heparin and its derivatives are complex, structurally uncharacterized oligosaccharide mixtures containing variations in chain length and chemical modifications. The structural heterogeneity makes it difficult to define the relationship of oligosaccharide structure to its biological function. Furthermore, the lack of structurally homogeneous HS oligosaccharides hampers the efforts to exploit the characteristics of HS for use as a therapeutic agent [13]. To address this issue, a chemoenzymatic method to synthesize structurally specific HS oligosaccharides with high efficiency has been developed [14-16]. In accordance with particular aspects of the presently disclosed subject matter, it is demonstrated that certain oligosaccharides that possess anticoagulant activity and anti-inflammation activity (e.g., bind to HMGB1) are more effective in reducing IR-mediated liver injury compared to oligosaccharide that only bind to HMGB1 or only have anticoagulant activity. By using synthetic HS oligosaccharides, rather than heparin or LMWH, the hepatoprotective effect of oligosaccharides can be distinguished.
HS is a sulfated glycosaminoglycan abundant on the cell surface and in the extracellular matrix and has several biological activities including anticoagulation and anti-inflammation. Liver ischemia reperfusion injury is associated with coagulation and inflammatory responses. In accordance with the presently disclosed subject matter, HS oligosaccharides with defined sulfation patterns were synthesized and it was shown that synthetic anticoagulant HS oligosaccharides limit liver ischemia reperfusion injury in a mouse model. Using a small targeted HS library, in accordance with particular aspects of the presently disclosed subject matter, it was demonstrated that an oligosaccharide that possesses both anticoagulant activity and binding affinity to HMGB1, an inflammatory target, decreases injury greater than oligosaccharides that only bind to HMGB1 or only have anticoagulant activity. The presently disclosed HS oligosaccharides provides a new therapeutic option for decreasing liver damage resulting from ischemia reperfusion injury.
By way of elaboration and not limitation, in some embodiments of the presently disclosed subject matter, a hepatoprotection effect from an HS compound disclosed herein involves consideration of size and/or sulfation pattern. In one non-limiting example, a 12-mer oligosaccharide having a sulfation pattern providing anticoagulant activity and anti-inflammation activity provides a hepatoprotection effect. In another non-limiting example, a 12-mer oligosaccharide having a sulfation pattern providing an anti-inflammation activity provides a hepatoprotection effect when administered with a 6-mer having a sulfation pattern anticoagulant activity. In another non-limiting example, an 18-mer HS oligosaccharide having a sulfation pattern that nonanticoagulant and that provides anti-inflammation activity provides hepatoprotection.
In some embodiments of the presently disclosed subject matter, it is shown that a heparan sulfate (HS) 12-mer that can reduce the liver damage caused by ischemia reperfusion injury. The presently disclosed subject matter reduces inflammation responses that damage hepatocytes for liver transplantation and surgery. In some embodiments, the HS compound comprises about 5 to about 18 saccharide units, including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 saccharide units. Representative HS compounds are also disclosed in the Figures. 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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. As used herein the term “alkyl” refers to C1-20 inclusive, linear (i.e., “straight-chain”), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.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 (i.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.
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.
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.
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.
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.
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.
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.
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.
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.
“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 (—C6H10—); —CH═CH—CH═CH—; —CH═CH—CH2—; —(CH2)q—N(R)—(CH2)r—, 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.
“Arylene” refers to a bivalent aryl group.
“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.
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 subcombinations of A, B, C, and D.
The presently disclosed subject matter provides HS compounds, and compositions comprising the same, including pharmaceutical and/or therapeutic compositions comprising a HS compound, as disclosed herein. In some embodiments, a pharmaceutical composition can comprise one or more HS compounds, as disclosed herein. Methods of treating subjects with the HS compounds are also disclosed.
In some embodiments, the presently disclosed subject matter provides a heparan sulfate (HS) compound. In some embodiments, the presently disclosed subject matter provides a composition, such as a pharmaceutical composition, comprising one or more HS compounds as disclosed herein. Thus, in some embodiments, the HS compound is 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 liver ischemia reperfusion (I/R) injury in a subject is provided. In some embodiments, the method comprises providing a subject suffering from liver I/R injury or at risk of suffering liver I/R injury; and administering to the subject one or more HS compounds as disclosed herein. In some embodiments, the one or more HS compounds have sulfation patterns and/or size based on saccharide units that provides anti-inflammation and/or anti-coagulant activity administering the composition to the subject provides anti-inflammation and/or anti-coagulant activity in the subject.
In some embodiments, the HS compound comprises about 5 to about 18 saccharide units, optionally about 12 to about 18 saccharide units. In some embodiments, the HS compound comprises about 12 saccharide units. In some embodiments, the HS compound binds HMGB1. In some embodiments, the HS compound is 12-mer-1, 12-mer-2, 12-mer-3, or 12-mer-4, shown schematically in
By way of elaboration and not limitation, in some embodiments of the presently disclosed subject matter, a hepatoprotection effect from an HS compound disclosed herein involves consideration of size and/or sulfation pattern. In one non-limiting example, a 12-mer oligosaccharide having a sulfation pattern that provides anticoagulant activity and anti-inflammation activity provides a hepatoprotection effect. In some embodiments, an 11-mer or a 13-mer having the same or similar sulfation pattern should behave similarly. In another non-limiting example, a 12-mer oligosaccharide having a sulfation pattern that provides an anti-inflammation activity provides a hepatoprotection effect when administered with a 6-mer having a sulfation pattern that provides anticoagulant activity. Here as well, an 11-mer or a 13-mer having the same or similar sulfation pattern should behave similarly. In another non-limiting example, an HS compound on the larger end of the range of sizes disclosed herein, such as an 18-mer HS oligosaccharide having a sulfation pattern that is nonanticoagulant and that provides anti-inflammation activity provides hepatoprotection.
The presently disclosed subject matter reduces inflammation responses that damage hepatocytes for liver transplantation and surgery. In some embodiments, the HS compound comprises about 5 to about 18 saccharide units, including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 saccharide units. Representative HS compounds are also disclosed in the Figures. 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.
In some embodiments, it is demonstrated that both anti-inflammation (such as via HMGB1 inhibition) and anti-coagulant activity provides protection in an IR model. These activities can come from one molecule (for example, the HS compound referred to herein as 12-mer-1 or as 12-mer AXa) or from the combination dose of two molecules (for example, the HS compound referred to herein as 6-mer-AXa (shown schematically herein in
In some embodiments, when two or more HS compounds are administered, the two or more HS compounds are administered separately but at the same time. In some embodiments, the two or more HS compounds are administered at different times but sufficiently proximate to each other to have a desired therapeutic effect. In some embodiments, the two or more HS compounds are administered in a single composition or formulation.
In some embodiments, the HS compound comprises the following formula:
wherein R1 is —NHSO3H or —NHCOCH3, R2 is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle and n is an integer of 0-6.
In some embodiments, the HS compound comprises the following structure:
wherein R1 is —SO3H or —COCH3 and R2 is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the HS compound comprises the following structure:
wherein R1 is —SO3H or —COCH3 and R2 —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the HS compound comprises the following formula:
wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the HS compound comprises the following formula:
wherein R1 is —SO3H or —H and R2 is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
In some embodiments, the HS compound is a HS molecule that exists in non-anticoagulant heparin and low-molecular weight heparin, and comprises one of the following structural formulas:
In some embodiments, the presently disclosed subject matter provides a HS compound, comprising a 5-mer, a 6-mer or a 7-mer. In one example, the 6-mer is the HS compound referred to herein as 6-mer-AXa (shown schematically herein in
wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle; and wherein R1 and n are defined as follows:
in embodiment 1, R1=H, n=1;
in embodiment 2, R1=H, n=2;
in embodiments 3 (OSO3H) and 4 (OH),
in embodiments 5 (OSO3H) and 6 (OH),
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 the HS compound referred to herein as 6-mer-AXa (shown schematically herein in
wherein R is —H, alkyl, aryl, substituted alkyl, substituted aryl, or a functional handle.
Referring to
Referring to
In some embodiments a pharmaceutical composition can also contain a pharmaceutically acceptable carrier or adjuvant. 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.
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.
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.
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 unit-dose 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.
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.
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.
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 symptoms of liver ischemia reperfusion (I/R) 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.
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.
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.
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 liver ischemia reperfusion (I/R) conditions is desirable, particularly agricultural and domestic mammalian species.
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.
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, 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 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.
Liver ischemia reperfusion (IR) injury is a major complication of surgery during liver transplantation and hepatic tumor resection. Liver surgery often requires the use of the Pringle maneuver to reduce blood loss at the expense of potential IR injury. The initial injury starts with the ischemia phase where blood flow is disrupted to the tissue resulting in a lack of oxygen and flow of nutrients. Thus, subjects undergoing surgery during liver transplantation are representative subjects to be treated. However, any suitable subject as would be apparent to one of ordinary skill in the art upon a review of the instant disclosure can be treated, such as a subject suffering from liver I/R injury or at risk of suffering liver I/R injury.
The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.
The synthesis of the 12-mers and 6-mer-AXa has been previously described [15]. See also
The oligosaccharides were converted to biotinylated versions using the same method as described in PCT International Patent Application Serial No. PCT/US2018/059152. Briefly, 12-mers and 6-mer-AXa with a pNP tag (5-10 mg) and 0.5 mg Pd/C were dissolved in 20 mM NaOAc, pH 5.0 in a total volume of 4 ml. Reaction mixture was vacuumed and refilled with H2 three times. The reaction was then incubated at room temperature for 4 h. After that, it was filtered to remove charcoal. The filtered solution was adjusted to pH 8.5 using 500 mM Na2HPO4. Succinimidyl 6-azidohexanoate (20 molar equivalent of starting oligosaccharides) was added and incubated at 37° C. overnight. Reaction was purified by DEAE-HPLC column to generate azido tagged oligosaccharides. PBS (pH7.4) buffer was bubbled with N2 for 5 min to prepare the sample solution of 0.1 M CuSO4, 0.1 M Tris(3-hydroxypropyl-triazolylmethyl)amine (THPTA)(Sigma), 0.15 M sodium ascorbate, 0.01 M azido tagged oligosaccharides and 0.02 M biotin-PEG4-alkyne (Sigma). The mixture of 400 THPTA and 80 μl CuSO4 was vortexed, then 160 μl sodium ascorbate, 200 μl azido tagged oligomers and 200 μl biotin-PEG4-alkyne was added and bubbled with N2 for 2 min, then incubated at 37° C. overnight. The reaction was purified by DEAE -HPLC column to generate biotinylated products. The biotinylated 12-mers and 6-mer-AXa products were confirmed by ESI-MS.
Affinity Purification of HMGB1 from Liver Lysate
6-mer-AXa and 12-mer biotinylated oligosaccharides were prepared using to affinity purify HMGB1 from liver lysate following the method described in PCT International Patent Application Serial No. PCT/US2018/059152. Briefly, liver lysate was prepared by snap freezing tissue in liquid nitrogen at the time of sacrifice. The tissue was mechanically homogenized in buffer containing 200 mM MES, 500 mM phosphate, and 1 mM EDTA at pH 6 followed by three rounds of freeze thawing. The lysed sample were centrifuged at 10,000× g for 15 min 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 purification and biotinylated HS bound complex where achieved using avidin-Sepharose and increasing concentration of NaCl washes. The elution of each sample was separated by gel electrophoresis, transferred to nitrocellulose membrane, and blotted for HMGB1 using anti-HMGB1 primary antibody (Abcam) followed by anti-rabbit HRP (Abcam).
Assays were based on a previously published method [16]. Briefly, human FXa (Enzyme Research Laboratories) was diluted to 50 U m1−1 with PBS. The chromogenic substrate S-2765 (Diapharma) was diluted to 1 mg ml−1 in water. For in vitro studies, fondaparinux (available under the trade name ARIXTRA) and 12-mer oligosaccharides were dissolved in PBS at various concentrations (0-131 nM). 16 μl of sample was incubated with 60 μl of 35 μl ml−1 antithrombin (Cutter Biologics) for 2 min at room temperature. Next, 100 μl of FXa was added and incubated for 4 min 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 min. PBS serves as a control sample. The maximum slope for each sample was convert to percent FXa activity by dividing by the maximum slope for the control sample.
For ex vivo studies, mouse plasma collected after the 6 hour reperfusion period and assayed the same as described above.
Liver ischemia-reperfusion (IR) surgery was performed by the Animal Surgery Core Laboratory of the McAllister Heart Institute, University of North Carolina Chapel Hill, N.C. The mouse experiments were approved by the UNC Animal Care and Use Committees and complied with National Institutes of Health guidelines. Male C57BL/6J mice, approximately 8 weeks old, were used in the IR surgeries. Mice received a subcutaneous (SC) injection of 1 mg/kg oligosaccharide or the equivalent volume of saline 30 minutes prior to the surgical procedure. For the combination treatment of 12-mer-3+6-mer-AXa, equal concentration of each oligosaccharide was combined into a single solution. Under ketamine/xylazine anesthesia, an abdominal midline incision was made to expose the portal vein. A clamp was placed on the portal vein and bile duct to three major liver lobes to cause a 70% hepatic ischemia. Visible blanching of the ischemic liver lobes confirmed correct placement of the clamp. A temporary stitch closure of the muscle and skin over the clamp was used to prevent dehydration during the ischemia phase. Mice stayed on a heating pad and under anesthesia during the ischemia phase (60 minutes). The clamp was removed after 60 minutes and the ischemia liver lobes regained their red color as blood began to reperfusion the tissue, an indicator of correct reperfusion occurring. The incisions were closed in two layers with 5-0 silk sutures and the mice returned to their active state (no anesthesia used during the reperfusion phase). After 6 hours, the mice were re-anaesthetized, blood was drawn via cardiac puncture and ischemia liver lobes were harvested for histology (fixed in 10% formalin).
Plasma ALT was measured using the ALT Infinity reagent (Thermo Fisher) following the manufacturer's instructions. Plasma TNF-a was measured using Mouse TNF-α 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. Plasma syndecan-1 levels were determined using Mouse Syndecan-1 ELISA (CellSciences) according to manufacturer's instructions. Plasms IL-6 levels were determined using Mouse IL-6 ELISA (R&D Systems).
Ischemia liver tissues were fixed in 10% neutral buffered formalin for 24 hours at room temperature, paraffin-embedded, and sectioned. Liver sections (4 μm) were stained with hematoxylin-eosin (H&E) or immunostained with monoclonal antibodies 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 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 ImageJ. For neutrophil quantitation, five 100× images were randomly selected for each sample and the average neutrophils/field were reported.
Ischemia liver lobe was mechanically homogenized in 50 mM CTAB 50 mM potassium phosphate pH 6 at a ratio of 100 μl buffer per 10 mg tissue. Samples were centrifuged at 15,000×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 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 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.
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., graphpad.com/scientific-software/prism/)
A partial liver IR injury mouse model was used to evaluate the in vivo efficacy of the oligosaccharides. In this model, a clamp was used to induced ischemia in 70% of the liver (
HMGB1 has been implicated in the damaging inflammation response following liver IR [17,19]. In a recent report, HMGB1 binding to HS oligosaccharides of specific residue-repeat lengths was explored [20]. In this Example a panel of 12-mers that vary in sulfation degrees and 2-O-sulfo iduronic acid residues was tested. The access to these 12-mers allowed for the further dissection of the effect of sulfations or 2-O-sulfo iduronic acid residues on HMGB1 binding (
Referring to
Referring to
Based on the ability to bind to HMGB1, 12-mer-1 and 12-mer-3 were used in the in vivo model of liver IR. The compounds were administered 30 minutes prior to ischemia. Anticoagulant activity of 12-mer-1 was confirmed in the plasma (
In liver IR, neutrophils are rapidly recruited during the reperfusion phase to the post-ischemic tissue [8]. After neutrophils migrate into the liver, they release cytotoxic compounds including reactive oxygen species and proteases to clear damaged tissue [21]. Neutrophils and their potent cargo are key effectors in sterile inflammation due to the lack of specificity for damage vs healthy tissue. As a result, neutrophil recruitment continues and perpetuates inflammation.
Neutrophil-derived proteases including elastase, MMP-9, cathepsin G, proteinase-3, and myeloperoxidase (MPO) are reportedly implicated 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 oxidative stress in the tissue by reacting with hydrogen peroxide [8]. MPO activity was measured in the ischemic liver lysate (
Next, whether hepatoprotection requires anticoagulant activity was investigated. To accomplish this, a 6-mer-AXa oligosaccharide (
Unlike acetaminophen-induced liver injury, liver IR is reported to involve coagulation disturbances in addition to inflammation and thus described as thromboinflammation [3]. Anticoagulant HS oligosaccharides were ineffective in acetaminophen-induced liver injury [20]; however, since liver IR involves thromboinflammation anticoagulant 12-mer-1 was included in this study to explore how anticoagulant activity and HMGB1 binding effected hepatoprotection in liver IR injury. In doing so, it is demonstrated that synthetic HS oligosaccharides are a therapeutic in another disease model.
In EXAMPLES 1-5, the structure-activity relationship of HS oligosaccharides for HMGB1 binding was explored by screening a panel of 12-mer oligosaccharides with various sulfation patterns. 12-mer-1 and 12-mer-3, which are both highly sulfated oligosaccharides, were the only successful compounds to pull down HMGB1 from liver lysates. However, in vivo it was observed that 12-mer-1 but not 12-mer-3 decreased ALT and necrosis in the ischemic liver lobe. 12-mer-l′s anti-inflammatory activity is associated with the ability to bind to HMGB1, decrease tissue MPO, and decrease neutrophil accumulation in ischemic liver lobe. Interestingly, binding the HMGB1 is not sufficient for hepatoprotection as demonstrated by 12-mer-3 in vivo. It was shown that both anticoagulation and anti-inflammatory activity play a role to achieve hepatoprotection by using 6-mer-AXa in combination with 12-mer-3 or alone in the IR model. In this way, anticoagulation in the absence or presence of HMGB1 binding was examined Treatment with 6-mer-AXa alone did not decrease the concentration of ALT that has statistical significance, however the combination treatment did. While it is not desired to be bound by any particular theory of operation, it appears that the 12-mer-1's hepatoprotective effect can be attributed to the dual activities of anticoagulation and anti-inflammation as both mechanisms are essential in the pathophysiology of liver IR.
EXAMPLES 1-5 demonstrate that 2-mer-1 is an active anti-inflammatory agent as well as an anticoagulant. In addition to 12-mer-1's protective mechanism in liver IR, 12-mer-1 also has several favorable drug-like properties. Renal clearance of 12-mer-1 is considered since most liver transplant patients also have impaired renal function [23]. The renal clearance impairment of 12-mer-1 was demonstrated using a kidney IR model [16]. However, 12-mer-1 is amenable to dose adjustments since it is a homogeneous compound with uniform anticoagulant activity and potentially a safe option for renally impaired patients. Bleeding issues have been reported, with 9% of liver transplant recipients on heparin therapy requiring surgical intervention for bleeding complications [24]. LMWH lowers the bleeding risk but it is incompletely reversed by protamine [16]. 12-mer-1 anticoagulant activity is reversible by protamine, which adds an additional benefit to ameliorate bleeding complications [16]. Thus, controlling the dose and having the potential of reversibility by protamine are aspects of 12-mer-1. Additionally, 12-mer-1 displayed no toxicity in a rat model at elevated doses [20]. Therefore, the anti-thromboinflammatory properties, reversibility by protamine, lack of toxicity and ability to precisely control the dose make 12-mer-1 an appealing therapeutic for liver transplant/IR patients. Pharmacokinetic studies investigate the relationship of 12-mer-1 dose and response against liver IR injury.
Heparin and de-sulfated heparin bind to P-selectin [J. Wang; Geng, J., Thromb Haemost 90, 7 (2003)], a tethering molecule for neutrophils in most organs and tissues. Interestingly, neutrophil recruitment to the liver has several differences compared to the classical model. For example, there is little evidence for the requirement of selectin or β2-integrin mediated adhesion for neutrophil migration to the liver [8]. Rather, neutrophils are physically trapped in the liver sinusoids where nearly 80% of leukocyte trafficking takes place [S. L. Maas, O. Soehnlein, J. R. Viola, Frontiers in Immunology 9, (2018)]. Therefore, without wishing to be bound by any particular theory of operation, it appears that the decrease of neutrophil accumulation after treatment with 12-mer AXa is not due to selectin inhibition.
Dalteparin, a low molecular weight heparin, decreases liver IR injury in rats [22]. Interestingly, in this study they observed no protection when using a selective factor Xa inhibitor, DX9065a, suggesting that dalteparin's protective effect is not solely due to anticoagulation. Dalteparin decreased MPO levels suggesting an effect on neutrophil recruitment. However, due to the incomplete structural characterization of dalteparin, further biochemical analysis is very difficult if not impossible. The chemoenzymatic synthesis technology generates heparin oligosaccharides with or without anticoagulant activity. As heparin is becoming increasingly recognized for its anti-inflammatory properties [25], the presently disclosed subject matter helps to solve the issues of using a heterogenous mixture of oligosaccharides for characterization of biological effects.
The chemoenzymatic synthesis technology generates structurally defined HS oligosaccharides. As heparin is becoming increasingly recognized for its anti-inflammatory properties [25], the presently disclosed subject matter contributes to the transformation of the therapeutic field from one using heterogeneous mixtures of oligosaccharides to a new class of homogeneous, precision-based oligosaccharide therapeutics.
Background:
18-mer with repeating units of N-sulfo glucosamine and 2-O-sulfo iduronic acid was used in the liver ischemia reperfusion (IR) mouse model (
IR Procedure:
1 mg/kg of 18-mer or equal volume of sterile saline was administered by subcutaneous injection 30 minutes prior to start of the ischemia phase. Ischemia to 70% of the liver was caused by clamping the portal vein and bile duct for 60 minutes. After the clamp was removed, the reperfusion phase last 6 hours before the animals were sacrificed for collection of liver tissue and blood. For surgical controls, a sham operation was performed such that the animal experienced the same anesthesia, abdominal midline incision, and suturing as the IR mice (
Results:
Treatment with 18-mer decreased liver injury after IR. Plasma alanine aminotransferase (ALT) is a marker of liver injury. 18-mer treatment reduced ALT levels compared to the IR group (
For histological evidence of 18-mer's protection, the ischemia liver lobes were stained with H&E for quantification of necrotic area. 18-mer significantly decreased the necrotic area compared to IR (
In addition to decreasing necrotic area, 18-mer also had a profound effect on many inflammatory mediators (
Conclusion:
18-mer decreases liver IR injury possibly by inhibiting HMGB1-mediated neutrophil infiltration and attenuating sterile inflammation. It is likely that other compounds with a similar structure, including 16-mer with the same repeating disaccharide units of N-sulfo glucosamine and 2-O-sulfo iduronic acid, will also be effective in vivo. Additionally, the compounds can be modified to have N-, 6-O-sulfo glucosamine and glucuronic acid residues, which also may be effective in vivo. Also, the compounds can be functionalized with different chemical handles to studying binding in vitro and further elucidate the protective mechanism in liver IR (
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.
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.
This application is a continuation of PCT International Patent Application No. PCT/US2020/060581, filed on Nov. 13, 2020, herein incorporated by reference in its entirety, which claims benefit of U.S. Provisional Patent Application Ser. No. 62/934,845, filed Nov. 13, 2019, herein incorporated by reference in its entirety.
This invention was made with government support under Grant Numbers HL094463, HL144970, GM128484, and HL142604 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62934845 | Nov 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2020/060581 | Nov 2020 | US |
| Child | 17744407 | US |
