WO2013101314A1 - Composition of novel carbohydrate drug for treatment of human diseases - Google Patents
Composition of novel carbohydrate drug for treatment of human diseases Download PDFInfo
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- WO2013101314A1 WO2013101314A1 PCT/US2012/055311 US2012055311W WO2013101314A1 WO 2013101314 A1 WO2013101314 A1 WO 2013101314A1 US 2012055311 W US2012055311 W US 2012055311W WO 2013101314 A1 WO2013101314 A1 WO 2013101314A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/738—Cross-linked polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0045—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Galacturonans, e.g. methyl ester of (alpha-1,4)-linked D-galacturonic acid units, i.e. pectin, or hydrolysis product of methyl ester of alpha-1,4-linked D-galacturonic acid units, i.e. pectinic acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
Definitions
- aspects of the invention relate to a pharmaceutical-grade polysaccharide, pharmaceutical compositions thereof and to methods of manufacturing the same.
- Other aspects of the invention relate to methods of treating disease conditions, such as medical conditions related to inflammation and fibrosis and cancer linked at least in part to aberrant or increased expression of galectin proteins, using these compounds and compositions.
- modified pectins and method of manufacturing modified pectins for use as parenteral or enteral medicinal compounds. Moreover, there is a need to for these compounds to have the required pharmacological properties to inhibit inflammation and fibrosis while remaining nontoxic to other cells and tissues.
- aspects of the invention relate to a compound or composition comprising a compound in an acceptable pharmaceutical carrier for parenteral or enteral administration, for use in therapeutic formulations.
- the compound is a polysaccharide and may be chemically defined as arabinogalacto-rhamnogalacturonan (herein referred to as Compound G), a selectively depolymerized, branched heteropolymer whose backbone is predominantly comprised of 1 ,4-linked galacturonic acid (GalA) and methyl galacturonate (MeGalA) residues, with a lesser backbone composition of alternating 1 ,4-linked GalA and 1 ,2-linked rhamnose (Rha), which in-turn is linked to any number of oligomer side chains, including predominantly 1 ,4- -D-galactose (Gal), 1 ,5-a-L- arabinose (Ara) residues or combination thereof.
- Other side chain minor constituents may include xylose (Xyl), glucose (Glu), fucose (Fuc) or any combination of the foregoing.
- the arabinogalacto-rhamnogalacturonan compound of the present invention is capable of reducing the secretion of TNF-alpha cytokine from monocytes stressed with endotoxin. In some embodiments, the compound G is capable of reducing the secretion of TNF alpha by activated macrophages by at least 25%.
- the 1 ,4-linked galacturonic acid and methyl galacturonate residues backbone can represent between 55 to 85 molar percent of the total carbohydrate molar content
- the branched heteropolymer of alternating a -1 ,2 linked rhamnose and a -1 ,4-linked GalA residues can represent between 1 and 6 molar percent of the total carbohydrate molar content
- the oligomer 1 ,4- ⁇ -D-galactose of the primary branching can represent between 6 to 15 molar percent of the total carbohydrate molar content
- the oligomer 1 ,5- a -L-arabinose of the primary branching can represent between 2 to 8 molar percent of the total carbohydrate molar content, as characterized by gas chromatography/mass spectrometry.
- the oligomer of 1 ,4- ⁇ -D-galactose residues, 1 ,5- ⁇ -L-arabinose residues or combinations thereof represents at least 8 molar percent of the total carbohydrate molar content.
- the 1 ,4- ⁇ -D-galactose and 1 ,5- a -L-arabinose residues are present in a 2:1 or a 3:1 ratio in the arabinogalacto-rhamnogalacturonan compound of the present invention.
- the compound of the present invention has a degree of methoxylation ranging from 40% to 70% of the maximum of 87%.
- the compound of the present invention has a methyl galacturonate to galacturonic acid ratio ranging from 2:1 to 1 :2.
- the compound of the present invention has a methyl galacturonate plus galacturonic acid ratio to galactose ranging from 4:1 to 7:1 .
- the molar percent of the 1 ,4- -D-Gal and 1 ,5-a-L- Ara residues in the compound of the present invention can exceed 10 % of the total molar carbohydrates with approximate ratio ranging from 1 :1 to 3:1 respectively.
- the compound is a polysaccharide chemically defined as galactoarabino-rhamnogalacturonate (Compound G), a branched heteropolymer with average molecular weight distribution of 2,000 to 80,000, or 20,000 to 70,000, or 5,000 to 55,000 Daltons, as determined by SEC-RI and/or the SEC- MALLS method.
- Compound G galactoarabino-rhamnogalacturonate
- the compound can be a highly soluble modified polysaccharide sufficiently reduced in average molecular weight, for example from about 2,000 to about 80,000 D, so as to be compatible with therapeutic formulations for pluralistic administration via routes including but not limited to intravenous, subcutaneous, intra-articular, inhaled, and oral.
- the compound can be substantially free of microbial endotoxin, agricultural pesticides, agricultural herbicides, copper, heavy metals, proteins, nitrogenous compounds or any combination of the foregoing.
- the compound can be synthesized from natural, highly branched, minimally processed and high methoxylated USP pectin which may come from any plant source, including but not limited to, citrus fruits, apple, or beet.
- the compound can be synthesized from natural, highly branched, minimally processed and high methoxylated USP pectin like one manufactured from apple pomace containing 8-12% pectin.
- the compound can be synthesized under a sufficiently controlled and specific hydrolysis of the glycosidic-linked methoxylated a - 1 ,4-linked GalA while preserving the side-chains with enriched amounts of 1 ,4- ⁇ -D- Gal and 1 ,5-a-L-Ara.
- Amounts of 1 ,4- ⁇ -D-Gal and 1 ,5-a-L-Ara can be quantitatively determined by GC-MS (Gas chromatography-mass spectroscopy) and AELC-PAD (anion exchange liquid chromatography-pulsed amperometric detector) methods.
- the compound can be produced by a process comprising depolymerization catabolized by targeted peroxidation cleavage of glycosidic bonds by ionized OH sup- generated from ascorbic acid and/or peroxide in presence or absence of additional reduced form of a transition metal ion, like Cu sup.++. at 1 to 100 mM.
- a transition metal ion like Cu sup.++. at 1 to 100 mM.
- Other transition metals like Ca. sup.++ or Fe.sup.++ can also be used for this purpose.
- the depolymerized compound can be exposed to pH of between 8 to10 for 10 to 30 minutes at temperature of 2 to 60°C to initiate controlled limited demethoxylation to generate a depolymerized compound with a degree of methoxylation of 40 to 70 percent in comparison to initial levels of maximum 87% and can be referred to as middle-methoxylated compound. Complete methoxylation of galacturonic acid is considered to be approximately DE 87%.
- the depolymerized composition can be exposed to multiple washes of hot acidic alcohol (e.g at temperatures ranging from 30 to 80°C) to remove any residual endotoxin, copper and heavy metals, agricultural contaminates and other impurities.
- the compound does not induce decreased viability when used to treat LX2 immortalized human hepatic stellate cells.
- the compound is capable of reducing expression of galectin-3 at the cell surface or substantial decrease in secretion of galectin-3 in the media when used to treat stressed LX2 immortalized human hepatic stellate cells producing galectin-3.
- aspects of the invention relate to an arabinogalacto-rhamnogalcturonan compound
- the compound does not inhibit cancer cell proliferation in a cancer cell and is not cytotoxic to monocytes or activated monocytes at concentrations up to 500 g/mL.
- the 1 ,4-linked galacturonic acid and the methyl galacturonate residues backbone can represent between 55 to 85 molar percent of the total carbohydrate molar content
- the branched heteropolymer of alternating a -1 ,2 linked rhamnose and a -1 ,4-linked GalA residues can represent between 1 and 3 molar percent of the total carbohydrate molar content
- the oligomer 1 ,4- ⁇ -D-galactose of the primary branching can represent between 6 to 15 molar percent of the total carbohydrate molar content
- the oligomer 1 ,5- a -L-arabinose of the primary branching can represent between 2 to 8 molar percent of the total carbohydrate molar content, as characterized by gas chromatography/mass spectrometry.
- the 1 ,4- ⁇ -D-galactose residues, 1 ,5- a -L- arabinose residues or combination thereof can represent at least 8 molar percent of the total carbohydrate molar content.
- the 1 ,4- ⁇ -D-galactose and 1 ,5- a -L-arabinose residues can be present in a 2:1 ratio.
- the compound can have an average molecular weight ranging from 5 kDa to 55 kDa or ranging from 2 kDa to 70 kDa.
- the compound can have a degree of methoxylation ranging from 40% to 70% out of maximum 87%.
- the compound can have a methyl galacturonate to galacturonic acid ratio ranging from 2:1 to 1 :2.
- the compound can have a methyl galacturonate plus galacturonic acid ratio to galactose ranging from 4:1 to 8:1 .
- the compound does not induce decreased viability when used to treat LX2 immortalized human hepatic stellate cells.
- the compound is capable of inducing substantial decrease in expression of galectin-3 at the cell surface or substantial decrease in secretion of galectin-3 in the media when used to treat stressed LX2 immortalized human hepatic stellate cells producing galectin-3.
- compositions comprising a compound in an acceptable pharmaceutical carrier for use in therapeutic formulations.
- the composition can be administered parenterally via an intravenous, subcutaneous, or oral route.
- the composition can further comprise a therapeutic agent.
- the therapeutic agent can be an anti-oxidant compound, an anti-inflammatory agent, vitamins, a neutraceutical supplement or combinations thereof.
- the composition can be used in the treatment of nonalcoholic steatohepatitis, fibrosis, inflammatory and autoimmune disorders, neoplastic conditions or of cancer.
- the composition can be used in the treatment of liver fibrosis, kidney fibrosis, lung fibrosis, or heart fibrosis.
- the invention relates to a composition or a compound utilized in treating or a method of treating inflammatory and fibrotic disorders in which galectins are at least in part involved in the pathogenesis, including but not limited to enhanced anti-fibrosis activity in organs, including but not limited to liver, kidney, lung, and heart.
- the invention relates to a composition or a compound that has therapeutic activity or a method to reduce the pathology and disease activity associated with nonalcoholic steatohepatitis (NASH) including but not limited to steatosis (fat accumulation in hepatocytes), ballooning degeneration of hepatocytes, inflammatory infiltrate in the liver, and deposition of collagen or fibrosis.
- NASH nonalcoholic steatohepatitis
- the invention relates to a composition or a compound utilized in treating or a method of treating inflammatory and autoimmune disorders in which galectins are at least in part involved in the pathogenesis including but not limited to arthritis, rheumatoid arthritis, asthma, and inflammatory bowel disease.
- the invention relates to a composition or a compound utilized in treating or a method of treating neoplastic conditions (e.g. cancers) in which galectins are at least in part involved in the pathogenesis by inhibiting processes promoted by the increase in galectins, including but not limited to tumor cell invasion, metastasis, and neovascularization.
- neoplastic conditions e.g. cancers
- galectins are at least in part involved in the pathogenesis by inhibiting processes promoted by the increase in galectins, including but not limited to tumor cell invasion, metastasis, and neovascularization.
- the invention relates to a composition or a compound utilized in enhancing or a method for enhancing the ability of tumor infiltrating T-cells, which are inhibited at least in part by the effect of tumor derived galectin proteins, to more effectively identify and kill tumor cells and thereby slow, stop or reverse the progression of tumors.
- a therapeutically effective amount of the depolymerized compound or of the composition can be compatible and effective in combination with a therapeutically effective amount of various anti-inflammatory drugs, vitamins, other pharmaceuticals and nutraceuticals drugs, without limitation.
- a therapeutically effective amount of the depolymerized compound or of the composition can be compatible and effective in combination with a therapeutically effective amount of various anti-oxidant compounds such as glycyrrhizin, ascorbic acid, L-glutathione, cysteamine and the like or combinations thereof.
- a therapeutically effective amount of the compound or of the composition can be non-toxic and does not induce apoptosis in cultured cell lines when added to cell culture media including but not limited to the cell lines LX-2, or other stellate cells.
- a therapeutically effective amount of the compound or of the composition is not cytotoxic to mammalian cultured cells when added to cell culture media including but not limited to B16-F10 melanoma cells, THP- 1 monocyte/macrophage cells, primary peripheral blood mononuclear cells (PBMC )and MRC-5 lung fibroblast cells.
- cell culture media including but not limited to B16-F10 melanoma cells, THP- 1 monocyte/macrophage cells, primary peripheral blood mononuclear cells (PBMC )and MRC-5 lung fibroblast cells.
- a therapeutically effective amount of the compound or of the composition can have an anti-inflammatory effect on cell lines as measured by production of pro-inflammatory cytokines including but not limited to TNF- alpha.
- efficacy of the compound or of the composition for treatment of liver fibrosis can be determined by administering the compound or composition to animal models of fibrosis including but not limited to rats injected intraperitoneally with the chemical toxin thioacetamide, resulting in at least 5% to 25% reduction in liver collagen content as determined by morphometric quantification.
- efficacy of the composition for treatment of NASH can be determined by administering the compound or composition to animal models of NASH including but not limited to mice rendered diabetic and fed a high fat diet, resulting in at least 5% reduction in hepatocellular fat accumulation, at least a 5% reduction in the number of hepatocytes with ballooning degeneration, at least 5% reduction in liver infiltration of inflammatory cells, and at least a 5% reduction in liver collagen content as determined by morphometric quantification (assessed by staining positive for Sirius red).
- administering a therapeutically effective amount of the depolymerized compound or of the composition to an animal model of NASH can result in reduction of fibrosis as measured by standard histopathology of biopsies, reduction in disease activity by NAFLD grading, a decrease in the number of cells expressing alpha Smooth Muscle Actin, or a decrease in other inflammatory mediators or a decrease in lipid trafficking and metabolism enzymes including but not limited to CD36.
- administering a therapeutically effective amount of the depolymerized compound or of the composition can result in reduction of galectin-3 as measured by either level of mRNA or the expression of the galactose binding protein.
- administering a therapeutically effective amount of the depolymerized compound or of the composition can result in reduced growth, invasion, metastasis, or increased sensitivity to the innate immune system of syngeneic or xenotopic tumors in animals.
- FIGS. 1 shows a schematic of a manufacturing process flow chart that may be utilized in one embodiment to manufacture Compound G, the active pharmaceutical ingredient.
- FIG. 2 shows an illustration of the molecular structure of Compound G according to some embodiments.
- FIG. 3a shows a Zimm plot calculation of average molecular weight calculated for the selectively depolymerized compound of the instant invention (Compound G) as determined by Multi-Angel Laser Scattering Analyzer (MALS) method.
- FIG. 3b shows a Zimm plot calculation of dn/dc parameters calculated for the selectively depolymerized compound (Compound G) of the instant invention in a 10 nM EDTA buffered solution as determined by Multi-Angel Laser Scattering Analyzer (MALS) method.
- MALS Multi-Angel Laser Scattering Analyzer
- FIG. 4a shows the determination of molecular weight distribution of the selectively depolymerized compound (Compound G) of the instant invention using a size exclusion chromatography (SEC) method.
- SEC size exclusion chromatography
- MW polysaccharides molecular weight
- Rl Refractive Index
- Fig 4b shows the profile of the selectively depolymerized compound (Compound G) of the instant invention as characterized by Refractive Index (Rl) detector.
- FIG. 5a shows two dimensional NMR (2-D NMR) of the combined spectrum of D1 and C13 NMR for Compound G. This two dimensional NMR depicts a finger-printing identification of the glycosidic linkages of Compound G.
- FIG. 5b shows two dimensional NMR (2-D NMR) of the combined spectrum of D1 and C13 NMR for Compound S.
- Figure 5c shows an overlap comparison of these spectra demonstrating the differences between Compound G and Compound S.
- the circled signals are specific to Compound G.
- FIG. 6a-d is a depiction of various types of experiments demonstrating the lack of cytotoxicity and apoptosis of Compound G on cultured stellate liver cell line LX-2.
- FIG. 6a is a graph showing the incorporation of radiolabeled thymidine in cultured LX-2 cells.
- FIG. 6b is a graph showing the evaluation of cellular viability in cultured LX-2 cells.
- FIG. 6c depicts an assessment of apoptosis and cell cycle by FACS in cultured LX-2 cells.
- FIG. 6d shows an assessment of apoptosis by DNA fragmentation in cultured LX-2 cells using Annexin V apoptosis detection kit APC (eBioscience).
- FIG. 6a-d is a depiction of various types of experiments demonstrating the lack of cytotoxicity and apoptosis of Compound G on cultured stellate liver cell line LX-2.
- FIG. 6a is a graph showing the incorporation of radio
- FIG. 7 is a graph showing the relative cytotoxicity on the monocyte/macrophage cell line THP-1 following 3 days in the presence of Connpound G of the present invention (square), Connpound D, a galactomannan product (diamond) and digitoxin (triangle).
- FIG. 8 is a graph showing the relative cytotoxicity on cultured melanoma cell line B16-F10 following 3 days in the presence of Compound G of the present invention (diamond), Compound D, a galactomannan product (square) and 5- Fluoruracil (triangle).
- FIG. 9 is a graph showing the lack of cytotoxicity of Compound G on cultured peripheral blood mononuclear cells (PBMC), a primary cell culture.
- PBMC peripheral blood mononuclear cells
- FIG. 10 is a graph showing the lack of cytotoxicity on growth of lung fibroblast (MRC-5 * ) cells after 4 days growth in present of Compounds G and Compound D. Cytotoxicity was measured using MTS assay.
- FIG. 1 1 is a graph showing the relative anti-inflammatory effect of three modified polysaccharides including compound G of the current invention, on secretion of TNF-alpha by PBMC cells stressed with microbial endotoxin (50 ng/ml).
- FIG. 12 is a comparison of Compound G of the present invention and Compound H on the secretion of TNF-alpha by PBMC cells stressed with microbial endotoxin (50 ng/ml).
- FIG. 13 is a photograph of immunostaining of galectin-3 with MAb-HRP depicting the anti-galectin suppression effect of Compound G on secretion of galectin- 3 by LX-2 cells.
- FIG. 14 is a depiction of an experimental rat fibrosis model induced by thioacetamide injection (TAA model, a chemical toxicity liver fibrosis model).
- FIG. 15 is a photographic depiction of histological regression of fibrosis (Sirius Red staining for collagen) after 4 weekly treatments with Compound G of TAA- induced liver fibrosis.
- FIG. 16 is a graphical and statistical comparison of percentage of collagen in liver (as a measure of fibrosis) in the TAA model after 4 weekly treatments with vehicle control, Compound D and Compound G. Sirius Red staining was used for quantitative measurement of percent collagen in the histological slides.
- FIG. 17 is a depiction of the mouse fatty-liver fibrosis model, a NASH experimental model associated with metabolic disorder (severe diabetes) and high fat diet. Also shown is the experimental design of treatment with Compound G and Compound D.
- FIG. 18 is a graph showing the change in body weight of NASH mice treated with vehicle, Compound D, or Compound G.
- FIGS. 19A-B show the statistical significance of the extent of depicting the extent of liver cell steatosis, ballooning degeneration, and lobular inflammation (as summarized in a NAFLD activity score) in mice treated with vehicle, Compound D, and Compound G.
- FIGS. 20A-B show the statistical significance of the extent of percent collagen in liver sections as measured by the Sirius Red staining method, for NASH mice treated with vehicle, Compound D, and Compound G.
- Pectins are comprised of diversified large carbohydrate polymers that are composed of a backbone of polymeric galacturonic acid with periodic interspersed rhamnose molecules and periodic branched chains of various carbohydrates including galactose, arabinose, fucose, glucose, and others.
- the galacturonic acid molecules are naturally modified by methoxylation at a high percentage of the residues.
- the large pectin carbohydrate polymers have been studied in their native form (directly after extraction from plants) and after various degrees of modification which hydrolyze and chemically modify the polymers. While modified pectins are widely used in the food industry for their ability to gel and provide consistency to various food preparations, they have also been evaluated as potential medicinal compounds.
- Plant-derived pectin materials both unaltered and modified, have been extensively evaluated when administered orally to animals and humans.
- a variety of effects of orally administered unaltered and modified pectins have been described including increased satiety, weight loss, modification of bowel motility, and effects on bowel function including salutatory effects on constipation and diarrhea.
- Ingested pectin material is not digested by normal gut enzymes nor absorbed by the intestine. Therefore, there is little to no pectin that is absorbed into the blood stream after oral absorption.
- modified pectins that could be utilized as parenteral compounds.
- Pectin derived from a mixture of citrus fruit has been evaluated in cell culture models and in animal models of disease, particular in cancer cells and models of cancer in animals.
- the use of modified citrus pectin have been shown to cause cytotoxicity and apoptosis in animal cell lines.
- modified citrus pectin has been proposed to be useful in malignant conditions (See US 8,128,966).
- Galectins also known as galaptins or S-lectin
- S-lectin are a family of lectins which bind beta-galactoside. Galectin as general name was proposed in 1994 for a family of animal lectins (Barondes, S.H., et al.: Galectins: a family of animal beta- galactoside-binding lectins. Cell 76, 597-598, 1994), The family is defined by having at least one characteristic carbohydrate recognition domain (CRD) with an affinity for beta-galactosides and sharing certain sequence elements.
- CCD characteristic carbohydrate recognition domain
- galectins within the same peptide chain, some galectins have a CRD with only a few additional amino acids, whereas others have two CRDs joined by a link peptide, and one (galectin-3) has one CRD joined to a different type of domain.
- the galectin carbohydrate recognition domain is a beta-sandwich of about 135 amino acids. The two sheets are slightly bent with 6 strands forming the concave side and 5 strands forming the convex side. The concave side forms a groove in which carbohydrate is bound (Leffler H, Carlsson S, Hedlund M, Qian Y, Poirier F (2004). "Introduction to galectins". Glycoconj. J. 19 (7-9): 433-40).
- galectins A wide variety of biological phenomena have been shown to be related to galectins, e.g., development, differentiation, morphogenesis, tumor metastasis, apoptosis, RNA splicing, etc. However, relatively little is known about the mechanism by which galectins exert these functions, particularly in terms of carbohydrate recognition.
- carbohydrate domain binds to galactose residues associated with glycoproteins.
- At least fifteen mammalian galectin proteins have been identified which have one or two carbohydrate domain in tandem.
- Galectin proteins are found in the intracellular space where they have been assigned a number of functions and are secreted into the extracellular space. In the extracellular space, galectin proteins can have multiple functions including promoting interactions between glycoproteins that may lead to reduced function, or enhanced functions, or in the case of integral membrane glycoprotein receptors, modification of cellular signaling (Sato et al "Galectins as danger signals in host- pathogen and host-tumor interactions: new members of the growing group of "Alarmins.” In “Galectins,” (Klyosov, et al eds.), John Wiley and Sons, 1 15-145 , 2008, Liu et al "Galectins in acute and chronic inflammation," Ann.
- Galectin proteins in the extracellular space can additionally promote cell-cell and cell matrix interactions (Wang et al., "Nuclear and cytoplasmic localization of galectin-1 and galectin-3 and their roles in pre-mRNA splicing.” In “Galectins” (Klyosov et al eds.), John Wiley and Sons, 87-95, 2008).
- Galectins have been shown to have domains which promote homodimerization. Thus, galectins are capable of acting as a "molecular glue" of sorts between glycoproteins. Galectins are found in multiple cellular compartments, including the nucleus and cytoplasm, and are secreted into the extracellular space where they interact with cell surface and extracellular matrix glycoproteins. The mechanism of molecular interactions can depend on the localization. While galectins can interact with glycoproteins in the extracellular space, the interactions of galectin with other proteins in the intracellular space generally occurs via protein domains. In the extracellular space the association of cell surface receptors may increase or decrease receptor signaling or the ability to interact with ligands.
- Galectin proteins are markedly increased in a number of animal and human disease states, including but not limited to diseases associated with inflammation, fibrosis, autoimmunity, and neoplasia. Galectins have been directly implicated in the disease pathogenesis, as described below.
- diseases states that may be dependent on galectins include, but are not limited to, acute and chronic inflammation, allergic disorders, asthma, dermatitis, autoimmune disease, inflammatory and degenerative arthritis, immune-mediated neurological disease, fibrosis of multiple organs (including but not limited to liver, lung, kidney, pancreas, and heart), inflammatory bowel disease, atherosclerosis, heart failure, ocular inflammatory disease, a large variety of cancers.
- galectins are important regulatory molecules in modulating the response of immune cells to vaccination, exogenous pathogens and cancer cells.
- Galectins show an affinity for galactose residues attached to other organic compounds, such as in lactose [( -D-Galactosido)-D-glucose], N-acetyl- lactosamine, poly-N-acetyllactosamine, galactomannans, fragments of pectins, as well as other galactose containing compounds. It should be noted that galactose by itself does not bind to galectins, or binds so weakly that the binding can hardly be detected.
- Pectin and modified pectin have been shown to bind to galectin proteins presumably on the basis of containing galactose residues that are presented in the context of a macromolecule, in this case a complex carbohydrate rather than a glycoprotein in the case of animal cells.
- Galectin proteins have been shown to be markedly increased in inflammation, fibrotic disorders, and neoplasia (Ito et al. "Galectin-1 as a potent target for cancer therapy: role in the tumor microenvironment", Cancer Metastasis Rev. PMID: 22706847 (2012), Nangia-Makker et al. Galectin-3 binding and metastasis," Methods Mol Biol. 878: 251 -266, 2012, Canesin et al. Galectin-3 expression is associated with bladder cancer progression and clinical outcome," Tumour Biol. 31 : 277-285, 2010, Wanninger et al.
- compositions comprising a chemically modified pectin derived from apple pectin and methods of manufacturing such modified apple pectin having an activity in a cellular assay of inflammatory fibrosis have been previously described [see US 8,236,780, incorporated herein by reference in its entirety].
- modified pectin has been shown to reduce the induction of liver derived fibrogenic cells while having no effect on viability of the cells.
- aspects of the invention relate to a chemically modified pectin or modified pectin composition and methods of producing a modified pectin or modified pectin composition having an anti-inflammatory and/or anti-fibrogenic effects.
- the modified pectin is a polysaccharide chemically defined as galactoarabino-rhamnogalacturonate.
- compositions for parenteral or enteral administration to a subject are disclosed herein.
- the composition can comprise a pectin derivative or modified pectin compound of the invention in an acceptable pharmaceutical carrier.
- pharmaceutically acceptable carrier refers to a carrier or adjuvant that may be administered to a subject (e.g., a patient), together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount or an effective mount of the compound.
- “Pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, e.g., human albumin or cross-linked gelatin polypeptides, coatings, antibacterial and antifungal compounds, isotonic, e.g., sodium chloride or sodium glutamate, and absorption delaying compounds, and the like that are physiologically compatible.
- the carrier is suitable for oral, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidural administration (e.g., by injection or infusion).
- the active compound can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.
- the modified pectin compound is obtained from a pharmaceutical-grade pectin.
- the modified pectin of the present invention can have an enhanced anti-inflammatory and/or an enhanced anti-fibrogenic effects when compared to previous pectin compositions.
- the modified pectin of the present invention can inhibit secretion of Tumor necrosis factor (TNF-a, cachexin), a protein involved in systemic inflammation and a major member of a group of cytokines that stimulate the acute phase reaction. It is produced chiefly by activated macrophages.
- the modified pectin of the present invention does not cause cytotoxicity.
- the modified pectin or modified pectin composition has potent anti-inflammatory and anti-fibrosis properties without exhibiting cytotoxicity. This represents a novel and unexpected combination of effects which suggest that the pharmaceutical-grade modified pectin can have important effects in the treatment of human inflammatory diseases, fibrotic diseases, neoplastic diseases or combinations thereof.
- methods for treating e.g., controlling, relieving, ameliorating, alleviating, or slowing the progression of
- methods for preventing e.g., delaying the onset of or reducing the risk of developing
- the methods include administering to the subject an effective amount of a compound of the invention, or a composition comprising the compound of the invention, to the subject.
- the term "effective dose” refers to the amount of a compound that, alone or in combination with an amount of a therapeutic agent, when administered as a parental, subcutaneous, inhaled, intra-articular, ocular, or oral formulation or to an animal or human with a galectin-dependent inflammatory, fibrotic or neoplastic disease results in reduction in disease activity, as defined below in various embodiments.
- administering refers to oral, or parenteral including intravenous, subcutaneous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraorbital, intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
- the galactoarabino-rhamnogalacturonate compound has terminal galactose residues that can constitute the main binding entity to galectin proteins.
- the remaining side chain composition may stabilize or enhance its interaction with galectin proteins and thereby potentiate the compound's therapeutic action. Interaction with galectins may not be the only mechanism by which Compound G exerts its therapeutic action.
- the branched galactoarabino-rhamnogalacturonate compound of the invention can interact with galectin proteins and can have wide therapeutic effects that have not been seen with galactose or small molecules derivatives of galactose.
- the compound of the instant invention is a polysaccharide chemically defined as galactoarabino-rhamnogalacturonate (also referred herein as "Compound G”), a branched heteropolymer having a backbone characterized as a majority homopoly 1 ,4-linked galacturonic acid (GalA) molecules and methyl galacturonate (MeGalA) residues with intermittent short regions of alternating a -1 ,4-linked GalA and a -1 ,2-linked rhamnose (Rha), whereas the Rha units are further linked to side-chains.
- GalA galactoarabino-rhamnogalacturonate
- MeGalA methyl galacturonate
- backbone refers to the major chain of a polysaccharide, or the chain originating from the major chain of a starting polysaccharide, having saccharide moieties sequentially linked by either alpha or beta glycosidic bonds.
- a backbone may comprise additional monosaccharide moieties connected thereto at various positions along the sequential chain.
- Compound G is a chemically controlled modified natural branched heteropolymer of plant polysaccharides, some of which are available commercially as USP pectin material. These natural pectin materials are of high average molecular weight ranging from 40,000 to over 1 ,000,000 Daltons (D) and are crude mixtures. Most common USP pectins are from citrus fruit, apple and beets, although others are also available.
- the Compound G composition can have a an average molecular weight range of 2,000 to 80,000 D. In some embodiments, the average molecular weight of the compound G can range from 20,000 to 70,000 D.
- the galactoarabino-rhamnogalacturonate may have an average molecular weight of 5,000 to 55,000 D.
- Such galactoarabino-rhamnogalacturonate compounds can be obtained preferably through chemical and physical treatments and purification from natural pectic substance of apple pectin. However compounds similar to Compound G may be obtained from citrus, sugar beet pectin and other plants under appropriate extraction and chemical processing.
- the selectively depolymerized galactoarabino- rhamnogalacturonate (Compound G), a branched heteropolymer, can have an average molecular weight of 2,000 to 80,000, or 20,000 to 70,000, or 5,000 to 55,000 Daltons, as determined by SEC-RI and MALLS method.
- the term "depolymerization” refers to partial, selective or complete hydrolysis of the polysaccharide backbone occurring, for example, when the polysaccharide is treated chemically resulting in fragments of reduced size when compared with the original polysaccharide.
- aspects of the invention relate to methods for producing selectively depolymerized galactoarabino-rhamnogalacturonate.
- the process of the instant invention includes methods intended to preserve the structure and/or beta galactose characteristics and galactose binding abilities of the side chains comprised primarily of galactose and arabinose, and to enhance the prevalence of galactose binding moieties in the galactoarabino-rhamnogalacturonate compound.
- the methods can generate a galactoarabino-rhamnogalacturonate compound having a reduced average molecular weight, so as to be compatible with therapeutic formulations for pluralistic administration via routes, including but not limited to, intravenous, subcutaneous, inhaled, intra-articular, ocular, and oral.
- the compound can be synthesized from natural, highly branched, minimally processed and high methoxylated USP pectin which may come from any plant source, including but not limited to, citrus fruits, apple, or beet.
- the compound can be synthesized from natural, highly branched, minimally processed and high methoxylated USP pectin like one manufactured from apple pomace containing 8-12% pectin.
- the compound can be synthesized under a sufficiently controlled and specific hydrolysis of the glycosidic-linked methoxylated a - 1 ,4-linked GalA while preserving the side-chains with enriched amounts of 1 ,4- ⁇ -D- Gal and 1 ,5-a-L-Ara.
- Amounts of 1 ,4- ⁇ -D-Gal and 1 ,5-a-L-Ara can be quantitatively determined by GC-MS (Gas chromatography-mass spectroscopy) and AELC-PAD (anion exchange liquid chromatography-pulsed amperometric detector) methods.
- the compound can be produced by a process comprising depolymerization catabolized by targeted peroxidation cleavage of glycosidic bonds (also known as beta elimination reaction) by ionized OH sup- generated from ascorbic acid and/or peroxide in presence of a reduced form of a transition metal ion, like Cu sup.++.at 1 to 100 mM.
- a transition metal ion like Cu sup.++.at 1 to 100 mM.
- Other transition metals like Ca. sup.++ or Fe.sup.++ can also be used for this purpose.
- the depolymerized compound can be exposed to pH range of 8 to 10 for 10 to 30 minutes at temperature of 2 to 60° C to initiate controlled partial demethoxylation to generate a selectively middle depolymerized compound with a degree of methoxylation of 40 to 70 percent in comparison to initial levels of about 87%.
- the resulting compound can be referred as middle-methoxylated compound.
- Complete methoxylation of galacturonic acid is considered to be approximately DE 87%.
- the selectively-depolyme zed polysaccharide of the present invention can have an endotoxin level of no more than 100 EU or no more than 300 EU as assessed by LAL method.
- the selectively-depolymehzed polysaccharide of the present invention can have no more than 0.05% Nitrogenous impurities, as assessed by total nitrogen.
- the depolymerized composition can be exposed to multiple washes of hot acidic alcohol (30-80°C) to remove any residual endotoxin, copper and heavy metals, agricultural contaminates and other impurities.
- the therapeutic activity of the compound of the invention can be derived from multiple beta-galactose and arabinose moieties present on the compound.
- Such moieties can mimic cellular glycoproteins on cell surfaces and the extracellular matrix that bind to galectin proteins (e.g. galactose-binding proteins) which are highly expressed in inflammatory, fibrogenic, and tumorgenic processes.
- galectin proteins e.g. galactose-binding proteins
- the expression of galectin-3 has been proven to be critical to inflammation and fibrogenesis in multiple organs, including but not limited to liver, kidney, heart, and lung. Modulating their activity may thus lead to inhibition and reversing of pathologic processes.
- the selectively-depolymerized polysaccharide of the present invention has no cytotoxicity and does not induce apoptosis in cell culture systems.
- the selectively-depolymerized polysaccharide of the present invention differs from other known preparations of modified pectins that have been reported (See US 8,128,966).
- the selectively-depolymerized polysaccharide of the present invention can have an anti-inflammatory effect on peripheral blood mononuclear cells (PMBC) and other inflammatory cell lines.
- PMBC peripheral blood mononuclear cells
- Compound G can reduce expression or response to inducers of cytokine genes or proteins, including but not limited to, TNF-alpha.
- the process of inflammation and repair can involve multiple cell types including cells of the immune system and many inflammatory mediators in a complex and interconnected cascade of events. Acute inflammation can be terminated or can progress to a chronic phase which may lead to fibrosis, a late stage of damage seen in a variety of human organ disease including but not limited to liver, lung, kidney, heart and pancreas.
- Galectin-3 knock out null mice are resistant to fibrosis of the liver in response to hepatotoxins, resistant to lung fibrosis in response to intra-tracheal bleomycin, and can be used as models of kidney fibrosis, heart fibrosis, and chronic pancreatitis (Henderson et al., "The regulation of inflammation by galectin- 3," Immunol Rev. 230: 160-171 , 2009, lacobini et al., "Galectin-3 ablation protects mice from diet-induced NASH: a major scavenging role for galectin-3 in liver,.” J. Hepatol.
- Fibrosis refers to any tissue disorder, including, but not limited to, such cellular disorders as, for example, cirrhosis, kidney fibrosis, liver fibrosis, ovarian fibrosis, lung fibrosis, gastrointestinal or stomach fibrosis, and fibroids.
- fibrosis refers to both the pathological process leading from tissue injury through its encapsulation by extracellular matrix, and the result of the process, which is a pathological formation of scar tissue.
- Cirrhosis refers to any tissue disorder, including such cellular disorders including, but not limited to, renal cirrhosis, liver cirrhosis, ovarian cirrhosis, lung cirrhosis, gastrointestinal or stomach cirrhosis.
- the term “cirrhosis” refers to an advanced stage of fibrosis, defined by the presence of encapsulated nodules.
- “cirrhosis” is considered to be a type of fibrosis, and is included within the meaning of the term “fibrosis” used herein.
- molecular markers As used herein, “molecular markers”, “biochemical markers”, “biomarkers”, or “markers” are used interchangeably and refer to individual molecules of biological origin, which can be monitored as a “readout” of specific metabolic events. These events are accompanied by formation of the “markers”, the quantitative level of which can often be used as an indication to advancement of the event.
- liver fibrosis The advanced stage of liver fibrosis is cirrhosis, defined by the presence of hepatocellular nodules encapsulated by broad bands of fibrous tissue. Fibrosis is a systematic and coordinated response to chronic injury, developing through a series of highly coordinated molecular events, collectively called fibrogenesis. For example, fibrosis can develop as a result of chronic mammalian liver injury. The steps immediately following chronic liver injury can result in the activation of hepatic stellate cells. The stellate cells' activation can lead to proliferation, fibrogenesis and cirrhosis.
- the activation events in stellate cells can be identified by specific molecular markers, such as collagen I, alpha 1 -smooth muscle actin, beta PDGF-receptor (a proliferation biomarker), matrix metalloproteinases and their inhibitors MMP2, MMP9, TIMP1 and TMP2 (markers on matrix degradation), and a variety of cytokines, including but not limited to, TFG-beta1 (a marker of fibrogenesis).
- Development of fibrosis can be evaluated by the quantitative level of the respective markers. Reduction of fibrosis can be evaluated by the decrease of the level of the respective markers during various stages of fibrosis.
- the pathophysiologic spectrum of fibrosis may be associated with serum biomarkers including but not limited to hyaluronic acid and other breakdown products of collagens, cytokeratin-18 and other cytoskeletal cellular proteins, tissue inhibitor of metalloprotease I and II, other liver derived collagen, matrix proteases or combinations thereof. These compounds and/or biomarkers may be measured in serum or liver tissue using immunoassays and the levels correlated with severity of disease and treatment.
- the pathophysiologic spectrum of fibrosis also may be associated with serum biomarkers, including, but not limited to, reactive oxygen products of lipid or protein origin, lipid molecules or conjugates, or combinations thereof. These biomarkers can be measured by various means including immunoassays and electrophoresis and their levels correlated with severity of disease and treatment. Additional biomarkers may include global shifts in proteomic analysis of serum or urine proteins.
- the pathophysiologic spectrum of fibrosis also may be associated with serum biomarkers of NASH, a chronic metabolic inflammatory disorder that leads to fibrosis and is described in this application.
- These biomarkers can be cytokines, including but not limited to, TNF-alpha, TGF-beta or IL-8, or a metabolic profile of serum components that is indicative of NASH presence or severity (these include serum and urine markers) or combinations thereof.
- a profile of one or more of the cytokines biomarkers, as measured by immunoassay or proteomic assessment by LC mass spec, may provide an assessment of activity of the disease and a marker to follow in therapy of the disease.
- the pathophysiologic spectrum of fibrosis in the liver also may be associated with histopathological findings on liver biopsy, that include but are not limited to, evidence of collagen deposition (including but not limited to peri-sinusoidal, portal, central collagen deposition or combinations thereof), portal to central bridging collagen deposition, hepatocellular nodules that distort the normal architecture, hepatocellular atypia consistent with malignant transformation or combinations thereof.
- the pathophysiologic spectrum of fibrosis in the liver may also be associated with other pathological histological findings on liver biopsy that are associated with the underlying cause of chronic liver disease that results in fibrosis.
- Such findings can include, but are limited to, abnormalities in hepatocytes (including, but not limited to, ballooning degeneration and intracellular hyaline and macrovesicular or microvesicular fat or combinations thereof), endothelial cells, macrophages, or bile duct cells and the infiltration of multiple types of inflammatory cells, such as lymphocytes, monocytes, and/or neutrophils, or any combination of the foregoing.
- the pathophysiologic spectrum of fibrosis can also include histopathological findings on liver biopsy that are related to the underlying disease of NASH.
- Such findings can include, but are not limited to, evidence of intra- hepatocellular fat, hepatocellular toxicity including but not limited to hyaline bodies, inflammatory cell infiltrates (including but not limited to lymphocytes and various subsets of lymphocytes and neutrophils), changes in bile duct cells, changes in endothelial cells, number of Kupffer cell macrophages, collagen deposition (including but not limited to peri-sinusoidal, portal and central collagen deposition and portal to central bridging collagen deposition, hepatocellular nodules that distort the normal architecture, hepatocellular atypia consistent with malignant transformation, and various scales and methods that combine various sets of observations for grading the severity of NASH or any combinations of the foregoing.
- Such histological assessments can be the sine-qua-none of NASH diagnosis and
- the pathophysiologic spectrum of fibrosis can also include histopathological findings on liver biopsy that examine the expression of or change in expression of various molecules and their localization in liver tissue or various cell types.
- Suitable molecules include, but are not limited to, various cytokine proteins.
- Cytokine proteins of interest can include, but are not limited to, TGF-beta, inflammatory mediators, reactive metabolite scavenger transport proteins, including but not limited to, CD36, and galectin proteins, including but not limited to galectin-3 protein, or any combinations of the foregoing.
- Clinical manifestations of fibrosis can include, but are not limited to, clinical testing of stage and severity of the disease, clinical signs and symptoms of disease, and/or medical complications resulting from fibrosis.
- Clinical testing of stage and severity of liver fibrosis can include, but are not limited to, hematologic testing (including, but not limited to, red blood cell count and/or morphology, white blood cell count and/or differential and/or morphology, and/or platelet count and morphology), serum or plasma lipids, including but not limited to, triglycerides, cholesterol, fatty acids, lipoprotein species and lipid peroxidation species, serum or plasma enzymes (including but not limited to aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (AP), gamma glutamyltranspeptidase (GGTP), lactate dehydrogenase (LDH) and isoforms, serum or plasma albumin and other proteins indicative of liver synthetic capacity
- Clinical testing also includes but is not limited to non-invasive and invasive testing that assesses the architecture, structural integrity or function of the liver including but not limited to computerized tomography (CT scan), ultrasound (US), ultrasonic elastography (FibroScan) or other measurements of the elasticity of liver tissue, magnetic resonance scanning or spectroscopy, magnetic resonance elastography, percutaneous or skinny needle or transjugular liver biopsy and histological assessment (including but not limited to staining for different components using affinity dyes or immunohistochemistry), measurement of hepatic portal-venous wedge pressure gradient, or other non-invasive or invasive tests that may be developed for assessing severity of fibrosis in the liver tissue or any combinations of the foregoing.
- CT scan computerized tomography
- US ultrasound
- FibroScan ultrasonic elastography
- histological assessment including but not limited to staining for different components using affinity dyes or immunohistochemistry
- measurement of hepatic portal-venous wedge pressure gradient or other non-invasive or invasive tests that may
- Clinical signs and symptoms of advanced fibrosis that has progressed to cirrhosis can include fatigue, muscle weight loss, spider angiomata, abdominal pain, abdominal swelling, ascites, gastrointestinal bleeding, other bleeding complications, easy bruising and ecchymoses, peripheral edema, hepatomegaly, nodular firm liver, somnolence, sleep disturbance, confusion, and/or coma.
- Medical complications of fibrosis are related to cirrhosis and include ascites, peripheral edema, esophageal and other gastrointestinal tract varices, gastrointestinal bleeding, other bleeding complications, emaciation and muscle wasting, hepatorenal syndrome, and hepatic encephalopathy.
- Nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are common liver disorders in the United States and Europe. Histopathologically, these disorders resemble alcoholic liver disease, but can occur in people who drink little or no alcohol.
- the pathological changes in the liver include, but are not limited to, fat accumulation in hepatocytes, evidence of hepatocellular degeneration, infiltrates of inflammatory cells, deposition of excess fibrous tissue, hepatocellular nodule formation, cirrhosis, hepatocellular carcinoma and combinations thereof.
- NAFLD fat accumulation in hepatocytes with minimal inflammation. NAFLD is usually identified on the basis of a liver biopsy performed because of mildly elevated liver transaminase levels in the patient's serum or the suspicion of fatty liver on non-invasive testing such as computerized tomography or ultrasound.
- NASH is fatty liver with the addition of the development of infiltration of inflammatory cells (including but not limited to neutrophils or lymphocytes) within the lobule, central vein and portal areas and evidence of damage to hepatocytes, including but not limited to, ballooning degeneration.
- This inflammatory state of NASH may result in the deposition of fibrous tissue, including but not limited to, collagen, which can lead to cirrhosis, nodule formation, and/or hepatocellular carcinoma.
- NASH can be severe and can lead to the deposition of fibrotic material in the liver which can result in severe scarring and/or cirrhosis and, in some cases, hepatocellular carcinoma.
- the compound of the present invention may be used in the treatment of NASH or fibrosis.
- the compound of the present invention may be effective through manipulation of galectin proteins that are involved in pathogenesis and progression of diseases.
- the compound of the present invention i.e. Compound G
- the compound of the present invention has an anti-fibrotic effect in rat fibrosis model where fibrosis is induced by chemical toxin thioacetamide (TAA) which have similar pathology to the effect of chronic consumption of alcohol that lead to fibrosis, cirrhosis and increase occurrence of hepatocarcinoma.
- TAA chemical toxin thioacetamide
- the compound of the present invention has an anti-inflammatory effect by reducing secretion of TNF alpha as depicted in an in-vitro model using PBMC cell stressed with microbial endotoxin to produce TNF alpha, a major cytokine, a biomarker and an inflammatory protein (FIG. 16).
- the Compound G is proposed as therapy alone or in combination with other compounds listed above as treatment for human NASH.
- the compound of the present invention can be used for ameliorating or reversing hepatocyte fat accumulation, intra-portal and intra-lobular inflammatory infiltrate, and/or fibrosis, including but not limited to collagen deposition in the peri-sinusoidal space, cirrhosis, and for preventing progression to hepatocellular carcinoma.
- chronic liver diseases can include, but are not limited to, chronic hepatitis virus infection (hepatitis B, C, and D), chronic alcohol abuse, biliary diseases (including, but not limited to, sclerosing cholangitis), primary biliary cirrhosis, genetic storage diseases, and metal storage diseases (including but not limited to hemochromatosis and Wilsons disease).
- Compound G may be effective in all chronic liver diseases that lead to fibrosis regardless of the underlying etiology.
- Compound G may be effective in the treatment of fibrosis in organs other than the liver, including, but not limited to, lung, kidney, heart and pancreas occurs through chronic inflammation leading to collagen deposition by cell types other than stellate cells which are specific to the liver.
- the cells responsible for fibrosis in other organs are myofibroblasts that have precursor cells that include, but are not limited to, resident tissue fibroblasts, circulating fibrocytes, or epithelial cells generated through a process called EMT— epithelial mesenchymal transformation.
- Lung fibrosis can occur as a result of chronic inflammatory process in a variety of diseases including, but not limited to, idiopathic pulmonary fibrosis, chronic obstructive lung disease, and chronic infections.
- Fibrosis in the lung usually leads to stiffness of the lung tissues resulting in reduced function of the lung, including but not limited to, reduced total lung capacity, vital capacity, forced expiratory volume, and diffusion capacity. These reduced functions can lead to clinical symptoms, including but not limited to, shortness of breath, reduced exercise tolerance, and reduced gas exchange resulting in low blood oxygen levels. The ultimate result can be lung failure which requires a lung transplant. There are no current pharmacological therapies for lung fibrosis.
- Kidney fibrosis can occur as a result of multiple underlying diseases, including but not limited to, diabetes, obstruction of the urinary tract, hypertension, vascular disease, and autoimmune diseases.
- Kidney fibrosis can progress to inhibit kidney function which results in reduced urine output and the accumulation of toxic metabolites in the blood stream. Kidney failure associated with kidney fibrosis requires external support through dialysis or a kidney transplant. There are no known current pharmacological therapies for kidney fibrosis.
- Heart failure is associated with multiple diseases, including but not limited to, chronic hypertension, coronary artery disease, valvular heart disease, and hypertrophic heart disease.
- Heart failure can occur in part because of the deposition of fibrous tissue in the heart muscle, a process that has been shown to be associated causally with an increased expression of galectin-3.
- Progressive heart failure can result in reduced contractility of the heart, dilation of the heart chambers, reduced cardiac output with multiple resultant symptoms including edema, shortness of breath, reduced kidney function, mental confusion, and others.
- heart failure can only be treated with mechanical cardiac assist devices or heart transplantation.
- Galectin proteins have been shown to have multiple functions in cancer cells including, but not limited to, enhancing invasiveness, causing resistance to chemotherapy, promoting metastasis, enhancing neovascularization, and allowing evasion of the immune system.
- galectins include but not linnited cancers of the skin (squamous and melanoma), mouth, head and neck, lymphatic system, blood cells, alimentary tract (esophagus, stomach, small intestine, colon and rectum), pancreas, biliary tree, liver, lung, breast, kidney, ovary, testes, cervix, uterus, and neurological system
- aspects of the invention relate to a compound, or a composition comprising the compound, utilized for the treatment of inflammatory and fibrotic disorders in which galectins are involved in the pathogenesis, including but not limited to enhanced anti-fibrosis activity in organs, including but not limited to liver, kidney, lung, and heart.
- Other aspects of the invention relate to the methods of treating inflammatory and fibrotic disorders in which galectins are involved in the pathogenesis.
- the invention relates to a compound, a composition that has therapeutic activity or a method to reduce the pathology and disease activity associated with nonalcoholic steatohepatitis (NASH) including, but not limited to, steatosis (fat accumulation in hepatocytes), ballooning degeneration of hepatocytes, inflammatory infiltrate in the liver, and deposition of collagen or fibrosis.
- NASH nonalcoholic steatohepatitis
- the invention relates to a compound, or a composition comprising the compound, utilized in treating inflammatory and autoimmune disorders in which galectins are involved in the pathogenesis including, but not limited to, arthritis, rheumatoid arthritis, asthma, and inflammatory bowel disease (ulcerative colitis and Crohn's Disease).
- the invention relates to a compound, or a composition comprising the compound, utilized in treating neoplastic conditions (e.g. cancers) in which galectins are involved in the pathogenesis by inhibiting processes promoted by the increase in galectins, including, but not limited to, tumor cell invasion, metastasis, and neovascularization.
- neoplastic conditions e.g. cancers
- galectins are involved in the pathogenesis by inhibiting processes promoted by the increase in galectins, including, but not limited to, tumor cell invasion, metastasis, and neovascularization.
- the invention relates to a compound, or a composition comprising the compound, utilized in enhancing or a method for enhancing the ability of tumor infiltrating T-cells, which are inhibited by the effect of tumor derived galectin proteins, to more effectively identify and kill tumor cells and thereby slow, stop or reverse the progression of tumors.
- the invention relates to a compound, a composition comprising the compound utilized in combination with tumor immunotherapy which may be a vaccine directed towards specific tumor antigens or agents which activate or inhibit specific immune regulatory molecules including but not limited to CTLA4, OX40, PD-1 , or PD-L.
- An effective dose of the compound of the present invention or a composition comprising an effective dose of the compound can be administered via a variety of routes including, parenteral via an intravenous infusion given as repeated bolus infusions or constant infusion, intradermal injection, subcutaneously given as repeated bolus injection or constant infusion, intra-articular injection, inhaled in an appropriate formulation, or oral administration.
- the amount administered depends on the compound formulation, route of administration, etc. and is generally empirically determined in routine trials, and variations will necessarily occur depending on the target, the host, and the route of administration, etc.
- administering refers to oral, or parenteral including intravenous, subcutaneous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraorbital, intracardiac, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
- An effective parenteral dose of the compound of the present invention to an experimental animal can be within the range of 2 mg/kg up to 200 mg/kg body weight given intravenously.
- An effective subcutaneous injection dose of the compound of the present invention to an animal can be within the range of 2 mg/kg up to 200 mg/kg body weight, or by intraperitoneal 2 mg/kg up to 200 mg/kg or by oral administration 10 mg/kg or 50 mg/kg or 200 mg/kg or 1500 mg/kg body weight. Higher and lower doses can also be contemplated.
- An effective parenteral dose of the compound of the present invention to a human subject can be within the range of 0.2 mg/kg up to 20 mg/kg body weight given intravenously.
- An effective subcutaneous injection dose of the compound of the present invention to a human subject can be in the range of 0.2 mg/kg up to 50 mg/kg body weight or by oral administration 10 mg/kg up to 200 mg/kg body weight. Lower or higher doses than those recited above may be required.
- Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.
- An effective parental dose may be given daily (in one or divided doses), three times weekly, two times weekly, or monthly via intravenous, intradermal, subcutaneous or other routes as practiced by the medical professional to administrate drugs.
- An effective oral dose of the compound of the present invention to an experimental animal or human may be formulated with a variety of excipients and additives that enhance the absorption of the compound via the stomach and small intestine.
- An effective oral dose could be 10 times and up to 100 times the amount of the effective parental dose.
- An effective oral dose may be given daily, in one or divided doses or twice, three times weekly, or monthly.
- the compound of the present invention or compositions comprising the compound of the present invention may be administered orally; or by intravenous injection; or by injection directly into an affected tissue, as for example by injection into an arthritic joint.
- the compound or composition may be administered topically, as in the form of eye drops, nasal sprays, ointments or the like.
- other techniques such as transdermal delivery systems, inhalation or the like may be employed.
- the compounds described herein can be coadministered with one or more other therapeutic agents.
- the additional agents may be administered separately, as part of a multiple dose regimen, from the compounds of this invention (e.g., sequentially, e.g., on different overlapping schedules with the administration of the compound of the invention.
- these agents may be part of a single dosage form, mixed together with the compounds of this invention in a single composition.
- these agents can be given as a separate dose that is administered at about the same time that the compound of the invention.
- compositions include a combination of the compound of this invention and one or more additional therapeutic or prophylactic agents
- both the compound and the additional agent can be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen.
- a therapeutically effective amount of the depolymerized compound or of the composition can be compatible and effective in combination with a therapeutically effective amount of various anti-oxidant compounds (e.g. glycyrrhizin, ascorbic acid, L-glutathione, cysteamine etc) as described in US Patent No 7,078,064.
- various anti-oxidant compounds e.g. glycyrrhizin, ascorbic acid, L-glutathione, cysteamine etc
- An effective dose given to an animal or human subject with NASH and liver fibrosis means the amount of a compound that, alone or in combination with amount of other therapeutic agent, when administered as a parental, subcutaneous, inhaled, intra-articular, ocular, or oral formulation results in at least a 10% reduction in hepatocellular fat, hepatocytes with ballooning degeneration, inflammatory cell infiltrated, at least a one point reduction in the NAFLD activity score, or at least a 10% reduction in collagen deposition in the liver assessed by histological staining with Sirius red, or slowing the progression of deposition of fibrotic tissue in the liver by at least 10%.
- An effective dose given to a human subject with NASH and liver fibrosis can result in at least a 10% reduction in serum biomarkers associated with NASH, or at least a 10% improvement in hepatocyte fat content or liver stiffness as assessed by ultrasound or MR elastography, or at least a 10% improvement in liver function tests that measure metabolic function or shunting in the liver, or at least a 10% reduction in clinical symptoms and complications resulting from liver fibrosis and cirrhosis including but not limited to symptoms and complications resulting from reduced metabolic and elimination processes (including but not limited to bilirubin), reduced liver synthetic capacity (including but not limited to albumin and coagulation proteins), portal hypertension, and hepatic encephalopathy.
- An effective dose given to a human subject with NASH and liver fibrosis means an improvement in clinical parameters or reduced progression of clinical parameters when compared to a control untreated group of human subjects including but not limited to at least a 10% reduction in serum biomarkers associated with NASH, or at least a 10% improvement in hepatocyte fat content or liver stiffness as assessed by ultrasound or MR elastography, or at least a 10% improvement in liver function tests that measure metabolic function or shunting in the liver, or at least a 10% reduction in clinical symptoms and complications resulting from liver fibrosis and cirrhosis including but not limited to symptoms and complications resulting from reduced metabolic and elimination processes (including but not limited to bilirubin), reduced liver synthetic capacity (including but not limited to albumin and coagulation proteins), portal hypertension, and hepatic encephalopathy.
- reduced metabolic and elimination processes including but not limited to bilirubin
- reduced liver synthetic capacity including but not limited to albumin and coagulation proteins
- portal hypertension and hepatic encephalopathy.
- An effective dose given to an animal or human subject with liver fibrosis or cirrhosis due to a disorder other than NASH means the amount of a compound that, alone or in combination with amount of other therapeutic agent, when administered as a parental, subcutaneous, inhaled, intra-articular, ocular, or oral formulation results in, for example, at least a 10% reduction in collagen deposition in the liver assessed by histological staining with Sirius red, or slowing the progression of deposition of fibrotic tissue in the liver by at least 10%.
- An effective dose given to a human subject with liver fibrosis or cirrhosis due to a disorder other than NASH means the amount of a compound that, alone or in combination with amount of other therapeutic agent, when administered as a parental, subcutaneous, inhaled, intra-articular, ocular, or oral formulation results in, but is not limited to, at least a 10% reduction in serum biomarkers associated with liver fibrosis, or at least a 10% improvement in liver stiffness as assessed by ultrasound or MR elastography, or at least a 10% improvement in liver function tests that measure metabolic function or shunting in the liver, or at least a 10% reduction in clinical symptoms and complications resulting from liver fibrosis and cirrhosis including but not limited to symptoms and complications resulting from reduced metabolic and elimination processes (including but not limited to bilirubin), reduced liver synthetic capacity (including but not limited to albumin and coagulation proteins), portal hypertension, and hepatic encephalopathy.
- reduced metabolic and elimination processes including but not limited to bilirubin
- An effective dose given to a human subject with liver fibrosis or cirrhosis due to a disorder other than NASH means an improvement in clinical parameters or reduced progression of clinical parameters when compared to a control untreated group of human subjects including but not limited to at least a 10% reduction in serum biomarkers associated with fibrosis, or at least a 10% improvement in liver stiffness as assessed by ultrasound or MR elastography, or at least a 10% improvement in liver function tests that measure metabolic function or shunting in the liver, or at least a 10% reduction in clinical symptoms and complications resulting from liver fibrosis and cirrhosis including but not limited to symptoms and complications resulting from reduced metabolic and elimination processes (including but not limited to bilirubin), reduced liver synthetic capacity (including but not limited to albumin and coagulation proteins), portal hypertension, and hepatic encephalopathy.
- reduced metabolic and elimination processes including but not limited to bilirubin
- reduced liver synthetic capacity including but not limited to albumin and coagulation proteins
- portal hypertension and he
- An effective dose given to an animal or human subject with kidney fibrosis means an improvement in clinical parameters or reduced progression of clinical parameters when compared to a control untreated group of animals or human subjects including but not limited to at least a 10% reduction in kidney fibrosis assessed by histology, or a least a 10% improvement in proteinuria, or at least a 10% improvement in glomerular filtration rate, or at least a 10% improvement in clinical signs and symptoms related to renal insufficiency.
- An effective dose given to an animal or human subject with lung fibrosis means an improvement in clinical parameters or reduced progression of clinical parameters when compared to a control untreated group of animals or human subjects including but not limited to at least a 10% reduction in lung fibrotic tissue assessed on histology, 10% improvement in lung volumes, or at least a 10% improvement in expiratory volumes, or at least a 10% improvement in clinical signs and symptoms related to pulmonary insufficiency.
- An effective dose given to an animal or human subject with heart fibrosis means an improvement in clinical parameters or reduced progression of clinical parameters when compared to a control untreated group of animals or human subjects including but not limited to at least a 10% reduction in heart fibrotic tissue assessed on histology, 10% improvement in heart contractility, or at least a 10% improvement in cardiac output, or at least a 10% improvement in clinical signs and symptoms related to heart failure.
- An effective dose given to an animal or human subject with pancreatic fibrosis means an improvement in clinical parameters or reduced progression of clinical parameters when compared to a control untreated group of animals or human subjects including but not limited to at least a 10% reduction in pancreas fibrotic tissue assessed on histology, 5% improvement in synthesis or secretion of pancreatic exocrine enzymes, or at least a 5% improvement in pancreatic endocrine enzymes including but not limited to insulin, or at least a 10% improvement in clinical signs and symptoms related to pancreatic insufficiency.
- An effective dose given to an animal or human subject with cancer means an improvement in clinical parameters or reduced progression of clinical parameters when compared to a control untreated group of animals or human subjects including but not limited to at least a 10% reduction in size of tumors, or at least a 10% reduction in number of cancer metastases, or at least a 10% increase in activity of immune system against the cancer cells, or at least a 10% improvement in clinical signs and symptoms related to cancer including but not limited to improvement in progression free survival, or overall survival, or reduced adverse effects of therapy, or improved quality of life.
- Compound G refers to a compound prepared in accordance with Example 1 described herein
- Compound D refers to a reference polysaccharide prepared in accordance with the methods disclosed in US Patent No. 7,893,252, incorporated herein by reference in its entirety
- Compound H refers to a high molecular depolymerized pectin polysaccharide (90,000 to 140,000 D), one not prepared in accordance with Example 1 described herein, but rather prepared according with the methods disclosed in US Patent No. 8,236,780, incorporated herein by reference in its entirety.
- Compound S refers to a polysaccharide manufactured from USP Citrus pectin according to the methods disclosed in US Patent No. 8,128,966. The invention provides manufacturing process of modified pectin, and use in cancer treatment. And it is incorporated herein by reference in its entirety.
- Compound T refers to a commercial MCP "Thorne Research Fractionated Pectin Powder 5 OZ" purchased on Amazon.com.
- the selectively depolymerized product of the instant invention was prepared by a process illustrated in FIG. 1 .
- Apple pectin USP HM 50 kg was dissolved and heated in water to 35- 85 °C. 1 M HCI or NaOH was added in order to pH-adjust the solution to pH 5-7 and mixed well. The mixing was continued for 2 hours at the 35-85 °C set-point. 1 M NaOH or HCI was added as needed to readjust pH to between 5 and 7. Solution was cooled to 30° C. At 30 °C, pH was adjusted to between 5 and 7. [000194] CuSO 4 is added to the pH-adjusted pectin solution so as to result in a final 1 mM CuSO 4 concentration. The 1 mM CuSO 4 solution was mixed for 30 minutes at a temperature of between 10°C and 30°C.
- the filtrate was concentrated 1 .5 - 3X under vacuum, and then pH- adjusted to between 3 and 5. Hot ethanol or isopropanol was added on a 50% weight. The mixture was stirred 1 -2 hours to precipitate product, and the mixture was then filtered. The filtrate was discarded, leaving a white to off-white precipitate.
- a Multi-Angle Laser Light Scattering detection system can be used to generate a ZIMM plot that independently predicts the molecular weight of polymers.
- the principle of the MALLS method is based on the fact that light is more strongly scattered by large molecules than by small molecules.
- the output of the light scattering detector is proportional to the multiplication of the concentration and the average molecular weight of macromolecules. Therefore, the shape of the light scattering peak is asymmetric.
- Molecular weight versus the elution volume is obtained and average molecular weights and average molecular weight distributions can be calculated.
- FIGS. 3A - 3B illustrate a determination of the average molecular weight of a selectively depolymerized compound of the instant invention by MALLS, indicating an average molecular weight of approximately 37 kilodaltons (kD) in EDTA buffer solution with a standard deviation of 8%.
- SEC Size Exclusion Chromatography
- Rl Refractive Index
- Glycosyl composition analysis was performed by combined gas chromatography followed by mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis.
- GC/MS mass spectrometry
- EXAMPLE 5 Determination of Glvcosidic linkages and Structure by H1 and C13 NMR.
- NMR spectroscopy reveals individual molecular signatures and linkages, providing a type of analytical fingerprint for complex carbohydrate molecules.
- Two dimensional NMR spectra were evaluated to reveal the molecular fingerprint of the composition of the instant invention, Compound G, and comparison to Compound S.
- Compound G The major components in Compound G are 4-linked galacturonic acid, 4- linked methyl galacturonate, and 4-linked galactose. Rhamnose is clearly present in the HSQC spectrum. Table 4 contains the NMR spectral assignment of Compound G. TABLE 4
- FIG 5a shows the two dimensional NMR spectrum for Compound G and FIG 5b shows the two dimensional NMR spectrum for Compound S.
- FIG 5c shows the overlapping spectra of Compound G and Compound S, revealing the marked differences in the two dimensional NMR spectra of these two compounds. The positions that are circled are in the spectrum of Compound G, but not in Compound S. This indicates important structural differences between Compound G and Compound S.
- modified pectin compounds that have been reported (U.S. 8,128,966 and U.S. 2012/0149658) is their cytotoxicity in cell lines of various types, including the induction of apoptosis.
- US Patent 8,128,966 discloses modified pectins that induce apoptosis in cancer cells, such as B16-F10 melanoma cell line.
- LX-2 Human hepatic stellate cell line, LX-2 is routinely used as a tool for analysis of hepatic fibrosis.
- the LX-2 cells proliferate normally in 2% FC serum rich media. However, once stress in 0.1 % FC serum, LX-2 cells go through similar pathological changes as established in fibrotic liver. Cytotoxicity was assessed using Compound D, Compound H, and Compound G.
- FIG 6a shows the results of a proliferation assay utilizing the incorporation of tritium-labeled thymidine into growing LX-2 cells. There was no difference from control cells in thymidine incorporation at 48 hours of culture when cells were incubated with 1 mg/ml of Compound D, Compound H, or Compound G.
- FIG 6b shows the results of a cell viability assay utilizing a vital dye which is taken up by non viable cells and excluded from viable cells. There was no difference from control cells in cell viability at 48 hours of culture when cells were incubated with 1 mg/ml of Compound D, Compound H, or Compound G.
- Annexins are a family of calcium-dependent phospholipid binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses is asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected fluorescently labeled Annexin V in a calcium-dependent manner.
- FIG 6c shows the results of FACS (Fluorescence activated cell sorting) analysis of LX-2 cells to assess apoptosis which showed no evidence of apoptosis in cells treated with Compound D or Compound G versus control cells (vehicle treated).
- FACS Fluorescence activated cell sorting
- FIG 6d shows that there was no evidence of DNA fragmentation in cells treated with Compound D (D1 or D2), Compound H or Compound G as compared to control.
- THP-1 Acute Monocytic Leukemia
- the THP-1 cells were suspended in assay media containing 10%FBS. About 25,300 cells/well were transferred at 100ul/well to 96 well plates. At 24 hours the culture media was changed to fresh media and the cells were incubated over night. Test compounds were serially diluted in assay media containing 10% FBS and transfer 100 ul/well to the growing monocyte cells THP-1 . The final assay volume was 200 ul/well containing 10% FBS, 2X Gentamicin, and the following test articles: Compound G of the present invention and Compound D, a galactomannan product or in presence of digitoxin. The cells were incubated for 3 days with the test articles.
- cytotoxicity/growth was measured by adding 15 ul of Promega "CellTiter 96® Aqueous One Solution” to the 96 wells and viability was monitored at OD 490nm after 1 -7.5 hours.
- the CellTiter 96® AQueous One Solution Cell Proliferation Assay is a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays.
- the CellTiter 96® AQueous One Solution Reagent contains a tetrazolium compound [3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES).
- PES has enhanced chemical stability, which allows it to be combined with MTS to form a stable solution for detection cell viability in in-vitro culture media.
- FIG. 7 shows that increasing amounts of Compound G applied to THP-1 cells did not result in cytotoxicity.
- the melanoma cell line B16-F10 was previously used as an assay for activity of modified pectins. Thus, B16-F10 cells were evaluated for the effect of Compound G on cytotoxicity.
- the melanoma cell line B16-F10 (ATCC® Number: CRL-6475TM) are a mixture of spindle-shaped and epithelial-like cells from skin melanoma of mouse (Mus musculus, Strain: C57BL/6J (See Fidler IJ. Biological behavior of malignant melanoma cells correlated to their survival in vivo. Cancer Res. 35: 218-224, 1975.)
- Cytotoxicity / growth was measured by adding 20 ul of Promega "CellTiter 96® Aqueous One Solution” to the 96 wells and monitored at OD 490nm after 1 hour.
- the CellTiter 96® AQueous One Solution Cell Proliferation Assay is a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays.
- the CellTiter 96® AQueous One Solution Reagent contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES).
- PES has enhanced chemical stability, which allows it to be combined with MTS to form a stable solution for detection cell viability in in-vitro culture media.
- Figure 8 shows that increasing amounts of Compound G applied to B16- F10 cells did not result in cytotoxicity. This represents a clear difference from the function of other modified pectin compounds previously described.
- PBMC cells are primary cell extracted from whole blood using ficoll, a hydrophilic polysaccharide.
- Peripheral blood mononuclear cells PBMC are integrally involved in inflammatory and fibrotic processes. Thus, PBMC were evaluated for the effect of Compound G on cytotoxicity.
- FIG. 9 shows that Compound G from three different lots applied to PBMCs did not result in cytotoxicity.
- Figure 10 shows that increasing amounts of Compound G applied to cultured lung fibroblast cells did not result in cytotoxicity.
- the MRC-5 cell line is a normal fibroblast that was derived from normal lung tissue (Homo sapiens (human) male (ATCC Catalog No. CCL-171TM).
- Compound G of the present invention Compound D, a galactomannan product were tested using a CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS)- Promega USA.
- MTS CellTiter 96® AQueous One Solution Cell Proliferation Assay
- EXAMPLE 7 In-Vitro Assay For Anti-Inflammatory Effect Using PBMC Primary Cells
- Compound G was assessed for its biological activity in inflammatory conditions. Multiple cells are involved in and activated in the inflammatory process. One key cell type in this process are Peripheral Blood Mononuclear Cells (PMBCs), a primary cell routinely use as anti-inflammatory in-vitro model, which are activated by inflammatory mediators, recruited to sites of inflammation, transform into tissue macrophages, and enhance the inflammatory process. Therefore, PBMCs were used as an in vitro model to evaluate the effect of Compound G.
- PMBCs Peripheral Blood Mononuclear Cells
- the in-vitro assay for inflammation was developed by stressing PBMC with microbial endotoxin (microbial lipopolysaccharides) and measuring by secretion of TNF alpha, a major cytokine and biomarker for inflammation.
- PBMC were re- suspended in assay media containing 10% FBS (Gibco lot# 749413), 2X Gentamicin, and L-Glutamine.
- PMBC were transferred 90 ⁇ /well to assay plate (169,000 cells/well).
- 90 ⁇ /well of assay media was added to assay plate to a total volume of 180 ⁇ /well for approx. 16 hours.
- LPS microbial toxin
- h-TNF- ⁇ was analyzed using an ELISA Development Kit (PeproTech, cat# 900-K25, lot# 0509025), with Human TNF-a as standard on an ELISA plate.100 ⁇ of ABTS Liquid Substrate was added to each well and OD was read at 405 nm with a wavelength correction set at 650nm.
- FIG. 1 1 demonstrates graphically results of Plate 364 with h-TNF alfa ELISA.
- Compound G at 0.5 mg/mL reduced 50% of TNF-alpha secretion by stressed PBMC primary cells. The reduction of TNF-alpha secretion is significantly higher than either Compound D or Compound S reference polysaccharides tested.
- the ability of Compound D, Compound H, and Compound G to inhibit TNF-alpha secretion in PBMC were directly compared, as shown in FIG 12.
- Compound H was produced using the method disclosed in US Patent No. 8,236,780.
- Compound D and Compound H did not show any ability to reduce TNF-alpha secretion from PBMC cells, in fact Compound H appeared increase TNF-alpha.
- Compound G was shown to be a potent inhibitor of an in vitro model of inflammation. Such activity was shown to be absent from Compound S or Compound H, modified pectin compounds made through different processes from Compound G. Therefore, results form Examples 6 and 7 showed that Compound G is modified pectin having unique non-cytotoxic and anti-inflammatory properties that were not described for other known pectin-derived compounds.
- EXAMPLE 8 ln-vitro Induction of Fibrogenesis in liver LX-2 stellate Cell line.
- LX-2 Human hepatic stellate cell line, LX-2 is routinely used as a tool for analysis of hepatic fibrosis.
- the LX-2 cells proliferate normally in 2% FC serum rich media. However, once stress in 0.1 % FC serum they go through a similar pathological changes as established in fibrotic liver
- the Compound G may also modulate, increase or decrease in LX-2 stellate cells molecules or biomarkers that are involved in fibrogenesis, including but not limited to collagen I, II, III, IV, metalloproteases, inhibitors of metalloproteases, and cytokines.
- the Compound G compound may also modulate expression of cytokines and lipids and reactive oxygen species in liver macrophages, or Kupffer cells.
- the Compound G compound may also modulate the expression of hepatocellular genes, uptake and metabolism of lipids and reactive oxygen species.
- the effect of Compound G on the expression of galectin-3 in both intra and extra-cellular compartments was assessed, as shown in FIG 13.. While rich serum media had produced negligible amount of galectin-3, when LX-2 cells (human stellate cell line) were stressed with growth media depleted of fetal calf serum (in only 0.1 % serum media), a model which have been shown to be an in-vitro model for fibrogenesis, galectin-3 was expressed reaching maximum at about 5 to 7 days post culture in 0.1 % serum media. An immunochemistry staining technique demonstrated increased expression of galectin-3 at day 6 post-stress in 0.1 % serum, while the addition of compound G was shown to significantly suppress the expression of galectin-3.
- EXAMPLE 11 Assessment of Therapy in a TAA-lnduced Liver Fibrosis Model
- FIG. 16 shows a statistically significant reduction in fibrotic area measured by digital morphometric analysis of Sirius red-stained liver sections when animals were treated with Compound G and Compound D when compared to vehicle controls, with greater reduction seen with Compound G when compared to Compound
- mice in which diabetes was induced and a high fat diet was administered were used as an experimental model. This is a proven model in which the mice consistently develop NASH with hepatocyte fat accumulation, evidence of hepatocyte toxicity, portal and lobular inflammatory infiltrates, peri-sinusoidal fibrosis, advanced fibrosis with nodule formation, cirrhosis, and ultimately hepatocellular carcinoma in a certain percentage of animals. NASH mice were treated biweekly IV at 9-12 weeks of study, as shown in Figure 17a-b.
- Figure 18 shows that mice in all groups gained weight over the time of the experiment with no differences between groups. This result indicates at a gross level that there was little toxic effect of the treatments on the animals and any changes detected are unlikely due to the overall health of the animals. Overall there were 2 deaths in the vehicle group (2/12, 17%) and one in the Compound D treated group (1/12, 8%), all related to liver disease as determined by postmortem examination by the veterinarian. There were no deaths in the Compound G treated group. This in vivo activity of lack of toxicity in the mice correlates with the lack of cytotoxicity seen in vitro cell line experiments.
- the NAFLD activity score was used to evaluate disease severity and gives points for three aspects of NASH pathology including, steatosis (0 ( ⁇ 5%), 1 (5- 33%), 2 (33-66%), or 3 (>66%)), hepatocyte ballooning (0 (none), 1 , (few), or 3 (many)), and lobular inflammation (0 (no foci), 1 ( ⁇ 2 foci/200x field), 2 (2-4 foci/200x field), or 3 (>4 foci/200x field)).
- the total number of points is the NAFLD activity score.
- Figure 19 shows a graphical depiction with statistical values of the NAFLD activity score in the three experimental groups. There was an improvement in NAFLD activity score in animals treated with Compound G and less of an effect with Compound D.
- FIG 20 shows a graphical depiction with statistical values of the percent collagen in the three experimental groups.
- Sirius red is a histological stain that has a specific affinity for collagen fibers, staining them red, and is therefore a quantitative tool for assessing the degree of fibrosis in liver biopsies.
- the area of Sirius red staining on liver histopathological sections from each of the three treatment groups was assessed using computer assisted morphometric analysis. Animals in both the early and late treatment groups had a marked reduction in collagen proportional area when treated with Compound G. Treatment with Compound D had an intermediate effect on collagen proportional area between vehicle control and Compound G.
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014043708A1 (en) | 2012-09-17 | 2014-03-20 | Traber Peter G | Method for enhancing specific immunotherapies in cancer treatment |
EP2755480A1 (en) * | 2011-09-16 | 2014-07-23 | Galectin Therapeutics Inc. | Galacto-rhamnogalacturonate compositions for the treatment of non-alcoholic steatohepatitis and non-alcoholic fatty liver disease |
US8828971B2 (en) | 2012-10-10 | 2014-09-09 | Galectin Therapeutics, Inc. | Galactose-pronged carbohydrate compounds for the treatment of diabetic nephropathy and associated disorders |
EP2858651A4 (en) * | 2012-06-06 | 2016-02-17 | Galectin Therapeutics Inc | Galacto-rhamnogalacturonate compositions for the treatment of diseases associated with elevated inducible nitric oxide synthase |
US9649327B2 (en) | 2011-12-28 | 2017-05-16 | Galectin Therapeutics, Inc. | Composition of novel carbohydrate drug for treatment of human diseases |
WO2021064729A1 (en) * | 2019-10-02 | 2021-04-08 | Patchor Ltd. | Injectable composition for the treatment of musculoskeletal disorders and methods of use thereof |
US11576924B2 (en) | 2017-05-12 | 2023-02-14 | Galectin Sciences, Llc | Compounds for the prevention and treatment of diseases and the use thereof |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008011216A2 (en) * | 2006-05-16 | 2008-01-24 | Pro-Pharmaceuticals, Inc. | Galactose-pronged polysaccharides in a formulation for antifibrotic therapies |
WO2014130648A1 (en) | 2013-02-20 | 2014-08-28 | Galectin Therapeutics, Inc. | Method for treatment of pulmonary fibrosis |
GB201502668D0 (en) * | 2015-02-17 | 2015-04-01 | Glycomar Ltd And Albert Bartlett & Sons Airdrie Ltd | Method for isolation of polysaccharides |
FR3036943A1 (en) * | 2015-06-02 | 2016-12-09 | Echosens | NON-INVASIVE HEPATIC LESION DETECTION DEVICE |
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US20190365790A1 (en) * | 2017-01-13 | 2019-12-05 | Sivanaray Inc. | Compounds, Compositions and Methods for Prevention or Treatment of Liver and Lipid-related Conditions |
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US20210138080A1 (en) | 2018-06-29 | 2021-05-13 | Glykos Biomedical Oy | Conjugates |
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WO2022016105A1 (en) * | 2020-07-17 | 2022-01-20 | Kaleido Biosciences, Inc. | Oligosaccharide compositions and methods of use |
CN116211884B (en) * | 2023-02-16 | 2024-05-17 | 海孵(海南自贸区)医疗科技有限责任公司 | Nasal cavity spray and preparation method thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060211653A1 (en) * | 1998-05-13 | 2006-09-21 | Yawei Ni | Pharmaceutical compositions comprising Aloe pectins, and methods for their production and use |
US20110046086A1 (en) * | 2008-04-07 | 2011-02-24 | Korea Institute Of Radiological & Medical Sciences | Composition Comprising Polysaccharide Extracted from Panax Ginseng Preventing and Treating Liver Diseases |
Family Cites Families (42)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2132577A (en) | 1934-08-28 | 1938-10-11 | Gen Foods Corp | Method of preparing pectin |
US2444266A (en) | 1944-03-31 | 1948-06-29 | Us Agriculture | Pectic materials and method of preparing same |
US2503258A (en) | 1948-01-23 | 1950-04-11 | Us Agriculture | Neutralization of pectinic acids with sodium bicarbonate |
US4016351A (en) | 1976-02-04 | 1977-04-05 | Eschinasi Emile H | Pectic substances with varying methoxyl content and process therefor |
US4268533A (en) | 1979-11-21 | 1981-05-19 | General Foods Corporation | Quick-set low methoxyl pectin composition |
EP0181351A4 (en) * | 1984-05-09 | 1989-06-21 | Univ Australian | A method for the modulation of the immune response. |
US4686106A (en) | 1986-06-16 | 1987-08-11 | General Foods Corporation | Pectin food product |
US5071970A (en) | 1988-11-23 | 1991-12-10 | University Of Florida | Process for producing pectin with a high-to-medium methoxyl content from beet pulp |
US5605938A (en) | 1991-05-31 | 1997-02-25 | Gliatech, Inc. | Methods and compositions for inhibition of cell invasion and fibrosis using dextran sulfate |
US7491708B1 (en) | 1993-03-01 | 2009-02-17 | David Platt | Modified pectin |
JP3360899B2 (en) | 1993-10-08 | 2003-01-07 | 日本医薬品工業株式会社 | Cancer prevention / treatment agent |
US5498702A (en) | 1993-12-16 | 1996-03-12 | California Natural Products | Treated pectinic acid process and product |
US5834442A (en) | 1994-07-07 | 1998-11-10 | Barbara Ann Karmanos Cancer Institute | Method for inhibiting cancer metastasis by oral administration of soluble modified citrus pectin |
US5681923A (en) | 1995-10-06 | 1997-10-28 | Platt; David | Tumor derived carbohydrate binding protein |
US6632797B2 (en) | 1996-06-24 | 2003-10-14 | Matti Siren | Method of treating angiogenesis |
KR100252194B1 (en) | 1997-10-10 | 2000-04-15 | 박호군 | Novel pectin type polysaccharide from Angelica gigas Nakai, purification process thereof |
US5929051A (en) * | 1998-05-13 | 1999-07-27 | Carrington Laboratories, Inc. | Aloe pectins |
US6500807B1 (en) | 1999-02-02 | 2002-12-31 | Safescience, Inc. | Modified pectin and nucleic acid composition |
IT1316986B1 (en) | 2000-01-25 | 2003-05-26 | Sigma Tau Ind Farmaceuti | GLYCOSAMINOGLICAN DERIVATIVES PARTIALLY DESULPHATED NONANTICOAGULANTS WITH ANTIANGIOGENIC ACTIVITY. |
US6770622B2 (en) | 2001-06-08 | 2004-08-03 | Gary A. Jarvis | N-terminally truncated galectin-3 for use in treating cancer |
US6680306B2 (en) | 2001-06-21 | 2004-01-20 | Glycogenesys, Inc. | Method for enhancing the effectiveness of cancer therapies |
US20030004132A1 (en) | 2001-06-22 | 2003-01-02 | Yan Chang | Method and material for treating immune diseases |
US6890906B2 (en) | 2001-11-21 | 2005-05-10 | Glycogenesys, Inc. | Method for controlling angiogenesis in animals |
US20040121981A1 (en) | 2001-11-21 | 2004-06-24 | Glycogenesys, Inc. | Method for controlling angiogenesis in animals |
KR100594353B1 (en) | 2002-05-28 | 2006-07-03 | 주식회사 엠디바이오알파 | Active fraction having anti-cancer and anti-metastic activity isolated from leaves and stems of Ginseng |
JP2004107295A (en) * | 2002-09-20 | 2004-04-08 | National Agriculture & Bio-Oriented Research Organization | Histamine release inhibitor |
US20040223971A1 (en) | 2003-04-07 | 2004-11-11 | Glycogenesys, Inc. | Composition and uses of galectin antagonists |
DE602004014485D1 (en) * | 2003-04-07 | 2008-07-31 | Prospect Therapeutics Inc | COMPOSITION AND APPLICATIONS OF GALECTIN ANTAGONISTS |
US20050008572A1 (en) | 2003-04-29 | 2005-01-13 | Ales Prokop | Nanoparticular tumor targeting and therapy |
US20050053664A1 (en) | 2003-09-08 | 2005-03-10 | Eliezer Zomer | Co-administration of a polysaccharide with a chemotherapeutic agent for the treatment of cancer |
EP1765874A1 (en) | 2004-03-26 | 2007-03-28 | Glycogenesys, Inc. | Modified pectins, compositions and methods related thereto |
ITFI20040174A1 (en) * | 2004-08-03 | 2004-11-03 | Protera S R L | ARYTHOLPHONAMIDIC DERIVATIVES OF HYDROXAMIC ACID WITH INHIBITORY ACTION OF METALLOPROTEINASE |
SI2439273T1 (en) | 2005-05-09 | 2019-05-31 | Ono Pharmaceutical Co., Ltd. | Human monoclonal antibodies to programmed death 1(PD-1) and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics |
WO2008011216A2 (en) * | 2006-05-16 | 2008-01-24 | Pro-Pharmaceuticals, Inc. | Galactose-pronged polysaccharides in a formulation for antifibrotic therapies |
US9646327B2 (en) | 2011-08-09 | 2017-05-09 | Yahoo! Inc. | Disaggregation to isolate users for ad targeting |
KR101974675B1 (en) | 2011-09-16 | 2019-05-02 | 갈렉틴 테라퓨틱스, 인크. | Galacto-rhamnogalacturonate compositions for the treatment of non-alcoholic steatohepatitis and non-alcoholic fatty liver disease |
AU2012322706A1 (en) * | 2011-10-12 | 2014-05-22 | Teva Pharmaceutical Industries Ltd. | Treatment of multiple sclerosis with combination of laquinimod and fingolimod |
CN104245717B (en) | 2011-12-28 | 2017-10-24 | 卡莱克汀医疗有限公司 | For the composition for the new Carbohydrate drugs for treating human diseases |
CN104619329B (en) * | 2012-06-06 | 2018-01-26 | 卡莱克汀医疗有限公司 | For treating the galactolipin rhamnose galacturonic ester composition of the disease associated with elevated nitric oxide synthase type |
EP2900061B1 (en) | 2012-09-17 | 2020-01-22 | Galectin Therapeutics Inc. | Method for enhancing specific immunotherapies in cancer treatment |
MX363178B (en) | 2012-10-10 | 2019-03-13 | Galectin Therapeutics Inc | Galactose-pronged carbohydrate compounds for the treatment of diabetic nephropathy and associated disorders. |
WO2014130648A1 (en) | 2013-02-20 | 2014-08-28 | Galectin Therapeutics, Inc. | Method for treatment of pulmonary fibrosis |
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- 2012-09-14 CN CN201280068334.1A patent/CN104245717B/en active Active
- 2012-09-14 CA CA2861492A patent/CA2861492C/en active Active
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060211653A1 (en) * | 1998-05-13 | 2006-09-21 | Yawei Ni | Pharmaceutical compositions comprising Aloe pectins, and methods for their production and use |
US20110046086A1 (en) * | 2008-04-07 | 2011-02-24 | Korea Institute Of Radiological & Medical Sciences | Composition Comprising Polysaccharide Extracted from Panax Ginseng Preventing and Treating Liver Diseases |
Non-Patent Citations (1)
Title |
---|
See also references of EP2797942A4 * |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2755480A1 (en) * | 2011-09-16 | 2014-07-23 | Galectin Therapeutics Inc. | Galacto-rhamnogalacturonate compositions for the treatment of non-alcoholic steatohepatitis and non-alcoholic fatty liver disease |
EP2755480A4 (en) * | 2011-09-16 | 2015-04-29 | Galectin Therapeutics Inc | Galacto-rhamnogalacturonate compositions for the treatment of non-alcoholic steatohepatitis and non-alcoholic fatty liver disease |
US9974802B2 (en) | 2011-12-28 | 2018-05-22 | Galectin Therapeutics, Inc. | Composition of novel carbohydrate drug for treatment of human diseases |
US11413303B2 (en) | 2011-12-28 | 2022-08-16 | Galectin Therapeutics, Inc. | Methods for treatment of arthritis |
US10799525B2 (en) | 2011-12-28 | 2020-10-13 | Galectin Therapeutics, Inc. | Composition of novel carbohydrate drug for treatment of human diseases |
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EP2858651A4 (en) * | 2012-06-06 | 2016-02-17 | Galectin Therapeutics Inc | Galacto-rhamnogalacturonate compositions for the treatment of diseases associated with elevated inducible nitric oxide synthase |
AU2013271585B2 (en) * | 2012-06-06 | 2018-02-01 | Galectin Therapeutics, Inc. | Galacto-rhamnogalacturonate compositions for the treatment of diseases associated with elevated inducible nitric oxide synthase |
US9763974B2 (en) | 2012-06-06 | 2017-09-19 | Galectin Therapeutics, Inc. | Galacto-rhamnogalacturonate compositions for the treatment of diseases associated with elevated inducible nitric oxide synthase |
US9872909B2 (en) | 2012-09-17 | 2018-01-23 | Galeotin Therapeutics, Inc. | Method for enhancing specific immunotherapies in cancer treatment |
US10398778B2 (en) | 2012-09-17 | 2019-09-03 | Galectin Therapeutics, Inc. | Method for enhancing specific immunotherapies in cancer treatment |
WO2014043708A1 (en) | 2012-09-17 | 2014-03-20 | Traber Peter G | Method for enhancing specific immunotherapies in cancer treatment |
US8828971B2 (en) | 2012-10-10 | 2014-09-09 | Galectin Therapeutics, Inc. | Galactose-pronged carbohydrate compounds for the treatment of diabetic nephropathy and associated disorders |
US11576924B2 (en) | 2017-05-12 | 2023-02-14 | Galectin Sciences, Llc | Compounds for the prevention and treatment of diseases and the use thereof |
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