heparitin-sulfate and phosphomannopentaose-sulfate

The heparan sulfate mimetic PI-88 (muparfostat) is a complex mixture of sulfated oligosaccharides that was identified in the late 1990s as a potent inhibitor of heparanase. In preclinical animal models it was shown to block angiogenesis, metastasis and tumor growth, and subsequently became the first heparanase inhibitor to enter clinical trials for cancer. It progressed to Phase III trials but ultimately was not approved for use. Herein we summarize the preparation, physicochemical and biological properties of PI-88, and discuss preclinical/clinical and structure-activity relationship studies. In addition, we discuss the PI-88-inspired development of related HS mimetic heparanase inhibitors with improved properties, ultimately leading to the discovery of PG545 (pixatimod) which is currently in clinical trials.

Topics: Animals; Antineoplastic Agents; Glucuronidase; Heparitin Sulfate; Humans; Neoplasms; Neovascularization, Pathologic; Oligosaccharides; Structure-Activity Relationship

Heparanase (Hpse) is an endo-β-d-glucuronidase that degrades the glycosaminoglycan heparan sulfate (HS) in basement membranes (BMs) to facilitate leukocyte migration into tissues. Heparanase activity also releases HS-bound growth factors from the extracellular matrix (ECM), a function that aids wound healing and angiogenesis. In disease states, the degradation of HS in BMs by heparanase is well recognized as an invasive property of metastatic cancer cells. Recent studies by our group, however, have identified unexpected new roles for heparanase and HS. First, we discovered that in Type 1 diabetes (T1D) (i) HS in the pancreatic islet BM acts as a barrier to invading cells and (ii) high levels of HS within the insulin-producing islet beta cells themselves are critical for beta cell survival, protecting the cells from free radical-mediated damage. Furthermore, catalytically active heparanase produced by autoreactive T cells and other insulitis mononuclear cells was shown to degrade intra-islet HS, increasing the susceptibility of islet beta cells to free radical damage and death. This totally novel molecular explanation for the onset of T1D diabetes opens up new therapeutic approaches for preventing disease progression. Indeed, administration of the heparanase inhibitor, PI-88, dramatically reduced T1D incidence in diabetes-prone NOD mice, preserved islet beta cell HS and reduced islet inflammation. Second, in parallel studies it has been shown that heparanase and HS can be transported to the nucleus of cells where they impact directly or indirectly on gene transcription. Based on ChIP-on-chip studies heparanase was found to interact with the promoters and transcribed regions of several hundred genes and micro-RNAs in activated Jurkat T cells and up-regulate transcription, with many of the target genes/micro-RNAs being involved in T cell differentiation. At the molecular level, nuclear heparanase appears to regulate histone 3 lysine 4 (H3K4) methylation by influencing the recruitment of demethylases to transcriptionally active genes. These studies have unveiled new functions for heparanase produced by T lymphocytes, with the enzyme mediating unexpected intracellular effects on T cell differentiation and insulin-producing beta cell survival in T cell-dependent autoimmune T1D.

Topics: Animals; Cell Proliferation; Diabetes Mellitus, Type 1; Enzyme Inhibitors; Extracellular Matrix; Free Radicals; Gene Expression Regulation; Glucuronidase; Heparitin Sulfate; Humans; Islets of Langerhans; Mice; Oligosaccharides; Signal Transduction; T-Lymphocytes

The heparan sulfate (HS) mimetic PI-88 is a promising inhibitor of tumor growth and metastasis expected to commence phase III clinical evaluation in 2007 as an adjuvant therapy for postresection hepatocellular carcinoma. Its anticancer properties are attributed to inhibition of angiogenesis via antagonism of the interactions of angiogenic growth factors and their receptors with HS. It is also a potent inhibitor of heparanase, an enzyme that plays a key role in both metastasis and angiogenesis. A series of PI-88 analogs have been prepared with enhanced chemical and biological properties. The new compounds consist of single, defined oligosaccharides with specific modifications designed to improve their pharmacokinetic properties. These analogs all inhibit heparanase and bind to the angiogenic fibroblast growth factor 1 (FGF-1), FGF-2, and vascular endothelial growth factor with similar affinity to PI-88. However, compared with PI-88, some of the newly designed compounds are more potent inhibitors of growth factor-induced endothelial cell proliferation and of endothelial tube formation on Matrigel. Representative compounds were also tested for antiangiogenic activity in vivo and were found to reduce significantly blood vessel formation. Moreover, the pharmacokinetic profile of several analogs was also improved, as evidenced primarily by lower clearance in comparison with PI-88. The current data support the development of HS mimetics as potent antiangiogenic anticancer agents.

Topics: Animals; Biomimetic Materials; Carcinoma, Hepatocellular; Cell Proliferation; Chemotherapy, Adjuvant; Clinical Trials, Phase III as Topic; Endothelial Cells; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Heparin Lyase; Heparitin Sulfate; Humans; Neoplasm Metastasis; Neovascularization, Pathologic; Oligosaccharides; Vascular Endothelial Growth Factor A

The sulfated oligosaccharide PI-88 is a potent antiangiogenic, antitumor and anti-metastatic agent derived from yeast. It is primarily composed of sulfated phosphomannopentaose and phosphomannotetraose oligosaccharide units and is presently under evaluation in Phase II clinical trials for anticancer efficacy. PI-88 inhibits the heparan sulfate-degrading enzyme heparanase, exhibits antiangiogenic activity and has anticoagulant properties mediated by heparin cofactor II. It also inhibits vascular smooth muscle cell proliferation, kinase signalling and arterial intimal thickening following balloon injury. Many heparan sulfate-binding growth factors require heparan sulfate as a co-receptor in order to effectively deliver growth signals to cells. Thus, the antiangiogenic and antirestenotic activity of PI-88 may be at least partially due to this highly sulfated oligosaccharide competing with the interaction of growth factors, such as FGF-2 and VEGF, with cell surface heparan sulfate. This heparan sulfate mimetic has, therefore, multiple functions and therapeutic potential in a variety of vascular disorders.

Topics: Angiogenesis Inhibitors; Anticoagulants; Antineoplastic Agents; Coronary Restenosis; Heparitin Sulfate; Humans; Molecular Mimicry; Oligosaccharides

Topics: Animals; Antineoplastic Agents; Carbohydrate Sequence; Cloning, Molecular; DNA, Complementary; Endopeptidases; Enzyme Precursors; Extracellular Matrix; Gene Expression Regulation, Enzymologic; Glucuronidase; Heparitin Sulfate; Humans; Molecular Sequence Data; Neoplasms; Oligosaccharides

Heparanase is the only confirmed endoglycosidase that cleaves heparan sulfate (HS), a ubiquitous glycosaminoglycan with various essential roles in multiple pathological processes. Thus, the development of heparanase inhibitors has become an attractive strategy for drug discovery, especially in tumour therapy, in which HS mimetics are the most promising compounds. The various biological effects of heparanase also suggest a role for HS mimetics in many non-cancer indications, such as type 1 diabetes. However, the potential benefits of HS mimetics in obesity-related type 2 diabetes have not been elucidated.. In this study, we investigated muparfostat (PI-88), a developed HS mimetic currently enrolled in Phase III clinical trials, in obese mouse models and in vitro cultured murine hepatocytes.. Daily administration of muparfostat for 4 weeks caused hyperlipidaemia and aggravated hepatic steatosis in obese mice models, but not in lean animals. In cultured hepatocytes, muparfostat did not alter lipid accumulation. Acute tests suggested that muparfostat binds to lipoprotein lipase in competition with HS on vascular endothelial cell surfaces, thereby reducing the degradation of circulating triglycerides by lipoprotein lipase and subsequent uptake of fatty acids into vascular endothelial cells and causing hyperlipidaemia. This hyperlipidaemia aggravates hepatic steatosis and causes liver injury in muparfostat-treated obese mice.. The binding activity of HS mimetics to lipoprotein lipase should be investigated as an additional pharmacological effect during heparanase inhibitor drug discovery. This study also provides novel evidence for an increased risk of drug-induced liver injury in obese individuals.

Topics: Animals; Diabetes Mellitus, Type 2; Endothelial Cells; Fatty Liver; Heparitin Sulfate; Lipoprotein Lipase; Mice; Mice, Obese

Heparan sulfates (HS) bind a diversity of protein ligands on the cell surface and in the extracellular matrix and thus can modulate cell signaling. The state of sulfation in glucosamines and uronic acids within the chains strongly influences their binding. We have previously cloned and characterized two human extracellular endoglucosamine 6-sulfatases, HSulf-1 and HSulf-2, which selectively liberate the 6-O sulfate groups on glucosamines present in N, 6-O, and 2-O trisulfated disaccharides of intact HS and heparins. These enzymes serve important roles in development and are upregulated in a number of cancers. To determine whether the Sulfs act on the trisulfated disaccharides that exist on the cell surface, we expressed HSulfs in cultured cells and performed a flow cytometric analysis with the RB4CD12, an anti-HS antibody that recognizes N- and O-sulfated HS saccharides. The endogenously expressed level of the cell surface RB4CD12 epitope was greatly diminished in CHO, HEK293, and HeLa cells transfected with HSulf-1 or HSulf-2 cDNA. In correspondence with the RB4CD12 finding, the N, 6-O, and 2-O trisulfated disaccharides of the HS isolated from the cell surface/extracellular matrix were dramatically reduced in the Sulf-expressed HEK293 cells. We then developed an ELISA and confirmed that the RB4CD12 epitope in immobilized heparin was degraded by purified recombinant HSulf-1 and HSulf-2, and conditioned medium (CM) of MCF-7 breast carcinoma cells, which contain a native form of HSulf-2. Furthermore, HSulf-1 and HSulf-2 exerted activity against the epitope expressed on microvessels of mouse brains. Both HSulf activities were potently inhibited by PI-88, a sulfated heparin mimetic with anti-cancer activities. These findings provide new strategies for monitoring the extracellular remodeling of HS by Sulfs during normal and pathophysiological processes.

Topics: Animals; Brain; Cells, Cultured; Cloning, Molecular; Cricetinae; Cricetulus; Enzyme Inhibitors; Epitopes; Heparitin Sulfate; Humans; Mice; Microvessels; Oligosaccharides; Recombinant Proteins; Structure-Activity Relationship; Sulfatases; Sulfotransferases

Heparanase-1 (HPR1), an endoglycosidase that specifically degrades heparan sulfate (HS) proteoglycans, is overexpressed in a variety of malignancies. Our present study sought to determine whether oncogene BRAF and RAS mutations lead to increased HPR1 expression. Reverse transcription-polymerase chain reaction analysis revealed that HPR1 gene expression was increased in HEK293 cells transiently transfected with a mutant BRAF or RAS gene. Flow cytometric analysis revealed that B-Raf activation led to loss of the cell surface HS, which could be blocked by two HPR1 inhibitors: heparin and PI-88. Cotransfection of a BRAF or RAS mutant gene with HPR1 promoter-driven luciferase reporters increased luciferase reporter gene expression in HEK293 cells. Knockdown of BRAF expression in a BRAF-mutated KAT-10 tumor cell line led to the suppression of HPR1 gene expression, subsequently leading to increased cell surface HS levels. Truncational and mutational analyses of the HPR1 promoter revealed that the Ets-relevant elements in the HPR1 promoter were critical for BRAF activation-induced HPR1 expression. Luciferase reporter gene expression driven by a four-copy GA binding protein (GABP) binding site was significantly lower in BRAF siRNA-transfected KAT-10 cells than in the control siRNA-transfected cells. We further showed that BRAF knockdown led to suppression of the expression of the GABPβ, an Ets family transcription factor involved in regulating HPR1 promoter activity. Taken together, our study suggests that B-Raf kinase activation plays an important role in regulating HPR1 expression. Increased HPR1 expression may contribute to the aggressive behavior of BRAF-mutated cancer.

Topics: Binding Sites; Blotting, Western; Cell Line, Tumor; Enzyme Activation; GA-Binding Protein Transcription Factor; Gene Expression Regulation, Enzymologic; Glucuronidase; HEK293 Cells; Heparin; Heparitin Sulfate; Humans; Luciferases; Microscopy, Fluorescence; Mutation; Oligosaccharides; Promoter Regions, Genetic; Proto-Oncogene Proteins B-raf; ras Proteins; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference

Many viruses, including flaviviruses, display affinity for cell surface heparan sulfate (HS) proteoglycans with biological relevance in virus attachment/entry. This raises the possibility of the application of HS mimetics in antiviral therapy. We have evaluated the antiviral effect of the sulfated polysaccharides, suramin, pentosan polysulfate (PPS) and PI-88, which are currently approved or in trial for clinical use, against dengue virus (DEN) and the encephalitic flaviviruses, Japanese encephalitis virus, West Nile virus, and Murray Valley encephalitis virus. A flow cytometry-based method for the measurement of inhibition of virus infectivity was developed, which showed the in vitro antiviral activity of the three compounds, albeit with differences in efficiency which were virus-dependent. The 50% effective concentration (EC(50)) values for DEN inhibition were in the order: PPS

Topics: Animals; Antiviral Agents; Cell Line; Dengue; Dengue Virus; Disease Models, Animal; Drug Evaluation, Preclinical; Encephalitis Viruses, Japanese; Encephalitis, Arbovirus; Female; Flavivirus Infections; Heparitin Sulfate; Injections, Intraperitoneal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oligosaccharides; Pentosan Sulfuric Polyester; Suramin; Treatment Outcome

Although a number of sulfated polysaccharides have been shown to inhibit infection of cells by herpes simplex virus (HSV), little is known about their effects on the cell-to-cell spread of the virus. These compounds act by inhibiting the virus binding to cells, and their antiviral potencies usually increase with increasing molecular weight and sulfation density. We report that the low molecular weight HS-mimetic, PI-88, which is a mixture of highly sulfated mannose-containing di- to hexa-saccharides, inhibited HSV infection of cells and cell-to-cell spread of HSV-1 and HSV-2. Compared to a relatively large heparin polysaccharide, PI-88 demonstrated weaker inhibition of HSV infectivity but more efficient reduction of cell-to-cell spread of HSV. A tetrasaccharide fraction of PI-88 was the minimum fragment necessary to inhibit HSV-1 infectivity, while a trisaccharide was sufficient to reduce cell-to-cell spread. A reduction in HSV lateral spread was also observed in cells incubated with another low molecular weight compound, pentosan polysulfate but not with much larger polysaccharide chondroitin sulfate E. Some differences as regards the effects of PI-88, heparin, protamine, poly-L-lysine and sodium chlorate on intercellular spread of HSV-1 and HSV-2 were found. We conclude that structurally different sulfated oligosaccharides are preferred for inhibition of HSV infectivity and the cell-to-cell spread. The latter was efficiently inhibited by a relatively small but densely sulfated PI-88 oligosaccharide, very likely due to the capability of the compound to access the narrow intercellular space.

Topics: Alphaherpesvirinae; Antiviral Agents; Cell Line; Heparitin Sulfate; Molecular Weight; Oligosaccharides; Sulfates

Percutaneous transluminal coronary angioplasty is a frequently used interventional technique to reopen arteries that have narrowed because of atherosclerosis. Restenosis, or renarrowing of the artery shortly after angioplasty, is a major limitation to the success of the procedure and is due mainly to smooth muscle cell accumulation in the artery wall at the site of balloon injury. In the present study, we demonstrate that the antiangiogenic sulfated oligosaccharide, PI-88, inhibits primary vascular smooth muscle cell proliferation and reduces intimal thickening 14 days after balloon angioplasty of rat and rabbit arteries. PI-88 reduced heparan sulfate content in the injured artery wall and prevented change in smooth muscle phenotype. However, the mechanism of PI-88 inhibition was not merely confined to the antiheparanase activity of this compound. PI-88 blocked extracellular signal-regulated kinase-1/2 (ERK1/2) activity within minutes of smooth muscle cell injury. It facilitated FGF-2 release from uninjured smooth muscle cells in vitro, and super-released FGF-2 after injury while inhibiting ERK1/2 activation. PI-88 inhibited the decrease in levels of FGF-2 protein in the rat artery wall within 8 minutes of injury. PI-88 also blocked injury-inducible ERK phosphorylation, without altering the clotting time in these animals. Optical biosensor studies revealed that PI-88 potently inhibited (Ki 10.3 nmol/L) the interaction of FGF-2 with heparan sulfate. These findings show for the first time the capacity of this sulfated oligosaccharide to directly bind FGF-2, block cellular signaling and proliferation in vitro, and inhibit injury-induced smooth muscle cell hyperplasia in two animal models. As such, this study demonstrates a new role for PI-88 as an inhibitor of intimal thickening after balloon angioplasty. The full text of this article is available online at http://www.circresaha.org.

Topics: Angioplasty, Balloon; Animals; Binding, Competitive; Carotid Arteries; Carotid Artery Injuries; Cell Division; Enzyme Activation; Fibroblast Growth Factor 2; Heparitin Sulfate; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Models, Biological; Muscle, Smooth, Vascular; Oligosaccharides; Rabbits; Rats; Rats, Wistar; Signal Transduction; Tunica Intima; Tunica Media; Whole Blood Coagulation Time