Structural Identification And Characterization Of A Multicomponent Glycosaminoglycan Drug

Sulodexide is a multicomponent glycosaminoglycan drug extracted from porcine intestinal with high purity.It is mainly composed of 80%of fast mobility heparin(FMH),20%of dermatan sulfate(DS),and no more than 2%of slow mobility heparin(SMH).Sulodexide possesses various biological activities,like anticoagulation,antithrombotic,anti-vascular disease and hypolipidemic.Comparing to heparin,the most commonly utilized anticoagulant drug in clinical practice,sulodexide has higher antithrombotic effect,longer half-life,higher lipolytic activity,but lower blood clotting and low hemorrhage parameters.In addition,sulodexide can be absorbed completely not only via intravenous injection and intramuscular injection,but also via oral administration with high bioavailability and good tolerance.The FMH,SMH and DS fractions of sulodexide are all belonging to the family of glycosaminoglycans(GAGs),which is a category of highly sulfated and negatively charged liner polysaccharide constructed of repeating disaccharides building block units of hexuronic acid(HexA)and hexosamine(HexN).The HexA residues are either glucuronic acid(GlcA)or iduronic acid(IdoA),while the HexN residues are two conformational isomers,N-acetyl-glucosamine(GlcN)and N-acetyl-galactosamine(GalN).The name of FMH and SMH are defined based on their electrophoretic mobility,both of them are consist of HexA alternating 1,4-linked to GlcN.The HexA can be modified with a 2-O-sulfo group.The GlcN can be N-acetylated(GlcNAc),N-sulfated(GlcNS),3-O-sulfated(GlcN3S)and 6-O-sulfated(GlcN6S).The average molecular weight of FMH is 7,000 Da,while SMH is 15,000 Da.DS is composed primarily of IdoA(1?3)linked 2-acetamido-2-deoxy-galactopyranosyl 4-sulfate(GalNAc4S)with the average molecular weight about 25,000 Da.Due to the multicomponent of FMH,SMH and DS,large molecular weight of each component and the high heterogeneity of structure,conventional analytical methods,such as nuclear magnetic resonance(NMR),high performance liquid chromatography(HPLC),electrophoresis and mass spectrometry(MS),are not able to characterize the structures of each component in sulodexide by now.This study,aiming at the challenge of the structural analysis of multicomponent and complicated structure of sulodexide,established a novel approach of isolating and characterizing sulodexide.FMH,SMH and DS were firstly separated through agarose gel electrophoresis based on their differences in electrophoretic mobility,and extracted by vivapure spin column,digested into building blocks utilizing the specificity of enzyme,and then characterized and quantified by LC-MS.By this method,31 building blocks and special structures were identified including 19 species from FMH fraction,10 species from SMH fraction and 13 species from DS fraction.The combination of electrophoresis and LC-MS in this approach,not only distinguished the SMH,FMH and DS fractions in sulodexide directly,but also realized the characterization of fine structures of these components.These building blocks could help us have a better understanding of the structural information and specialty about sulodexide.Moreover,this study can provides powerful evidence for mechanism investigation of sulodexide.Meanwhile,the research of fine structures is a powerful tool of evaluating the differences between the innovator and generic products of sulodexide in their raw material sources and production process,which can provide crucial information for drug quality control and drug safety assurance.