Synthetic oligosaccharides can replace animal-sourced low-molecular weight heparins.
ABSTRACT: Low-molecular weight heparin (LMWH) is used clinically to treat clotting disorders. As an animal-sourced product, LMWH is a highly heterogeneous mixture, and its anticoagulant activity is not fully reversible by protamine. Furthermore, the reliability of the LMWH supply chain is a concern for regulatory agencies. We demonstrate the synthesis of heparin dodecasaccharides (12-mers) at the gram scale. In vitro experiments demonstrate that the anticoagulant activity of the 12-mers could be reversed using protamine. One of these, labeled as 12-mer-1, reduced the size of blood clots in the mouse model of deep vein thrombosis and attenuated circulating procoagulant markers in the mouse model of sickle cell disease. An ex vivo experiment demonstrates that the anticoagulant activity of 12-mer-1 could be reversed by protamine. 12-mer-1 was also examined in a nonhuman primate model to determine its pharmacodynamic parameters. A 7-day toxicity study in a rat model showed no toxic effects. The data suggest that a synthetic homogeneous oligosaccharide can replace animal-sourced LMWHs.
Project description:The global supply of unfractionated heparin (UFH) and all commercially available low molecular weight heparins (LMWH) remain dependent on animal sources, such as porcine intestine or bovine lung. Recent experience has shown that contamination of the supply chain (with over-sulfated chondroitin sulfates) can result in lethal toxicity. Fondaparinux is currently the only commercially available synthetic analog of heparin. We recently described a new class of chemoenzymatically synthesized heparin analogs. One of these compounds (S12-mer) is a dodecasaccharide consisting of an antithrombin-binding moiety with repeating units of IdoA2S-GlcNS6S and two 3-O-sulfate groups that confer the ability to bind protamine.We sought to further characterize this new compound in vitro using biochemical and global coagulation assays and in vivo using thrombosis and hemostasis assays.The anticoagulant activities of the Super 12-mer (S12-mer) and Enoxaparin in anti-factor Xa and plasma-based thrombin generation assays were roughly equivalent with a 50% reduction in peak thrombin generation occurring at approximately 325nM. When protamine was titrated against a fixed concentration of S12-mer in plasma or blood, the S12-mer displayed a significant restitution of thrombin generation and clot formation. In vivo, S12-mer inhibited venous thrombosis to a similar extent as Enoxaparin, with similar bleeding profiles.These data show that the S12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the ability to ensure purity and homogeneity from batch to batch, the S12-mer is a promising new synthetic heparin analog with a potentially enhanced safety profile.
Project description:Heparin, a commonly used anticoagulant drug, is a mixture of highly sulfated polysaccharides with various molecular weights (MWs). The unique sulfation pattern dictates the anticoagulant activity of heparin. Commercial heparins are categorized into three forms according to their average MW: unfractionated heparin (UFH, MWavg 14,000), low-MW heparin (LMWH, MWavg 3500-6500) and the synthetic pentasaccharide (fondaparinux, MW 1508.3). UFH is isolated from porcine intestine while LMWH is derived from UFH by various methods of depolymerization, which generate a wide range of oligosaccharide chain lengths. Different degradation methods result in structurally distinct LMWH products, displaying different pharmacological and pharmacokinetic properties. In this report, we utilized a chemoenzymatic method to synthesize LMWH with the emphasis on controlling the size distribution of the oligosaccharides. A tetrasaccharide primer and a controlled enzyme-based polymerization were employed to build a narrow size oligosaccharide backbone. The oligosaccharide backbones were further modified by a series of sulfation and epimerization steps in order to obtain a full anticoagulation activity. Determination of the anticoagulation activity in vitro and ex vivo indicated that the synthetic LMWH has higher potency than enoxaparin, a commercial LMWH drug in clinical usage.
Project description:Heparin, a high-molecular weight acidic polysaccharide, has raised much interest in the field of biomedical research due to its multiple bio-functions. The anticoagulant application of heparin in routine clinical practice, however, has been limited as the large molecular size of heparin can reduce its subcutaneous bioavailability and lead to severe adverse consequences such as thrombocytopenia. Here, we report a highly efficient and convenient method to depolymerize high-molecular weight, unfractionated heparin (UFH), into low molecular weight heparin (LMWH) by combining physical ultrasonic treatment with the chemical Fenton reaction, referred to as sono-Fenton. We found that this combination treatment synergistically degraded UFH into a LMWH of 4.87?kDa within 20?min. We characterized the mechanism of sono-Fenton heparin degradation through multiple approaches, including HPLC-SAX, disaccharide composition, FT-IR, NMR and top-down analysis, and found that the uronic acid residue in heparin was the most susceptible site attacked by OH radicals produced in the sono-Fenton process. Importantly, the LMWH prepared by this method had significantly higher anticoagulant activity than UFH and other LMWHs. This approach represents an effective method to produce heparin with improved activity and should be potentially useful for heparin production in the pharmaceutical industry.
Project description:Low molecular weight heparin (LMWH) is being investigated as a potential preventative therapy against preeclampsia. There is evidence suggesting that LMWH may prevent preeclampsia through anticoagulation-independent mechanisms. In this study, we compared the in vitro placental, endothelial, and anti-inflammatory effects of a LMWH (dalteparin) with a non-anticoagulant, glycol-split heparin derivative (gsHep). In contrast with dalteparin, gsHep did not interact with antithrombin III, possess significant anti-Factor Xa activity, or significantly prolong in vitro plasma clotting time. However, dalteparin and gsHep were otherwise mechanistically similar, both interacting with soluble fms-like tyrosine kinase-1 (sFlt1) and promoting secretion of the pro-angiogenic protein placental growth factor, but not the anti-angiogenic sFlt1, from healthy placental villous explants. Placental explant media pre-treated with dalteparin or gsHep significantly stimulated endothelial cell tube formation compared to untreated explants. Lastly, dalteparin and gsHep both significantly suppressed inflammation by inhibiting complement activation and leukocyte adhesion to endothelial cells that were activated using serum from preeclamptic women. Our data suggest that non-anticoagulant heparin derivatives may be utilized as a tool to distinguish the anticoagulationindependent mechanisms of LMWH, and provide insight into the role of anticoagulation in the prevention of preeclampsia.
Project description:<h4>Background</h4>Heparins are usually produced from animal tissues. It is now possible to synthesize heparins. This provides the abilities to overcome shortages of heparin, to optimize biological effects, and to reduce adverse drug effects. Heparins interact with platelet factor 4 (PF4), which can induce an immune response causing thrombocytopenia. This side effect is called heparin-induced thrombocytopenia (HIT). We characterized the interaction of PF4 and HIT antibodies with oligosaccharides of 6-, 8-, 10-, and 12-mer size and a hypersulfated 12-mer (S12-mer).<h4>Methods</h4>We utilized multiple methodologies including isothermal calorimetry, circular dichroism spectroscopy, single molecule force spectroscopy (SMFS), enzyme immunosorbent assay (EIA), and platelet aggregation test to characterize the interaction of synthetic heparin analogs with PF4 and anti-PF4/heparin antibodies.<h4>Results</h4>The synthetic heparin-like compounds display stronger binding characteristics to PF4 than animal-derived heparins of corresponding lengths. Upon complexation with PF4, 6-mer and S12-mer heparins showed much lower enthalpy, induced less conformational changes in PF4, and interacted with weaker forces than 8-, 10-, and 12-mer heparins. Anti-PF4/heparin antibodies bind more weakly to complexes formed between PF4 and heparins ? 8-mer than with complexes formed between PF4 and heparins ? 10-mer. Addition of one sulfate group to the 12-mer resulted in a S12-mer, which showed substantial changes in its binding characteristics to PF4.<h4>Conclusions</h4>We provide a template for characterizing interactions of newly developed heparin-based anticoagulant drugs with proteins, especially PF4 and the resulting potential antigenicity.
Project description:Background ?The noninterventional XALIA study compared rivaroxaban with standard anticoagulation for deep vein thrombosis treatment. This substudy describes the demographics, clinical characteristics, and outcomes of the patients with cancer. Methods ?Therapy type, dose, and duration were at the physician's discretion. The cohorts identified were rivaroxaban (rivaroxaban alone or after heparin or fondaparinux for ?48 hours); early switchers (rivaroxaban after heparin or fondaparinux for >48 hours to 14 days and/or a vitamin K antagonist [VKA] for 1-14 days); standard anticoagulation (heparin or fondaparinux and a VKA); low-molecular-weight heparin (LMWH) alone; and miscellaneous (other heparins, fondaparinux alone, VKA alone). Primary outcomes were major bleeding, recurrent venous thromboembolism, and all-cause mortality. Results ?In XALIA, 587 patients (11.4% of the XALIA cohort) were with cancer: 146 (24.9%) rivaroxaban, 30 (5.1%) early switchers, 141 (24.0%) standard anticoagulation, 223 (38.0%) LMWH, and 47 (8.0%) miscellaneous. Patients with gastrointestinal or lung cancer more commonly received LMWH than rivaroxaban; the opposite occurred in patients with breast or genitourinary cancer. Rates of primary outcome in the rivaroxaban group were as follows: major bleeding, 1.4% ( n ?=?2); recurrent venous thromboembolism, 3.4% ( n ?=?5); and all-cause mortality, 4.8% ( n ?=?7). Conclusion ?In XALIA, physicians treated cancer-associated thrombosis with various anticoagulant regimens, most commonly LMWH. In addition, the choice of anticoagulant varied with cancer type. In rivaroxaban-treated patients, rates for the primary outcomes were low, suggesting that patients administered rivaroxaban were a good prognosis group.
Project description:Seven pharmaceutical heparins were investigated by oligosaccharide mapping by digestion with heparin lyase 1, 2, or 3, followed by high performance liquid chromatography analysis. The structure of one of the prepared mapping standards, ?UA-Gal-Gal-Xyl-O-CH(2)CONHCH(2)COOH (where ?UA is 4-deoxy-?-l-threo-hex-4-eno-pyranosyluronic acid, Gal is ?-d-galactpyranose, and Xyl is ?-d-xylopyranose) released from the linkage region using either heparin lyase 2 or heparin lyase 3 digestion, is reported for the first time. A size-dependent susceptibility of site cleaved by heparin lyase 3 was also observed. Heparin lyase 3 acts on the undersulfated domains of the heparin chain and does not cleave the linkages within heparin's antithrombin III binding site. Thus, a novel low molecular weight heparin (LMWH) is afforded on heparin lyase 3 digestion of heparin due to this unique substrate specificity, which has anticoagulant activity comparable to that of currently available LMWH.
Project description:Glioblastoma multiforme (GBM) is the most common primary brain tumor that is invariably lethal. Novel treatments are desperately needed. In various cancers, heparin and its low molecular weight derivatives (LMWHs), commonly used for the prevention and treatment of thrombosis, have shown therapeutic potential. Here we systematically review preclinical and clinical studies of heparin and LMWHs as anti-tumor agents in GBM. Even though the number of studies is limited, there is suggestive evidence that heparin may have various effects on GBM. These effects include the inhibition of tumor growth and angiogenesis in vitro and in vivo, and the blocking of uptake of extracellular vesicles. However, heparin can also block the uptake of (potential) anti-tumor agents. Clinical studies suggest a non-significant trend of prolonged survival of LMWH treated GBM patients, with some evidence of increased major bleedings. Heparin mimetics lacking anticoagulant effect are therefore a potential alternative to heparin/LMWH and are discussed as well.
Project description:An intimate interplay with platelets is an initial key issue for tumor cells in terms of hematogenous metastasis. Tumor cells activate platelets by different pathways and receive, upon forming a platelet cloak, protection from immune surveillance and support in metastatic niche creation. Therapeutic intervention with this early interaction is promising to antagonize the whole metastatic cascade. Here we aimed to investigate the capability of low molecular weight heparin (LMWH), unfractionated heparin (UFH), and a non-anticoagulant heparin derivative or FXa inhibitor fondaparinux to interfere with platelet activation by tumor cells. Coagulation-dependent and independent pathways of platelet activation by three tumor cell lines, and interference therewith were analyzed by fluorigenic thrombin formation assay, platelet aggregometry, ATP and VEGF release and endothelial tube formation assay. LMWH and UFH were found to repress various routes of platelet activation, reflected by attenuated endothelial tube formation. This confirms the duality of anti-coagulative and anti-adhesive properties of heparin. While non-anticoagulative heparin (RO-heparin) depressed platelets' ATP and VEGF release by contact inhibition sufficiently, fondaparinux just attenuated tissue factor mediated thrombin generation. Concluding, these data suggest that LMWH as a guideline-based drug for anticoagulative strategies in oncology is promising to provide additional benefit for interference with metastatic activities.
Project description:<h4>Background and purpose</h4>Cerebral venous thrombosis (CVT) is a rare but life-threatening disease. Timely and proper treatments are the keys in saving patients' life and preventing from permanent neurological deficits. We performed this network meta-analysis to evaluate the role of anticoagulation in CVT, especially for the patients accompanied with hemorrhagic stroke.<h4>Methods</h4>PubMed, Embase, Web of Science, Cochrane Database, and Chinese Biomedical (CBM) databases were searched comprehensively to select eligible articles (up to 30 June 2017). Network meta-analysis was performed based on classical frequency statistics.<h4>Results</h4>Around 14 studies comprising 1135 cases were included. Overall analysis showed that low-molecular weight heparin (LMWH) and unfractionated heparin (UFH) were more effective (LMWH vs placebo: OR 4.76, 95%CI: 2.56-8.33; UFH vs placebo: OR 4.12, 95%CI: 2.17-8.33), and safe (LMWH vs placebo: OR 0.22, 95%CI: 0.069-0.65; UFH vs placebo: OR 0.28, 95%CI: 0.058-0.99) than placebo in the management of CVT. Besides, LMWH showed more advantages than UFH; As for the patients accompanied with hemorrhagic stroke, LMWH and UFH were also better than placebo (efficacy: LMWH vs placebo: OR 20, 95%CI: 5.56-100; UFH vs placebo: OR 12.5, 95%CI: 3.7-33.3; safety: LMWH vs placebo: OR 0.18, 95%CI: 0.04-0.77; UFH vs placebo: OR 0.16, 95%CI: 0.04-0.6) in the management of CVT. In addition, LMWH was more effective than UFH for the patients accompanied with hemorrhagic stroke.<h4>Conclusion</h4>Anticoagulant treatment with heparin is safe and beneficial for patients with CVT, even for those accompanied with hemorrhagic stroke. Besides, LMWH is better than UFH in the management of CVT.