Ultra-low Molecular Weight Heparin Research Articles

Design of an Ultralow Molecular Weight Heparin That Resists Heparanase Biodegradation.

Heparanase, an endo-β-d-glucuronidase produced by a variety of cells and tissues, cleaves the glycosidic linkage between glucuronic acid (GlcA) and a 3-O- or 6-O-sulfated glucosamine, typified by the disaccharide -[GlcA-GlcNS3S6S]-, which is found within the antithrombin-binding domain of heparan sulfate or heparin. As such, all current forms of heparin are susceptible to degradation by heparanase with neutralization of anticoagulant properties. Here, we have designed a heparanase-resistant, ultralow molecular weight heparin as the structural analogue of fondaparinux that does not contain an internal GlcA residue but otherwise displays potent anticoagulant activity. This heparin oligosaccharide was synthesized following a chemoenzymatic scheme and displays nanomolar anti-FXa activity yet is resistant to heparanase digestion. Inhibition of thrombus formation was further demonstrated after subcutaneous administration of this compound in a murine model of venous thrombosis. Thrombus inhibition was comparable to that observed for enoxaparin with a similar effect on bleeding time.

A rechargeable anti-thrombotic coating for blood-contacting devices

Despite the potential of anti-thrombogenic coatings, including heparinized surfaces, to improve the performance of blood-contacting devices, the inevitable deterioration of bioactivity remains an important factor in device failure and related thrombotic complications. As a consequence, the ability to restore the bioactivity of a surface coating after implantation of a blood-contacting device provides a potentially important strategy to enhance its clinical performance. Here, we report the regeneration of a multicomponent anti-thrombogenic coating through use of an evolved sortase A to mediate reversible transpeptidation. Both recombinant thrombomodulin and a chemoenzymatically synthesized ultra-low molecular weight heparin were repeatedly and selectively immobilized or removed in a sequential, alternating, or simultaneous manner. The generation of activated protein C (aPC) and inhibition of activated factor X (FXa) was consistent with the molecular composition of the surface. The fabrication of a rechargeable anti-thrombogenic surface was demonstrated on an expanded polytetrafluoroethylene (ePTFE) vascular graft with reconstitution of the surface bound coating 4 weeks after in vivo implantation in a rat model.

A highly active heparinase I from Bacteroides cellulosilyticus: Cloning, high level expression, and molecular characterization.

As one of the most extensively studied glycosaminoglycan lyases, heparinase I has been used in producing low or ultra-low molecular weight heparin. Its’ important applications are to neutralize the heparin in human blood and analyze heparin structure in the clinic. However, the low productivity and activity of the enzyme have greatly hindered its applications. In this study, a novel Hep-I from Bacteroides cellulosilyticus (BcHep-I) was successfully cloned and heterologously expressed in E. coli BL21 (DE3) as a soluble protein. The molecular mass and isoelectric point (pI) of the enzyme are 44.42 kDa and 9.02, respectively. And the characterization of BcHep-I after purified with Ni-NTA affinity chromatography suggested that it is a mesophilic enzyme. BcHep-I can be activated by 1 mM Ca2+, Mg2+, and Mn2+, while severely inhibited by Zn2+, Co2+, and EDTA. The specific activity of the enzyme was 738.3 U·mg-1 which is the highest activity ever reported. The Km and Vmax were calculated as 0.17 mg·mL-1 and 740.58 U·mg-1, respectively. Besides, the half-life of 300 min at 30°C showed BcHep-I has practical applications. Homology modeling and substrate docking revealed that Gln15, Lys74, Arg76, Lys104, Arg149, Gln208, Tyr336, Tyr342, and Lys338 were mainly involved in the substrate binding of Hep-I, and 11 hydrogen bonds were formed between heparin and the enzyme. These results indicated that BcHep-I with high activity has great potential applications in the industrial production of heparin, especially in the clinic to neutralize heparin.

Research progress of heparinase in the field of medicine

Heparinases can produce biologically active oligosaccharides by specifically cleaving the α-(1,4) glycosidic linkages of heparin and heparan sulphate. Heparinases are divided into heparinase and heparanase. Because heparinase is an effective biocatalyst, more and more researchers pay attention to the application of heparinase in medical field in the recent years. Combined with the related research work in our group, the application value of heparinase in the medical field was summarized, such as the determination of the structure of heparin, the preparation of low-molecular-weight heparin and ultra-low-molecular-weight heparin, tumor therapy and as a heparin antagonist. In addition, we summarized the definition, source of heparinase and its application in the medicine field. Heparinases have a great application prospect in the field of medicine.

Primary prophylaxis for venous thromboembolism in ambulatory cancer patients receiving chemotherapy.

Venous thromboembolism (VTE) often complicates the clinical course of cancer. The risk is further increased by chemotherapy, but the trade-off between safety and efficacy of primary thromboprophylaxis in cancer patients treated with chemotherapy is uncertain. This is the second update of a review first published in February 2012. To assess the efficacy and safety of primary thromboprophylaxis for VTE in ambulatory cancer patients receiving chemotherapy compared with placebo or no thromboprophylaxis. For this update the Cochrane Vascular Information Specialist searched the Cochrane Vascular Group Specialised Register (June 2016). In addition, the Information Specialist searched the Cochrane Central Register of Controlled Trials (CENTRAL) (2016, Issue 5). Clinical trials registries were searched up to June 2016. Randomised controlled trials comparing any oral or parenteral anticoagulant or mechanical intervention to no thromboprophylaxis or placebo, or comparing two different anticoagulants. We extracted data on methodological quality, participant characteristics, interventions, and outcomes including symptomatic VTE and major bleeding as the primary effectiveness and safety outcomes, respectively. We identified five additional randomised controlled trials (2491 participants) in the updated search, considering in this update 26 trials with a total of 12,352 participants, all evaluating pharmacological interventions and performed mainly in people with locally advanced or metastatic cancer. The quality of the evidence ranged from high to very low across the different outcomes and comparisons. The main limiting factors were imprecision and risk of bias. One large trial of 3212 participants found a 64% (risk ratio (RR) 0.36, 95% confidence interval (CI) 0.22 to 0.60) reduction of symptomatic VTE with the ultra-low molecular weight heparin (uLMWH) semuloparin relative to placebo, with no apparent difference in major bleeding (RR 1.05, 95% CI 0.55 to 2.00). When compared with no thromboprophylaxis, LMWH significantly reduced the incidence of symptomatic VTE (RR 0.54, 95% CI 0.38 to 0.75; no heterogeneity, Tau2 = 0.00%) with a non-statistically significant 44% higher risk of major bleeding events (RR 1.44, 95% CI 0.98 to 2.11). In participants with multiple myeloma, LMWH was associated with a significant reduction in symptomatic VTE compared with the vitamin K antagonist warfarin (RR 0.33, 95% CI 0.14 to 0.83), while the difference between LMWH and aspirin was not statistically significant (RR 0.51, 95% CI 0.22 to 1.17). Major bleeding was observed in none of the participants treated with LMWH or warfarin and in less than 1% of those treated with aspirin. Only one study evaluated unfractionated heparin against no thromboprophylaxis but did not report on VTE or major bleeding. When compared with placebo, warfarin was associated with a non-statistically significant reduction of symptomatic VTE (RR 0.15, 95% CI 0.02 to 1.20). Antithrombin, evaluated in one study involving paediatric patients, had no significant effect on VTE or on major bleeding when compared with no antithrombin. The direct oral factor Xa inhibitor apixaban was evaluated in a phase II dose-finding study that suggested a low rate of major bleeding (2.1% versus 3.4%) and symptomatic VTE (1.1% versus 13.8%) in comparison with placebo. In this second update, we confirmed that primary thromboprophylaxis with LMWH significantly reduced the incidence of symptomatic VTE in ambulatory cancer patients treated with chemotherapy. In addition, the uLMWH semuloparin, which is not commercially available, significantly reduced the incidence of symptomatic VTE. The risk of major bleeding associated with LMWH, while not reaching statistical significance, suggest caution and mandate additional studies to determine the risk-to-benefit ratio of LMWH in this setting. Despite the encouraging results of this review, routine prophylaxis in ambulatory cancer patients cannot be recommended before safety issues are adequately addressed. We need additional studies investigating targeted primary prophylaxis in people with specific types or stages of cancer associated with a higher risk of VTE.

Analysis of heparin oligosaccharides by capillary electrophoresis-negative-ion electrospray ionization mass spectrometry.

Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins. Graphical abstract Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins.

Heparin thromboprophylaxis in critically ill patients: Is it really changing outcome?

Heparin thromboprophylaxis in critically ill patients: Is it really changing outcome?

Chemoenzymatic synthesis of heparan sulfate and heparin.

Heparan sulfate is a polysaccharide that plays essential physiological functions in the animal kingdom. Heparin, a highly sulfated form of heparan sulfate, is a widely prescribed anticoagulant drug worldwide. The heparan sulfate and heparin isolated from natural sources are highly heterogeneous mixtures differing in their polysaccharide chain lengths and sulfation patterns. The access to structurally defined heparan sulfate and heparin is critical to probe the contribution of specific sulfated saccharide structures to the biological functions as well as for the development of the next generation of heparin-based anticoagulant drugs. The synthesis of heparan sulfate and heparin, using a purely chemical approach, has proven extremely difficult, especially for targets larger than octasaccharides having a high degree of site-specific sulfation. A new chemoenzymatic method has emerged as an effective alternative approach. This method uses recombinant heparan sulfate biosynthetic enzymes combined with unnatural uridine diphosphate-monosaccharide donors. Recent examples demonstrate the successful synthesis of ultra-low molecular weight heparin, low-molecular weight heparin and bioengineered heparin with unprecedented efficiency. The new method provides an opportunity to develop improved heparin-based therapeutics.

Heparin Dodecasaccharide Containing Two Antithrombin-binding Pentasaccharides: STRUCTURAL FEATURES AND BIOLOGICAL PROPERTIES

The antithrombin (AT) binding properties of heparin and low molecular weight heparins are strongly associated to the presence of the pentasaccharide sequence AGA*IA (A(NAc,6S)-GlcUA-A(NS,3,6S)-I(2S)-A(NS,6S)). By using the highly chemoselective depolymerization to prepare new ultra low molecular weight heparin and coupling it with the original separation techniques, it was possible to isolate a polysaccharide with a biosynthetically unexpected structure and excellent antithrombotic properties. It consisted of a dodecasaccharide containing an unsaturated uronate unit at the nonreducing end and two contiguous AT-binding sequences separated by a nonsulfated iduronate residue. This novel oligosaccharide was characterized by NMR spectroscopy, and its binding with AT was determined by fluorescence titration, NMR, and LC-MS. The dodecasaccharide displayed a significantly increased anti-FXa activity compared with those of the pentasaccharide, fondaparinux, and low molecular weight heparin enoxaparin.

Extended venous thromboembolism prophylaxis in patients undergoing hip fracture surgery – the SAVE-HIP3 study

There is currently limited information available on the benefits and risks of extended thromboprophylaxis after hip fracture surgery. SAVE-HIP3 was a randomised, double-blind study conducted to evaluate the efficacy and safety of extended thromboprophylaxis with the ultra-low molecular-weight heparin semuloparin compared with placebo in patients undergoing hip fracture surgery. After a seven- to ten-day open-label run-in phase with semuloparin (20 mg once daily subcutaneously, initiated post-operatively), patients were randomised to once-daily semuloparin (20 mg subcutaneously) or placebo for 19 to 23 additional days. The primary efficacy endpoint was a composite of any venous thromboembolism (VTE; any deep-vein thrombosis and non-fatal pulmonary embolism) or all-cause death until day 24 of the double-blind period. Safety parameters included major and clinically relevant non-major bleeding, laboratory data, and treatment-emergent adverse events (TEAEs). Extended thromboprophylaxis with semuloparin demonstrated a relative risk reduction of 79% in the rate of any VTE or all-cause death compared with placebo (3.9% vs 18.6%, respectively; odds ratio 0.18 (95% confidence interval 0.07 to 0.45), p < 0.001). Two patients in the semuloparin group and none in the placebo group experienced clinically relevant bleeding. TEAE rates were similar in both groups. In conclusion, the SAVE-HIP3 study results demonstrate that patients undergoing hip fracture surgery benefit from extended thromboprophylaxis.

Neuroprotective Effects of Ultra-Low-Molecular-Weight Heparin on Cerebral Ischemia/Reperfusion Injury in Rats: Involvement of Apoptosis, Inflammatory Reaction and Energy Metabolism

Previous experiments showed that ultra-low-molecular-weight heparin (ULMWH) reduced the infarct and neurologic deficit in rats followed by transient cerebral ischemia, but the mechanisms of its neuroprotective effect are unclear. This study reported the effect of ULMWH on energy metabolism, inflammatory reaction and neuronal apoptosis. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 24 h. ULMWH (0.5, 1 mg/kg, i.v.) was administered after the MCAO and reperfusion. 24 h after the reperfusion, Spectrophotometric assay was used to determine the activity of ATPase and the content of lactic acid in the brain. The ICAM-1 and Caspase-3 genes were investigated by RT-PCR. Furthermore, the apoptotic percentage of cells in hippocampus was quantified by flow cytometry. Compared with the model group, ULMWH significantly decreased lactic acid content and increased ATPase activity in ischemic brain. At the same time, ULMWH inhibited the neural apoptosis and decreased the expressions of ICAM-1 and Caspase-3 mRNA in hippocampus. These findings suggest that ULMWH exhibits a neuroprotective effect against cerebral ischemia/reperfusion injury, partly through improving energy metabolism, inhibiting apoptosis and attenuating inflammatory reaction.

Effect of Heparin and Its Derivatives On the Progression of Tumor Growth in Mouse Lewis Lung Carcinoma Model.

Abstract 2274 Background:Unfractionated heparin and it derivatives are known to produce pleotropic effects including the anticoagulant, anti-inflammatory and antitumor actions. Preclinical evidence suggests that heparins have an effect on tumor progression independent of their anticoagulant activity. Heparin and low molecular weight heparin have also been shown to exhibit interactions with growth factors and other cellular receptors. Recently an ultra low molecular weight heparin namely, semuloparin has also been found to be effective in the management of cancer associated thrombosis (Save ONCO trial). This study was designed to investigate whether heparin and its derivatives are able to inhibit tumor growth in a Lewis Lung carcinoma model. Methods:Female C57BL/6 mice were obtained at 6–8 weeks of age and were implanted with 5×105 LN7 tumor cells by dorsal subcutaneous injection in the upper back. When tumors were initially palpable, at 7–10 days of growth, mice were treated with subcutaneous injections of heparin, a low molecular weight heparin (LMWH), namely enoxaparin, an ultra LMWH, semuloparin or saline, daily for two weeks at approximately 1 cm away from the tumor growth, in a dose range of 1.0 – 0.25 mg/kg. After the treatment period, animals were sacrificed and the spleens and tumors were removed and tumor weight, tumor volume, spleen weight and spleen size were measured. Blood samples were drawn through heart puncture for ex vivo analysis. VEGF levels were measured using a commercially available ELISA method. Results:At the 1.0 and 0.5 mg/kg dosages, both enoxaparin (p<0.01) and semuloparin (p<0.01) showed a decrease in tumor volume compared to the saline control animals. At the 1.0 mg/kg dosage although heparin was effective in reducing the tumor size, the mortality was higher in this group due to bleeding. At 0.5 mg/kg heparin was not different from the saline control in terms of reducing tumor growth. In addition, at a dosage of 0.25 mg/kg, only semuloparin showed a significant difference in reducing the tumor size in comparison to saline control (p<0.01). Similar results were observed for the tumor weight. There were no significant differences noted in spleen weight or spleen size among these agents. The mortality rates in the mice treated with enoxaparin and semuloparin were relatively lower (<10%) and were comparable between the two groups. Ex vivo analysis of blood showed a significant difference in the VEGF levels in the semuloparin group in comparison to enoxaparin and heparin. Conclusions:These studies suggest that heparin and its derivatives are capable of inhibiting tumor growth in a dose dependent manner. Enoxaparin and semuloparin are more effective at reducing tumor growth compared to heparin. In addition, both the enoxaparin and semuloparin were safer than heparin at the 1.0 and 0.5 mg dosages as bleeding was observed in the heparin treated animals. Semuloparin downregulated the VEGF levels in comparison to the other groups suggesting that this mechanism potentially plays a role in the control of the tumor growth. Reported clinical studies have shown that semuloparin is safer and effective for the prevention of venous thromboembolism in cancer patients and compares favorably to enoxaparin in terms of antithrombotic effect and safety profile. Therefore, semuloparin may be a better alternate for the management of cancer associated thrombosis. Disclosures:No relevant conflicts of interest to declare.

Primary Venous Thromboembolism Prophylaxis in Patients with Solid Tumors Receiving Chemotherapy: A Meta-Analysis

Abstract 1157 Background:Venous thromboembolism [VTE] is the second highest cause of mortality among patients with cancer. However, pharmacological thromboprophylaxis for patients with solid tumor is only recommended during hospitalization. Primary outpatient thromboprophylaxis is not a widely accepted practice. Objective:Determine safety and efficacy of outpatient primary VTE prophylaxis in patients with solid tumors. Data sources:A systematic review was conducted using MEDLINE and EMBASE up to June 2012. Key search words included venous thromboembolism, malignancy, anticoagulants, and chemotherapy. Studies were considered for our meta-analysis if they included outpatient primary pharmacological thromboprophylaxis in adult patients with active solid cancer. All the information was independently reviewed by 2 of the authors [MP, SJ] and a third reviewer resolved discrepancies. The measure of association was calculated with R (R: A Language and Environment for Statistical, R Development Core Team, www.R-project.org), R META package (Version 0.8–2, Author: Guido Schwarzer). The Q statistic was calculated and a formal test of homogeneity was conducted. Random-effects model was preferred in case of heterogeneity. Results:A total of 1371 abstracts were reviewed and 29 manuscripts were fully abstracted. Eight randomized controlled trials including 6706 patients were analyzed. There were less VTE events with outpatient prophylaxis: odds ratio [OR] of 0.53 (95% CI, 0.40–0.70). Six studies used low or ultra-low molecular weight heparin and two studies used warfarin. In the subgroup analysis of heparin based primary prophylaxis, there was a significant reduction in VTE events [OR 0.47, 95% CI, 0.34–0.64], no significant heterogeneity [FIG 1]. In addition, there was no difference in major bleeding events between groups [OR 1.48, 95% CI, 0.89–2.46]. Five studies reported mortality data; there was significant heterogeneity between studies. Conclusions:Heparin based outpatient VTE prophylaxis in patients with solid tumors reduced by half the risk of VTE with no significant differences in major bleeding events. The current publications do not allow a meaningful grouped analysis of survival data, improved patient selection is necessary in order to adequately target VTE prevention strategies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

98IN - Thrombosis in Cancer Patients

ABSTRACT Arterial thrombosis is not a specific cancer related disorder except in some patients on some antiangiogenic drugs, but remains an infrequent event compared to venous thromboembolism (VTE). VTE is the second most frequent cause of death in cancer patients and is a predictor of mortality in hospitalized cancer patients. The death rate is about 30% in cancer patients when VTE occurs within 3 months of diagnosis. About 15% of patients suffering from cancer will have a VTE during the course of their disease and about 10% of patients with an idiopathic (?) VTE develop a cancer in the following two years. Risk factors for VTE in oncology are patient dependent, treatment dependent, type of cancer and stage dependent, The pathophysiology of VTE is complex. In addition to the standard causes, biomarkers and a clinical risk assessment score can be useful in deciding on time and duration of VTE prophylaxis and treatment. Prophylactic treatment in the ambulatory cancer patient needs to be discussed as newer anticoagulants like ultra low molecular weight heparin have been used recently in clinical trials and other novel agents (dabigatran, rivaroxaban and apixaban) marketed for orthopedic surgery and atrial fibrillation have become routine. The latter drugs are given orally, have negligeable interference with other medications or with foodstuffs and do not need laboratory surveillance. Approval for market release in VTE therapy in the general medical situation is pending and will certainly be used in the oncological setting if cost compatible. Various guidelines (ESMO, ASCO, NCCN …) are concurrent in their recommendations depending on disease activity, anti-cancer treatments (surgery, chemotherapy …) and the patient's clinical situation. These will be discussed in regards to the ambulatory and hospitalized patient, for first episode and for recurrent VTE. Some data are available though not presently recommended, for anticoagulant therapy in oncology for improving survival. Disclosure The author has declared no conflicts of interest.

The effect of tissue factor pathway inhibitor release and interactions with growth factors on the antitumor effects of ultra low molecular weight heparin semuloparin.

e13117 Background: Heparins are known to produce anti-tumor effects beside other pharmacologic actions. More recently an ultra LMWH has been developed for the prevention of thrombosis in cancer patients. Initial studies demonstrated that this agent also exhibits anti-tumor effects in animal models. To understand the mechanism of anti-tumor effects of this agent studies were designed to fractionate this ULMWH in to various AT affinity fractions. Methods: An ULMWH (semuloparin, Sanofi-aventis) and its high (HAF) and low (LAF) affinity fractions were prepare by AT affinity chromatography. These agents were administered at 1 mg/kg to primates via intravenous route. Blood samples were collected for a period of up to 6 hours. TFPI release and anti-Xa activities were measured. The interactions of ULMWH and its fractions with various growth factors such as FGF, VEGF, EGF and beta TGF were measured using a mass spectral method employing SELDI technique. The anti-tumor effects of each of these agents were measured using the mouse model of Lewis lung carcinoma. Results: In comparison to ULMWH the LAF showed minimal anti Xa activity whereas HAF exhibited a higher activity. All agents exhibited a similar release of the TFPI; however, HAF exhibited slightly higher release. All agents also showed varying degrees of binding to growth factors as measured by peak intensities. All agents produce anti-tumor effects in the mouse model resulting in a significant reduction in tumor size. Conclusions: Semuloparin has been clinically proven to prevent venous thromboembolism in patients with cancer (SAVE ONCO). These effects are attributable to the inhibition of factor Xa and the generation of thrombin. The fact that LAF mediates its anti-tumor effects without producing anti Xa effects suggest that these may be related to the release of TFPI. Furthermore the ULMWH, HAF and LAF all interact with growth factors suggest that these interactions may also contribute to anti-tumor effects of semuloparin, therefore, beside antithrombotic effects this agent also exhibits oncostatic effects.

Effect of heparin and its derivatives on the progression of tumor growth in mouse Lewis lung carcinoma model.

e13115 Background: Preclinical evidence suggests that heparins have an effect on tumor progression independent of their anticoagulant activity. Heparins have also been shown to exhibit interactions with growth factors and other cellular receptors. This study was designed to investigate whether heparin and its derivatives are able to inhibit tumor growth. Methods: Female C57BL/6 mice were obtained at 6-8 weeks of age and were implanted with 5X105 LN7 tumor cells by dorsal subcutaneous injection of in the upper back. When tumors were first palpable, after 7-10 days of growth, mice were treated with subcutaneous injections of heparin, a low molecular weight heparin (LMWH), namely enoxaparin, an ultra LMWH, semuloparin or saline, daily for two weeks in a dose range of 1.0 – 0.25 mg/kg. After the treatment period, animals were sacrificed and the spleens and tumors were removed and their weight, volume, spleen weight and size were measured. Results: At the 1.0 and 0.5 mg/kg dosages, both enoxaparin (p&lt;0.01) and semuloparin (p&lt;0.01) showed a decrease in tumor volume compared to the saline control animals. At the 1.0 mg/kg dosage, the mortality was high in the heparin group due to bleeding. At 0.5 mg/kg heparin was not different from the saline control. In addition, at a dosage of 0.25 mg/kg, only semuloparin showed a difference compared to the saline control (p&lt;0.01). Similar results were observed for the tumor weight. There were no significant differences noted in spleen weight or spleen size among these agents. The mortality rates between the mice treated with enoxaparin and semuloparin were comparable. Conclusions: These studies suggest that heparin and its derivatives are capable of inhibiting tumor growth in a dose dependent manner. Enoxaparin and semuloparin are more effective at reducing tumor growth compared to heparin. Clinical studies have shown that semuloparin is safe and effective for the prevention of venous thromboembolism in cancer patients and compares favorably to enoxaparin in terms of antithrombotic effect and safety profile. Therefore, semuloparin may be a better alternate for the prevention of cancer associated thrombosis.

Non‐antithrombin affinity Semuloparin and it's component oligosaccharides release endogenous TFPI in a molecular weight dependent fashion

Semuloparin (S) is an ultra low molecular weight heparin (uLMWH) with a unique composition. The purpose of this study was to determine the effect of non‐AT affinity semuloparin (NATAS) and it's oligosaccharide components on TFPI release. Non‐human primates (n=4–6; Macaca mulatta) were administered with 1.0 mg/kg IV, S, it's non‐AT affinity fraction and isolated hexasaccharide (H), dodecasaccharide (D) and octasaccharide (O) components. Citrated plasma samples were drawn at baseline and 15 minutes post‐administration. TFPI antigen levels by ELISA and tissue factor mediated thrombin generation using a fluorogenic assay were measured. S produced a sizable release of TFPI (156 ± 15 ng/ml). NATAS produced a slightly lower but statistically significant release of TFPI (142 ± 12 ng/ml). The oligosaccharide components produced a molecular weight dependent release of TFPI, with H producing minimal TFPI release (86 ± 8 ng/ml) vs. saline (78 ± 9 ng/ml). D produced a statistically significant increase in the release of TFPI (116+15 ng/ml). While S exhibited a measurable anti‐Xa response and produced a strong inhibition of thrombin generation (84% inhibition) whereas NATAS and it's fractions produced &lt; 20% inhibition of thrombin generation. These results suggest that the NATAS and it's oligosaccharide components enhance TFPI antigen release which may mediate some of it's pharmacodynamic actions.

Evaluation of the Antithrombotic and Hemorrhagic Effects of a Novel Ultra Low Molecular Weight Heparin (ULMWH)

Semuloparin (S) (Sanofi‐Aventis, Paris, France) is an ULMWH exhibiting high anti‐Xa (aXa) activity due to preservation of antithrombin‐binding sites. Antithrombotic (jugular vein clamping model) and hemorrhagic (tail transection model) effects of S, the LWMH enoxaparin (E) and heparin (H) were compared in Sprague‐Dawley rats treated subcutaneously (SC) with doses of 1.0 and 2.5 mg/kg (n=5). Blood samples collected prior to euthanasia were analyzed using aPTT, aXa and Heptest (HT) assays. 1.0 mg/kg SC H significantly prolonged bleeding time compared to control (C). All agents had equivalent antithrombotic effects. S and E significantly increased HT prolongation and aXa activity compared to C and H (HT: p&lt;0.001 A vs H; p&lt;0.05 E vs H; Xa: p&lt;0.001 A vs H; p&lt;0.01 E vs H). At 2.5 mg/kg SC, only S did not significantly prolong bleeding time compared to C. Only H significantly increased aPTT compared to C. Prolongation of HT and aXa activity was observed with all agents. S exhibited a more potent aXa activity than H (p&lt;0.001 S vs H). All agents demonstrated a dose‐dependent antithrombotic effect. Bleeding time was lower with S at both dosages, suggesting a better safety profile. AXa activity with S was closely related to its antithrombotic activity. These studies suggest that S represents a new alternative for the prevention of thrombotic disorders with an improved bene&amp;#64257;t/risk pro&amp;#64257;le compared with standard H and E.

Chemoenzymatic synthesis of homogeneous ultralow molecular weight heparins.

Ultralow molecular weight (ULMW) heparins are sulfated glycans that are clinically used to treat thrombotic disorders. ULMW heparins range from 1500 to 3000 daltons, corresponding from 5 to 10 saccharide units. The commercial drug Arixtra (fondaparinux sodium) is a structurally homogeneous ULMW heparin pentasaccharide that is synthesized through a lengthy chemical process. Here, we report 10- and 12-step chemoenzymatic syntheses of two structurally homogeneous ULMW heparins (MW = 1778.5 and 1816.5) in 45 and 37% overall yield, respectively, starting from a simple disaccharide. These ULMW heparins display excellent in vitro anticoagulant activity and comparable pharmacokinetic properties to Arixtra, as demonstrated in a rabbit model. The chemoenzymatic approach is scalable and shows promise for a more efficient route to synthesize this important class of medicinal agent.

Modulation of ultra-low-molecular-weight heparin on [Ca2+]i in nervous cells

Heparin is an effective competitive antagonist of inositol 1,4,5-trisphosphate receptors (IP3Rs). It binds to IP3Rs and affects calcium homeostasis. Ultra-low-molecular-weight heparin (ULMWH) is heparin's derivative, the present study was designed to test the effects of ULMWH on intracellular calcium concentration ([Ca2+]i) in primary cultured neurons. [Ca2+]i was measured by Multilabel Counter Victor-1420 using Fura-2/AM as the calcium fluorescent probe. The results indicated that ULMWH decreased the resting [Ca2+]i with or without extracellular Ca2+. They had no effects on high K+-induced elevation of intracellular Ca2+ level indicating that ULMWH had no effect on external Ca2+ influx mediated by voltage-dependent calcium channels. However, they partially reduced the increase in [Ca2+]i induced by glutamate. Furthermore, ULMWH significantly inhibited the inositol 1,4,5-trisphosphate (IP3)-induced increase in [Ca2+]i both in cellular and subcellular level. These results suggest that ULMWH may reduce [Ca2+]i in neurons through suppressing Ca2+ release from IP3-sensitive stores.