Ultra Low Molecular Weight Heparins Research Articles

Cloning and Expression of Heparinase Gene from a Novel Strain Raoultella NX-TZ-3-15.

Heparin is a class of highly sulfated, acidic, linear, and complex polysaccharide that belongs to the heparin/heparan sulfate (HS) glycosaminoglycans family. Enzymatic depolymerization of heparin by heparinases is a promising strategy for the production of ultra-low molecular weight heparins (ULMWHs) as anticoagulants. In the present study, a novel heparinase-producing strain Raoultella NX-TZ-3-15 was isolated and identified from soil samples. Herein, the heparinase gene MBP-H1 was cloned to the pBENT vector to enable expression in Escherichia coli. The optimized conditions made the activity of recombinant heparinase reach the highest level (2140 U/L). The overexpressed MBP-H1 was purified by affinity chromatography and a purity of more than 90% was obtained. The condition for biocatalysis was also optimized and three metal ions Ca2+, Co2+, and Mg2+ were utilized to activate the reaction. In addition, the kinetics regarding the new fusion heparinase was also determined with a Vm value of 11.29μmol/min and a Km value of 31.2μmol/L. In short, due to excellent Km and Vmax, the recombinant enzyme has great potential to be used in the clinic in medicine and industrial production of low or ultra-low molecule weight heparin.

Filter-entrapment enrichment pull-down assay for glycosaminoglycan structural characterization and protein interaction

Heparins are the most pharmaceutically important polysaccharides. These heparin-based anticoagulant/antithrombotic agents include unfractionated heparins, low molecular weight heparins (LMWHs) and ultralow molecular weight heparins (ULMWHs). Heparins exhibit their pharmacological and biological activities through interaction with heparin-binding proteins. The prototypical heparin-binding protein is antithrombin III (AT), responsible for heparin’s anticoagulant/antithrombotic activity. This study describes a filter-trapping method to isolate the chains in enoxaparin, a LMWH, which bind to AT. We demonstrate this method using the ULMWH, fondaparinux, which consists of a single well defined AT binding site. The interacting chains of enoxaparin are then characterized by activity assays, top-down liquid chromatography-mass spectrometry, and capillary zone electrophoresis mass spectrometry. This filter-trapping assay is an improvement over affinity chromatography for isolating heparin chains interacting with heparin binding proteins.

Heparin and anticoagulation.

Heparin, a sulfated polysaccharide, has been used as a clinical anticoagulant for over 90 years. Newer anticoagulants, introduced for certain specialized applications, have not significantly displaced heparin and newer heparin-based anticoagulants in most medical procedures. This chapter, while reviewing anticoagulation and these newer anticoagulants, focuses on heparin-based anticoagulants, including unfractionated heparin, low molecular weight heparins and ultra-low molecular weight heparins. Heparin's structures and its biological and therapeutic roles are discussed. Particular emphasis is placed on heparin's therapeutic application and its adverse effects. The future prospects are excellent for new heparins and new heparin-based therapeutics with improved properties.

Anti-heparanase activity of ultra-low-molecular-weight heparin produced by physicochemical depolymerization

Heparanase is an endo-β-D-glucuronidase that plays an important role in cancer progression, in particular during tumor angiogenesis and metastasis. Inhibiting this enzyme is considered as one of the most promising approaches in cancer therapy. Heparin is a complex glycoaminoglycan known as a strong inhibitor of heparanase. It is primarily used in clinical practice for its anticoagulant activities, which may not be compatible with its use as anti-angiogenic agent. In this study, we described the production of ultra-low-molecular-weight heparins (ULMWH) by a physicochemical method that consists in a hydrogen peroxide-catalyzed radical hydrolysis assisted by ultrasonic waves. We assessed the structural characteristics, anticoagulant and anti-heparanase activities of the obtained heparin derivatives and compared them with three commercial low-molecular-weight heparins (LMWH), glycol-split non-anticoagulant heparins and heparins produced by enzymatic methods. ULMWH generated by the physicochemical method were characterized by high anti-heparanase and moderate anticoagulant activities. These heparin derivatives might be potential candidates for cancer therapy when a compromise is needed between anti-heparanase and anticoagulant activities.

Pharmacological effects and clinical applications of ultra low molecular weight heparins

Heparin, one of the common anticoagulants, is clinically used to prevent and treat venous thromboembolism (VTE). Though it has been the drug of choice for many advanced medical and surgical procedures with a long history, the adverse events, such as bleeding, heparin-induced thrombocytopenia (HIT), allergic reactions, follow. Therefore, low molecular weight heparins (LMWHs) and ultra low molecular weight heparins (ULMWHs), with lower molecular weights, higher anti-FXa activity, longer half-life times and lower incidence of adverse events than unfractionated heparin (UFH), were researched and developed. Fondaparinux, a chemically synthesized ULMWH of pentasaccharide, has the same antithrombin III (AT-III)-binding sequence as found in UFH and LMWH. In addition, AVE5026 and RO-14, another two ULMWHs, are obtained by selective chemical depolymerization. In this paper, we review the preparation process, pharmacological effects and clinical applications of fondaparinux, AVE5026 and RO-14.

Preparation and application of a ‘clickable’ acceptor for enzymatic synthesis of heparin oligosaccharides

A ‘clickable’ disaccharide was prepared by treating the aldehyde precursor with hydroxylamine, followed by the catalytic hydrogenation and diazotransfer reaction. This disaccharide was successfully applied to the elongation of the backbone construction of ultralow molecular weight (ULMW) heparins using two bacterial glycosyl transferases, N-acetyl glucosaminyl transferase from Escherichia coli K5 (KfiA) and heparosan synthase-2 (pmHS2) from Pasteurella multocida.

Ultra-low-molecular-weight heparins: Precise structural features impacting specific anticoagulant activities

Ultra-low-molecular-weight heparins (ULMWHs) with better efficacy and safety ratios are under development; however, there are few structural data available. The main structural features and molecular weight of ULMWHs were studied and compared to enoxaparin. Their monosaccharide composition and average molecular weights were determined and preparations studied by nuclear magnetic resonance spectroscopy, scanning ultraviolet spectroscopy, circular dichroism and gel permeation chromatography. In general, ULMWHs presented higher 3-O-sulphated glucosamine and unsaturated uronic acid residues, the latter being comparable with their higher degree of depolymerisation. The analysis showed that ULMWHs are structurally related to LMWHs; however, their monosaccharide/oligosaccharide compositions and average molecular weights differed considerably explaining their different anticoagulant activities. The results relate structural features to activity, assisting the development of new and improved therapeutic agents, based on depolymerised heparin, for the prophylaxis and treatment of thrombotic disorders.

Oral and parenteral anticoagulants

Well-documented drawbacks of traditional anticoagulants have lead to the quest for an ideal anticoagulant resulting in a surge of novel anticoagulant molecules. These newer agents directly target specific steps in coagulation cascade and include newer low molecular weight heparins (adomiparin), ultra low molecular weight heparins (semuloparin, RO-14), inhibitors of activated factor II (dabigatran, AZD0837), X (rivaroxaban, apixaban, edoxaban, betrixaban), IX (REG1,2), XI (antisense oligonucleotides, BMS 262084, clavatadine A), VII/tissue factor (tifacogin, PCI 274836, and BMS 593214), V (recomodulin, solulin), VIII (TB402), dual thrombin/factor X inhibitors (EP21709, tanogitran), and newer vitamin K antagonists (tecarfarin). Direct thrombin inhibitors and Factor X inhibitors are the most clinically advanced. This article discusses the recent advances in the development of novel targets of anticoagulants. Medline, EMBASE, cochrane database, medscape, SCOPUS, and clinicaltrials.gov were searched using terms "anticoagulants", "blood coagulation inhibitors", "anticoagulants and venous thromboembolism", "anticoagulants and atrial fibrillation", and "'antithrombins." Journal articles published from 2007 to 2012 discussing pharmacology and/or clinical trials were screened.

Comparative Studies on the HIT Antibody Mediated Platelet Aggregation / Serotonin Release by Synthetic Pentasaccharide and Two Chemoenzymatically Synthesized Heptasaccharides

Abstract 2231Synthetic oligosaccharides such as the Pentasaccharide (Arixtra) and its derivatives are antithrombotic agents which are clinically used in the management of thrombotic indications. These agents are claimed to be devoid of triggering the generation of HIT antibodies and therefore do not produce HIT syndrome. Several additional synthetic oligosaccharides are also developed for the management of thrombotic indications. More recently, two novel ultra low molecular weight heparins (ULMWHs) were synthesized chemoenzymatically. These ULMWHs are both heptasaccharides with AT pentasaccharide-binding sites within their structures which is comparable to the pentasaccharide. The IC50 of the anti-Xa effects of the agents are comparable to pentasaccharide, ranging from 0.7 to 1.0 ug/ml in comparison to pentasaccharide which is 0.8 ug/ml. All agents produced comparable anticoagulant effects in the Heptest clotting time. The purpose of this study is to compare the effects of the pentasaccharide and the two heptasaccharides namely ULMWH1 and ULMW2 in the HIT mediated platelet aggregation and serotonin release assays. In addition platelet factor 4 release in whole blood was also studied. The HIT mediated platelet aggregation studies were carried out utilizing a HIT antibody positive pool plasma preparation. PRP collected from 10 individual donors (250ul) was mixed with 200ul of HIT pool plasma and equilibrated at 37° C for 3 minutes. 50ul of 1, 10, and 100 ug/ml of each of these agents was added to trigger the platelet aggregation responses. Enoxaparin was used as a positive control in the same concentration ranges. The serotonin release assay was carried out using the standard method in the same concentration range monitoring the release of 14C serotonin with each of these agents. The PF4 release was also measured using an ELISA method for serotonin measurement in whole blood samples incubated with each of these agents at concentrations of 0, 10 and 100ug/ml. The pentasaccharide and the two heptasaccharides did not produce any aggregation of platelets in the HIT aggregation assay at all concentrations whereas Enoxaparin at concentrations of > 1ug/ml produces positive aggregation responses. In the 14C assay none of the agents produced any release of serotonin however Enoxaparin produced 14C release at all concentration studied. Similarly, the pentasachhardide and heptasaccharides did not produce any platelet factor 4 from the whole blood incubation studies, however Enoxaparin produced a measurable release of platelet factor 4. Interestingly, unlike Enoxaparin, the anti-Xa and heptest effects of these agents were not neutralized by platelet factor 4 or protamine sulfate. These results demonstrate that the pentasaccharide and chemoenzymatically synthesized ULMWH1 and ULMWH2 do not meditate HIT antibody induced aggregation and serotonin release. Therefore, these heptasaccharides may exhibit comparable safety profile to the pentasaccharide in heparin compromised patients. Disclosures:No relevant conflicts of interest to declare.

Biological Activities of Two Ultra-Low Molecular Weight Heparins, Semuloparin and Bemiparin, Reveal That a Common Potency Standard Is Inadequate

Abstract 4324 Introduction:Ultra-low molecular weight heparins (U-LMWHs) are being developed to improve the safety and efficacy of antithrombotic therapy. Two of these agents, bemiparin (Rovi, Madrid, Spain) and semuloparin (Sanofi-Aventis, Paris, France), are produced by distinct methods. Bemiparin is produced by esterification of porcine mucosal heparin followed by alkaline hydrolysis and fractionation. Semuloparin is prepared by a highly selective depolymerization reaction using a phosphazene base which preserves the AT binding sequences from destruction. While both bemiparin and semuloparin are effective at reducing the incidence of post-surgical VTE, some biological differences have been observed in humans. The objective of this investigation was to determine whether a common standard could be used to define their potency. Methods:Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. Anticoagulant activity was assessed using aPTT and ACT assays. Anti-Xa and anti-IIa activities were determined using amidolytic assays. Constrained and unconstrained curves were determined for the anti-Xa and anti-IIa data to assess parallelism of the concentration-response curves. Thrombin generation was measured using a fluorometric substrate kinetic method (Technothrombin TGA assay; Technoclone GmbH, Vienna, Austria). Platelet function was assessed using platelet aggregometry and the serotonin release assay (SRA). Results:A significantly larger prolongation of the aPTT was observed with bemiparin at concentrations >1 μ g/ml. Differences in anticoagulant activity measured by the Heptest assay were not observed at concentrations below 2.5 μ g/ml. When supplemented to whole blood samples at higher concentrations, bemiparin and semuloparin are able to prolong the kaolin ACT. Anti-FXa activity for bemiparin and semuloparin was the same over the concentration range tested. Bemiparin produced more anti-FIIa activity at each concentration tested. Depletion of AT led to a complete loss of anti-FXa and anti-FIIa activities for both agents while depletion of heparin cofactor-II had minimal impact on anti-IIa activity. Parallelism assessment is used to identify similarity or difference of products in biological assays. The constrained and unconstrained models using anti-FXa concentration-response data were equivalent (p=0.422), indicating that bemiparin and semuloparin were equivalent (IC50 ratio = 0.97, confidence interval= 0.95–0.98, CV = 0.7%). In contrast, the constrained and unconstrained models using anti-FIIa concentration-response data were not equivalent (p<0.001), indicating that the in vitro anti-FIIa activities of the two agents is not equivalent (IC50 ratio = 1.25, confidence interval = 1.17–1.34, CV = 3.4%). At clinically relevant concentrations, bemiparin more effectively inhibited thrombin generation than semuloparin. Platelet aggregation in response to collagen, ADP and arachidonic acid was not affected by either agent at concentrations up to 10 μ g/ml. At a concentration of 5 μ g/ml, bemiparin was able to completely inhibit thrombin-induced aggregation (0.5 U/ml), whereas no effect was observed with semuloparin. Cross-reactivity of bemiparin and semuloparin with anti-PF4/heparin HIT antibodies was tested using platelet aggregation and the SRA. The level of maximal aggregation was comparable in the presence of bemiparin and semuloparin, however, the rate of aggregation was significantly slower in the presence of semuloparin compared to bemiparin. The typical bell-shaped concentration-response curve was observed with both drugs in the SRA. Although the peak release was comparable for the two drugs (65.7 ± 16.0% for semuloparin vs. 75.5 ± 6.6% for bemiparin), the peak serotonin release was observed at lower concentrations for bemiparin (1 μ g/ml) than for semuloparin (10 μ g/ml) suggesting a higher sensitivity of bemiparin to (stronger interaction with) the anti-PF4/heparin antibodies. Conclusions:These data demonstrate that the molecular profiling of heparin-derived agents is insufficient to determine drug class. Additional structural characterization and multiple biologic activities relevant to clinical safety and efficacy need to be considered as well. As such, the use of a common reference standard for potency determination of ULMWH is not valid. Disclosures:Rigal: Sanofi-Aventis: Employment. Bayol:Sanofi-Aventis: Employment. Viskov:Sanofi-Aventis: Employment.

A Novel Polymer Based Antidote for Reversing the Anticoagulation Effect of Clinically Used Heparins,

Abstract 3359 IntroductionAnticoagulation is one of the most widely used and essential clinical practices in modern medicine. Heparins are universally used for the prevention of blood coagulation in surgical procedures and for the treatment of diseases such as venous thromboembolism (VTE). Unfractionated heparin (UFH), low and ultra-low molecular weight heparins (LMWHs&ULMWHs) and synthetic pentasaccharides such as fondaparinux and idraparinux are the most commonly used clinical anticoagulants. Heparin is the second most widely used drug after insulin. However, it is associated with bleeding complications and heparin induced thrombocytopenia. Hence a careful monitoring and neutralization of heparins is essential. Protamine is the only clinically approved antidote to UFH, but it has several side effects and is not effective against LMWHs and synthetic pentasaccharides. Hence there is an unmet clinical need to develop safer and more efficient antidotes for all these anticoagulants. Here, we report a novel polymer based antidote, heparin binding synthetic polyvalent cationic macromolecule (HBSPCM), that completely neutralizes UFH and LMWHs in vitro and in vivo and is highly biocompatible and non-toxic in the required therapeutic dose range. ExperimentalHBSPCMs were synthesized by the polymerization of glycidol and methoxy polyethylene glycol and functionalized with multifunctional tertiary amines as binding groups. Blood compatibility of HBSPCM was evaluated by activated partial thromboplastin time (APTT), prothrombin time (PT), thromboelastography (TEG), platelet and complement activation assays. Cell viability of HBSPCM was evaluated in human umbilical vein endothelial cells and fibroblast cells. Single dose tolerability in mice was studied by injecting escalating doses of HBSPCM and monitoring the body weights over a period of 29 days. HBSPCMs were tested for in vitro heparin neutralization by measuring the APTT in human plasma. An anti-fXa assay was used to study the in vivo neutralization of heparins by HBSPCM in a rat model. Pharmacokinetics and biodistribution of 3H-labeled HBSPCM was studied by bolus i.v. injection in female Balb/c mice and measuring the radioactivity in major organs at different time points. Results and DiscussionThe newly designed antidotes, HBSPCMs, alone do not show any adverse effect on blood coagulation, platelet and complement activation and cytotoxicity that reveal their excellent blood and cell compatibilities. When injected in mice, HBSPCMs were well-tolerated up to the maximum injected dose of 200 mg/kg, which is ten-fold higher than the maximum tolerated dose of protamine (20 mg/kg) (Figure 1). HBSPCMs were 20-times more efficient than protamine for neutralizing heparins in vitro, and unlike protamine they do not show anticoagulant effect at higher concentrations. In vivo studies in rats revealed complete neutralization of both UFH and LMWHs by HBSPCMs, and the neutralization activities for LMWHs were significantly higher than that of protamine (Figure 2). HBSPCMs and their heparin complexes showed rapid clearance through urine, without significant accumulation in major organs. [Display omitted] [Display omitted] Conclusions and SignificanceIn order to overcome the challenges associated with heparin based anticoagulation therapy, there is an increasing demand to develop safer, stable, effective, economical and universal antidotes which could neutralize all the available heparin anticoagulants. The developed polymer based antidote, HBSPCM, represents a major breakthrough towards this goal and could be a potential replacement for protamine. This polymer based therapeutic agent opens the scope for the development of non-toxic antidotes for all heparin based drugs. Disclosures:No relevant conflicts of interest to declare.

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.

A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin

IntroductionLower low-molecular-weight heparins are being developed to improve on the safety and efficacy of antithrombotic therapy. Semuloparin and bemiparin are two depolymerized heparins produced by distinct manufacturing processes. The objective of this investigation was to determine whether a common standard could be used to define their potency. Materials and MethodsActivities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. ResultsThe activity of semuloparin and bemiparin was comparable in FXa-based assays (anti-FXa, Heptest). However, bemiparin produced a stronger effect in the aPTT, ACT and anti-thrombin assays. Assessment of the parallelism of the concentration-response curves indicated that bemiparin and semuloparin are not equivalent in terms of anti-FIIa activity. Bemiparin had a stronger inhibitory effect on thrombin induced platelet aggregation, and a stronger interaction with HIT antibodies. ConclusionsThese data demonstrate that depolymerized heparins can exhibit a range of biologic activities making them unique agents. Pharmacopoeial parameters such as anti-IIa and anti-Xa potency and molecular weight are insufficient to characterize such agents.

Comparative Studies on An Anti-Xa Enriched Ultra Low Molecular Weight Heparin with Bemiparin

AbstractAbstract 4394Most low molecular weight heparins (LMWHs) have a mean molecular weight in the range of 4–6 kDa and anti-Xa/IIa ratios of 3–6. Further depolymerization of porcine mucosal heparin results in the generation of Ultra low molecular weight heparins (ULMWHs) with a molecular weight range of 2–4 kDa with proportionately decreased anti-Xa and anti-IIa activities. Bemiparin (Rovi, Madrid, Spain) represents one such ULMWH. AVE 5026 (Sanofi-Aventis, Paris, France) is a unique ULMWH (2.5 kDa) which exhibits higher affinity to antithrombin (AT) and therefore, enhanced anti-Xa activity. Because of the compositional differences between these two agents, it was hypothesized that each of these agents will have distinct anticoagulant, antiprotease and thrombin generation effects. Each of these agents was supplemented to native whole blood. Anticoagulant activity was measured using ACT, TEG, PT, APTT, thrombin time and Heptest assays. Similar studies were carried out in plasma. Amidolytic assays were used to determine the anti-Xa and anti-IIa activities. Both agents were also tested for the interactions with heparin cofactor II (HC II) and AT and were compared in the HIT antibody screening assay using platelet aggregation. In whole blood clotting assays bemiparin showed a strong anticoagulant activity in comparison to AVE 5026. Both agents also exhibited assay dependent differences in the APTT, heptest and thrombin time assays. AVE 5026 exhibited a higher anticoagulant activity in the heptest whereas bemiparin showed a stronger anticoagulant effect in the other clot based assays. In the amidolytic anti-Xa assay, AVE 5026 showed an activity of 156U/mg compared to 86 U/mg for bemiparin. In the anti-IIa assay bemiparin showed a higher activity (10 U/mg) in comparison to AVE 5026 (3.2 U/mg). The calculated Xa/IIa ratio of AVE 5026 was > 48, whereas it was 8.6 for bemiparin. While bemiparin exhibited interactions with HC II, AVE 5026 did not show significant activity in the tested concentrations (anti-IIa – IC50: 1.10±.45 μ M and >3.44±.00 μ M, respectively). On the other hand, AVE 5026 exhibited stronger interactions with AT in comparison to bemiparin (anti-FXA – IC50: .223±.03 μ M and .894±.06 μ M, respectively). Interestingly, heparinase digestion of the two products resulted in a complete loss of anti-IIa activity, but residual anti-Xa activity was found. AVE 5026 exhibited stronger anti-Xa interactions even after heparinase digestion. In the heparin induced platelet aggregation assay at 2.5 μ g/ml, bemiparin showed a relatively higher prevalence of positive interactions with HIT antibodies, whereas AVE 5026 showed a much lower prevalence (slope; AVE 5026 compared bemiparin, p=0.012). Bemiparin exhibited greater platelet factor 4 neutralization in comparison to AVE 5026. These studies clearly demonstrate that while bemiparin behaves like a typical ULMWH, AVE 5026 behaves differently in the different assays. Moreover, the oligosaccharide composition of the two products, in terms of distribution profile structure, is also different. Therefore, AVE 5026 does not represent a typical depolymerized ULMWH and is expected to exhibit a distinct pharmacologic and clinical profile. Disclosures:Hoppensteadt:Sanofi-Aventis: Research Funding.

Unfractionated and Low-Molecular-Weight Heparins, Basic Mechanism of Action and Pharmacology

Unfractionated heparin has enjoyed sole anticoagulant status for nearly 50 years. Despite a dramatic growth in the development and the introduction of many newer anticoagulant and antithrombotic drugs and polytherapeutic approaches during the past decade, unfractionated and low-molecular-weight heparins remain the drugs of choice for many indications, including surgical anticoagulation, interventional cardiology, and in several additional considerations. Unfractionated heparin has a major role in the areas of vascular medicine and surgery, and it is the only parenteral anticoagulant drug that can be empirically neutralized by such agents as protamine sulfate. The development of low and ultra low-molecularweight heparins, which are a class of depolymerized heparin derivatives with distinct pharmacologic profiles that are largely determined by their composition, represents a refinement for the use of heparin. These drugs produce their major effects by combining with antithrombin Ill and exerting antithrombin and anti-Xa inhibition. The low-molecular-weight heparins also increase non-antithrombin III-dependent effects, such as tissue factor pathway inhibitor release, modulation of adhesion molecules, and the release of profibrinolytic and antithrombotic mediators from the blood vessels. Each of the low-molecular-weight heparins has different cumulative effects, and each product exhibits a distinct profile. Initially developed for the prophylaxis of postsurgical deep vein thrombosis, these drugs are now also used for the treatment of both venous and arterial thrombotic disorders. To a large extent, the low-molecular-weight heparins have replaced unfractionated heparin in most of the subcutaneous indications. This has resulted in a dramatic evolution in anticoagulant management that allows patients with thrombotic disorders to be treated in an outpatient setting. Thus, the introduction of low-molecular-weight heparins represents a major advance in improving the use of heparin. Generic versions of these drugs are likely to be developed as their patents expire. Currently, there are no clear guidelines for the acceptance of the generic versions of branded products. To avoid safety and efficacy-related problems, a generic drug must meet both the chemical and biologic equivalence criterion. Synthetically and biosynthetically derived agents such, as pentasaccharide, will also be introduced for clinical use; however, these drugs will have a narrower therapeutic spectrum due to their monotherapeutic nature. Heparin and its derivatives will continue to have a crucial role in the management of thrombotic and cardiovascular disorders in years to come.

Inhibition of allergic late airway responses by inhaled heparin-derived oligosaccharides.

Inhaled heparin has been shown to inhibit allergic bronchoconstriction in sheep that develop only acute responses to antigen (acute responders) but was ineffective in sheep that develop both acute and late airway responses (LAR) (dual responders). Because the antiallergic activity of heparin is molecular-weight dependent, we hypothesized that heparin-derived oligosaccharides (<2, 500) with potential anti-inflammatory activity may attenuate the LAR in the dual-responder sheep. Specific lung resistance was measured in 24 dual-responder sheep before and serially for 8 h after challenge with Ascaris suum antigen for demonstration of early airway response (EAR) and LAR, without and after treatment with inhaled medium-, low-, and ultralow-molecular-weight (ULMW) heparins and "non-anticoagulant" fractions (NAF) of heparin. Airway responsiveness was estimated before and 24 h postantigen as the cumulative provocating dose of carbachol that increased specific lung resistance by 400%. Only ULMW heparins caused a dose-dependent inhibition of antigen-induced EAR and LAR and postantigen airway hyperresponsiveness (AHR), whereas low- and medium-molecular-weight heparins were ineffective. The effects of ULMW heparin and ULMW NAF-heparin were comparable and inhibited the LAR and AHR even when administered "after" the antigen challenge. The ULMW NAF-heparin failed to inhibit the bronchoconstrictor response to histamine, carbachol, and leukotriene D(4), excluding a direct effect on airway smooth muscle. In six sheep, segmental antigen challenge caused a marked increase in bronchoalveolar lavage histamine, which was not prevented by inhaled ULMW NAF-heparin. The results of this study in the dual-responder sheep demonstrate that 1) the antiallergic activity of inhaled "fractionated" heparins is molecular-weight dependent, 2) only ULMW heparins inhibit the antigen-induced EAR and LAR and postantigen AHR, and 3) the antiallergic activity is mediated by nonanticoagulant fractions and resides in the ULMW chains of <2,500.

New antithrombotic agents for the prevention and treatment of deep vein thrombosis.

Besides low molecular weight heparins (LMWHs) a number of new antithrombotic agents have been evaluated mainly in the prevention of deep vein thrombosis (DVT) and, to a lesser extent, in the treatment of established DVT. They include the Pentasaccharide, a synthetic ultra LMWH, Dermatan Sulphate, a glycosaminoglycan which activates heparin cofactor II, Orgaran, a mixture of Heparan and of Dermatan Sulphate, Hirulog and Hirudin, two direct thrombin inhibitors. The efficacy and safety of these compounds have been studied in comparison with a placebo or with unfractionated heparin but not with LMWH which is considered as a gold standard for these clinical indications. It is thus difficult at present to appreciate the advantages of these new antithrombotic agents over conventional LMWH therapy.

Mode of Action of Unfractionated and Low Molecular Weight Heparins on the Generation of Thrombin in Plasma

Heparins, unfractionated and low molecular weight, act primarily by their scavenging of thrombin (S-type heparins). Via the feedback effect on factor VIII this has a secondary effect on prothrombin conversion in the intrinsic pathway (activated partial thromboplastin time). The anti-Xa action of a heparin will not significantly inhibit prothrombin conversion, except in the case of ultra low molecular weight heparins (P-type heparins) that have no significant antithrombin activity. These P-type heparins need, therefore, be given at high doses to have an antithrombotic effect. In platelet-rich plasma heparins retard platelet activation by lowering thrombin levels. Activated platelets neutralize up to 0.5 U/ml of unfractionated heparin, but low molecular weight heparin is much less affected.