Low Molecular Weight Heparin Derivatives Research Articles

Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N-sulfoheparosan with N-sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products.

Heparin, Low Molecular Weight Heparin, and Non-Anticoagulant Derivatives for the Treatment of Inflammatory Lung Disease.

Unfractionated heparin has multiple pharmacological activities beyond anticoagulation. These anti-inflammatory, anti-microbial, and mucoactive activities are shared in part by low molecular weight and non-anticoagulant heparin derivatives. Anti-inflammatory activities include inhibition of chemokine activity and cytokine synthesis, inhibitory effects on the mechanisms of adhesion and diapedesis involved in neutrophil recruitment, inhibition of heparanase activity, inhibition of the proteases of the coagulation and complement cascades, inhibition of neutrophil elastase activity, neutralisation of toxic basic histones, and inhibition of HMGB1 activity. This review considers the potential for heparin and its derivatives to treat inflammatory lung disease, including COVID-19, ALI, ARDS, cystic fibrosis, asthma, and COPD via the inhaled route.

Abstract NTOC-077: ORALLY ACTIVE LOW MOLECULAR WEIGHT HEPARIN TAUROCHOLATE AND DEOXYCHOLATE CONJUGATE FOR THE INHIBITION OF THE PROGRESSION OF METASTATIC OVARIAN CANCER

Abstract PURPOSE: Ovarian cancer is one of the major aggressive cancer which is not manageable blocking a single pathway. Recently, chemically modified low molecular weight heparin (LMWH) derivatives have shown good anti-cancer activity targeting various biochemical parameters, such as, transforming growth factor beta 1 (TGFβ-1), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), heparin-like growth factor (HGF), CXCL-12, and CXCL-9. In this study, we have focused on TGFβ-1 and VEGF that are known to promote metastatic ovarian cancer progression and use an orally LMWH-taurocholate conjugated with tetrameric deoxycholic acid, namely LHTD4, to successfully inhibit the progression at different stages of ovarian metastasis cancer. EXPERIMENTAL PROCEDURES: We performed the binding affinity of TGFβ-1 and VEGF with LHTD4. We carried out in vitro phosphorylation assays of the consecutive receptors of TGFβ-1 and VEGF (TGF-β-1R1 and VEGFR2, respectively). Effects of LHTD4 on in vitro SKOV3-LUC cell migration (induced by TGFβ-1 and VEGF), spheroid formation, and the epithelial to mesenchymal transition (EMT) were investigated. We prepared a formulation of LHTD4 and a synthetic bile acid-based absorption enhancer, DCK (L-lysine- deoxycholylethylamine), to enable the oral absorption. The in vivo anti-metastatic effect of LHTD4 was evaluated in an intraperitoneal adhesion model and an orthotopic SKOV3-LUC ovarian cancer model. We measured the luciferase activity of the mice every week to check the progression of the cancer. RESULTS: LHTD4 inhibited the migration of SKOV3-LUC cells 10-fold greater than the control group. LHTD4 also blocked the VEGF induced SKOV3-LUC spheroid formation. LHTD4 effectively inhibited ligand-induced VEGFR2 and TGFβ-1R1 phosphorylated. The expressions of EMT marker proteins such as Vimentin and Snail were prevented by LTHD4 treatment in in vitro studies with TGFβ-1 treated SKOV3-LUC cells. In orthotopic model ovarian metastasis model, the treatment with LHTD4 (5 mg/kg daily, orally) significantly inhibited the growth and organ metastasis progression compared to the control: LHTD4-treated mice showed 20-times lower luciferase activity than that of control mice. Finally, the intraperitoneal adhesion of the SKOV3-LUC cells was also decreased robustly in mice which received LHTD4 daily. CONCLUSION: LHTD4 is one of the promising candidate for the inhibition of the progression of epithelial ovarian cancer. We report that the oral formulation of LHTD4/DCK complex could be a good choice for the treatment of metastatic ovarian cancer as it can target multiple pathways of metastasis. LHTD4 primarily showed anti-metastatic effects via blocking of TGFβ-1 and VEGF signaling pathway at different stages of metastasis progress. Citation Format: Farzana Alam, Mohammad Rezwanul Haque, Taslim A. Al-Hilal, Fakhrul Ahsan, Youngro Byun. ORALLY ACTIVE LOW MOLECULAR WEIGHT HEPARIN TAUROCHOLATE AND DEOXYCHOLATE CONJUGATE FOR THE INHIBITION OF THE PROGRESSION OF METASTATIC OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-077.

Orally active desulfated low molecular weight heparin and deoxycholic acid conjugate, 6ODS-LHbD, suppresses neovascularization and bone destruction in arthritis

The regulation of angiogenesis is an interesting area to consider for novel therapeutic approaches to rheumatoid arthritis (RA). Chemically modified heparins have been developed as possible candidates for angiogenesis inhibitor; however, they have a major clinical drawback in exhibiting poor oral bioavailability. Here, orally absorbable O-desulfated low molecular weight heparin (ODS-LMWH) derivatives were newly synthesized by conjugating 2-O- or 6-O-desulfated LMWH with deoxycholic acid (DOCA) or bisDOCA (a dimer of DOCA), and their physicochemical properties, antiangiogenic potency and pharmacokinetic profiles were assessed. After selecting the best candidate among those derivatives, its therapeutic efficacy on arthritis was investigated in a murine collagen antibody-induced arthritis (CAIA) model. ODS-LMWH derivatives significantly inhibited the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs) and basic fibroblast growth factor (bFGF)-induced angiogenesis in the Matrigel plug assay. Among all the compounds, 6ODS-LHbD showed the highest oral bioavailability in rats (19.3%). In the CAIA mouse model, 6ODS-LHbD (10mg/kg, p.o., S.I.D.) significantly inhibited neovascularization in the joint, the increase of hind-paw thickness, and the structural damage in the bone. Therefore, 6ODS-LHbD would be a promising candidate for an orally active drug for the treatment of RA.

1616P - Low Molecular Weight Heparin (LMWH) Resistance in Cancer Patients

ABSTRACT Introduction Cancer patients have an increased risk of developing venous thrombo-embolism (VTE). Treatment of VTE involves the administration of heparin, low molecular weight heparin (LMWH) or coumarin derivatives. Heparin resistance is a situation in which the administration of heparin does not result in an increase of the activated partial thromboplastin time (APTT) and blood anticoagulation. While heparin resistance is a known entity, LMWH resistance is not well documented, and anti factor Xa activity is not tested routinely in cancer patients treated with LMWH. Here, we report the pharmacokinetics of LMWH in cancer patients suffering from VTE and receiving treatment with enoxaparin at standard doses of 1 mg/kg twice daily. Patients/methods Patients suffering from malignancy and VTE, and treated with the LMWH, enoxaparin at standard dose of 1mg/kg q12 hr were enrolled. Ambulatory patients were admitted to the oncology day care unit for 8 hours in order to facilitate repeated blood testing. Blood samples were obtained before the injection of LMWH and 1, 2, 3, 4, 6 and 8 hours after LMWH subcutaneous administration, and tested for anti Xa activity as a surrogate marker of bioavailable LMWH levels. The trial was approved by the ethics committee of Shaare Zedek Medical Center. ClinicalTrials.gov Identifier: NCT00716898. Study funding: Israel Cancer Association. Results Eleven patients were enrolled; one was excluded from analysis due to complete remission at time of VTE diagnosis. Peak anti Xa activity was achieved after 2, 3, 4, 6, and 8 hours in 2, 3, 2, 2, and 1 patient respectively. 60% of the patients (n = 6) did not reach the therapeutic anti Xa activity target (0.6 -1.0 IU/ml) at 4 hours after subcutaneous administration of LMWH. Average anti Xa activity at 4 hours was 0.62 ± 0.29 IU/ml as opposed to 1.1 IU/ml in historical controls of non-oncology patients. Conclusions Our results show that a substantial number of cancer patients suffering from VTE and treated with standard dose enoxoparin do not reach therapeutic target anti Xa activity. If confirmed in a larger study, our results suggests that cancer patients suffering from VTE should be tested for anti Xa activity and LMWH dose should be titrated accordingly in order to achieve effective anticoagulation. Disclosure All authors have declared no conflicts of interest.

High antiangiogenic and low anticoagulant efficacy of orally active low molecular weight heparin derivatives

Heparin, an anticoagulant that is widely used clinically, is also known to bind to several kinds of proteins through electrostatic interactions because of its polyanionic character. These interactions are mediated by the physicochemical properties of heparin such as sequence composition, sulfation patterns, charge distribution, overall charge density, and molecular size. Although this electrostatic character mediates its binding to many proteins related with tumor progression, thereby providing its antiangiogenic property, the administration of heparin for treating cancer is limited in clinical applications due to several drawbacks, such as its low oral absorption, unsatisfactory therapeutic effects, and strong anticoagulant activity which induces hemorrhaging. Here, we evaluated novel, orally active, low molecular weight heparin (LMWH) derivatives (LHD) conjugated with deoxycholic acid (DOCA) that show reduced anticoagulant activity and enhanced antiangiogenic activity. The chemical conjugate of LMWH and DOCA was synthesized by conjugating the amine group of N-deoxycholylethylamine (EtDOCA) with the carboxylic groups of heparin at various DOCA conjugation ratios. The LMWH–DOCA conjugate series (LHD1, LHD1.5, LHD2, and LHD4) were further formulated with poloxamer 407 as a solubilizer for oral administration. An in vitro endothelial tubular formation and in vivo Matrigel plug assay were performed to verify the antiangiogenic potential of LHD. Finally, we evaluated tumor growth inhibition of oral LHD administration in a SCC7 model as well as in A549 human cancer cell lines in a mouse xenograft model. Increasing DOCA conjugation ratios showed decreased anticoagulant activity, eventually to zero. LHD could block angiogenesis in the tubular formation assay and the Matrigel plug assay. In particular, oral administration of LHD4, which has 4 molecules of DOCA per mole of LMWH, inhibited tumor growth in SCC7 mice model as well as A549 mice xenograft model. LHD4 was orally absorbable, showed minimal anticoagulant activity and inhibits tumor growth via antiangiogenesis. These findings demonstrate the therapeutic potential of LHD4 as a new oral anti-cancer drug.

Anti-inflammatory effects of low molecular weight heparin derivative in a rat model of carrageenan-induced pleurisy

Low molecular weight heparin derivatives are characterized by low anti-coagulant activity and marked anti-inflammatory effects that allow for these molecules to be viewed as a new class of non-steroidal anti-inflammatory drugs (NSAIDs). We show here that K5NOSepiLMW, an O-sulphated heparin-like semi-synthetic polymer of the D-glucuronic acid-N-acetyleparoson disaccharide unit with low molecular weight, has marked anti-inflammatory effects in a rat model of acute inflammation, the carrageenan-induced pleurisy, commonly used to test NSAID efficacy. A 30-min. pre-treatment with K5NOSepiLMW (0.1, 0.5 and 1 mg/kg b.wt., given intrapleurally) attenuated the recruitment of leucocytes in the lung tissue and the pleural exudate, inhibited the induction of inducible nitric oxide synthase and cyclooxygenase-2 (COX-2), thereby abating the generation of nitric oxide and pro-inflammatory prostaglandins such as PgE(2) and PGF(1alpha), reduced the inflammation-induced nitroxidative lung tissue injury, as shown by tissue thiobarbituric acid-reactive substances and nitrotyrosine, and blunted the local generation of cytokines such as interleukin-1beta and tumour necrosis factor-alpha. All these parameters were markedly increased by intrapleural carrageenan in the absence of any pre-treatment. The anti-inflammatory action of K5NOSepiLMW is specific, as judged by the lack of therapeutic effects of B4/110, a biologically inactive cognate polysaccharide, given in the place of the authentic molecule. Moreover, K5NOSepiLMW showed similar effects as celecoxib (1 mg/kg b.wt), a COX-2 inhibitor and well-known NSAID. This study provides further insight into the mechanisms underlying the beneficial effects of heparin derivatives in inflammation and identifies K5NOSepiLMW as a novel, promising anti-inflammatory drug.

Enzymatic synthesis of glycosaminoglycan heparin.

Heparin and its low molecular weight heparin derivatives, widely used as clinical anticoagulants, are acidic polysaccharide members of a family of biomacromolecules called glycosaminoglycans (GAGs). Heparin and the related heparan sulfate are biosynthesized in the Golgi apparatus of eukaryotic cells. Heparin is a polycomponent drug that currently is prepared for clinical use by extraction from animal tissues. A heparin pentasaccharide, fondaparinux, has also been prepared through chemical synthesis for use as a homogenous anticoagulant drug. Recent enabling technologies suggest that it may now be possible to synthesize heparin and its derivatives enzymatically. Moreover, new technologies including advances in synthetic carbohydrate synthesis, enzyme-based GAG synthesis, micro- and nano-display of GAGs, rapid on-line structural analysis, and microarray/microfluidic technologies might be applied to the enzymatic synthesis of heparins with defined structures and exhibiting selected activities. The advent of these new technologies also makes it possible to consider the construction of an artificial Golgi to increase our understanding of the cellular control of GAG biosyntheses in this organelle.

A caution regarding the use of low-molecular weight heparin in pediatric otogenic lateral sinus thrombosis

Lateral sinus thrombosis (LST), a rare complication of otitis media, is managed by antibiotics, surgery and anticoagulation. Traditionally, post-operative anticoagulation has been achieved by intravenous unfractionated heparin followed by oral warfarin. Fractionated, or low-molecular weight heparin derivatives (LMWH) have been introduced recently. There has been minimal literature to date regarding their use for the management of LST. We present use of the LMWH enoxaparin (Lovenox) for otogenic LST in two children, both of whom experienced hemorrhagic complications. On this basis and in the context of a literature review, we urge caution when using LMWH for pediatric otogenic LST.

Does low-molecular-weight heparin influence cancer-related mortality?

Does low-molecular-weight heparin influence cancer-related mortality?

Microscale isolation and analysis of heparin from plasma using an anion-exchange spin column

Heparin, a linear highly sulfated polysaccharide, is a member of the glycosaminoglycan (GAG)1 family [1,2]. Since the 1930s, heparin has been widely used as a clinical anticoagulant [3,4]. More recently, low-molecular weight heparin (LMWH) derivatives have come into widespread use as anticoagulant and antithrombotic drugs [5]. Although heparin’s clinical use predated the establishment of the US. Food and Drug Administration (FDA), the approval of LMWH required a number of pharmacological studies on these new drugs. The pharmacologically relevant concentration of heparin and LMWH in plasma is between 0.1 and 1.0 anti-factor XaU/ml, corresponding to approximately 1–10 µg/ml of drug [6]. Plasma is a complex mixture, making the direct analysis of heparin or LMWH at these concentrations exceedingly challenging. Thus, most of the pharmacological studies of heparin and LMWH have relied on bioassays such as anti-factor Xa activity to evaluate these agents, providing only pharmacodynamic data. This inability to obtain pharmacokinetic data on LMWH is a serious concern because the plasma anti-factor Xa activity of LMWH does not correlate well with its therapeutic efficacy [7].

Randomized Comparison of Low Molecular Weight Heparin and Coumarin Derivatives on the Survival of Patients With Cancer and Venous Thromboembolism

Experimental studies and indirect clinical evidence suggest that low molecular weight heparins may have antineoplastic effects. We investigated the influence of a low molecular weight heparin dalteparin on the survival of patients with active cancer and acute venous thromboembolism. Survival data were examined in a posthoc analysis in patients with solid tumors and venous thromboembolism who were randomly assigned to dalteparin or a coumarin derivative for 6 months in a multicenter, open-label, randomized, controlled trial. All-cause mortality at 12 months was compared between treatment groups in patients with and without metastatic malignancy. The effect of dalteparin on survival was compared between the two patient subgroups. During the 12-month follow-up period, 356 of 602 patients with solid tumors and acute venous thromboembolism died. Among patients without metastatic disease, the probability of death at 12 months was 20% in the dalteparin group, as compared with 36% in the oral anticoagulant group (hazard ratio, 0.50; 95% CI, 0.27 to 0.95; P = .03). In patients with metastatic cancer, no difference in mortality between the treatment groups was observed (72% and 69%, respectively; hazard ratio, 1.1; 95% CI, 0.87 to 1.4; P = .46). The observed effects of dalteparin on survival were statistically significantly different between patients with and without metastatic disease (P = .02). The use of dalteparin relative to coumarin derivatives was associated with improved survival in patients with solid tumors who did not have metastatic disease at the time of an acute venous thromboembolic event. Additional studies are warranted to investigate these findings.

Antifactor Xa activity correlates to thrombin generation time, platelet contractile force and clot elastic modulus following ex vivo enoxaparin exposure in patients with and without renal dysfunction

Antifactor Xa activity is the gold standard monitoring parameter for low molecular weight heparin (LMWH) derivatives. It is frequently measured in high-risk populations, such as patients with renal dysfunction. Despite antifactor Xa monitoring, however, bleeding in renal dysfunction patients receiving LMWH remains a problem. This study determined the relationship between antifactor Xa activity and three novel coagulation monitoring parameters: thrombin generation time (TGT), platelet contractile force (PCF) and clot elastic modulus (CEM). This study also assessed the effect of renal dysfunction on these relationships. This was an ex vivo pharmacodynamic study of the relationship between antifactor Xa activity and TGT, PCF and CEM in subjects both with and without renal dysfunction. Thirty subjects completed this study (10 controls, 10 chronic kidney disease subjects, and 10 end-stage renal disease subjects receiving hemodialysis). Blood samples obtained from participants were spiked with increasing enoxaparin concentrations (0.25, 0.5, 1.0 and 3.0 IU mL(-1)). Samples were analyzed for TGT, PCF and CEM. The relationship between antifactor Xa activity and TGT, PCF and CEM was determined by Pearson's correlation. The effect of renal dysfunction on the relationship between antifactor Xa activity and TGT, PCF and CEM was determined by analysis of covariance. There is strong correlation between antifactor Xa activity and TGT, CEM and PCF. The presence of renal dysfunction significantly prolongs the TGT, and decreases the CEM relative to controls. These results suggest that patients with renal dysfunction have a greater pharmacodynamic response to LMWH, independent of the pharmacokinetics of LMWH.

Modulation of antithrombin-protease interactions by semisynthetic low-molecular-weight heparins with different sulfation patterns.

Heparin, a natural glycosaminoglycan (GAG), is widely used for the treatment of thrombotic diseases. Most of its side effects are related to its ability to bind to different proteins, thus interfering with its target biological activity. To gain insight into structure-activity relationships, we investigated the interaction of a homogeneous series of sulfated polysaccharides, derived from controlled desulfation of a supersulfated low-molecular-weight heparin (LMWH) with the target enzymes human antithrombin (AT) and thrombin (T). In addition, we analyzed the activation process of the serpin AT against T and factor Xa (FXa). A nonlinear correlation between the strength of the AT-heparin complex and the polysaccharide sulfation degree was observed, whereas only a modest modulation of T binding to heparin occurred. The efficiency of the LMWH derivatives in activating AT toward the proteases was generally high for derivatives exhibiting a low dissociation constant. Only the supersulfated heparin showed a serpin activation ability higher than expected from the affinity studies. Examination of the sulfation pattern in the light of the above results suggests a key role of the substitution of the iduronic acid residue in the heparin-mediated serpin binding and activation processes. Indeed, sulfation at position 2 of the uronic acid is beneficial, whereas 2,3-disubstitution generates unfavorable contacts between the GAG and AT. Glucosamine sulfation at position 6 appears to grant increased catalytic efficiency. These results indicate that chemical modification of the heparin sulfation pattern can be used to modulate binding specificity and activity toward its biological targets.

Heparin oligosaccharides that pass the blood-brain barrier inhibit beta-amyloid precursor protein secretion and heparin binding to beta-amyloid peptide.

We have previously demonstrated that full-length heparin stimulates the synthesis and secretion of beta-amyloid precursor protein (APP) through an amyloidogenic pathway in neuroblastoma cells. In the present study, heparin was chemically depolymerized, and the effect of low-molecular-weight (LMW) heparin on APP secretion was investigated. In contrast to full-length heparin, LMW heparin had no significant effect on APP secretion. However, LMW heparin fragments, especially heparin disaccharides, were able to inhibit efficiently the stimulatory effect of heparin on APP secretion. LMW heparin derivatives also inhibit the binding of heparin to the beta-amyloid peptide (1-28). Using an in vitro model, we further demonstrated the passage of LMW heparin derivatives through the blood-brain barrier. This study suggests that LMW heparin derivatives or analogues may be effective as therapeutic agents to prevent or slow the process of amyloidogenesis in Alzheimer's disease.

INHIBITION OF TRANSPLANT ARTERIOSCLEROSIS IN RAT AORTIC GRAFTS BY LOW MOLECULAR WEIGHT HEPARIN DERIVATIVES

The effects of low molecular weight heparin derivatives with a low anticoagulant activity on transplant arteriosclerosis (TA) in a rat aortic transplant model were investigated. TA was induced by ischemia in the syngeneic transplants and primarily by immunological mechanisms in the allogeneic transplants. Treatment with the heparin derivatives, OAM 71262 or LA-heparin, was administered in a dosage of 250 micrograms/kg/hr by mini-osmotic pumps during 8 weeks. No immunosuppressive regimen was given to the recipient rats in either model. All rats were killed 8 weeks after aortic grafting. The grafts were examined for intimal and medial changes using an image analysis system. Heparin derivatives had a beneficial effect on both the intimal thickening and the medial injury in the syngeneic transplants, but not in the allogeneic grafts. In the syngeneic LA-heparin treated grafts, the thickness of the intima was less than that in the syngeneic control grafts (P < 0.05). In the syngeneic transplants, a significant increase was observed in the media after treatment with OAM 71262 (P < 0.01) as well as those with LA-heparin (P < 0.001). In the syngeneic grafts treated with both heparin derivatives, a significant reduction in the antigen expression of alpha-actin-containing smooth muscle cells in the intima, transforming growth factor-beta 1 both in the media and adventitia, and platelet-derived growth factor-beta receptors in the adventitia was observed immunohistochemically. In summary, low molecular weight heparin derivatives with low anticoagulant activity partially inhibited ischemia-induced syngeneic TA, whereas no such effect could be demonstrated in nonimmunosuppressed recipients with allogeneic grafts.

Gradient Polyacrylamide Gel Electrophoresis for Determination of Molecular Weights of Heparin Preparations and Low-Molecular-Weight Heparin Derivatives

The Mr values of pharmaceutical heparins and low-molecular-weight (LMW) heparin derivatives were examined as part of a collaborative study to develop methods for their characterization. Standard methods of Mr determination rely on gel permeation high-performance liquid chromatography (HPLC). We report the use of gradient polyacrylamide gel electrophoresis (PAGE) to determine the Mr values of pharmaceutical heparins and LMW heparin derivatives. This approach offers certain advantages over the HPLC method. Gradient PAGE analysis was performed in parallel, on multiple samples, with the same standard curve. HPLC was performed serially. Gradient PAGE gave higher resolution than HPLC, and thus, a mixture of easily obtained standards was used in place of individual standards for the construction of a standard curve. Heparin and various LMW heparin samples were analyzed by both gradient PAGE and conventional gel permeation HPLC methods. The number-average Mr, weight-average Mr, and polydispersity were examined by both techniques and found to be similar. This study demonstrates that gradient PAGE analysis is a sensitive method for the determination of the Mr values of heparin and LMW heparin.

Low molecular weight heparin (enoxaparin) reduces restenosis after iliac angioplasty in the hypercholesterolemic rabbit

Smooth muscle cell proliferation is central to the process of restenosis. Attempts to inhibit the events leading to this proliferation have met with little success. In addition to its known antithrombotic effects, heparin also has inhibitory effects on smooth muscle cell proliferation. These effects appear to be unrelated to its anticoagulant properties and are retained in low molecular weight heparin derivatives. Although the use of heparin for as long as 18 to 24 h after coronary angioplasty in humans has not prevented restenosis, longer treatment periods have not been assessed.This study examines the effect of treatment with a low molecular weight heparin (enoxaparin) in a hypercholesterolemic rabbit iliac artery model. Control rabbits had a mean iliac artery diameter of 0.70 ± 0.06 mm, which increased to 1.73 ± 0.09 mm after balloon angioplasty. At follow-up angiography 4 weeks later, the mean vessel diameter was 0.56 ± 0.12 mm. Animals treated with low dose enoxaparin (1 mg/kg per day) for 4 weeks and high dose enoxaparin (10 mg/kg per day) for either 2 or 4 weeks had similar mean luminal diameters before and immediately after angioplasty. At follow-up angiography, the mean luminal diameter was 0.82 ± 0.17 mm for low dose enoxaparin, 1.04 ± 0.20 mm for 2 week high dose enoxaparin (p = 0.03 versus control) and 1.19 ± 0.09 mm for 4 week high dose enoxaparin (p = 0.001 versus control).When defined as loss of 50% of the initial gain achieved with angioplasty, restenosis was found in all control vessels. In the 2 week high dose enoxaparin group, only two of nine vessels had restenosis and in the 4 week high dose group, three of nine vessels had restenosis (p = 0.001 versus control). These results show that antiproliferative agents such as low molecular weight heparin can inhibit restenosis in animal models and suggest that they may be useful in humans.

Physicochemical Characterization of the First World Health Organization International Standard for Low Molecular Weight Heparin Derivatives

High-field (300 MHz) 1H NMR spectral analysis and particle size distribution analysis employing the quasielastic light scattering (QELS) technique were performed on samples of the 1st International Standard for low molecular weight (LMW) heparin derivatives recently selected by the World Health Organization (WHO). We propose that the results of these analyses, which showed that the material is highly homogeneous in particle size and retains spectral features characteristic of its porcine mucosal origin, form an appropriate basis for physicochemical comparison between the “Standard” and other LMW heparin preparations.

Chemical Composition, Particle Size Range, and Biological Activity of some Low Molecular Weight Heparin Derivatives

Several low molecular weight (LMW) heparin sodium derivatives from different sources, as well as some related regular heparin sodium preparations, were examined for chemical composition by high field (300 MHz) 1H NMR spectroscopy, for particle size range by quasi‐elastic light scattering (QELS) methods, and for anti‐coagulation potency and anti‐factor Xa activity by the standard U.S. Pharmacopeial assays described for regular heparin. The NMR spectra provided insight into possible modes of depolymerization used to generate the LMW heparins, as well as into the presence of dermatan sulfate or other chemical contaminants. The QELS analysis permitted the heparin preparations to be characterized and compared by virtue of their distinctive particle size distributions.