A Unique Enoxaparin Derived from Bovine Intestinal Heparin: A Single Purification Step of the Starting Material Assures a Bovine Enoxaparin Like the Standard from Porcine Origin.

Low-molecular-weight heparin represent a significant advancement in anticoagulant therapy with enoxaparin being a prominent example obtained exclusively through the fragmentation of porcine intestinal heparin. However, escalating demand and limited resources have raised concerns about enoxaparin supplementation. The current challenge involves exploring alternative heparin sources for large-scale enoxaparin production with bovine intestinal heparin emerging as a promising option. Our study demonstrates that enoxaparin derived from the available bovine heparin preparation differs significantly from the reference compound. Yet, the implementation of a straightforward purification step yields a preparation termed "high-anticoagulant bovine heparin". Fragmentation of this purified product through ß-elimination produces enoxaparin akin to the standard from a porcine origin. To ensure physicochemical similarity, we employed various spectroscopic, enzymatic, and chromatographic tests to compare the new bovine-derived enoxaparin with the original porcine compound. Biological activity was confirmed through in vitro coagulation assays and assessments using an animal model of venous thrombosis. Our study affirms that the ß-elimination reaction cleaves the bovine heparin chain without preferential breaks in regions with different sulfation patterns. Additionally, we scrutinized decasaccharides purified from enoxaparin preparations, providing a comprehensive demonstration of the similarity between products obtained from porcine and bovine heparin. In summary, our findings indicate that an enoxaparin equivalent to the original porcine-derived product can be derived from bovine heparin, given that the starting material undergoes a simple purification step.

A low-anticoagulant heparin suppresses metastatic dissemination through the inhibition of tumor cell-platelets association.

Metastasis is the leading cause of cancer-related deaths. Despite this relevance, there is no specific therapy targeting metastasis. The interaction of the tumor cell with platelets, forming microemboli is crucial for successful hematogenous dissemination. Heparin disrupts it by a P-selectin-mediated event. However, its clinical use for this purpose is hindered by the requirement of high doses, leading to anticoagulant-related side effects. In this study, we obtained a low-anticoagulant heparin through the fractionation of a pharmaceutical bovine heparin. This derivative was referred to as LA-hep and we investigated its efficacy in inhibiting metastases and explored its capacity of suppressing the interaction between tumor cells and platelets. Our data revealed that LA-hep is as efficient as porcine unfractionated heparin in attenuating lung metastases from melanoma and colon adenocarcinoma cells in an assay with a single intravenous administration. It also prevents platelet arrest shortly after cell injection in wild-type mice and suppresses melanoma-platelets interaction in vitro. Moreover, LA-hep blocks P-selectin's direct binding to tumor cells and platelet aggregation, providing further evidence for the role of P-selectin as a molecular target. Even in P-selectin-depleted mice which developed a reduced number of metastatic foci, both porcine heparin and LA-hep further inhibited metastasis burden. This suggests evidence of an additional mechanism of antimetastatic action. Therefore, our results indicate a dissociation between the heparin anticoagulant and antimetastatic effects. Considering the simple and highly reproducible methodology used to purify LA-hep along with the data presented here, LA-hep emerges as a promising drug for future use in preventing metastasis in cancer patients.

Polysaccharide rich fractions from barks of Ximenia americana inhibit peripheral inflammatory nociception in mice Antinociceptive effect of Ximenia americana polysaccharide rich fractions

Abstract Ximenia americana L., Olacaceae, barks are utilized in folk medicine as analgesic and anti-inflammatory. The objective was to evaluate the toxicity and antinociceptive effect of polysaccharides rich fractions from X. americana barks. The fractions were obtained by extraction with NaOH, followed by precipitation with ethanol and fractionation by ion exchange chromatography. They were administered i.v. or p.o. before nociception tests (writhing, formalin, carragenan-induced hypernociception, hot plate), or during 14 days for toxicity assay. The total polysaccharides fraction (TPL-Xa: 8.1% yield) presented 43% carbohydrate (21% uronic acid) and resulted in two main fractions after chromatography (FI: 12%, FII: 22% yield). FII showed better homogeneity/purity, content of 44% carbohydrate, including 39% uronic acid, arabinose and galactose as major monosaccharides, and infrared spectra with peaks in carbohydrate range for COO- groups of uronic acid. TPL-Xa (10 mg/kg) and FII (0.1 and 1 mg/kg) presented inhibitory effect in behavior tests that evaluate nociception induced by chemical and mechanical, but not thermal stimuli. TPL-Xa did not alter parameters of systemic toxicity. In conclusion, polysaccharides rich fractions of X. americana barks inhibit peripheral inflammatory nociception, being well tolerated by animals.

Sperm and Egg Jelly Coat from Sea Urchin Lytechinus variegatus Collected in Rio de Janeiro Contain Distinct Sialic Acid-Rich Polysaccharides

This work found the occurrence of a distinct sialic acid-rich polysaccharide in the sperm surface of the sea urchin Lytechinus variegatus, which differed significantly from a similar molecule found in the egg jelly. The sperm polysaccharide extracted by protease digestion was purified using anion exchange chromatography and characterized using agarose gel electrophoresis, gas chromatography/mass spectrometry and NMR spectroscopy. This polysaccharide was highly sulfated and composed almost exclusively of N-acetylneuraminic acid. In contrast, the sialic acid-rich polysaccharide from the egg jelly was composed of N-glycolylneuraminic acid and contains several other hexoses in its structure. This new molecule could help to characterize in further detail the mechanism of fertilization in the sea urchin model system. Sulfated polysaccharides from the jelly coat of sea urchins showed species-specificity in inducing the sperm acrosome reaction, providing an example of a signal transduction event regulated by the sulfated polysaccharide. The new sialic acid-rich polysaccharide found in the sperm head could represent a new molecule involved in the biology of the sea urchin fertilization.

Debate propositivo sobre os biossimilares de enoxaparina no Brasil / Propositional debate on biosimilar enoxaparin in Brazil / Debate de una propuesta sobre los biosimilares de enoxaparina en Brasil

Algumas patentes das heparinas de baixo peso molecular expiraram e outras estão vencendo. Versões biossimilares desses fármacos estão disponíveis para o uso clínico em vários países. Entretanto, ainda persiste ceticismo sobre a possibilidade de se obter preparações semelhantes ao medicamento original em razão do complexo processo para gerar heparina de baixo peso molecular. Nosso laboratório analisou, nos últimos anos, amostras de enoxaparina disponíveis para uso clínico no Brasil. Já analisamos 30 lotes distintos e 70 produtos acabados. Essas preparações foram avaliadas quanto à estrutura química, distribuição de peso molecular, atividade anticoagulante in vitro e efeitos farmacológicos em modelos animais de trombose e sangramento. Claramente, nossos resultados indicam que as preparações biossimilares de enoxaparina são semelhantes ao medicamento original. Nossos resultados indicam que essas versões biossimilares de enoxaparina são uma alternativa terapêutica válida, mas que requerem regulamentação adequada para assegurar o atendimento de requisitos regulatórios apropriados.

Some patents of low-molecular-weight heparins (LMWHs) have expired and others are about to expire. Biosimilar versions of those drugs are available for clinical use in several countries. However, skepticism persists about the possibility of obtaining preparations similar to the original drug, because of the complexity of the process to generate LMWHs. In recent years, our laboratory has analyzed biosimilar samples of enoxaparin available for clinical use in Brazil (30 different batches and 70 finished products). Those preparations were assessed regarding their chemical structure, molecular weight distribution, in vitro anticoagulant activity, and pharmacological effects in animal models of thrombosis and bleeding. Our results have clearly shown that biosimilar preparations of enoxaparin are similar to the original drug. Our results have shown that those biosimilar versions of enoxaparin are a valid therapeutic alternative, which are, however, in need of appropriate regulation to ensure compliance with regulatory requirements.

Algunas patentes de las heparinas de bajo peso molecular caducaron y otras van por el mismo camino. Versiones biosimilares de esos fármacos están disponibles para el uso clínico en varios países. Sin embargo, todavía persiste el escepticismo sobre la posibilidad de obtener preparaciones similares al medicamento original en razón del complejo proceso para producir la heparina de bajo peso molecular. En los últimos años, nuestro laboratorio analizó muestras de enoxaparina disponibles para el uso clínico en Brasil. Ya hemos analizado 30 lotes distintos y 70 productos acabados. Esas preparaciones fueron evaluadas en cuanto a la estructura química, distribución de peso molecular, actividad anticoagulante in vitro y efectos farmacológicos en modelos animales de trombosis y sangramiento. Lógicamente que nuestros resultados indican que las preparaciones biosimilares de enoxaparina son similares al medicamento original. Nuestros resultados dan fe de que esas versiones biosimilares de enoxaparina son una alternativa terapéutica válida, pero que requieren una reglamentación adecuada para garantizar la atención de los requisitos reglamentarios pertinentes.

Sulfated fucan from marine alga inhibits HeLa cells infection by HTLV-1 free particles: semi-quantitative analysis

A sulfated fucan from Laminaria abyssalis marine alga prevented the interaction of HTLV-1 particles, purified from the MT-2 cell line, with HeLa cells. The infection obtained using a concentrated virus suspension was detected only by amplification of the newly synthesized HTLV-1 proviral cDNA by the nested-polymerase chain reaction (PCR). The sulfated polysaccharide was not toxic to the cells at a concentration of 100 µg/mL and prevented infection by the viral particles when added to the cell monolayers. The proviral cDNA was only detected when the sulfated polysaccharide was added to the cells three hours post-infection, indicating that the inhibitory activity occurred in the initial stages of virus-cell interaction. Our results demonstrate, for the first time, the ability of a sulfated fucan from marine algae to inhibit virus transmission through free virus particles.

Controle da qualidade das preparações de heparina disponíveis no Brasil: implicações na cirurgia cardiovascular / Heparin quality control in the Brazilian market: implications in the cardiovascular surgery

INTRODUÇÃO: A mudança na marca da heparina rotineiramente utilizada nas cirurgias cardíacas no Brasil tem sido acompanhada por aumento do número de casos de discrasia sanguínea, aumento de reoperações e efeitos adversos em nossa Instituição e em outras. MÉTODOS: Foram avaliadas no Laboratório de Tecido Conjuntivo do HUCFF/UFRJ, quatro preparações disponíveis e comparadas à heparina retirada do mercado (Liquemine) e ao padrão de controle internacional. As preparações de heparina foram submetidas à ressonância nuclear magnética para avaliação da integridade estrutural, bem como avaliação de sua eficácia anticoagulante. RESULTADOS: Houve diferença significativa quanto à atividade anticoagulante entre as amostras. Também se observou a presença de contaminação com dermatam sulfato, amostras degradadas quimicamente e com significativa alteração do peso molecular. CONCLUSÃO: Das amostras estudadas, nenhuma atendeu aos requisitos de segurança para utilização em cirurgias cardíacas com circulação extracorpórea. Nenhuma delas apresentou a qualidade semelhante ao Liquemine, não mais disponível no mercado brasileiro.

INTRODUCTION: The change in the heparin solution trade mark in Brazil that had been commonly used in cardiac surgery has shown increased number in the coagulopathy, re-exploration and other side effects in our Institution and others. METHODS: All four different heparin solutions available in the Brazilian market were studied in the Connective Tissue Lab, HUCFF, UFRJ and compared to the Liquemine (out of the market) and the international control solution. All samples were evaluated by magnetic nuclear resonance as well as their anticoagulant effectiveness. RESULTS: There were significant differences among them regarding the anticoagulant activity. It was also observed contamination with other dermatan sulfate, samples chemically degraded and with significant change in the molecular weight. CONCLUSION: Among the studied samples, none of them can offer security in cardiac surgeries on pump. None of them has demonstrated similar quality to Liquemine, which is not available in the Brazilian market.

Implications of naturally occurring variations in arterial glycosaminoglycans for the pathogenesis of atherosclerosis

Proteoglycans and their constituent glycosaminoglycan (GAG) side chains participate in the pathogenesis of atherosclerosis by holding and modifying plasma lipoproteins (LDL). Of the GAGs existing in arteries, only chondroitin sulfate and dermatan sulfate interact with LDL.In addition, these LDL-binding GAGs show some naturally occurring variations that have direct consequences to their participation in atherogenesis. These variations are: (1) Polydispersity: GAGs normally vary widely in molecular weight and in human aortas there are longer chains of chondroitin sulfate/dermatan sulfate presenting stronger affinity to LDL; (2) Ageing: steric factors play a role in GAG-LDL interaction, and with ageing there is an increase in the relative content of the 6-sulfated isomer of aortic chondroitin sulfate; this isomer binds to LDL, whereas the 4-sulfated isomer does not: (3) Anatomic heterogeneity: The risk to develop atherosclerotic lesions is different among arteries, and the composition and LDL binding affinity of chondroitin sulfate/dermatan sulfate from normal arteries correlate with their susceptibility to atherosclerosis. The participation of GAGs in atherosclerosis should therefore be viewed as variable.