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.

Use of infrared thermography in Quarter Horse submitted to team roping / Uso da termografia infravermelha em equinos da raça Quarto de Milha submetidos à prova de laço em dupla

This study aimed to verify whether the body and local temperatures change after high-intensity, short-duration exercise (team roping) and whether different pieces of training influence these changes. To this end, twelve animals, males and females, aged 3-6 years, with an average weight of 450 kg, were used. The horses were divided into two groups: regular training (RTG) and sporadic training (STG). The surface temperatures were assessed using a specific thermal camera. Temperatures of the ocular, thoracolumbar, distal tendon (thoracic and pelvic limbs) and croup regions were measured 30 min before, immediately after, and one, two, six and 24 hours after competition simulation. In the RTG, there was an increase in surface eye temperature two hours after exercise, returning to baseline level 24 hours later. In the STG, increase in eye temperature occurred immediately after exercise and returned to baseline level two hours later. Temperature of the pelvic limb tendons and croup (right side) rose immediately after exercise and did not return to baseline level 24 hours later. Team roping exercise increased the surface temperature of the distolateral thoracic and pelvic limb, croup and thoracolumbar regions in both groups and the eye temperature in the STG. Training frequency influenced the surface temperature profile in the distolateral pelvic limb, croup and thoracolumbar regions.(AU)

Os objetivos do presente estudo foram verificar se as temperaturas corpóreas e locais se alteram após exercício de alta intensidade e curta duração (prova de laço em dupla) e se treinamentos distintos podem influenciar nestas alterações. Foram utilizados 12 animais, machos e fêmeas, com idade entre 3 e 6 anos e peso médio de 450kg. Os animais foram divididos em dois grupos: treino regular (GTR) e treino esporádico (GTE). As aferições da temperatura por meio de termografia infravermelha foram feitas por uma câmera termal específica. As medições das temperaturas das regiões ocular, toracolombar, tendíneas distais (membros torácicos e pélvicos) e garupa foram realizadas 30 minutos antes, imediatamente depois, uma, duas, seis e 24 horas após a simulação de competição. No GTR houve aumento de temperatura ocular duas horas após o exercício, retornando ao basal apenas 24 horas depois. No GTE o aumento ocorreu imediatamente após o exercício e retornando ao basal duas horas depois. As temperaturas da região dos tendões dos membros pélvicos e garupa (lado direito) elevaram-se imediatamente após o exercício e não retornaram ao basal após 24 horas. O exercício de laço em dupla aumentou as temperaturas superficiais nas regiões distolateral de membros torácicos e pélvicos, garupa e região toracolombar de ambos os grupos e da temperatura ocular do GTE. A frequência de treinamento influenciou o perfil de temperatura superficial na região distal de membros pélvicos, garupa e toracolombar.(AU)

Use of infrared thermography in Quarter Horse submitted to team roping / Uso da termografia infravermelha em equinos da raça Quarto de Milha submetidos à prova de laço em dupla

This study aimed to verify whether the body and local temperatures change after high-intensity, short-duration exercise (team roping) and whether different pieces of training influence these changes. To this end, twelve animals, males and females, aged 3-6 years, with an average weight of 450 kg, were used. The horses were divided into two groups: regular training (RTG) and sporadic training (STG). The surface temperatures were assessed using a specific thermal camera. Temperatures of the ocular, thoracolumbar, distal tendon (thoracic and pelvic limbs) and croup regions were measured 30 min before, immediately after, and one, two, six and 24 hours after competition simulation. In the RTG, there was an increase in surface eye temperature two hours after exercise, returning to baseline level 24 hours later. In the STG, increase in eye temperature occurred immediately after exercise and returned to baseline level two hours later. Temperature of the pelvic limb tendons and croup (right side) rose immediately after exercise and did not return to baseline level 24 hours later. Team roping exercise increased the surface temperature of the distolateral thoracic and pelvic limb, croup and thoracolumbar regions in both groups and the eye temperature in the STG. Training frequency influenced the surface temperature profile in the distolateral pelvic limb, croup and thoracolumbar regions.(AU)

Os objetivos do presente estudo foram verificar se as temperaturas corpóreas e locais se alteram após exercício de alta intensidade e curta duração (prova de laço em dupla) e se treinamentos distintos podem influenciar nestas alterações. Foram utilizados 12 animais, machos e fêmeas, com idade entre 3 e 6 anos e peso médio de 450kg. Os animais foram divididos em dois grupos: treino regular (GTR) e treino esporádico (GTE). As aferições da temperatura por meio de termografia infravermelha foram feitas por uma câmera termal específica. As medições das temperaturas das regiões ocular, toracolombar, tendíneas distais (membros torácicos e pélvicos) e garupa foram realizadas 30 minutos antes, imediatamente depois, uma, duas, seis e 24 horas após a simulação de competição. No GTR houve aumento de temperatura ocular duas horas após o exercício, retornando ao basal apenas 24 horas depois. No GTE o aumento ocorreu imediatamente após o exercício e retornando ao basal duas horas depois. As temperaturas da região dos tendões dos membros pélvicos e garupa (lado direito) elevaram-se imediatamente após o exercício e não retornaram ao basal após 24 horas. O exercício de laço em dupla aumentou as temperaturas superficiais nas regiões distolateral de membros torácicos e pélvicos, garupa e região toracolombar de ambos os grupos e da temperatura ocular do GTE. A frequência de treinamento influenciou o perfil de temperatura superficial na região distal de membros pélvicos, garupa e toracolombar.(AU)

Converting the Distinct Heparins Sourced from Bovine or Porcine Mucosa into a Single Anticoagulant Drug.

Unfractionated heparin (UFH) and their low-molecular-weight derivatives are sourced almost exclusively from porcine mucosa (HPI); however, a worldwide introduction of UFH from bovine mucosa (HBI) has been recommended to reinforce the currently unsteady supply chain of heparin products. Although HBI has different chemical composition and about half of the anticoagulant potency of HPI (∼100 and ∼180 international unit [IU]/mg, respectively), they have been employed as interchangeable UFHs in some countries since the 1990s. However, their use as a single drug provoked several bleeding incidents in Brazil, which precipitated the publication of the first monographs exclusive for HBI and HPI by the Brazilian Pharmacopoeia. Nevertheless, we succeed in producing with high-resolution anion-exchange chromatography a novel HBI derivative with anticoagulant potency (200 IU/mg), disaccharide composition (enriched in N,6-disulfated α-glucosamine) and safety profile (bleeding and heparin-induced thrombocytopaenia potentials and protamine neutralization) similar to those seen in the gold standard HPI. Therefore, we show that it is possible to equalize the composition and pharmacological characteristics of these distinct UFHs by employing an easily implementable improvement in the HBI manufacturing.

Heparins Sourced From Bovine and Porcine Mucosa Gain Exclusive Monographs in the Brazilian Pharmacopeia.

Most of the unfractionated heparin (UFH) consumed worldwide is manufactured using porcine mucosa as raw material (HPI); however, some countries also employ products sourced from bovine mucosa (HBI) as interchangeable versions of the gold standard HPI. Although accounted as a single UFH, HBI, and HPI have differing anticoagulant activities (~100 and 200 IU mg-1, respectively) because of their compositional dissimilarities. The concomitant use of HBI and HPI in Brazil had already provoked serious bleeding incidents, which led to the withdrawal of HBI products in 2009. In 2010, the Brazilian Pharmacopeia (BP) formed a special committee to develop two complementary monographs approaching HBI and HPI separately, as distinct active pharmaceutical ingredients (APIs). The committee has rapidly agreed on requirements concerning the composition and presence of contaminants based on nuclear magnetic resonance and anion-exchange chromatography. On the other hand, consensus on the anticoagulant activity of HBI was the subject of long and intense discussions. Nevertheless, the committee has ultimately agreed to recommend minimum anti-FIIa activities of 100 IU mg-1 for HBI and 180 IU mg-1 for HPI. Upon the approval by the Brazilian Health Authority (ANVISA), the BP published the new monographs for HPI and HBI APIs in 2016 and 2017, respectively. These pioneer monographs represent a pivotal step toward the safest use of HBI and HPI as interchangeable anticoagulants and serve as a valuable template for the reformulation of pharmacopeias of other countries willing to introduce HBI.

Use of infrared thermography in Quarter Horse submitted to team roping

ABSTRACT: This study aimed to verify whether the body and local temperatures change after high-intensity, short-duration exercise (team roping) and whether different pieces of training influence these changes. To this end, twelve animals, males and females, aged 3-6 years, with an average weight of 450 kg, were used. The horses were divided into two groups: regular training (RTG) and sporadic training (STG). The surface temperatures were assessed using a specific thermal camera. Temperatures of the ocular, thoracolumbar, distal tendon (thoracic and pelvic limbs) and croup regions were measured 30 min before, immediately after, and one, two, six and 24 hours after competition simulation. In the RTG, there was an increase in surface eye temperature two hours after exercise, returning to baseline level 24 hours later. In the STG, increase in eye temperature occurred immediately after exercise and returned to baseline level two hours later. Temperature of the pelvic limb tendons and croup (right side) rose immediately after exercise and did not return to baseline level 24 hours later. Team roping exercise increased the surface temperature of the distolateral thoracic and pelvic limb, croup and thoracolumbar regions in both groups and the eye temperature in the STG. Training frequency influenced the surface temperature profile in the distolateral pelvic limb, croup and thoracolumbar regions.

RESUMO: Os objetivos do presente estudo foram verificar se as temperaturas corpóreas e locais se alteram após exercício de alta intensidade e curta duração (prova de laço em dupla) e se treinamentos distintos podem influenciar nestas alterações. Foram utilizados 12 animais, machos e fêmeas, com idade entre 3 e 6 anos e peso médio de 450kg. Os animais foram divididos em dois grupos: treino regular (GTR) e treino esporádico (GTE). As aferições da temperatura por meio de termografia infravermelha foram feitas por uma câmera termal específica. As medições das temperaturas das regiões ocular, toracolombar, tendíneas distais (membros torácicos e pélvicos) e garupa foram realizadas 30 minutos antes, imediatamente depois, uma, duas, seis e 24 horas após a simulação de competição. No GTR houve aumento de temperatura ocular duas horas após o exercício, retornando ao basal apenas 24 horas depois. No GTE o aumento ocorreu imediatamente após o exercício e retornando ao basal duas horas depois. As temperaturas da região dos tendões dos membros pélvicos e garupa (lado direito) elevaram-se imediatamente após o exercício e não retornaram ao basal após 24 horas. O exercício de laço em dupla aumentou as temperaturas superficiais nas regiões distolateral de membros torácicos e pélvicos, garupa e região toracolombar de ambos os grupos e da temperatura ocular do GTE. A frequência de treinamento influenciou o perfil de temperatura superficial na região distal de membros pélvicos, garupa e toracolombar.

Avaliação da lactatemia e das enzimas musculares de equinos Quarto de Milha submetidos à prova de laço em dupla com diferentes tipos de treinamento / Evaluation of lactatemia and muscle enzymes in Quarter Horses subjected to team ropping with different types of training

O objetivo do trabalho foi avaliar a influência de diferentes tipos de treinamento sobre o condicionamento físico de equinos por meio da determinação do lactato sanguíneo e da atividade sérica de creatina quinase, aspartato aminotransferase e lactato desidrogenase após exercício físico de alta intensidade e curta duração. Amostras de sangue venoso foram obtidas de 16 equinos da raça Quarto de Milha, divididos em dois grupos: grupo de treinamento regular (GTR) e grupo de treinamento esporádico (GTE), em sete diferentes momentos: 30 minutos antes do exercício (M0), imediatamente após (M1), 30 minutos (M2), uma (M3), duas (M4), seis (M5) e 24 (M6) horas após o exercício. Para a análise estatística, os dados foram testados quanto à normalidade e homogeneidade de variâncias. Para comparar os grupos e os momentos em cada grupo foram utilizados testes paramétricos (ANOVA) para a análise das atividades séricas das enzimas musculares e não paramétricos (Mann-Whitney e Friedmann) para a análise do lactato sanguíneo (P<0,05). Não houve diferença significativa entre os grupos para nenhuma variável. No entanto, dentro dos grupos experimentais foi possível observar diferenças significativas entre os momentos avaliados, em relação ao lactato e à LDH. No GTE, foram observadas diferenças significativas quanto ao lactato, entre o M0 e o M1, com valores respectivos de 0,90 mmol/L (mín. 0,8 - máx. 1,6) e 3,65mmo/L (mín. 1,0 - máx. 5,7) e quanto à LDH, onde os valores descritos no M6 diferiram significativamente de M0, M1, M2, M3 e M4. No GTR, diferenças significativas entre os momentos experimentais foram observadas em relação à LDH, sendo que os valores observados no M6 foram os menores e diferiram significativamente daqueles encontrados no M1, M2, M3 e M4. Em conclusão, não houve diferença entre o condicionamento físico dos animais treinados regularmente e aqueles treinados esporadicamente. A baixa magnitude das elevações das concentrações de CK, AST e LDH após o exercício e o rápido retorno aos valores basais, inclusive do lactato, observados em ambos os grupos, sugere que todos os animais avaliados estavam condicionados e aptos a realizar tal atividade física.(AU)

The aim of this study was to evaluate the influence of different types of training on physical fitness through the determination of blood lactate and serum creatinine kinase, aspartate aminotransferase, and lactate dehydrogenase activity after high intensity and short duration physical exercise. Venous blood samples were obtained from 16 Quarter Horses, divided into two groups: the regular training group (GTR) and sporadic training group (GTE), in seven different moments: 30 minutes before exercise (M0), immediately after the exercise (M1), 30 minutes (M2), one hour (M3), two hours (M4), six hours (M5) and 24 hours (M6) after the exercise. For statistical analysis, data was tested for normality and homogeneity of variances. To compare the groups and times in each group, parametric tests (ANOVA) were used for muscular enzymes activity and not parametric tests (Mann-Whitney and Friedmann) were used to analyze blood lactato (P<0.05). There was no significant difference between groups for any variable. However, within the experimental groups it was possible to observe significant differences between the evaluated moments, in relation to lactate and LDH. In the GTE, significant differences were observed for lactate between M0 and M1, with respective values ​​of 0.90 mmol/L (min. 0.8, max. 1.6) and 3.65 mmol/L (min. 1.0, max. 5,7) and for LDH, where the values ​​described in M6 differed significantly from M0, M1, M2, M3 and M4. In the GTR, significant differences between the experimental moments were observed in relation to LDH, and the values ​​observed in M6 were the lowest and differed significantly from those found in M1, M2, M3 and M4. In conclusion, there was no difference between the fitness of animals regularly trained and those trained sporadically. The low magnitude of elevations of serum CK, AST and LDH activity after exercise and the quick return to baseline values, including the blood lactate observed in both groups, suggest that all of evaluated animals were conditioned and able to perform such physical activity.(AU)

Avaliação da lactatemia e das enzimas musculares de equinos Quarto de Milha submetidos à prova de laço em dupla com diferentes tipos de treinamento / Evaluation of lactatemia and muscle enzymes in Quarter Horses subjected to team ropping with different types of training

O objetivo do trabalho foi avaliar a influência de diferentes tipos de treinamento sobre o condicionamento físico de equinos por meio da determinação do lactato sanguíneo e da atividade sérica de creatina quinase, aspartato aminotransferase e lactato desidrogenase após exercício físico de alta intensidade e curta duração. Amostras de sangue venoso foram obtidas de 16 equinos da raça Quarto de Milha, divididos em dois grupos: grupo de treinamento regular (GTR) e grupo de treinamento esporádico (GTE), em sete diferentes momentos: 30 minutos antes do exercício (M0), imediatamente após (M1), 30 minutos (M2), uma (M3), duas (M4), seis (M5) e 24 (M6) horas após o exercício. Para a análise estatística, os dados foram testados quanto à normalidade e homogeneidade de variâncias. Para comparar os grupos e os momentos em cada grupo foram utilizados testes paramétricos (ANOVA) para a análise das atividades séricas das enzimas musculares e não paramétricos (Mann-Whitney e Friedmann) para a análise do lactato sanguíneo (P<0,05). Não houve diferença significativa entre os grupos para nenhuma variável. No entanto, dentro dos grupos experimentais foi possível observar diferenças significativas entre os momentos avaliados, em relação ao lactato e à LDH. No GTE, foram observadas diferenças significativas quanto ao lactato, entre o M0 e o M1, com valores respectivos de 0,90 mmol/L (mín. 0,8 - máx. 1,6) e 3,65mmo/L (mín. 1,0 - máx. 5,7) e quanto à LDH, onde os valores descritos no M6 diferiram significativamente de M0, M1, M2, M3 e M4. No GTR, diferenças significativas entre os momentos experimentais foram observadas em relação à LDH, sendo que os valores observados no M6 foram os menores e diferiram significativamente daqueles encontrados no M1, M2, M3 e M4. Em conclusão, não houve diferença entre o condicionamento físico dos animais treinados regularmente e aqueles treinados esporadicamente. A baixa magnitude das elevações das concentrações de CK, AST e LDH após o exercício e o rápido retorno aos valores basais, inclusive do lactato, observados em ambos os grupos, sugere que todos os animais avaliados estavam condicionados e aptos a realizar tal atividade física.(AU)

The aim of this study was to evaluate the influence of different types of training on physical fitness through the determination of blood lactate and serum creatinine kinase, aspartate aminotransferase, and lactate dehydrogenase activity after high intensity and short duration physical exercise. Venous blood samples were obtained from 16 Quarter Horses, divided into two groups: the regular training group (GTR) and sporadic training group (GTE), in seven different moments: 30 minutes before exercise (M0), immediately after the exercise (M1), 30 minutes (M2), one hour (M3), two hours (M4), six hours (M5) and 24 hours (M6) after the exercise. For statistical analysis, data was tested for normality and homogeneity of variances. To compare the groups and times in each group, parametric tests (ANOVA) were used for muscular enzymes activity and not parametric tests (Mann-Whitney and Friedmann) were used to analyze blood lactato (P<0.05). There was no significant difference between groups for any variable. However, within the experimental groups it was possible to observe significant differences between the evaluated moments, in relation to lactate and LDH. In the GTE, significant differences were observed for lactate between M0 and M1, with respective values ​​of 0.90 mmol/L (min. 0.8, max. 1.6) and 3.65 mmol/L (min. 1.0, max. 5,7) and for LDH, where the values ​​described in M6 differed significantly from M0, M1, M2, M3 and M4. In the GTR, significant differences between the experimental moments were observed in relation to LDH, and the values ​​observed in M6 were the lowest and differed significantly from those found in M1, M2, M3 and M4. In conclusion, there was no difference between the fitness of animals regularly trained and those trained sporadically. The low magnitude of elevations of serum CK, AST and LDH activity after exercise and the quick return to baseline values, including the blood lactate observed in both groups, suggest that all of evaluated animals were conditioned and able to perform such physical activity.(AU)

A unique fucosylated chondroitin sulfate type II with strikingly homogeneous and neatly distributed α-fucose branches.

Fucosylated chondroitin sulfates (FCSs) and sulfated fucans (SFs) are conspicuous components of the body wall of sea cucumbers (Holothuroidea). FCSs are composed of a central core of chondroitin sulfate (CS) decorated with branches of mono- or both mono- and disaccharides of α-fucose (FCS types I and II, respectively). FCSs type II have heterogeneous and irregularly distributed α-fucose branches; however, the novel FCS type II from Holothuria lentiginosa described herein via solution nuclear magnetic resonance has strikingly homogeneous α-fucose branches neatly distributed along its CS core. This FCS is built up of three distinct sequential units composed of the typical CS disaccharides of FCSs, rich in ß-galactosamine-4,6diS, decorated with branches of α-Fucp-2,4diS, α-Fucp-3,4diS or α-Fucp[1→3]α-Fucp-4S[1→ linked to the position 3- of the ß-glucuronic acid. Conformational analyses of these repetitive units revealed a fairly rigid structure despite of the high sulfate content of their α-fucose branches. We also determined the structure of the SF from H. lentiginosa as a repetitive tetrasaccharide sequence composed of →3]α-Fucp-2,4diS[1→3]α-Fucp[1→3]α-Fucp-2S[1→3]α-Fucp-2S[1→. Furthermore, we determined that the nonsulfated α-fucose units present in FCS type II did not interfere with their anticoagulant potencies and affinities to calcium. FCS is an autapomorphic molecular character of the class Holothuroidea and the composition of their α-fucose branches differs in a species-specific manner. Branches containing α-Fucp-2,4diS are the most common within the extant holothurians, being found in 90% of the FCSs characterized thus far.

Systematic Analysis of Pharmaceutical Preparations of Chondroitin Sulfate Combined with Glucosamine.

Glycosaminoglycans are carbohydrate-based compounds widely employed as nutraceuticals or prescribed drugs. Oral formulations of chondroitin sulfate combined with glucosamine sulfate have been increasingly used to treat the symptoms of osteoarthritis and osteoarthrosis. The chondroitin sulfate of these combinations can be obtained from shark or bovine cartilages and hence presents differences regarding the proportions of 4- and 6-sulfated N-acetyl ß-d-galactosamine units. Herein, we proposed a systematic protocol to assess pharmaceutical batches of this combination drug. Chemical analyses on the amounts of chondroitin sulfate and glucosamine in the batches were in accordance with those declared by the manufacturers. Anion-exchange chromatography has proven more effective than electrophoresis to determine the type of chondroitin sulfate present in the combinations and to detect the presence of keratan sulfate, a common contaminant found in batches prepared with shark chondroitin sulfate. 1D NMR spectra revealed the presence of non-sulfated instead of sulfated glucosamine in the formulations and thus in disagreement with the claims declared on the label. Moreover, 1D and 2D NMR analyses allowed a precise determination on the chemical structures of the chondroitin sulfate present in the formulations. The set of analytical tools suggested here could be useful as guidelines to improve the quality of this medication.

Structural and haemostatic features of pharmaceutical heparins from different animal sources: challenges to define thresholds separating distinct drugs.

Heparins extracted from different animal sources have been conventionally considered effective anticoagulant and antithrombotic agents despite of their pharmacological dissimilarities. We performed herein a systematic analysis on the physicochemical properties, disaccharide composition, in vitro anticoagulant potency and in vivo antithrombotic and bleeding effects of several batches of pharmaceutical grade heparins obtained from porcine intestine, bovine intestine and bovine lung. Each of these three heparin types unambiguously presented differences in their chemical structures, physicochemical properties and/or haemostatic effects. We also prepared derivatives of these heparins with similar molecular weight differing exclusively in their disaccharide composition. The derivatives from porcine intestinal and bovine lung heparins were structurally more similar with each other and hence presented close anticoagulant activities whereas the derivative from bovine intestinal heparin had a higher proportion of 6-desulfated α-glucosamine units and about half anticoagulant activity. Our findings reasonably indicate that pharmaceutical preparations of heparin from different animal sources constitute distinct drugs, thus requiring specific regulatory rules and therapeutic evaluations.

Structural and functional analyses of biosimilar enoxaparins available in Brazil.

Biosimilar enoxaparins have been available for clinical use in Brazil since 2009. Although their use has reduced costs of treatment expenses, their implementation still raises some concerns about efficiency, safety, regularity and reproducibility of batches. We undertook structural and functional analyses on over 90 batches of pharmaceutical-active ingredient, and 330 ones of the final products of biosimilar enoxaparins available in the Brazilian market between 2009 and 2014. Besides a nationwide-scale analysis, we have also employed methods that go beyond those recommended by the standard pharmacopeias. We have used high-resolution 2D NMR, detailed assessment of the anticoagulant and antithrombotic properties, check of side effects in experimental animals after continuous administration, and analyses of individual composing oligosaccharides. The 1D 1H NMR spectra of all batches of biosimilar enoxaparins are fairly coincident, and the resultant average spectrum is quite identical to that from the original drug. This structural equality was also assured by highly resolved 2D NMR spectra. The anticoagulant activity, determined by diverse assays and the in vivo antithrombotic and bleeding effects of the biosimilar version were confirmed as equal as of the parental enoxaparins. Structure and function of the composing oligosaccharides were identical in both enoxaparin types. No side effect was observed after continuous subcutaneous administration to rats for 30 days at the dose of 2 mg kg⁻¹ body weight. Biosimilar enoxaparins available in Brazil fulfilled the requirement of the five items defined by FDA-USA for approval of this type of drug.

Structural and functional analyses of bovine and porcine intestinal heparins confirm they are different drugs.

Anticoagulant heparins are mostly obtained from porcine intestine. Occasionally they are also obtained from bovine intestine. Structural and functional analyses of pharmaceutical-grade heparins from these two sources using multiple methods such as NMR spectroscopy, in vitro and in vivo assays of the anticoagulant, antithrombotic and bleeding effects, complemented by fractionation on anion exchange chromatography, confirm they are different drugs. Although bovine heparin is more heterogeneous and less sulfated, heparins from both sources are overall made of a similar mixture of fractions, however with different proportions. Therefore, high-anticoagulant composites from bovine origin, similar to porcine counterparts, can be properly obtained.

Residual keratan sulfate in chondroitin sulfate formulations for oral administration.

Chondroitin sulfate is a biomedical glycosaminoglycan (GAG) mostly used as a dietary supplement. We undertook analysis on some formulations of chondroitin sulfates available for oral administration. The analysis was based on agarose-gel electrophoresis, strong anion-exchange chromatography, digestibility with specific GAG lyases, uronic acid content, NMR spectroscopy, and size-exclusion chromatography. Keratan sulfate was detected in batches from shark cartilage, averaging ∼16% of the total GAG. Keratan sulfate is an inert material, and hazardous effects due to its presence in these formulations are unlikely to occur. However, its unexpected high percentage compromises the desired amounts of the real ingredient specified on the label claims, and forewarns the pharmacopeias to update their monographs. The techniques they recommended, especially cellulose acetate electrophoresis, are inefficient in detecting keratan sulfate in chondroitin sulfate formulations. In addition, this finding also alerts the manufacturers for improved isolation procedures as well as the supervisory agencies for better audits. Analysis based on strong anion-exchange chromatography is shown to be more reliable than the methods presently suggested by standard pharmacopeias.

Propositional debate on biosimilar enoxaparin in Brazil.

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.

Structure and haemostatic effects of generic versions of enoxaparin available for clinical use in Brazil: similarity to the original drug.

Patent protection for enoxaparin has expired. Generic preparations are developed and approved for clinical use in different countries. However, there is still skepticism about the possibility of making an exact copy of the original drug due to the complex processes involved in generating low-molecular-weight heparins. We have undertaken a careful analysis of generic versions of enoxaparin available for clinical use in Brazil. Thirty-three batches of active ingredient and 70 of the final pharmaceutical product were obtained from six different suppliers. They were analysed for their chemical composition, molecular size distribution, in vitro anticoagulant activity and pharmacological effects on animal models of experimental thrombosis and bleeding. Clearly, the generic versions of enoxaparin available for clinical use in Brazil are similar to the original drug. Only three out of 33 batches of active ingredient from one supplier showed differences in molecular size distribution, resulting from a low percentage of tetrasaccharide or the presence of a minor component eluted as monosaccharide. Three out of 70 batches of the final pharmaceutical products contained lower amounts of the active ingredient than that declared by the suppliers. Our results suggest that the generic versions of enoxaparin are a viable therapeutic option, but their use requires strict regulations to ensure accurate standards.

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.

Brown algae overproduce cell wall polysaccharides as a protection mechanism against the heavy metal toxicity.

Brown algae are often used as heavy metal biomonitors and biosorbents because they can accumulate high concentrations of metals. Cation-exchange performed by cell wall polysaccharides is pointed out as the main chemical mechanism for the metal sequestration. Here, we biochemically investigated if the brown alga Padina gymnospora living in a heavy metal contaminated area would modify their polysaccharidic content. We exposed non-living biomass to Cd and Pb and studied the metals adsorption and localization. We found that raw dried polysaccharides, sulfate groups, uronic acids, fucose, mannose, and galactose were significantly higher in contaminated algae compared with the control ones. Metal concentrations adsorbed by non-living biomass were rising comparatively to the tested concentrations. Electron microscopy showed numerous granules in the cell walls and X-ray microanalysis revealed Cd as the main element. We concluded that P. gymnospora overproduces cell wall polysaccharides when exposed to high metal concentrations as a defense mechanism.

Heparins from porcine and bovine intestinal mucosa: Are they similar drugs?

Increasing reports of bleeding and peri- or post-operative blood dyscrasias in Brazil were possibly associated with the use of heparin from bovine instead of porcine intestine. These two pharmaceutical grade heparins were analysed for potential differences. NMR analyses confirmed that porcine heparin is composed of mainly trisulfated disaccharides -->4-alpha-IdoA2S-1-->4-alpha-GlcNS6S-1-->. Heparin from bovine intestine is also composed of highly 2-sulfated alpha-iduronic acid residues, but the sulfation of the alpha-glucosamine units vary significantly: approximately 50% are 6- and N -disulfated, as in porcine heparin, while approximately 36% are 6-desulfated and approximately 14% N -acetylated. These heparins differ significantly in their effects on coagulation, thrombosis and bleeding. Bovine heparin acts mostly through factor Xa. Compared to porcine heparin on a weight basis, bovine heparin exhibited approximately half of the anticoagulant and antithrombotic effects, but similar effect on bleeding. These two heparins also differ in their protamine neutralisation curves. The doses of heparin from bovine intestine required for effective antithrombotic protection and the production of adverse bleeding effects are closer than those for porcine heparin. This observation may explain the increasing bleeding observed among Brazilian patients. Our results suggest that these two types of heparin are not equivalent drugs.