Fish oil supplementation during pregnancy decreases liver endocannabinoid system and lipogenic markers in newborn rats exposed to maternal high-fat diet.

PURPOSE: Maternal high-fat diet (HF) programs obesity, metabolic dysfunction-associated steatotic liver disease (MASLD), hypertriglyceridemia, and hyperglycemia associated with increased endocannabinoid system (ECS) in the liver of adult male rat offspring. We hypothesized that maternal HF would induce sex specific ECS changes in the liver of newborn rats, prior to obesity onset, and maternal fish oil (FO) supplementation would reprogram the ECS and lipid metabolism markers preventing liver triglycerides (TG) accumulation. METHODS: Female rats received a control (CT) (10.9% fat) or HF (28.7% fat) diet 8 weeks prior to mating and during pregnancy. A subgroup of HF dams received 3% FO supplementation in the HF diet (35.4% fat) during pregnancy (HFFO). Serum hormones and liver TG, ECS, lipid metabolism, oxidative stress and autophagy markers were assessed in male and female newborn offspring. RESULTS: Maternal HF diet increased liver cannabinoid receptor 1 (CB1) in males and decreased CB2 in females, with no effect on liver TG. Maternal FO supplementation reduced liver CB1 regardless of the offspring sex, but reduced TG liver content only in females. FO reduced the liver content of the endocannabinoid anandamide in males, and the content of 2-arachidonoylglycerol in both sexes. Maternal HF increased lipogenic and decreased lipid oxidation markers, and FO induced the opposite regulation in the liver of offspring. CONCLUSION: Prenatal HF and FO differentially modulate liver ECS in the offspring before obesity and MASLD development. These results suggest that maternal nutrition at critical stages of development can modulate the offspring's ECS, predisposing or preventing the onset of metabolic diseases.

Maternal high-fat diet decreases milk endocannabinoids with sex-specific changes in the cannabinoid and dopamine signaling and food preference in rat offspring.

Introduction: Maternal high-fat (HF) diet during gestation and lactation programs obesity in rat offspring associated with sex-dependent and tissue-specific changes of the endocannabinoid system (ECS). The ECS activation induces food intake and preference for fat as well as lipogenesis. We hypothesized that maternal HF diet would increase the lipid endocannabinoid levels in breast milk programming cannabinoid and dopamine signaling and food preference in rat offspring. Methods: Female Wistar rats were assigned into two experimental groups: control group (C), which received a standard diet (10% fat), or HF group, which received a high-fat diet (29% fat) for 8 weeks before mating and during gestation and lactation. Milk samples were collected to measure endocannabinoids and fatty acids by mass spectrometry. Cannabinoid and dopamine signaling were evaluated in the nucleus accumbens (NAc) of male and female weanling offspring. C and HF offspring received C diet after weaning and food preference was assessed in adolescence. Results: Maternal HF diet reduced the milk content of anandamide (AEA) (p<0.05) and 2-arachidonoylglycerol (2-AG) (p<0.05). In parallel, maternal HF diet increased adiposity in male (p<0.05) and female offspring (p<0.05) at weaning. Maternal HF diet increased cannabinoid and dopamine signaling in the NAc only in male offspring (p<0.05), which was associated with higher preference for fat in adolescence (p<0.05). Conclusion: Contrary to our hypothesis, maternal HF diet reduced AEA and 2-AG in breast milk. We speculate that decreased endocannabinoid exposure during lactation may induce sex-dependent adaptive changes of the cannabinoid-dopamine crosstalk signaling in the developing NAc, contributing to alterations in neurodevelopment and programming of preference for fat in adolescent male offspring.

Chemical variability of Copaifera langsdorffii Desf. from environmentally contrasting populations.

Copaifera langsdorffii Desf. is recognised as one of most famous medicinal and economic species of Copaiba, occurring in several distinct biomes. An untargeted metabolomic approach was used to evaluate the chemical variability of C. langsdorffii from contrasting climates biomes (Atlantic Rainflorest and the semiarid Cerrado). Metabolomic analysis enabled the identification of 11 compounds, including glycosylated flavonoids and galloylquinic acid derivatives. Multivariate analysis highlighted that Cerrado population had a significantly higher concentrations of galloylquinic acid derivatives in comparison to the rainforest biome. Meanwhile, Atlantic Rainforest populations presented higher content of flavonols. Semiarid biome, reduced the concentration of flavonoids, mainly concerning quercetin and kaempferol derivatives, however, in this biome flavonoids were more diverse. Both chemical classes presented relevance to be used as geographical origin chemical markers by qualitative and quantitative features.

Adhesion of freshwater sponge cells mediated by carbohydrate-carbohydrate interactions requires low environmental calcium.

Marine ancestors of freshwater sponges had to undergo a series of physiological adaptations to colonize harsh and heterogeneous limnic environments. Besides reduced salinity, river-lake systems also have calcium concentrations far lower than seawater. Cell adhesion in sponges is mediated by calcium-dependent multivalent self-interactions of sulfated polysaccharide components of membrane-bound proteoglycans named aggregation factors. Cells of marine sponges require seawater average calcium concentration (10 mM) to sustain adhesion promoted by aggregation factors. We demonstrate here that the freshwater sponge Spongilla alba can thrive in a calcium-poor aquatic environment and that their cells are able to aggregate and form primmorphs with calcium concentrations 40-fold lower than that required by marine sponges cells. We also find that their gemmules need calcium and other micronutrients to hatch and generate new sponges. The sulfated polysaccharide purified from S. alba has sulfate content and molecular size notably lower than those from marine sponges. Nuclear magnetic resonance analyses indicated that it is composed of a central backbone of non- and 2-sulfated α- and ß-glucose units decorated with branches of α-glucose. Assessments with atomic force microscopy/single-molecule force spectroscopy show that S. alba glucan requires 10-fold less calcium than sulfated polysaccharides from marine sponges to self-interact efficiently. Such an ability to retain multicellular morphology with low environmental calcium must have been a crucial evolutionary step for freshwater sponges to successfully colonize inland waters.

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.

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.

ß-1,6-linked Galactofuranose- rich peptidogalactomannan of Fusarium oxysporum is important in the activation of macrophage mechanisms and as a potential diagnostic antigen.

A peptidogalactomannan (PGM) from Fusarium oxysporum was structurally characterized by a combination of chemical and spectroscopic methods, including one and two-dimensional nuclear magnetic resonance (1D and 2D NMR). The galactomannan component consists of a main chain containing (1→6)-linked ß-D-galactofuranose residues with side chains containing (1→2)-linked α-D-Glcp, (1→2)-linked -ß-D-Manp (1→2) and ß-D-Manp terminal nonreducing end units and differs from that of Aspergillus fumigatus and Cladosporium resinae that present a main chain containing (1→6)-linked α-D-Manp residues presenting ß-D-Galf as side chains of 3-4 units that are (1→5)-interlinked. The importance of the carbohydrate moiety of the F. oxysporum PGM was demonstrated. Periodate oxidation abolished much of the PGM antigenic activity. A strong decrease in reactivity was also observed with de-O-glycosylated PGM. In addition, de-O-glycosylated PGM was not able to inhibit F. oxysporum phagocytosis, suggesting that macrophages recognize and internalize F. oxysporum via PGM. F. oxysporum PGM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PGM led to a significant increase of TNF-α cytokine levels, suggesting that their removal could exposure another PGM motifs able to induce a higher secretion of TNF-α levels. Interestingly, F. oxysporum conidia, intact and de-O-linked PGM were not able to induce IL-10 cytokine release. The difference in patient serum reativity using a PGM from F. oxysporum characterized in the present study as compared with a PGM from C. resinae, that presents the same epitopes recognized by serum from patients with aspergillosis, could be considered a potential diagnostic antigen and should be tested with more sera.

Chelate-soluble pectin fraction from papaya pulp interacts with galectin-3 and inhibits colon cancer cell proliferation.

Colorectal cancer has an overexpression of galectin-3 that is related to cancer progression. A decreased risk of colon cancer can be related to consumption of dietary fibers, but the entire mechanism by which this protection occurs remains unclear. Pectin is a type of dietary fiber that possesses ß-galactosides and can bind and inhibit galectin-3-mediated effects. Papaya fruit has a massive cell wall disassembling during ripening that naturally changes its pectin structure. Our work shows that different points in the ripening time of papaya fruit exhibit pectins (chelate-soluble fractions; CSF) that can or cannot inhibit galectin-3. The fraction that inhibits galectin-3 (3CSF) also diminishes the proliferation of colon cancer cell lines, and it is derived from an intermediate point of papaya ripening. Therefore, we related this to a papaya pectin structure-dependent effect, and the papaya fruit seems to have a pectin structure that is promising in decreasing the risk of colon cancer development.

An arabinogalactan-glycoconjugate from Genipa americana leaves present anticoagulant, antiplatelet and antithrombotic effects.

Glycoconjugates extracted from Genipa americana leaves (PE-Ga) were separated into two fractions, denominated as PFI and PFII (total carbohydrate: 23-36%/uronic acid: 9-30%; protein:4-5%; polyphenols:0.776-0.812 mg/g), mainly composed by arabinose, galactose and uronic acid and presenting high (PFI) and low (PFII) molecular weight (based on polyacrylamide electrophoresis gel and gel permeation chromatography). Uronic acid was also detected by FT-IR (wavenumbers: 1410 and 1333 cm-1) and NMR (α-GalpA). Deproteinization of glycoconjugates showed reduced protein and polyphenol levels with loss of its biological effects. PE-Ga and PFII prolonged clotting time-aPTT (3.6 and 1.8x), while PE-Ga and PFI inhibited by 48% (100 µg/µL) the ADP-induced platelet aggregation. In vivo, these glycoconjugates at 1 mg/kg inhibited (37-53%) venous thrombus formation (4.7 ± 0.1 mg) and increased bleeding time (PE-Ga and PFI:3.0x; PFII:1.7x vs. PBS:906 ± 16.7 s). In conclusion, the arabinogalactan-rich glycoconjugate of G. americana leaves, containing uronic acid, present antiplatelet, anticoagulant (intrinsic/common pathway) and antithrombotic effects, with low hemorrhagic risk.

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.

Improved anticoagulant effect of fucosylated chondroitin sulfate orally administered as gastro-resistant tablets.

Fucosylated chondroitin sulfate (FucCS) is a potent anticoagulant polysaccharide extracted from sea cucumber. Its anticoagulant activity is attributed to the presence of unique branches of sulfated fucose. Although this glycosaminoglycan exerts an antithrombotic effect following oral administration, high doses are necessary to achieve the maximum effect. The diminished activity of FucCS following oral administration is likely due to its degradation in the gastrointestinal tract and its limited ability to cross the intestinal cell membranes. The latter aspect is particularly difficult to overcome. However, gastro-resistant tablet formulation may help limit the degradation of FucCS in the gastrointestinal tract. In the present work, we found that the oral administration of FucCS as gastro-resistant tablets produces a more potent and prolonged anticoagulant effect compared with its administration as an aqueous solution, with no significant changes in the bleeding tendency or arterial blood pressure. Experiments using animal models of arterial thrombosis initiated by endothelial injury demonstrated that FucCS delivered as gastro-protective tablets produced a potent antithrombotic effect, whereas its aqueous solution was ineffective. However, there was no significant difference between the effects of FucCS delivered as gastro-resistant tablets or as aqueous solution in a venous thrombosis model, likely due to the high dose of thromboplastin used. New oral anticoagulants tested in these experimental models for comparison showed significantly increased bleeding tendencies. Our study provides a framework for developing effective oral anticoagulants based on sulfated polysaccharides from marine organisms. The present results suggest that FucCS is a promising oral anticoagulant.

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.

NMR-based conformation and dynamics of a tetrasaccharide-repeating sulfated fucan substituted by different counterions.

The sulfated fucan from the sea urchin Lytechinus variegatus is composed of the repetitive sequence [-3)-α-l-Fucp-4( OSO3-)-(1-3)-α-l-Fucp-2,4-di( OSO3-)-(1-3)-α-l-Fucp-2( OSO3-)-(1-3)-α-l-Fucp-2( OSO3-)-(1-]n . Conformation (of rings and chains) and dynamics of this tetrasaccharide-repeating sulfated fucan substituted by Na(+) , Ca(2+) , and Li(+) as counterions have been examined through experiments of liquid-state nuclear magnetic resonance spectroscopy. Scalar coupling and nuclear Overhauser effect (NOE)-based data have confirmed that all composing units occur as (1) C4 chair conformer regardless of the cation type, unit position within the repeating sequence, and sulfation type. Chain conformation determined by NOE signal pattern assisted by molecular modeling for a theoretical octasaccharide has shown a similar linear 3D structure for the three differently substituted forms. Data derived from spin-relaxation measurements have indicated a contribution of counterion type to dynamics. The calcium-based preparation has shown the highest mobility while the sodiated one showed the lowest mobility. The set of results from this work suggests that counterion type can affect the physicochemical properties of the structurally well-defined sulfated fucan. The counterion effect seems to impact more on the structural mobility than on average conformation of the studied sulfated glycan in solution.

Carbohydrate-Carbohydrate Interactions Mediated by Sulfate Esters and Calcium Provide the Cell Adhesion Required for the Emergence of Early Metazoans.

Early metazoans had to evolve the first cell adhesion mechanism addressed to maintain a distinctive multicellular morphology. As the oldest extant animals, sponges are good candidates for possessing remnants of the molecules responsible for this crucial evolutionary innovation. Cell adhesion in sponges is mediated by the calcium-dependent multivalent self-interactions of sulfated polysaccharides components of extracellular membrane-bound proteoglycans, namely aggregation factors. Here, we used atomic force microscopy to demonstrate that the aggregation factor of the sponge Desmapsamma anchorata has a circular supramolecular structure and that it thus belongs to the spongican family. Its sulfated polysaccharide units, which were characterized via nuclear magnetic resonance analysis, consist preponderantly of a central backbone composed of 3-α-Glc1 units partially sulfated at 2- and 4-positions and branches of Pyr(4,6)α-Gal1→3-α-Fuc2(SO3)1→3-α-Glc4(SO3)1→3-α-Glc→4-linked to the central α-Glc units. Single-molecule force measurements of self-binding forces of this sulfated polysaccharide and their chemically desulfated and carboxyl-reduced derivatives revealed that the sulfate epitopes and extracellular calcium are essential for providing the strength and stability necessary to sustain cell adhesion in sponges. We further discuss these findings within the framework of the role of molecular structures in the early evolution of metazoans.

Development and characterization of a new hydrogel based on galactomannan and κ-carrageenan.

A new hydrogel based on two natural polysaccharides was prepared in aqueous medium with 1.7% (w/v) galactomannan (from Cassia grandis seeds) and different concentrations of κ-carrageenan (0.3, 0.4 and 0.5%w/v), CaCl2 (0.0, 0.1 and 0.2M) and pH (5.0, 5.5 and 6.0), using a full factorial design based on rheological parameters. The best formulation was obtained with 1.7% (w/v) galactomannan and 0.5% (w/v) κ-carrageenan, containing 0.2M CaCl2 at pH 5.0. Nuclear magnetic resonance and scanning electron microscopy where used in order to characterize the hydrogel formulation. A shelf life study was carried out with this formulation along 90 days-period of storage at 4 °C, evaluating pH, color, microbial contamination and rheology. This hydrogel showed no significant changes in pH, no microbial contamination and became more translucent along the aging. Analyses by nuclear magnetic resonance and rheology showed a larger organization of the polysaccharides in the hydrogel matrix. The results demonstrated that this hydrogel was stable with possible applications in medical and cosmetic fields.

Distinct structures of the α-fucose branches in fucosylated chondroitin sulfates do not affect their anticoagulant activity.

Fucosylated chondroitin sulfate (FCS) is a glycosaminoglycan found in sea cucumbers. It has a backbone like that of mammalian chondroitin sulfate (4-ß-d-GlcA-1→3-ß-d-GalNAc-1)n but substituted at the 3rd position of the ß-d-glururonic acid residues with α-fucose branches. The structure of these branches varies among FCSs extracted from different species of sea cucumbers, as revealed by solution NMR spectroscopy. Some species (Isostichopus badionotus and Patalus mollis) contain branches formed by single α-fucose residues but with variable sulfation patterns (2,4-, 3,4- and 4-sulfation). FCS from Ludwigothurea grisea is distinguished because it contains preponderant branches formed by disaccharide units containing non-sulfated and 3-sulfated α-fucose units at the reducing and non-reducing ends, respectively. Despite the structural variability on their α-fucose branches, these FCSs have similar anticoagulant action on assays using purified reagents. They have serpin-dependent and serpin-independent effects. Pharmacological assays using experimental animals showed that the three types of FCSs have similar antithrombotic effect and bleeding tendency. They also activate factor XII on the same range of concentration. Based on these observations, we proposed that only few sulfated α-fucose branches along the FCS chain are enough to assure the binding of this glycosaminoglycan to proteins of the coagulation system. Substitution with additional sulfated α-fucose does not increase further the activity. Overall, the use of FCSs with marked variability on their branches of α-fucose allowed us to establish correlations between structures vs biological effects of these glycosaminoglycans on a more refined basis. It opens new avenues for therapeutic intervention using FCSs.

Purified polysaccharides of Geoffroea spinosa barks have anticoagulant and antithrombotic activities devoid of hemorrhagic risks.

Polysaccharides were extracted from the barks of Geoffroea spinosa, purified using anion exchange chromatography and characterized by chemical and methylation analysis, complemented by infrared and NMR spectroscopies. These polysaccharides were tested for their anticoagulant, antithrombotic and antiplatelet activities and also for their effects on bleeding. Unfractionated polysaccharide contains low levels of protein and high levels of carbohydrate (including hexuronic acid). The purified polysaccharides (fractions FII and FIII) are composed of arabinose (Ara), rhamnose (Rha), hexuronic acid, small amounts of galactose, but no sulfate ester. They have highly complex structure, which was partially characterized. NMR and methylation analysis indicate that the polysaccharides have a core of α-Rhap and branches of 5-linked α-Araf. Residues of 4-linked α-GalpA are also found in the structure. The unfractionated (TPL) and fraction FIII, but not fractions FI and FII, prolonged the activated partial thromboplastin time (aPTT). TPL, FII and FIII inhibited the platelet aggregation induced by ADP. More significantly, both unfractionated and purified fractions exhibited potent antithrombotic effect (31-60%) and the fractions did not modify the bleeding tendency. These plant polysaccharides could be alternative source of new anticoagulant, antiplatelet and antithrombotic compounds devoid of the undesirable risk of hemorrhage.

Structure and rheological properties of a xyloglucan extracted from Hymenaea courbaril var. courbaril seeds.

Hymenaea courbaril var courbaril seed xyloglucan was efficiently extracted with 0.1M NaCl, followed by ethanol precipitation (yield=72±5% w/w). Its amorphous structure was identified by the pattern of X-ray diffraction. The monosaccharide composition was determined by GC/MS analysis of the alditol acetates and showed the occurrence of glucose:xylose:galactose:arabinose (40:34:20:6). One-(1D) and two-dimensional-(2D) NMR spectra confirmed a central backbone composed by 4-linked ß-glucose units partially branched at position 6 with non-reducing terminal units of α-xylose or ß-galactose-(1→2)-α-xylose disaccharides. The xyloglucan solution was evaluated by dynamic light scattering and presents a polydisperse and practically neutral profile, and at 0.5 and 1.0% (w/v) the solutions behave as a viscoelastic fluid. The polysaccharide did not show significant antibacterial or hemolytic activities. Overall our results indicate that xyloglucan from H. courbaril is a promising polysaccharide for food and pharmaceutical industries.

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.

Impact of sulfation pattern on the conformation and dynamics of sulfated fucan oligosaccharides as revealed by NMR and MD.

Sulfated fucans from sea urchin egg jelly express well-defined chemical structures that vary with species. This species specificity regulates the sperm acrosome reaction, a critical step to assure intra-specific fertilization. In addition, these polysaccharides are involved in other biological activities such as anticoagulation. Although sulfation patterns are relevant to the levels of response in both activities, conformation and dynamics of these glycans are also contributing factors. However, data about these features of sulfated fucans are very rare. To address this, we have employed nuclear magnetic resonance experiments combined with molecular dynamics on structurally defined oligosaccharides derived from two sulfated fucans. The results have indicated that the oligosaccharides are flexible in solution. Ring conformation of their composing units displays just the (1)C4 chair configuration. In a particular octasaccharide, composed of two tetrasaccharide sequences, inter-residual hydrogen bonds play a role to decrease dynamics in these repeating units. Conversely, the linking disaccharide [-3)-α-L-Fucp-2(OSO3(-))-(1-3)-α-L-Fucp-4(OCO3(-))-(1-] located right between the two tetrasaccharide units has amplified motions suggested to be promoted by electrostatic repulsion of sulfates on opposite sides of the central glycosidic bond. This conjunction of information about conformation and dynamics of sulfated fucan oligosaccharides provides new insights to explain how these glycans behave free in solution and influenced by sulfation patterns. It may also serve for future studies concerning structure-function relationship of sulfated fucans, especially those involving sea urchin fertilization and anticoagulation.