Ismael Nilo Lino de Queiroz

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.

Oligosaccharides from the 3-linked 2-sulfated alpha-L-fucan and alpha-L-galactan show similar conformations but different dynamics

Here we have performed an nuclear magnetic resonance-based study on the ring and chain conformations as well as dynamics of oligosaccharides generated by acid hydrolysis on two structurally related glycans, a 3-linked 2-sulfated alpha-L-galactan and a 3-linked 2-sulfated alpha-L-fucan. Results derived from scalar couplings have confirmed the 1C4 chair configuration to both alpha-L-fucose and alpha-L-galactose, and a similar solution 3D structure for the oligosaccharide chains of both sulfated glycans as seen on the basis of NOE patterns. Measurements of spin-relaxation rates have suggested, however, a slight difference dynamical property to these glycans. The fucose-based oligosaccharides showed an enhanced dynamical property if compared to the galactose-based oligosaccharides of same anomericity, sugar configuration, glycosidic bond and sulfation type. This distinction solely on the dynamical aspect has been driven therefore by the different sugar composition of the two studied sulfated glycans.

NMR structural biology of sulfated glycans

Sulfated fucans, sulfated galactans, and glycosaminoglycans are extensively studied worldwide in terms of both structure and biomedical functions. Liquid-state nuclear magnetic resonance (NMR) spectroscopy is the most employed analytical technique in structural analysis of these sulfated glycans. This is due to the fact that NMR-based analyses enable a series of achievements such as (i) accurate structure characterization/determination; (ii) measurements of parameters regarding molecular motion (dynamics); (iii) assessment of the 3D structures (usually assisted by computational techniques of Molecular Modeling and/or Molecular Dynamics) of the composing monosaccharides (ring conformers) and the overall conformational states of the glycan chains either free in solution or bound to proteins; and (iv) analysis of the resultant intermolecular complexes with functional proteins through either the protein or the carbohydrate perspective. In this review, after a general introduction about the principal NMR parameters utilized for achieving this set of structural information, discussion is given on NMR-based studies of some representative sulfated fucans, sulfated galactans, and glycosaminoglycans. Due to the growing number of studies concerning both structure and function of sulfated glycans and the widely use of NMR spectroscopy in such studies, a review paper discussing (i) the most experiments employed for analysis, (ii) procedures used in data interpretation, and (iii) the general aspects of the sulfated glycans, is timely in the literature.

Anticoagulant and Antithrombotic Properties of Three Structurally Correlated Sea Urchin Sulfated Glycans and Their Low-Molecular-Weight Derivatives

The anticoagulant and antithrombotic properties of three structurally correlated sea urchin-derived 3-linked sulfated α-glycans and their low molecular-weight derivatives were screened comparatively through various in vitro and in vivo methods. These methods include activated partial thromboplastin time, the inhibitory activity of antithrombin over thrombin and factor Xa, venous antithrombosis, the inhibition of platelet aggregation, the activation of factor XII, and bleeding. While the 2-sulfated fucan from Strongylocentrotus franciscanus was observed to be poorly active in most assays, the 4-sulfated fucan from Lytechinus variegatus, the 2-sulfated galactan from Echinometra lucunter and their derivatives showed multiple effects. All marine compounds showed no capacity to activate factor XII and similar low bleeding tendencies regardless of the dose concentrations used to achieve the highest antithrombotic effect observed. The 2-sulfated galactan showed the best combination of results. Our work improves the background about the structure-function relationship of the marine sulfated glycans in anticoagulation and antithrombosis. Besides confirming the negative effect of the 2-sulfated fucose and the positive effect of the 2-sulfated galactose on anticoagulation in vitro, our results also demonstrate the importance of this set of structural requirements on antithrombosis in vivo, and further support the involvement of high-molecular weight and 4-sulfated fucose in both activities.

A lectin fraction from green seaweed Caulerpa cupressoides inhibits inflammatory nociception in the temporomandibular joint of rats dependent from peripheral mechanisms

Temporomandibular disorders are the second most common cause of orofacial pain mediated by inflammatory compounds, which in many cases leads to chronic orofacial pain. This study assessed the antinociceptive and anti-inflammatory effects of a lectin from the green seaweed Caulerpa cupressoides (CcL) on hypernociception inflammatory in TMJ of rats and investigated the involvement of different mechanisms. Rats received i.v. CcL 30 min prior to injection of flogistic agentes or 0.9% saline into the left TMJ. Pretreatment with CcL (0. 1; 1 or 10 mg/kg) promoted a reduction (p < 0.05) of inflammatory hypernociception induced by 1.5% Formalin along with inhibition of inflammatory plasma extravasation, cytokines levels, ciclooxigenase-2, and intercellular adhesion molecule (ICAM-1). CcL was able to inhibit the nociceptive response induced by 1.5% Capsaicin, suggesting that CcL has an antinociceptive effect, acting directly on the primary nociceptive neurons. CcL also inhibited the nociceptive response induced by Carrageenan (100 μg/TMJ) or Serotonin (5-HT) (225 μg/TMJ). In conclusion, the results demonstrate that administration of CcL has a potential antinociceptive and anti-inflammatory effect, with a mechanism that is partially dependent on TNF-α, IL-1β, COX-2 and ICAM-1 inhibition and independently from the cannabinoide and opioid system and NO/cGMP/PKG/K+ATP channel pathway.

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+, Ca2+, 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 1C4 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.