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.

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.

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.

Update on Brazilian biosimilar enoxaparins.

INTRODUCTION: Brazil is among the first countries approving the commercialization and clinical use of biosimilar enoxaparins. Our research group has performed quality control assessments of these drugs over the last decade. Areas covered: We have not found noticeable differences between Brazilian biosimilar enoxaparins and the original product regarding their physicochemical properties, disaccharide composition, anticoagulant activity, bioavailability and safety. Expert commentary: In spite of clinical and pharmacological advantages of enoxaparin, subcutaneous formulations of unfractionated heparin are employed by the Brazilian public health system for prevention and treatment of thromboembolism. The underuse of both original and biosimilar enoxaparins in Brazil directly correlates with their high cost.

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.

Heparin from bovine intestinal mucosa: glycans with multiple sulfation patterns and anticoagulant effects.

Pharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide →4-α-IdoA2S-1→4-α-GlcNS6S-1→. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N -sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3- and 6-trisulfated α-glucosamine (lower proportions) and α-GlcNS-1→4-α-GlcA and α-IdoA2S-1→4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the α-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical heparin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro . Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.

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.

Effects of oversulfated and fucosylated chondroitin sulfates on coagulation. Challenges for the study of anticoagulant polysaccharides.

We report the effects of a chemically oversulfated chondroitin sulfate and a naturally fucosylated chondroitin sulfate on the coagulation system. The former has been recently identified as a contaminant of heparin preparations and the latter has been proposed as an alternative anticoagulant. The mechanism of action of these polymers on coagulation is complex and target different components of the coagulation system. They have serpin-independent anticoagulant activity, which preponderates in plasma. They also have serpin-dependent anticoagulant activity but differ significantly in the target coagulation protease and preferential serpin. Their anticoagulant effects differ even more markedly when tested as inhibitors of coagulation proteases using plasma as a source of serpins. It is possible that the difference is due to the high availability of fucosylated chondroitin sulfate whereas oversulfated chondroitin sulfate has strong unspecific binding to plasma protein and low availability for the binding to serpins. When tested using a venous thrombosis experimental model, oversulfated chondroitin sulfate is less potent as an antithrombotic agent than fucosylated chondroitin sulfate. These highly sulfated chondroitin sulfates activate factor XII in in vitro assays, based on kallikrein release. However, only fucosylated chondroitin sulfate induces hypotension when intravenously injected into rats. In conclusion, the complexity of the regulatory mechanisms involved in the action of highly sulfated polysaccharides in coagulation requires their analysis by a combination of in vitro and in vivo assays. Our results are relevant due to the urgent need for new anticoagulant drugs or alternative sources of heparin.

Slight differences in sulfation of algal galactans account for differences in their anticoagulant and venous antithrombotic activities.

We compared sulfated galactans (SGs) from two species of red algae using specific coagulation assays and experimental models of thrombosis. These polysaccharides have an identical saccharide structure and the same size chain, but with slight differences in their sulfation patterns. As a consequence of these differences, the two SGs differ in their anticoagulant and venous antithrombotic activities. SG from G. crinale exhibits procoagulant and prothrombotic effects in low doses (up to 1.0 mg/kg body weight), but in high doses (>1.0 mg/kg) this polysaccharide inhibits both venous and arterial thrombosis in rats and prolongs ex-vivo recalcification time. In contrast, SG from B. occidentalis is a very potent anticoagulant and antithrombotic compound in low doses (up to 0.5 mg/kg body weight), inhibiting venous experimental thrombosis and prolonging ex-vivo recalcification time, but these effects are reverted in high doses. Only at high doses (>1.0 mg/kg) the SG from B. occidentalis inhibits arterial thrombosis. As with heparin, SG from G. crinale does not activate factor XII, while the polysaccharide from B. occidentalis activates factor XII in high concentrations, which could account for its procoagulant effect at high doses on rats. Both SGs do not modify bleeding time in rats. These results indicate that slight differences in the proportions and/or distribution of sulfated residues along the galactan chain may be critical for the interaction between proteases, inhibitors and activators of the coagulation system, resulting in a distinct pattern in anti- and procoagulant activities and in the antithrombotic action.