Structural and functional analyses of biosimilar enoxaparins available in Brazil

Summary

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 vivoantithrombotic 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.

Keywords

Heparins - thrombosis - animal models - pharmacovigilance of heparin - low-molecular-weight heparin

• References

• 1 Weitz JI. Low-molecular-weight heparins. N Engl J Med 1997; 337: 688-698.
• 2 Harenberg J, Kakkar A, Bergqvist D. et al. Recommendations on biosimilar low-molecular-weight-heparins. J Thromb Haemost 2009; 07: 1222-1225.
• 3 Harenberg J, Walenga J, Torri G. et al. Update of the recommendations on biosimilar low-molecular-weight heparins from the scientific subcommittee on control ofanticoagulation of the ISTH. J Thromb Haemost 2013; 11: 1421-1425.
• 4 Gray E, Mulloy B. Biosimilar low-molecular-weight heparin products. J Thromb Haemost 2009; 07: 1218-1221.
• 5 Fareed J, Leong WL, Hoppensteadt DA. et al. Generic low-molecular-weight he-parins: some practicalconsiderations. Semin Thromb Hemost 2004; 30: 703-713.
• 6 Minghetti P, Cilurzo F, Fanzé S et al.. Low molecular weight heparins copies: are they considered to be generics or biosimilars?. Drug Disc Today 2013; 18: 305-311.
• 7 Glauser BF, Vairo BC, Oliveira CPM. et al. Generic versions of enoxaparin available for clinical use in Brazil are similar to the original drug. J Thromb Haemost 2011; 09: 1419-1422.
• 8 Glauser BF, Vairo BC, Oliveira S-NMCG. et al. Structure and haemostatic effects of generic versions of enoxaparin available for clinical use in Brazil: similarity to the original drug. Thromb Haemost 2012; 107: 302-314.
• 9 Aquino RS, Pereira MS, Vairo BC. et al. Heparins from porcine and bovine intestine mucosa: are they similar drugs?. Thromb Haemost 2010; 103: 1005-1015.
• 10 Sitkowski J, Bednarek E, Bocian W, Kozerski L. Assessment of oversulfated chondroitin sulfate in low molecular weight and unfractioned heparins diffusion ordered nuclear magnetic resonance spectroscopy method. J Med Chem 2008; 51: 7663-7665.
• 11 http://www.columbia.edu/cu/chemistry/groups/nmr/SpinWorks.html
• 12 Komatsu H, Takahata T, Tanaka M. et al. Determination of the molecular-weight distribution of low-molecular-weight heparins using high-performance gel permeation chromatography. Biol Pharm Bull 1993; 16: 1189-1193.
• 13 Olson ST, Björk I, Shore JD. Kinetic characterisation of heparin-catalysed and uncatalysed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol 1993; 222: 525-559.
• 14 Fonseca RJ, Oliveira SNMG, Pomin VH. et al. Effects of oversulfated and fuco-sylated chondroitin sulfate on coagulation. Challenges for the study of anticoagulant polysaccharides. Thromb Haemost 2010; 103: 994-1004.
• 15 Herbert JM, Bernat A, Maffrand JP. Importance of platelets in experiment venous thrombosis in the rat. Blood 1992; 80: 2281-2286.
• 16 Mascellani G, Guerrini M, Torri G, Liverani L. et al. Characterisation of di- and monosulfated, unsaturated heparin disaccharides with terminal N-sulfated 1,6-anhydro-beta-D-glucosamine or N-sulfated 1,6-anhydro-beta-D-mannosamine residues. Carbohydr Res 2007; 342: 835-842.
• 17 Guerrini M, Guglieri S, Naggi A. et al. Low molecular weight heparins: structural differentiation by bidimensional nuclear magnetic resonance spectroscopy. Semin Thromb Hemost 2007; 33: 478-487.
• 18 Casu B, Guerrini M, Naggi A. et al. Characterisation of sulfation patterns of beef and pig mucosal heparins by nuclear magnetic resonance spectroscopy. Arzneimittelforschung 1996; 46: 472-477.
• 19 Mulloy B, Forster MJ, Jones C. et al. The effect of variation of substitution on the solution conformation of heparin: a spectroscopic and molecular modelling study. arbohydr Res 1994; 255: 1-26.
• 20 Collignon F, Frydman A, Caplain H. et al. Comparison of the pharmacokinetic profiles of three low molecular mass heparin - dalteparin, enoxaparin and nadroparin - administered subcutaneously in healthy volunteers (dose for prevention of thromboembolism). Thromb Haemost 1995; 73: 630-640.
• 21 http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003928.pdf
• 22 http://portal.anvisa.gov.br/wps/wcm/connect/fdd0d20048bd2217a789af9a6e94f0d0/heparina.pdf
• 23 Kishimoto TK, Viswanathan K, Ganguly T. et al. Contaminated heparin associated with adverse clinical events and activation of the contact system. N Engl J Med 2008; 358: 2457-2467.
• 24 Pomin VH, Piquet AA, Pereira MS, Mouräo PAS. Residual keratan sulfate in chondroitin sulfate formulations for oral administration. Carbohydr Polymers 2012; 90: 839-846..
• 25 http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProviders/ucm220037.htm