ABSTRACT
Low-molecular-weight heparins (LMWHs) differ considerably in their influence on clotting tests and release of tissue factor pathway inhibitor (TFPI). Biosimilarity therefore becomes an issue when generic forms of LMWHs are developed. So far, no bioequivalence study with a generic LMWH has been reported. A generic enoxaparin (test) was compared with the originator (reference) in 20 volunteers after single-dose subcutaneous administration (40 mg enoxaparin sodium, 4000 IU/mL anti-factor Xa (anti-FXa; activity). Target variables were anti-FXa and anti-FIIa activity, activated partial thromboplastin time (aPTT), prothrombinase-induced clotting time (PiCT), and TFPI over 24 hours. The statistical evaluation of the anti-FXa activity profile demonstrated bioequivalence of test and reference with confidence intervals of area under the plasma concentration-time curve (AUC0-tlast) (93%-99%) and Amax (88%-95%). Confidence intervals of AUC(0-tlast) (89%-102%) and Amax (90%-103%) of anti-FIIa activity also fulfill bioequivalence criteria. The 90% confidence interval for the maximum concentration of TFPI ranged from 90% to 113%. The claim of similarity was also supported by aPTT and PiCT profiles. Bioequivalence with the originator enoxaparin could be demonstrated by ex vivo inhibition of FXa and FIIa activity, by coagulation tests (aPTT and PiCT), and by in vivo release of TFPI. Whether such data also prove biosimilarity of the generic enoxaparin needs to be determined.
Subject(s)
Anticoagulants/pharmacokinetics , Blood Coagulation/drug effects , Drugs, Generic/pharmacokinetics , Enoxaparin/pharmacokinetics , Adult , Anticoagulants/administration & dosage , Anticoagulants/pharmacology , Area Under Curve , Blood Coagulation Tests/methods , Cross-Over Studies , Double-Blind Method , Drugs, Generic/administration & dosage , Drugs, Generic/pharmacology , Enoxaparin/administration & dosage , Enoxaparin/pharmacology , Factor Xa Inhibitors , Female , Humans , Lipoproteins/drug effects , Male , Partial Thromboplastin Time , Prothrombin/antagonists & inhibitors , Therapeutic EquivalencyABSTRACT
Chemical and physical characteristics, building blocks, constitutive disaccharides, sulfation degree, and biological activities of heparins (UFHs) and of low molecular weight heparins (LMWHs) obtained by different depolymerization processes are examined comparatively in terms of structure characteristics, content of 1,6-anhydro rings, and other fingerprints. The heterogeneity of different LMWHs depends on different manufacturing processes and on particular specifications of pharmacopoeias. The reported examples prove that the variability among samples of LMWHs manufactured by the same process is quite limited. Most of the variability is derived from the parent UFH. In contrast, fingerprint groups and residues are specific to the depolymerization process and their extent can be roughly controlled through the process parameters.
Subject(s)
Anticoagulants/chemistry , Heparin, Low-Molecular-Weight/chemistry , Anticoagulants/standards , Drug Industry/standards , Heparin, Low-Molecular-Weight/standards , Reference StandardsABSTRACT
Modified heparin disaccharides were obtained by the alkaline treatment of a solution containing the disulfated heparin disaccharide DeltaHexA-alpha-(1-->4)-D-GlcNSO(3),6SO(3). Their structures were characterized by one- and two-dimensional NMR spectroscopy: DeltaHexA-alpha-(1-->4)-1,6-anhydro-GlcNSO(3), DeltaHexA-alpha-(1-->4)-1,6-anhydro-ManNSO(3) and DeltaHexA-alpha-(1-->4)-ManNSO(3),6OSO(3). NMR spectroscopy, in combination with HPLC, provided the composition of the mixture. Characteristic NMR signals of the disaccharides were identified, even at low levels, in a high field of (1)H-(13)C correlation NMR spectra (HSQC) of a low molecular weight heparin (LMWH) obtained by beta-elimination (alkaline hydrolysis) of heparin benzyl ester, providing a more complete structural profile of this class of compounds.
Subject(s)
Disaccharides/chemistry , Heparin/analogs & derivatives , Oligosaccharides/chemistry , Sulfates/chemistry , Carbohydrate Conformation , Chromatography, High Pressure Liquid , Heparin/chemistry , Heparin, Low-Molecular-Weight/chemistry , Magnetic Resonance Spectroscopy , Models, MolecularABSTRACT
Endogenous N-sulfated glycosaminoglycans (GAGs) comigrating with standard heparin and sensitive to nitrous acid treatment were isolated from plasma of healthy donors. The amount of these compounds was 7-10 microg/ml, and activated partial thromboplastin time, anti-Xa and anti-IIa activities were similar to those of standard heparin of high molecular mass. Analysis with gradient PAGE of the putative endogenous heparin showed a mean molecular mass of 12 kD. These N-sulfated GAGs could be isolated only after removal of binding peptides that impaired purification by ion-exchange chromatography. We used SDS-PAGE as a tool to separate peptides from endogenous GAGs. N-sulfated GAGs exited the gel before peptides when the electrophoresis was overrun. Endogenous GAGs could be recovered by ion-exchange chromatography of the SDS-PAGE buffer, 'free' from associating peptides. These results strongly support the hypothesis that endogenous heparin is associated in vitro with a variety of proteins and that this association could be responsible for modification of both heparin and protein activities.
