Anticoagulant Activity of Heparins from Different Animal Sources are Driven by a Synergistic Combination of Physical-chemical Factors

Abstract

Heparin has already been found in a variety of animal tissues but only few of them became effective sources for production of pharmaceutical preparations. Here, we correlate physical-chemical features and anticoagulant activities of structurally similar heparins employed in the past (from bovine lung, HBL), in the present (from porcine intestine, HPI) and in development for future use (from ovine intestine, HOI). Although they indeed have similar composition, our physical-chemical analyses with different chromatography and spectrometric techniques show that both HOI and HBL have molecular size notably lower than HPI and that the proportions of some of their minor saccharide components can vary substantially. Measurements of anticoagulant activities with anti-FIIa and anti-FXa assays confirmed that HPI and HOI have potency similar each other but significantly higher than HBL. Such a lower activity of HBL has been attributed to its reduced molecular size. Considering that HOI also has reduced molecular size, we find that its increased anticoagulant potency might result from an improved affinity to antithrombin (three times higher than HBL) promoted by the high content of N,3,6-trisulfated glucosamine units, which in turn are directly involved in the heparin-antithrombin binding. Therefore, the anticoagulant activity of different heparins is driven by a balance between different physical-chemical components, especially molecular size and fine-tuning composition. Although such minor but relevant chemical differences reinforce the concept that heparins from different animal sources should indeed be considered as distinct drugs, HOI could be approved for interchangeable use with the gold standard HPI and as a suitable start material for producing new LMWHs.

A Unique Enoxaparin Derived from Bovine Intestinal Heparin: A Single Purification Step of the Starting Material Assures a Bovine Enoxaparin Like the Standard from Porcine Origin

“…The chemical shift values of this system are equivalent to those previously reported for HBI. 15 Another observation is the presence of two spin systems of unsaturated hexuronic acid (Δ4,5UA) at the nonreducing ends of the molecule, designated as Ua and Uc, attributed to units linked to α-GlcN,6-diS and α-GlcNS, respectively.…”

“…These findings align with observations that pharmaceutical preparations of HBI have high proportions of 6-desulfated α-GlcNS, similarly reflected in bovine enoxaparin. 15 , 16 To address this limitation, an HBI obtained after an additional purification step (HABH) was utilized. Enoxaparin prepared from HABH exhibited a 1D 1 H ( Figure 2 and Figure 1SB,C ) and 2D 1 H– 1 H TOCSY (strips in Figure 2 B,C, Figure 2S ) NMR spectra like the product obtained from HPI.…”

“…The chemical shift values of this system are equivalent to those previously reported for HBI. 15 Another observation is the presence of two spin systems of unsaturated hexuronic acid (Δ4,5UA) at the nonreducing ends of the molecule, designated as Ua and Uc, attributed to units linked to α-GlcN,6-diS and α-GlcNS, respectively. These findings align with observations that pharmaceutical preparations of HBI have high proportions of 6-desulfated α-GlcNS, similarly reflected in bovine enoxaparin.…”

“…Notable differences in disaccharide composition and the anticoagulant effect between HPI and HBI have been observed. The most striking distinction lies in the higher content of α-GlcNH units N -sulfated but 6-desulfated in HBI than in HPI. − Nevertheless, some studies have reported the production of enoxaparin from HBI, although without a comprehensive structural analysis coupled with effectiveness tests in biological assays. , The absence of information regarding structural differences prompted our investigation into the production of enoxaparin from HBI and its biological activity.…”

Approaches to Assure Similarity between Pharmaceutical Heparins from Two Different Manufacturers

“…During the initial period of the clinical use of heparin, pharmaceutical preparations were evaluated by rudimentary biochemical methods and clotting assays. Notwithstanding refined methods have become routinely available in the last two decades, analysis of heparin is still challenging due to its heterogeneous nature; for example, minor components with a significant impact on anticoagulant activity require laborious methods for identification [12]. The demonstration of similarity among pharmaceutical heparins is far more complex than it is for proteins, which have routine methods for assuring their amino acid sequence and conformation [13].…”