Chemical and pharmacological aspects of neutralization of heparins from different animal sources by protamine

Abstract

Background Protamine neutralization is an essential step for the safe use and inactivation of the unfractionated heparin (UFH) that is widely employed in surgical and non-surgical procedures involving extracorporeal circulation. Objective To compare protamine neutralization of different pharmaceutical-grade UFHs prepared from porcine or bovine intestine (HPI and HBI, respectively). HBI has approximately half the anticoagulant potency of HPI, mostly as consequence of its fraction enriched with N-sulfated α-glucosamine disaccharides. Methods Protamine neutralization of HPI and HBI was evaluated with in vitro, ex vivo and in vivo assays. We also performed in-depth assessments of the complexation of protamine with these distinct UFHs by using nuclear magnetic resonance and mass spectroscopy. Results HPI and HBI interact similarly with protamine on a mass/mass basis; however, HBI requires more protamine than HPI to have its anticoagulant activity fully neutralized, because of its lower potency, which entails the use of higher doses. Nuclear magnetic resonance spectra revealed that HPI precipitates homogeneously with protamine. On the other hand, the low-sulfated fraction of HBI, enriched with N-sulfated α-glucosamine, precipitates at higher concentrations of protamine than the fraction more like HPI, with a preponderance of N,6-disulfated α-glucosamine disaccharides. Finally, mass spectroscopy spectra showed that some of the different peptide components of protamine interact preferentially with the heparins, irrespective of their animal origin. Conclusion Our results have important medical implications, indicating that protamine neutralization of HBI, determined exclusively by point-of-care coagulation assessments, must fail because of its lower-sulfated fraction with reduced anticoagulant activity that could remain in the circulation after the neutralization procedure.

Heparins Sourced From Bovine and Porcine Mucosa Gain Exclusive Monographs in the Brazilian Pharmacopeia

“…Thereafter, we have confirmed such structural differences by analyzing with state-of-the-art NMR techniques the compositions of approximately 500 batches of HPI and 400 batches of HBI manufactured, formulated and/or distributed by different Brazilian pharmaceutical companies (9, 10). The analysis of 1D 1 H NMR spectra of such a massive number of pharmaceutical preparations has revealed a remarkable batch-to-batch consistency for HBI and HPI and attested to their different disaccharide compositions (9).…”

“…Although clinically employed as interchangeable UFHs and considered as a single pharmaceutical compound (Heparin Sodium) by regulatory agencies, HBI and HPI have contrasting anticoagulant potencies due to their compositional differences (6–10). In this review, we summarize the major scientific, medical, and regulatory episodes leading to the development of the novel compendial monographs published by the Brazilian Pharmacopeia (BP), which consider heparins sourced from porcine or bovine mucosa as distinct active pharmaceutical ingredients (APIs).…”

“…(A) 1D 1 H NMR spectra of HBI (in blue) and HPI (in black) showing their characteristic signals, including the fingerprinting A1 and C1, ascribed to the anomeric protons (H1) of N ,6-disulfated and N -sulfated α-glucosamine units, respectively, and H1 and H5 of their respective 2-sulfated α-iduronic acid units (I1-A, I5-A and I1-C, I5-C). For information on the other signals check the section “Establishing chemical differences” and references (6–10, 13, 14). (B) Areas of the signals A1 and C1 (in gray) used to calculate the proportions of their correspondent disaccharides in HBI (in blue), HPI (in black) and mixtures (50% of each, in purple).…”

“…This explanation seemed plausible at that time because the sulfate-ester linked to the position 6 of the α-glucosamine is the most susceptible to solvolysis (16). However, in-depth analyses of further 1D and 2D NMR spectra allowed us to conclude that the real cause behind those structural differences was the raw material (porcine- or bovine-mucosa) employed to produce the UFHs (6–10). Independent investigations conducted by an Italian group had already demonstrated that HBI contained an increased proportion of 6-desulfated α-glucosamine, thereby strengthening our observation (17–19).…”

Imminent risk of a global shortage of heparin caused by the African Swine Fever afflicting the Chinese pig herd

“…Despite the advantages over products from other animal sources, the concomitant use of bovine‐ and porcine‐mucosa UFHs as the same anticoagulant can provoke serious medical incidents (bleeding), because of their contrasting anticoagulant potencies (~100 and ~180 IU mg −1 , respectively) . However, the Brazilian Pharmacopeia recently released novel monographs approaching the distinct pharmacological characteristics of these heparins separately, which makes their use as interchangeable UFHs safer and, therefore, should be used as a template for the reformulation of pharmacopeias of other countries willing to introduce bovine‐mucosa products .…”