Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin.

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N-sulfoheparosan with N-sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products.

Factors Influencing Outcome of Orbital Floor Reconstruction.

Introduction Orbital floor fractures are routinely encountered in facial trauma. Many factors influence the final outcome of the orbital floor surgery, time interval and the extent of other facial bone fractures are the two factors which can significantly influence the postoperative outcome following orbital floor reconstruction. Our study aims to find the ideal time for intervention and the association of other factors in the final outcome of orbital floor reconstruction. Methods A retrospective and prospective cohort study of patients who were operated at Pondicherry Institute of Medical Sciences for orbital floor fractures, between 2011 January and 2017 July. All the data were entered on an Excel work sheet and statistically analyzed. Results In our study 8 patients (8/29, 27.58%) had diplopia prior to surgery, 5 patients (5/29, 17.24%) had complete recovery following surgery and 3 patients (3/29, 10.34%) had persistence of diplopia postoperatively. Patients with diplopia operated prior to 7 days were found to have significant improvement in postoperative diplopia. Patients with 5 or more facial fractures were found to have persistence of diplopia, infraorbital numbness, and enophthalmos postoperatively. Conclusion Our study suggests that early intervention, before 7 days improves the outcome in patients with diplopia and provides a better result postoperatively. In our study preoperative diplopia and infraorbital numbness and postoperative persistence of enophthalmos, diplopia, and paresthesia were found more in patients with 5 or more facial bone fractures. Our study suggests a poor postoperative outcome when 5 or more facial bones are fractured.

Experience of Unilateral Cleft Lip Repair using the Anatomical Subunit Technique in an Omani Population over a Five-Year Period.

Objectives: This study aimed to report the authors' experience with the anatomical subunit technique for unilateral cleft lip repair, which has gained popularity worldwide. Methods: From July 2015 to April 2020, 114 consecutive cases of cleft lip underwent primary cleft lip repair with closed rhinoplasty by a single surgeon. The demographic data, severity and type of the cleft lip, surgical outcomes, including vermillion notching, were assessed by an independent senior surgeon. The rate of revision surgery was collected from the Al-Shifa® - 3Plus healthcare information system (Ministry of Health, Oman). Parents' satisfaction regarding scar quality and lip and nose appearance was collected and all data were statistically analysed. Results: A total of 82 cases satisfied the inclusion criteria. The mean age at surgery for cleft lip was 32 weeks. Among these, 35 cases (43%) were complete cleft lips and 47 cases (57%) were incomplete. Additionally, 43 children (52%) were born of consanguineous marriage. Six patients (7%) needed revision surgery. The digital survey was completed by 40 subjects (response rate: 48.8%) which showed 85% satisfaction rate with the postoperative scar and 77.5% satisfaction with the aesthetic appearance of the nose. Conclusion: The anatomical subunit technique resulted in a predictable outcome indicating a high rate of patient satisfaction with scar quality and nasal and lip symmetry in children with varying severity of cleft lip. The high percentage of consanguinity (52%) in this study highlights the need for more targeted national campaigns involving premarital counselling in the Omani population.

Enzymatic synthesis of low molecular weight heparins from N-sulfo heparosan depolymerized by heparanase or heparin lyase.

Low-molecular-weight heparin (LMWH) is prepared from the controlled chemical or enzymatic depolymerization of animal sourced heparins. It has been widely used as an anticoagulant. Concerns about the shortcomings of animal-derived heparin and the contamination of supply chain demand biochemical approaches for synthesizing LMWH. In the present study, two LMWHs were enzymatically synthesized from low molecular weight N-sulfated heparosan (LMW-NSH) cleaved by recombinant hydrolase, endo-ß-glucuronidase, (HepBp) or heparin lyase III (HepIII), followed by subsequent sulfotransferase modifications. Structural characterization shows that LMWH chains prepared using HepBp had a saturated uronic acid residue at their reducing ends, while chains of LMWH prepared using HepIII had an unsaturated uronic acid residue at their non-reducing end. Both LMWHs had anti-factor Xa and anti-factor IIa activities comparable to enoxaparin. This approach demonstrates that the hydrolase, HepBp, can be used to prepare a new type of LMWH that has no unsaturated uronic acid at its non-reducing end.

Chemobiocatalytic Synthesis of a Low-Molecular-Weight Heparin.

Heparin products are widely used clinical anticoagulants essential in the practice of modern medicine. Low-molecular-weight heparins (LMWHs) are currently prepared by the controlled chemical or enzymatic depolymerization of unfractionated heparins (UFHs) that are extracted from animal tissues. In many clinical applications, LMWHs have displaced UFHs and currently comprise over 60% of the heparin market. In the past, our laboratory has made extensive efforts to prepare bioengineered UFHs relying on a chemoenzymatic process to address concerns about animal-sourced UFHs. The current study describes the use of a novel chemoenzymatic process to prepare a chemobiosynthetic LMWH from a low-molecular-weight heparosan. The resulting chemobiocatalytic LMWH matches most of the United States pharmacopeial specifications for enoxaparin, a LMWH prepared through the base-catalyzed depolymerization of animal-derived UFH.

Preparation of Low Molecular Weight Heparin from a Remodeled Bovine Intestinal Heparin.

Bovine intestinal heparins are structurally distinct from porcine intestinal heparins and exhibit lower specific anticoagulant activity (units/mg). The reduced content of N-sulfo, 3-O-sulfo glucosamine, the central and critical residue in heparin's antithrombin III binding site, is responsible for bovine intestinal heparin's reduced activity. Previous studies demonstrate that treatment of bovine intestinal heparin with 3-O-sulfotransferase in the presence of 3'-phosphoadenosine-5'-phosphosulfate afforded remodeled bovine heparin with an enhanced activity reaching the United States Pharmacopeia's requirements. Starting from this remodeled bovine intestinal heparin, we report the preparation of a bovine intestinal low molecular weight heparin having the same structural properties and anti-factor IIa and anti-factor Xa activities of Enoxaparin. Moreover, this bovine intestinal heparin-derived "Enoxaparin" showed comparable platelet factor-4 binding affinity, suggesting that it should exhibit similarly low levels of heparin induced thrombocytopeneia, HIT.