Mutations in asparaginase II from E. coli and implications for inactivation and PEGylation.

All clinically-used asparaginases convert L-asparagine (L-Asn) to l-aspartate (L-Asp) and l-glutamine (L-Gln) to L-glutamate (L-Glu), which has been useful in reducing bioavailable asparagine and glutamine in patients under treatment for acute lymphoblastic leukemia. The E. coli type 2 L-asparaginase (EcA2) can present different sequences among varying bacterial strains, which we hypothesized that might affect their biological function, stability and interchangeability. Here we report the analysis of two EcA2 provided by the public health system of a middle-income country. These enzymes were reported to have similar specific activity in vitro, whereas they differ in vivo. Protein sequencing by LC-MS-MS and peptide mapping by MALDI-ToF-MS of their tryptic digests revealed that Aginasa™ share similar sequence to EcA2 from E. coli strain BL21(DE3), while Leuginase™ has sequence equivalent to EcA2 from E. coli strain AS1.357. The two amino acid differences between Aginasa™ (64D and 252 T) and Leuginase™ (64 N and 252S) resulted in structural divergences in solution as accessed by small-angle X-ray scattering and molecular dynamics simulation trajectories. The conformational variability further results in dissimilar surface accessibility with major consequences for PEGylation, as well as different susceptibility to degradation by limited proteolysis. The present results reveal that the sequence variations between these two EcA2 variants results in conformational changes associated with differential conformational plasticity, potentially affecting physico-chemical and biological properties, including proteolytic and immunogenic silent inactivation.

Amyloidogenicity of peptides targeting diabetes and obesity.

Since the discovery of insulin, a century ago, the repertoire of therapeutic polypeptides targeting diabetes - and now also obesity - have increased substantially. The focus on quality has shifted from impure and unstable preparations of animal insulin to highly pure, homologous recombinant insulin, along with other peptide-based hormones and analogs such as amylin analogs (pramlintide, davalintide, cagrilintide), glucagon and glucagon-like peptide-1 receptor agonists (GLP-1, liraglutide, exenatide, semaglutide). Proper formulation, storage, manipulation and usage by professionals and patients are required in order to avoid agglomeration into high molecular weight products (HMWP), either amorphous or amyloid, which could result in potential loss of biological activity and short- or long-term immune reaction and silent inactivation. In this narrative review, we present perspective of the aggregation of therapeutic polypeptides used in diabetes and other metabolic diseases, covering the nature and mechanisms, analytical techniques, physical and chemical stability, strategies aimed to hamper the formation of HMWP, and perspectives on future biopharmaceutical developments.

Polymeric microparticle systems for modified release of glucagon-like-peptide-1 receptor agonists.

Type 2 diabetes is a fast-growing worldwide epidemic. Despite the multiple therapies available to treat type 2 diabetes, the disease is not correctly managed in over half of patients, mainly due to non-compliance with prescribed treatment regimes. The development of analogues to the glucagon-like peptide 1 (GLP-1) has resulted in the extension of its half-life and associated benefits. Further benefits in the use of peptide-based GLP-1 receptor agonists have been achieved by the use of controlled-release systems based on polymeric microparticles. In this review, we focus on commercially available formulations and others that remain in development, discussing the preparation methods and the relationship between in vitro and in vivo kinetic release behaviours.

Sustained release and pharmacologic evaluation of human glucagon-like peptide-1 and liraglutide from polymeric microparticles.

The GLP1-receptor agonists exert regulatory key roles in diabetes, obesity and related complications. Here we aimed to develop polymeric microparticles loaded with homologous human GLP1 (7-37) or the analogue liraglutide. Peptide-loaded microparticles were prepared by a double emulsion and solvent evaporation process with a set of eight polymers based on lactide (PLA) or lactide-glycolide (PLGA), and evaluated for particle-size distribution, morphology, in vitro release and pharmacologic activity in mice. The resulting microparticles showed size distribution of about 30-50 µm. The in vitro kinetic release assays showed a sustained release of the peptides extending up to 30-40 days. In vivo evaluation in Swiss male mice revealed a similar extension of glycemic and body weight gain modulation for up to 25 days after a single subcutaneous administration of either hGLP1-microparticles or liraglutide-microparticles. Microparticles-loaded hGLP1 shows equivalent in vivo pharmacologic activity to the microparticles-loaded liraglutide.

Extrinsically magnetic poly(butylene succinate): An up-and-coming petroleum cleanup tool.

This work presents the synthesis and characterization of extrinsically magnetic poly(butylene succinate) (PBS). PBS is obtained from succinic acid (SA), which can be efficiently produced from renewable biomass by fermentation. Thus, the use of SA helps to remove CO2 from the atmosphere, constituting a good way to accumulate carbon credits. The magnetic PBS here presented was prepared by fusion using different amounts of maghemite. Obtained materials were characterized using Fourier transform infrared spectroscopy (FTIR), Thermogravimetric analysis (TGA), Differential scanning calorimetry (DSC), X-ray diffraction (XRD), Small angle X-ray scattering and magnetic force tests. Besides, the oil removal capability (OR) of the samples was also studied. All the magnetic composites were able to remove petroleum from the water. Among them, the one filled with the highest amount of magnetic particles was able to remove 11 g of oil per gram of composite. Also, XRD and SAXS results showed that PBS is a long size oriented material, which allows it to work as a thermoset, avoiding its dissolution in organic contaminant medium. As PBS can also be considered as a platform, these are promising results for the oil spill cleanup applications.