Quality Comparison of Biosimilar and Copy Filgrastim Products with the Innovator Product.

PURPOSE: Filgrastim, a recombinant human granulocyte-colony stimulating factor, is widely used to treat congenital and acquired neutropenia. Following patent expiration of the innovator filgrastim product, biosimilar filgrastim products have been approved in the EU and shown to be comparable with the innovator with respect to quality, safety and efficacy. In less regulated markets, copy filgrastim products are available but data about their quality are scarce. In the present study, we provide a head-to-head comparative study on the quality of biosimilar and copy filgrastim products. METHODS: Innovator filgrastim product, Neupogen®, two EU-licensed biosimilars, Zarzio® and Tevagrastim®, and two copy filgrastim products, Biocilin® and PDgrastim®, were subjected to peptide mapping, circular dichroism spectroscopy, fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance size-exclusion chromatography, reversed-phase ultra-performance liquid chromatography, endotoxin test, flow imaging microscopy and in vitro potency assay. RESULTS: Zarzio® and Tevagrastim® have comparable quality to Neupogen®, while Biocilin® showed a significantly lower and PDgrastim® a higher specific activity. Moreover, PDgrastim® showed a higher level of impurities and a lower thermo stability than the other products. CONCLUSIONS: Except for the deviating specific activities of the two copy filgrastim products, we found no substantial differences in product quality between the filgrastim products studied.

Development of a Parenteral Formulation of NTS-Polyplex Nanoparticles for Clinical Purpose.

Neurotensin (NTS)-polyplex is a nanoparticle system for targeted gene delivery that holds great promise for treatment of Parkinson's disease and various types of cancer. However, the high instability in aqueous suspension of NTS-polyplex nanoparticles is a major limitation for their widespread clinical use. To overcome this obstacle, we developed a clinical formulation and a lyophilization process for NTS-polyplex nanoparticles. The reconstituted samples were compared with fresh preparations by using transmission electron microscopy, dynamic light scattering, electrophoretic mobility, circular dichroism and transfection assays in vitro and in vivo. Our formulation was able to confer lyoprotection and stability to these nanoparticles. In addition, transmission electron microscopy (TEM) and size exclusion-high performance liquid chromatography (SEC-HPLC) using a radioactive tag revealed that the interaction of reconstituted nanoparticles with fetal bovine or human serum did not alter their biophysical features. Furthermore, the formulation and the lyophilization procedure guaranteed functional NTS-polyplex nanoparticles for at least six months of storage at 25 °C and 60% relative humidity. Our results offer a pharmaceutical guide for formulation and long-term storage of NTS-polyplex nanoparticles that could be applied to other polyplexes.

Effect of ionic strength on the aggregation kinetics of the amidated amyloid beta peptide Aß (1-40) in aqueous solutions.

In this work we study the effect of solution ionic strength on the structural evolution of amidated amyloid beta peptide Aß (1-40) oligomers at the early stages of fibril formation. By light scattering, we follow the time evolution of the structure and short-time dynamics of peptide structures at low ionic strengths. Our results allow identifying initial oligomer structures as the effective building blocks in the amyloid fibrils formation and indicate that the oligomers growth pathway, from compact structures to flexible chain-like structures, becomes faster as the solution ionic strength is increased. Furthermore, we find no evidence of structural branching what suggests that elongation of amyloid fibrils is dominated by linear association. To describe our results we adapt a phenomenological model based on population balance equations and linear polymer growth, where the parameters required are obtained from the experiments. Model calculations are in good agreement with experimentally-obtained estimates for the radius of gyration of Aß (1-40) oligomers, thus further supporting our findings. Additionally, we introduce a model for the effective interaction among initial Aß structures that captures the dependence of the effective association rates on solution ionic strength.

A bifunctional non-natural tetrapeptide modulates amyloid-beta peptide aggregation in the presence of Cu(ii).

Amyloid-beta peptide (Aß) aggregation is one of the hallmarks of Alzheimer's disease (AD), and metal ions such as Cu(ii) have been proposed to play a role in amyloid formation and the onset of this progressive neurodegenerative disorder. This study reports the design and characterization of a novel bifunctional non-natural tetrapeptide, Met-Asp-d-Trp-Aib, that is capable of binding copper, competing with Aß for Cu(ii), and modulating Aß aggregation. The study of this tetrapeptide provides further insights into the role of Cu(ii) in the Aß aggregation pathway, and into the design of compounds with therapeutic potential for Alzheimer's disease.

Calpain activation is involved in acute manganese neurotoxicity in the rat striatum in vivo.

Manganese is essential for life, yet chronic exposure to this metal can cause a neurodegenerative disease named manganism that affects motor function. In the present study we have evaluated Mn neurotoxicity after its administration in the rat striatum. The participation of the calcium-dependent protease calpain and the apoptosis-related protease caspase-3, in Mn-induced cell death was monitored in the striatum and globus pallidus. Mn induced the activation of both proteases, although calpain activation seems to be an earlier event. Moreover, while the broad-spectrum caspase inhibitor QVD did not significantly prevent Mn-induced cell death, the specific calpain inhibitor MDL-28170 did. The role of NMDA glutamate receptors on calpain activity was also investigated; blockage of these receptors by MK-801 and memantine did not prevent calpain activation, nor Mn-induced cell death. Finally, studies in striatal homogenates suggest a direct activation of calpain by Mn ions. Altogether the present study suggests that additional mechanisms to excitotoxicity are involved in Mn-induced cell death, placing calpain as an important mediator of acute Mn neurotoxicity in vivo.