Mixture of three amino acids as stabilizers replacing albumin in lyophilization of new third generation recombinant factor VIII GreenGene F

A formulation with stabilizers replacing albumin was developed for lyophilization of recombinant factor VIII (FVIII), GreenGene F (WHO INN: beroctocog alfa), to achieve stability and eliminate safety issues of blood‐derived albumin. L ‐Arginine (hydrophilic amino acid, positively charged side chain), L ‐glutamic acid (hydrophilic amino acid, negatively charged side chain), and L ‐isoleucine (hydrophobic amino acid, nonpolar) were selected as stabilizers, and the mixture of the three amino acids were optimized. The mixture had results comparative with albumin and other commonly used stabilizers showing good preservation of recombinant FVIII during lyophilization, robust stability with consistently high recovery of FVIIII, very low aggregate formation, and good storage stability without alterations in protein characteristics. In vivo test results showed that the efficacy was maintained and had no signs of toxicity. The study demonstrated that the three amino acid mixture acts as a good stabilizer for lyophilization of recombinant FVIII and as a safe excipient. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Copper Ion from Cu2O Crystal Induces AMPK-Mediated Autophagy via Superoxide in Endothelial Cells

Copper is an essential element required for a variety of functions exerted by cuproproteins. An alteration of the copper level is associated with multiple pathological conditions including chronic ischemia, atherosclerosis and cancers. Therefore, copper homeostasis, maintained by a combination of two copper ions (Cu⁺ and Cu²⁺), is critical for health. However, less is known about which of the two copper ions is more toxic or functional in endothelial cells. Cubic-shaped Cu₂O and CuO crystals were prepared to test the role of the two different ions, Cu⁺ and Cu²⁺, respectively. The Cu₂O crystal was found to have an effect on cell death in endothelial cells whereas CuO had no effect. The Cu₂O crystals appeared to induce p62 degradation, LC3 processing and an elevation of LC3 puncta, important processes for autophagy, but had no effect on apoptosis and necrosis. Cu₂O crystals promote endothelial cell death via autophagy, elevate the level of reactive oxygen species such as superoxide and nitric oxide, and subsequently activate AMP-activated protein kinase (AMPK) through superoxide rather than nitric oxide. Consistently, the AMPK inhibitor Compound C was found to inhibit Cu₂O-induced AMPK activation, p62 degradation, and LC3 processing. This study provides insight on the pathophysiologic function of Cu⁺ ions in the vascular system, where Cu⁺ induces autophagy while Cu²⁺ has no detected effect.