Hydrogel Coatings on Container Surfaces Reduce Protein Aggregation Caused by Mechanical Stress and Cavitation.

This work reports the ability of hydrogel coatings to protect therapeutic proteins from cavitation-induced aggregation caused by mechanical stress. Here, we show that polyacrylamide hydrogel coatings on container surfaces suppress mechanical shock-induced cavitation and the associated aggregation of intravenous immunoglobulin (IVIg). First, crosslinked polyacrylamide hydrogels were grown on the surfaces of borosilicate glass vials. Treatment with ultrasound showed that these hydrogel surfaces suppressed cavitation events to levels below those found for unfunctionalized borosilicate glass. Next, IVIg solutions were loaded into these vials and subjected to tumbling, horizontal shaking, and drop testing. Aggregation was quantified by bisANS fluorescence staining and particle counting by flow imaging microscopy (FIM). In all cases, the presence of polyacrylamide hydrogels on the vial surfaces reduced the amount of IVIg aggregation and the number of particulates. In addition, the polyacrylamide appeared to have a protective effect that prevented additional aggregates from forming at extended tumbling times. Finally, drop test studies showed that the polyacrylamide coatings suppressed detectable cavitation. This work reveals how even a simple hydrogel vial coating can have a profound effect on stabilizing protein therapeutics.

The Effect of Container Surface Passivation on Aggregation of Intravenous Immunoglobulin Induced by Mechanical Shock.

Aggregation of therapeutic proteins can result from a number of stress conditions encountered during their manufacture, transportation, and storage. This work shows the effects of two interrelated sources of protein aggregation: the chemistry and structure of the surface of the container in which the protein is stored, and mechanical shocks that may result from handling of the formulation. How different mechanical stress conditions (dropping, tumbling, and agitation) and container surface passivation affect the stability of solutions of intravenous immunoglobulin are investigated. Application of mechanical shock causes cavitation to occur in the protein solution, followed by bubble collapse and the formation of high-velocity fluid microjets that impinged on container surfaces, leading to particle formation. Cavitation was observed after dropping of vials from heights as low as 5 cm, but polyethylene glycol (PEG) grafting provided temporary protection against drop-induced cavitation. PEG treatment of the vial surface reduced the formation of protein aggregates after repeated dropping events, most likely by reducing protein adsorption to container surfaces. These studies enable the development of new coatings and surface chemistries that can reduce the particulate formation induced by surface adsorption and/or mechanical shock.

Enhanced morphine withdrawal and micro -opioid receptor G-protein coupling in A2A adenosine receptor knockout mice.

Much evidence supports the hypothesis that A2A adenosine receptors play an important role in the expression of morphine withdrawal and that the dopaminergic system might also be involved. We have evaluated morphine withdrawal signs in wild-type and A2A receptor knockout mice and shown a significant enhancement in some withdrawal signs in the knockout mice. In addition, micro -opioid and dopamine D2 receptor autoradiography, as well as micro -opioid receptor-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate ([35S]GTPgammaS) autoradiography was carried out in brain sections of withdrawn wild-type and knockout mice. No significant changes in D2 and micro -opioid receptor binding were observed in any of the brain regions analysed. However, a significant increase in the level of micro receptor-stimulated [35S]GTPgammaS binding was observed in the nucleus accumbens of withdrawn knockout mice. These data indicate that the A2A receptor plays a role in opioid withdrawal related to functional receptor activation.

Pyruvate limits zinc-induced rat oligodendrocyte progenitor cell death.

A growing body of evidence suggests that excessive Zn2+ release plays a key role in inducing neuronal death during central nervous system injury. However, the possible cytotoxicity of extracellular Zn2+ to oligodendrocyte lineage cells remains unknown. Employing cultures of rat oligodendrocyte progenitor cells (OPC), we report here that OPC are vulnerable to increased extracellular Zn2+ levels and that pyruvate limits Zn2+-induced OPC death. Zn2+-induced concentration-dependent (pEC50 = -4.1 +/- 0.1) OPC death, which was insensitive to both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (Evans Blue) and l-type Ca2+ channel (nicardipine) inhibition. Neither kainate nor nicardipine influenced OPC 65Zn2+ accumulation, in contrast with the Zn2+ ionophore, pyrithione. Cytotoxic extracellular Zn2+ concentrations failed to increase OPC reactive oxygen species production and the antioxidant reagents, trolox, N,N'-diphenyl-1,4-phenylenediamine and N-tert-butyl-alpha-phenylnitrone did not afford significant protection from Zn2+ insults. The apoptotic inducer staurosporine induced the appearance of known apoptotic markers [pyknotic nuclei and caspase-3 specific (120 kDa) alpha-fodrin cleavage fragment], events not reproduced with Zn2+ insults. Zn2+ insults were also insensitive to the pan-caspase inhibitor Z-VAD-fmk. However, pyruvate afforded significant OPC protection from lethal Zn2+ insults. We conclude that cultured OPC are vulnerable to Zn2+ insults, via a nonoxidative stress and noncaspase-3-based mechanism, involving Zn2+ inhibition of OPC glycolysis.