Is glutathione an important neuroprotective effector molecule against amyloid beta toxicity?

Cellular thiols are critical moieties in signal transduction, regulation of gene expression, and ultimately are determinants of specific protein activity. Whilst protein bound thiols are the critical effector molecules, low molecular weight thiols, such as glutathione, play a central role in cytoprotection through (1) direct consumption of oxidants, (2) regeneration of protein thiols and (3) export of glutathione containing mixed disulphides. The brain is particularly vulnerable to oxidative stress, as it consumes 20% of oxygen load, contains high concentrations of polyunsaturated fatty acids and iron in certain regions, and expresses low concentrations of enzymic antioxidants. There is substantial evidence for a role for oxidative stress in neurodegenerative disease, where excitotoxic, redox cycling and mitochondrial dysfunction have been postulated to contribute to the enhanced oxidative load. Others have suggested that loss of important trophic factors may underlie neurodegeneration. However, the two are not mutually exclusive; using cell based model systems, low molecular weight antioxidants have been shown to play an important neuroprotective role in vitro, where neurotrophic factors have been suggested to modulate glutathione levels. Glutathione levels are regulated by substrate availability, synthetic enzyme and metabolic enzyme activity, and by the presence of other antioxidants, which according to the redox potential, consume or regenerate GSH from its oxidised partner. Therefore we have investigated the hypothesis that amyloid beta neurotoxicity is mediated by reactive oxygen species, where trophic factor cytoprotection against oxidative stress is achieved through regulation of glutathione levels. Using PC12 cells as a model system, amyloid beta 25-35 caused a shift in DCF fluorescence after four hours in culture. This fluorescence shift was attenuated by both desferioxamine and NGF. After four hours, cellular glutathione levels were depleted by as much as 75%, however, 24 hours following oxidant exposure, glutathione concentration was restored to twice the concentration seen in controls. NGF prevented both the loss of viability seen after 24 hours amyloid beta treatment and also protected glutathione levels. NGF decreased the total cellular glutathione concentration but did not affect expression of GCS. In conclusion, loss of glutathione precedes cell death in PC12 cells. However, at sublethal doses the surviving fraction respond to oxidative stress by increasing glutathione levels, where this is achieved, at least in part, at the gene level through upregulation of GCS. Whilst NGF does protect against oxidative toxicity, this is not achieved through upregulation of GCS or glutathione.

Physicochemical characteristics of chlorofluorohydrocarbon based inhalation aerosols

The aim of this work was to gain a better understanding of the physiochemical factors which affect the formulation of suspension inhalation aerosols. This has been attempted by applying the principles of colloid science to aerosol formulation. Both a drug system and a model colloid system have been used. The adsorption of six nonionic and cationic surfactants onto Spherisorb has been investigated. The results were analysed by calculating the area occupied by one adsorbed molecule at the surface and by comparing these values for each surfactant. The amount of each surfactant adsorbed was correlated with the number of sites on that surfactant molecule which could interact with the surface. The stability of suspensions, produced by both the model colloid Spherisorb, and by the drug isoprenaline sulphate, after adsorption of the surfactants, has been assessed by measuring settling times and rising times. The most stable suspensions were found to be those which had the greatest amounts of long chain fatty acid surfactant adsorbed on their surface. A comparison was made between the effective stabilising properties of Span 85 and oleic acid on various drug suspensions. It was found that Span 85 gave the most stable suspensions. Inhalation aerosol suspensions of isoprenaline sulphate were manufactured using the same surfactants used in the adsorption and suspension stability studies and were analysed by measuring the particle size distributions of the suspension and the emitted doses. The results were found to correlate with the adsorption and suspension stability studies and it was concluded that a deflocculated suspension was preferable to a flocculated suspension in inhalation aerosols provided that the drug density was less than the propellant density. The application of this work to preformulation studies was also discussed.

Systematic identification of small molecule adjuvants

Introduction: Adjuvants potentiate immune responses, reducing the amount and dosing frequency of antigen required for inducing protective immunity. Adjuvants are of special importance when considering subunit, epitope-based or more unusual vaccine formulations lacking significant innate immunogenicity. While numerous adjuvants are known, only a few are licensed for human use; principally alum, and squalene-based oil-in-water adjuvants. Alum, the most commonly used, is suboptimal. There are many varieties of adjuvant: proteins, oligonucleotides, drug-like small molecules and liposome-based delivery systems with intrinsic adjuvant activity being perhaps the most prominent. Areas covered: This article focuses on small molecules acting as adjuvants, with the author reviewing their current status while highlighting their potential for systematic discovery and rational optimisation. Known small molecule adjuvants (SMAs) can be synthetically complex natural products, small oligonucleotides or drug-like synthetic molecules. The author provides examples of each class, discussing adjuvant mechanisms relevant to SMAs, and exploring the high-throughput discovery of SMAs. Expert opinion: SMAs, particularly synthetic drug-like adjuvants, are amenable to the plethora of drug-discovery techniques able to optimise the properties of biologically active small molecules. These range from laborious synthetic modifications to modern, rational, effort-efficient computational approaches, such as QSAR and structure-based drug design. In principal, any property or characteristic can thus be designed in or out of compounds, allowing us to tailor SMAs to specific biological functions, such as targeting specific cells or pathways, in turn affording the power to tailor SMAs to better address different diseases.