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Glutamate cysteine ligase and the age-related decline in cellular glutathione: The therapeutic potential of γ-glutamylcysteine.

A consistent underlying index of aging is a decline in the cellular levels of the tripeptide glutathione (GSH). GSH is an essential thiol antioxidant produced in the cytosol of all cells and plays a key role in protecting against oxidative stress by neutralising free radicals and reactive oxygen species (ROS). The decline in GSH has been associated with changes in the expression and activity of the rate-limiting enzyme glutamate cysteine ligase (GCL), which produces the intermediate dipeptide γ-glutamylcysteine (γ-GC). The molecular mechanisms that affect these age-related changes remain unclear due to the complexity of GCL regulation. Impairment of the transcriptional activity of Nrf2 has been demonstrated to contribute to GCL dysregulation in aged rats. However, considering the complex nature of GCL regulation, relatively little research has been conducted to investigate the age-associated post-transcriptional controls of the enzyme. Defining these unknown mechanisms may inform our understanding of the aetiology of many age-related diseases and assist in formulating appropriate therapeutic strategies. This review focuses on the suitability of treatment with exogenous γ-GC to raise GSH levels by circumventing the age-related dysregulation of the rate-limiting step of GSH, providing promise for future research for the treatment of chronic oxidative stress-related diseases.

Therapeutic approaches to modulating glutathione levels as a pharmacological strategy in Alzheimer's disease.

Accumulating evidence has suggested the involvement of oxidative stress in the pathogenesis of Alzheimers disease (AD). The main endogenous antioxidant, glutathione (GSH), has been shown to decline with ageing and in several age-related degenerative diseases, including AD. Potential options for replenishing GSH levels as a therapeutic target to treat these conditions include the administration of GSH itself, and low toxicity forms of the limiting amino acid for GSH synthesis; cysteine. However, passive GSH uptake is limited due to an unfavourable concentration gradient between the plasma and cytosol. Similarly, cysteine prodrugs have demonstrated limited efficacy to elevate depleted GSH levels in several in vivo and in vitro models of disease. It has been suggested that the decline in GSH levels in AD, may be associated with down regulation of GSH homeostasis rather than substrate limitation. Cellular GSH homeostasis is regulated by non-allosteric feedback inhibition exerted by GSH on glutamate cysteine ligase (GCL), which is responsible for the synthesis of the GSH precursor γ-glutamylcysteine (GGC). In conditions involving down regulated GSH homeostasis, GGC serves as a crucialrate-limiting substrate for GSH synthetase, the main enzyme responsible for condensing glycine with GGC to form the final thiol tripeptide, GSH. In this review, we focus on the therapeutic potential of GGC to elevate cellular GSH levels. We also discuss the efficacy of GGC prodrugs which would be taken up and converted by the unregulated GS to GSH, and the administration of modified GSH compounds, such as GSH esters that could potentially overcome the concentration gradient that prohibits passive GSH uptake, in AD.

Oral administration of γ-glutamylcysteine increases intracellular glutathione levels above homeostasis in a randomised human trial pilot study☆

Objective To determine if orally dosed γ-glutamylcysteine (γ-GC) can increase cellular glutathione (GSH) levels above homeostasis. Many chronic and age-related disorders are associated with down-regulation, or impairment, of glutamate cysteine ligase (GCL). This suggests that γ-GC supply may become limiting for the maintenance of cellular GSH at the normal levels required to effectively protect against oxidative stress and any resulting physiological damage. Methods GSH levels were measured in lymphocytes of healthy, non-fasting participants before and after single oral doses (2 and 4 g) of γ-GC. Blood samples were immediately processed using high speed fluorescence-activated cell sorting to isolate 106 lymphocytes that were then assayed for GSH content. Results A single 2 g dose of γ-GC increased lymphocyte GSH content above basal levels (53±47%, p<0.01, n=14) within 90 min of administration. A randomized dosage (2 and 4 g γ-GC) crossover design was used to explore the pharmacokinetics of this GSH increase. In general, for both dose levels (n=9), GSH increased from initial basal levels over 3 h (tmax) before reaching maximum GSH concentrations (Cmax) that were near two (2 g γ-GC) to three (4 g γ-GC) fold basal levels (0.4 nmol/106 lymphocytes). Beyond tmax, GSH levels progressively declined reaching near basal levels by 5 h. The GSH half-life was between 2 and 3 h with exposure (AUC) to increased GSH levels of 0.7 (2 g γ-GC) and 1.8 (4 g γ-GC) nmol.h/106 lymphocytes. Conclusions Oral γ-GC is a non-toxic form of cysteine that can be directly taken up by cells and transiently increase lymphocyte GSH above homeostatic levels. Our findings that γ-GC can increase GSH levels in healthy subjects suggests that it may have potential as an adjunct for treating diseases associated with chronic GSH depletion. This trial was registered at anzctr.org.au as ACTRN12612000952842.

Safety assessment of gamma-glutamylcysteine sodium salt.

γ-Glutamylcysteine (GGC) is a relatively unexplored option for the treatment of chronic glutathione depletion related disorders that involve down regulation of GGC synthetase. High purity GGC (sodium salt) has only recently become available and, given its reactive capacity, required an investigation of its safety profile. In this report, GGC sodium salt was demonstrated to be safe according to Organisation for Economic Cooperation and Development (OECD) toxicology protocols for acute and repeated doses. No mortalities or adverse effects were observed in Wistar rats following the acute oral (gavage) administration of 2000mg sodium GGC /kg body weight. No animal deaths occurred with daily administration (1000mg/kg sodium GGC) over 90days, with a post trial 28day observation period. GGC had no significant effect on feed consumption, body weights, physical appearance, neurological behaviour and urine chemistry. No consistent significant differences between treatment groups were observed in haematological and clinical chemistry parameters. Similarly, no post-mortem necroscopically identified abnormalities could be attributed to GGC. Based on these observations, sodium GGC can be classed as not acutely toxic at 2000mg/kg, with a no-observed-adverse-effect level (NOAEL) of at least 1000mg/kg/day for systemic toxicology from repeated dose oral gavage administration.