Preclinical pharmacokinetic analysis of NOV-002, a glutathione disulfide mimetic.

NOV-002 is a glutathione disulfide (GSSG) mimetic that is the subject of clinical investigation in oncology indications. GSSG is reduced by glutathione reductase (GR) to form glutathione (GSH), thereby maintaining redox homeostasis. The purpose of the study was to report the pharmacokinetic properties of NOV-002 and evaluate the effect that NOV-002 elicits in redox homeostasis. The pharmacokinetic analysis and tissue distribution of NOV-002 and GSH was evaluated in mice following a dose of 250 mg/kg, i.p. The redox potential and total protein thiol status was calculated. Here we show that NOV-002 is a substrate for GR and that GSH is a primary metabolite. Non-linear pharmacokinetic modeling predicted that the estimated absorption and elimination rate constants correspond to a half-life of approximately 13 min with an AUC of 1.18 µgh/mL, a C(max) of 2.16 µg/ml and a volume of distribution of 42.61 L/kg. In addition, measurement of the redox potential and total protein thiol status indicated the generation of a transient oxidative signal in the plasma compartment after administration of NOV-002. These results indicate that NOV-002 exerts kinetic and dynamic effects in mice consistent with the GSSG component as the active pharmacological constituent of the drug. A longer-lasting decrease in total plasma free thiol content was also seen, suggesting that the oxidative effect of the GSSG from NOV-002 was impacting redox homeostasis.

The disposition and bioavailability of 35S-GSH from 35S-GSSG in BSS PLUS in rabbit ocular tissues.

The purpose of this study was to evaluate the biodistribution and uptake of 35S-GSH into intraocular tissues following the administration of BSS PLUS containing 35S-GSSG by either an anterior chamber or intravitreal injection. This study evaluated the disposition and uptake of the 35S-radiolabel, the intracellular concentrations of 35S-GSH from extracellular 35S-GSSG, and the percentage of 35S-GSH to the total cellular GSH pool. Glutathione was analyzed by high-performance liquid chromatography (HPLC) using fluorescence detection after derivitizing the thiols in situ with monobromobimane. The effluent from the GSH peak was then collected for measurement of 35S-GSH. After an anterior chamber injection of 35S-BSS PLUS, 35S-radioactivity rapidly disappeared from the aqueous humor between 0.5 and 2 hours; corneal 35S-radioactivity remained constant over time. 35S-GSH was detected in the iris and ciliary body. However, in the cornea, 35S-GSH became the predominant radioactive thiol in the stroma, endothelium, and epithelium; the corneal stroma appeared to be a possible GSH reservoir for the adjacent corneal layers. After an intravitreal injection, 35S-radioactivity slowly decreased in the vitreous humor but was readily taken up by the tissues of the posterior segment, especially the retina and choroid, which showed the greatest concentrations of 35S-GSH of all tissues studied. The data from this study demonstrate that 35S-GSSG in BSS PLUS is metabolized and taken up by ocular cells and that 35S-GSH becomes incorporated into the intracellular GSH pool of ocular tissues.

Prophylactic action of melatonin against cyclophosphamide-induced oxidative stress in mice.

The present study investigated the prophylactic influence of melatonin against cyclophosphamide-induced oxidative stress in mouse tissues. Lipid peroxidation, reduced glutathione (GSH), glutathione disulphide (GSSG), glutathione peroxidase (GSH-Px) and serum phosphatase levels were analyzed in brain, spleen liver, lungs, kidney and testes. Fifteen days oral administration with melatonin (0.1 mg/kg bw per day) before treatment checked the augmentation of the level of lipid peroxidation, blood GSSG and acid phosphatase caused by an acute treatment with a radiomimetic drug, cyclophosphamide (75 mg/kg bw). Cyclophosphamide-induced depletion in the level of GSH, GSH-Px and alkaline phosphatase was made up statistically significant by chronic melatonin administration given orally. The results indicate the antioxidative properties of melatonin resulting into its prophylactic property against the cyclophosphamide-induced biochemical alterations. The finding support the idea that melatonin is a potent free-radical scavenger and antioxidant.

Import and metabolism of glutathione by Streptococcus mutans.

Glutathione (gamma-GluCysGly, GSH) is not found in most gram-positive bacteria, but some appear to synthesize it and others, including Streptococcus mutans ATCC 33402, import it from their growth medium. Import of oxidized glutathione (GSSG) by S. mutans 33402 in 7H9 medium was shown to require glucose and to occur with an apparent Km of 18+/-5 microM. GSSG, GSH, S-methylglutathione, and homocysteine-glutathione mixed disulfide (hCySSG) were imported at comparable rates (measured by depletion of substrate in the medium), as was the disulfide of gamma-GluCys. In contrast, the disulfide of CysGly was not taken up at a measurable rate, indicating that the gamma-Glu residue is important for efficient transport. During incubation with GSSG, little GSSG was detected in cells but GSH and gamma-GluCys accumulated during the first 30 min and then declined. No significant intracellular accumulation of Cys or sulfide was found. Transient intracellular accumulation of D/L-homocysteine, as well as GSH and gamma-GluCys, was observed during import of hCySSG. Although substantial levels of GSH were found in cells when S. mutans was grown on media containing glutathione, such GSH accumulation had no effect on the growth rate. However, the presence of cellular GSH did protect against growth inhibition by the thiol-oxidizing agent diamide. Import of glutathione by S. mutans ATCC 25175, which like strain 33402 does not synthesize glutathione, occurred at a rate comparable to that of strain 33402, but three species which appear to synthesize glutathione (S. agalactiae ATCC 12927, S. pyogenes ATCC 8668, and Enterococcus faecalis ATCC 29212) imported glutathione at negligible or markedly lower rates.