Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor.

AIMS: The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. RESULTS: GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cell-based NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. INNOVATION AND CONCLUSIONS: GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo.

Rasagiline improves learning and memory in young healthy rats.

The effect of rasagiline on learning and memory in Lister-Hooded rats was investigated in this study. Two cognitive tests were used: a 24-h temporal deficit novel object recognition test and a modified water maze task. Rasagiline (0.3 and 1 mg/kg) was administered subcutaneously 15 min before the cognitive tests. In a novel object recognition test, rasagiline treatment enhanced object recognition memory. A small effect was observed with 0.3 mg/kg rasagiline; at 1 mg/kg, rasagiline-treated animals spent twice as much time exploring the novel object. On the water maze test, the use of an on-demand platform allowed adjustment of the difficulty of this spatial learning task. This enabled the detection of a small positive effect of rasagiline (1 mg/kg) on spatial learning, which was not observed in earlier reports. For the first time, our study has showed the procognitive effect of rasagiline in young healthy rats. On the basis of these findings, a monoamine oxidase-B inhibitor would seem to be a potential symptomatic treatment for cognitive impairments affecting patients with neurodegenerative disorders.

Metabolic profiling of rat brain and cognitive behavioral tasks: potential complementary strategies in preclinical cognition enhancement research.

In this study, the correlation between the metabolic profiles of rats undergoing cognition enhancement drug therapy and their associated cognitive behavioral outcomes were investigated. Male Lister Hooded rats were administered either with donepezil, galantamine, or vehicle and subjected to Atlantis watermaze training and novel object recognition tests. An UPLC/MS/MS method was developed to profile 21 neurologically related metabolites in the rat brains. Pharmacologically induced behavioral changes were compared subsequently with the metabolic fluctuations of neurologically related metabolites from multiple neurotransmitter pathways using multivariate and univariate statistical analyses. Significant improvements in cognitive behavioral outcomes were demonstrated in the rats administered with donepezil and galantamine using both AWM training (P < 0.05) and NOR (P < 0.05) tests as compared to those dosed with the vehicle. This corroborated with the significant elevation of eight prominent biomarkers after the cognitive enhancement therapy. An orthogonal partial least-squares discriminant analysis model generated using only the 8 metabolites identified as discriminating the drug-dosed rats from the vehicle-dosed rats gave a Q(2) = 0.566, receiver operator characteristic (ROC) AUC = 1.000, using 7-fold cross validation. Our study suggests that metabolic profiling of rat brain is a potential complementary strategy to the cognitive behavioral tasks for characterizing neurobiological responses to cognition enhancement drug testing.

Effect of donepezil on reversal learning in a touch screen-based operant task.

Impairments in reversal learning, which are commonly observed in patients with psychiatric disorders, remain difficult to treat. There is still a debate over the beneficial effects of cholinergic enhancers on improving behavioural flexibility. The objective of this study was to investigate the effect of an acetylcholinesterase inhibitor, donepezil, on the performance of a rodent Probabilistic Reversal Learning task. Lister-Hooded rats were trained to retrieve food rewards by discriminating two computer-generated stimuli in an automated touch screen-based operant task. When a steady performance was achieved, the stimulus-reward rule was reversed. Each rat was given a 30-min training session daily for 24 days and donepezil was administered 30 min before each training session. Systemic treatment with donepezil had no effect on trial accuracy of the two-stimulus discrimination training. However, the donepezil group showed enhanced performance in the reversal learning. Our result showed that treatment with donepezil significantly enhanced Probabilistic Reversal Learning performance in healthy animals. On the basis of this finding, the inhibition of the central acetylcholinesterase would seem to be a potential therapeutic approach to treat behavioural inflexibility.

Cognitive enhancing effects of ghrelin receptor agonists.

RATIONALE: Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, has been shown to play a role in multiple physiological processes including appetite regulation, metabolism and, more recently, dendritic spine architecture, long-term potentiation and cognition. OBJECTIVE: The objective of this study was to determine the effects of two structurally non-peptide ghrelin receptor agonists (GSK894490A and CP-464709-18) on rodent cognition. METHODS: All experiments were performed in male Lister hooded rats. Effects of the test compounds on rat cognitive performance was determined using the novel object recognition test, a modified water maze paradigm and a scopolamine-induced deficit in cued fear conditioning. These tests were chosen as they each probe a relatively independent cognitive domain and therefore potentially have differing underlying neural substrates. RESULTS: Both compounds significantly improved performance in the novel object recognition and modified water maze tests but were unable to attenuate a scopolamine deficit in cued fear conditioning. CONCLUSIONS: These results demonstrate that the small-molecule ghrelin receptor agonists profiled here readily cross the blood/brain barrier and elicit pro-cognitive effects in recognition and spatial learning and memory tests. Based on these observations, the central ghrelin receptor would appear to be a chemically tractable receptor and perhaps should be considered as a new drug target for therapeutic approaches to treat diseases affecting cognition.

Effects of losartan on angiotensin receptors in the hypertrophic rat heart.

The effects of losartan on angiotensin receptors in hypertrophic rat hearts were studied. The study was prompted by inconsistent findings of either an increase or decrease in the mRNA of the AT1 receptor in the hearts of cardiac hypertrophic rats treated with losartan, and a paucity of information on the effects of losartan on functional angiotensin receptors in the heart. Losartan, administered i.p. to aortic coarcted rats, dose-dependently attenuated the cardiac hypertrophy. Significant effect was observed with a dose of 2.72 micromol/kg/day for four days. Hypertrophy was accompanied by an increase in [125I]-Sar1-Ile8-angiotensin II binding sites (due mainly to an increase in AT2 binding) and AT2 receptor protein in cardiac ventricles of aortic coarcted rats. Treatment with effective anti-hypertrophic doses of losartan dose-dependently downregulated the [125I]-Sar1-Ile8-angiotensin II binding sites, constitutive AT1 receptor protein, and the over expressed AT2 receptor protein. It was suggested that the anti-cardiac hypertrophic action of losartan resulted from its ability to suppress the expression of both the basal and enhanced cardiac angiotensin receptors. This raises the question as to whether such drastic action could form the therapeutic basis for the use of losartan in cardiac pathologies.

Structure-activity and structure-binding studies of des-Asp(1)-angiotensin I analogues on the rabbit pulmonary artery.

Structural modification of des-aspartate-angiotensin I (DAA-I), a pharmacologically active peptide, affected its actions on the precontracted cardiac and pulmonary sections of the rabbit pulmonary artery. The displacement of [125I]-Sar(1)-Ile(8)-angiotensin II by the DAA-I analogues from membrane homogenates of the whole pulmonary artery was also markedly reduced. Analogues that retained similar responses as DAA-I in the functional assays exhibited binding affinities of similar magnitude as DAA-I. Analogues that had no effect in the functional assay showed markedly reduced binding affinities. The first and fifth positions on DAA-I were identified as critical positions for activity as the replacement of Arg(2) and His(6) at these positions with alanine completely abolished activity and sharply reduced binding affinities. In contrast, the last two N-terminal amino acids of DAA-I can be modified substantially (D-amino acid and alanine substitution) without loss of activity or binding affinity. The identification of critical and noncritical amino acids would offer useful leads in the design of specific DAA-I antagonists.