Pharmacological characterizations of novel anti-dementia NMDA receptor antagonist memantine nitrates via multiple targets

Abstract

Background: Dementia, characterized by the cognitive function impairments, mainly affects the aged population. According to a report from World Health Organization, around 50 million patients with dementia in 2018, and nearly 10 million new cases every year. Although the problems are becoming more and more serious, the causes of dementia remain to be elucidated. Alzheimer's disease (AD) and vascular dementia (VaD) are the two most common forms of dementia, which contributing to 90% of the total cases of dementia today. Currently, only limited drugs are available for treating AD and VaD. Increasing evidences have shown that AD might be caused by multiple factors. Therefore, currently existing single-targeted drugs might only offer limited symptoms relieve effects of patients suffered from dementia. In the previous studies, our collaborators in Jinan University have designed and synthesized a series of novel compounds derived from memantine, a currently used anti-AD drug. The nitrate moiety has been introduced onto the backbone of memantine, providing multi-functional anti-dementia effects including neuroprotection and vasodilation. Some of the memantine nitrates have been proven to possess neuroprotective effects in vitro; however, the detail mechanism(s) is still unclear. My thesis study is aiming to further investigate the neuroprotective effects of these memantine nitrates, as well as the underlying mechanisms both in vitro and in vivo. Methodology: Various in vitro and in vivo models were employed to investigate the pharmacological characterizations of the memantine nitrates. MTT assays were used to evaluate the cell viability. Hoechst staining was used to differentiate apoptosis and necrosis induced by neurotoxins. Confocal microscopic fluorescent scanning and electrophysiological experiments were performed to explore the involvement of memantine nitrates on the blockage of NMDA receptor. Immunostaining is conducted to show the synapse integrality. Variety of chemical inhibitors and Western blotting were used to investigate the underlying signaling pathways on neuroprotection as offered by the memantine nitrates. Moreover, the 2-vessel occlusion (2VO) rat model was used to evaluate the anti-dementia effects and the improvement of cerebral blood flow (CBF) by memantine nitrates. Results: The neuroprotective effects of memantine nitrates were firstly evaluated. Three representative memantine nitrates (MN-06, MN-08 and MN-12) have exhibited neuroprotective effects against glutamate-induced excitotoxicity in rat primary cerebellar granule neurons. MN-08, the most promising candidate, has been selected for further mechanistic characterization. Our results revealed that MN-08 prevented glutamate-induced apoptosis through multiple targets. Western blotting assays have shown that 2 h pretreatment of MN-08 could prevent the activation of ERK and inhibition of Akt pathways caused by glutamate. In addition, the neuroprotective effects, as well as the regulations of Akt and GSK3β, exhibited by MN-08 could be abolished with the present of phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002. These results indicated that MN-08 provided the neuroprotective effects by inhibiting ERK pathway and attenuating the inhibited PI3-K/Akt pathway. In addition, the confocal microscopic calcium imaging demonstrated that the pretreatment of MN-08 prevented the calcium influx. Moreover, by using the patch clamp, MN-08 significantly inhibited MNDA-mediated calcium current in rat primary cultured hippocampal neurons in a concentration-dependent manner, indicating that MN-08 might block the calcium influx through the inhibition of NMDA receptor, which has been further confirmed by molecular docking simulation. Furthermore, MN-08 improved the CBF of the rat at 4th week after 2VO surgery. More importantly, the behavioral tests showed that MN-08 prevented the cognitive function impairments induced by 2VO surgery. Conclusion: Taken together, the derivatives of memantine nitrates, MN-08, might provide multi-functional anti-dementia effects including neuroprotection and vascular dilation. These data strongly suggest that memantine nitrates might be the potential candidates for the development of a new generation of anti-dementia drugs.