Anti-Amnesic and Neuroprotective Effects of Fluoroethylnormemantine in a Pharmacological Mouse Model of Alzheimer's Disease.

BACKGROUND: Current therapies in Alzheimer's disease (AD), including Memantine, have proven to be only symptomatic but not curative or disease modifying. Fluoroethylnormemantine (FENM) is a structural analogue of Memantine, functionalized with a fluorine group that allowed its use as a positron emission tomography tracer. We here analyzed FENM neuroprotective potential in a pharmacological model of AD compared with Memantine. METHODS: Swiss mice were treated intracerebroventricularly with aggregated Aß 25-35 peptide and examined after 1 week in a battery of memory tests (spontaneous alternation, passive avoidance, object recognition, place learning in the water-maze, topographic memory in the Hamlet). Toxicity induced in the mouse hippocampus or cortex was analyzed biochemically or morphologically. RESULTS: Both Memantine and FENM showed symptomatic anti-amnesic effects in Aß 25-35-treated mice. Interestingly, FENM was not amnesic when tested alone at 10 mg/kg, contrarily to Memantine. Drugs injected once per day prevented Aß 25-35-induced memory deficits, oxidative stress (lipid peroxidation, cytochrome c release), inflammation (interleukin-6, tumor necrosis factor-α increases; glial fibrillary acidic protein and Iba1 immunoreactivity in the hippocampus and cortex), and apoptosis and cell loss (Bcl-2-associated X/B-cell lymphoma 2 ratio; cell loss in the hippocampus CA1 area). However, FENM effects were more robust than observed with Memantine, with significant attenuations vs the Aß 25-35-treated group. CONCLUSIONS: FENM therefore appeared as a potent neuroprotective drug in an AD model, with a superior efficacy compared with Memantine and an absence of direct amnesic effect at higher doses. These results open the possibility to use the compound at more relevant dosages than those actually proposed in Memantine treatment for AD.

Memantine prodrug as a new agent for Alzheimer's Disease.

Hydrogen sulphide has recently drawn much attention due to its potent anti-inflammatory and neuroprotective roles in brain functions. The purpose of the current study was to exploit these beneficial properties of H2S to design a new agent for the treatment of Alzheimer's disease (AD). To pursue our aims, we replaced the free amine group of memantine with an isothiocyanate functionality as a putative H2S-donor moiety. The new chemical entity, named memit, was then tested in vitro to determine whether it retains the pharmacological profile of the "native drug", while also providing a source of H2S in the CNS. Indeed, Memit showed the ability to release H2S through a cysteine-mediated mechanism, thus generating memantine. Moreover, the new hybrid molecule exerts protective effects against neuronal inflammation and induces a drastic fall in ROS production. In addition, memit was also able to reduce the Aß(1-42) self-induced aggregation and exerted cytoprotective effect against Aß oligomers-induced damage in both human neurons and rat microglia cells. Finally, similarly to memantine, the new compound promotes autophagy, a complex process required for cellular homeostasis in cell survival that results to be altered in neurodegenerative diseases. In conclusion, our study revealed that memit is a prodrug of memantine. Further in vivo studies will be necessary to fully investigate the synergic or cumulative effects due to the H2S-releasing moiety and the native drug.

Triptolide derivatives as potential multifunctional anti-Alzheimer agents: Synthesis and structure-activity relationship studies.

Owning to the promising neuroprotective profile and the ability to cross the blood-brain barrier, triptolide has attracted extensive attention. Although its limited solubility and toxicity have greatly hindered clinical translation, triptolide has nonetheless emerged as a promising candidate for structure-activity relationship studies for Alzheimer's disease. In the present study, a series of triptolide analogs were designed and synthesized, and their neuroprotective and anti-neuroinflammatory effects were then tested using a cell culture model. Among the triptolide derivatives tested, a memantine conjugate, compound 8, showed a remarkable neuroprotective effect against Aß1-42 toxicity in primary cortical neuron cultures as well as an inhibitory effect against LPS-induced TNF-α production in BV2 cells at a subnanomolar concentration. Our findings provide insight into the different pharmacophores that are responsible for the multifunctional effects of triptolide in the central nervous system. Our study should help in the development of triptolide-based multifunctional anti-Alzheimer drugs.

Targeting nitric oxide and NMDA receptor-associated pathways in treatment of high grade glial tumors. Hypotheses for nitro-memantine and nitrones.

Glioblastoma multiforme (GBM) is a devastating brain cancer with no curative treatment. Targeting Nitric Oxide (NO) and glutamatergic pathways may help as adjunctive treatments in GBM. NO at low doses promotes tumorigenesis, while at higher levels (above 300 nM) triggers apoptosis. Gliomas actively secrete high amounts of glutamate which activates EGR signaling and mediates degradation of peritumoral tissues via excitotoxic injury. Memantine inhibits NMDA-subtype of glutamate receptors (NMDARs) and induces autophagic death of glioma cells in vitro and blocks glioma growth in vivo. Nitro-memantines may exert further benefits by limiting NMDAR signaling and by delivery of NO to the areas of excessive NMDAR activity leading NO-accumulation at tumoricidal levels within gliomas. Due to the duality of NO in tumorigenesis, agents which attenuate NO levels may also act beneficial in treatment of GBM. Nitrone compounds including N-tert-Butyl-α-phenylnitrone (PBN) and its disulfonyl-phenyl derivative, OKN-007 suppress free radical formation in experimental cerebral ischemia. OKN-007 failed to show clinical efficacy in stroke, but trials demonstrated its high biosafety in humans including elderly subjects. PBN inhibits the signaling pathways of NF-κB, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX). In animal models of liver cancer and glioblastoma, OKN-007 seemed more efficient than PBN in suppression of cell proliferation, microvascular density and in induction of apoptosis. OKN-007 also inhibits SULF2 enzyme, which promotes tumor growth via versatile pathways. We assume that nitromemantines may be more beneficial concomitant with chemo-radiotherapy while nitrones alone may act useful in suppressing basal tumor growth and angiogenesis.

Memantine-derived drugs as potential antitumor agents for the treatment of glioblastoma.

Glioblastoma is one of the most aggressive malignant primary brain cancer in adults. To date, surgery, radiotherapy and current pharmacological treatments are not sufficient to manage this pathology that has a high mortality rate (median survival 12-15months). Recently, anticancer multi-targeted compounds have attracted much attention with the aim to obtain new drugs able to hit different biological targets that are involved in the onset and progression of the disease. Here, we report the synthesis of novel memantine-derived drugs (MP1-10) and their potential antitumor activities in human U87MG glioblastoma cell line. MP1-10 were synthetized joining memantine, which is a NMDA antagonist, to different histone deacetylase inhibitors to obtain one molecule with improved therapeutic efficacy. Biological results indicated that MP1 and MP2 possessed more potent anti-proliferative effects on U87MG cells than MP3-10 in a dose-dependent manner. MP1 and MP2 induced significant cell death by apoptosis characterized by apoptotic morphological changes in Hoechst staining. Both drugs also exhibited non-genotoxic and only mild cytotoxic effects in human whole blood cells. However, only MP1, showing good chemico-physical properties (solubility, LogP) and enzymatic stabilities in gastric and intestinal fluids, can be considered a suitable candidate for in vivo pharmacokinetic studies.

Solid lipid nanoparticles loaded with lipoyl-memantine codrug: preparation and characterization.

Solid lipid nanoparticles (SLNs) are considered very attractive drug-delivery systems (DDS) able to enhance the efficacy of some therapeutic agents in several pathologies difficult to treat in a conventional way. Starting from these evidences, this study describes the preparation, physicochemical characterization, release, and in vitro cytotoxicity of stealth SLNs as innovative approach to improve solubility and absorption through the gastrointestinal tract of lipoyl-memantine (LA-MEM), a potential anti-Alzheimer codrug. Physico-chemical properties of LA-MEM loaded SLNs have been intensively investigated. Differential scanning calorimetry (DSC) was used to clarify the state and crystalline structure of the formulation. The results obtained from particles size analysis, polydispersity (PDI), and zeta potential measurements allowed the identification of the optimized formulation, which was characterized by a drug-lipid ratio 1:5, an average intensity diameter of 170nm, a PDI of 0.072, a zeta potential of -33.8mV, and an entrapment efficiency of 88%. Moreover, in vitro stability and release studies in both simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), and preliminary in vitro cytotoxicity studies revealed that LA-MEM loaded SLNs could represent potential candidate for an in vivo investigation as DDS for the brain since it resulted devoid of citotoxicity and able to release the free codrug.

Memantine-sulfur containing antioxidant conjugates as potential prodrugs to improve the treatment of Alzheimer's disease.

The approved treatments for Alzheimer's disease (AD) exploit mainly a symptomatic approach based on the use of cholinesterase inhibitors or N-methyl-D-aspartate (NMDA) receptor antagonists. Natural antioxidant compounds, able to pass through the blood-brain barrier (BBB), have been extensively studied as useful neuroprotective agents. A novel approach towards excitotoxicity protection and oxidative stress associated with excess ß amyloid (Aß) preservation in AD is represented by selective glutamatergic antagonists that possess as well antioxidant capabilities. In the present work, GSH (1) or (R)-α-lipoic acid (LA) (2) have been covalently linked with the NMDA receptor antagonists memantine (MEM). The new conjugates, proposed as potential antialzheimer drugs, should act both as glutamate receptor antagonists and radical scavenging agents. The physico-chemical properties and "in vitro" membrane permeability, the enzymatic and chemical stability, the demonstrated "in vitro" antioxidant activity associated to the capacity to inhibit Aß(1-42) aggregation makes at least compound 2 a promising candidate for treatment of AD patients.

19F and 1H MRI detection of amyloid beta plaques in vivo.

Formation of senile plaques composed of amyloid beta peptide, a pathological hallmark of Alzheimer disease, in human brains precedes disease onset by many years. Noninvasive detection of such plaques could be critical in presymptomatic diagnosis and could contribute to early preventive treatment strategies. Using amyloid precursor protein (APP) transgenic mice as a model of amyloid beta amyloidosis, we demonstrate here that an intravenously administered (19)F-containing amyloidophilic compound labels brain plaques and allows them to be visualized in living mice by magnetic resonance imaging (MRI) using (19)F and (1)H. Our findings provide a new direction for specific noninvasive amyloid imaging without the danger of exposure to radiation. This approach could be used in longitudinal studies in mouse models of Alzheimer disease to search for biomarkers associated with amyloid beta pathology as well as to track disease course after treatment with candidate medications.

NMR spectroscopy in studies of light-induced structural changes in mammalian rhodopsin: applicability of solution (19)F NMR.

We report high resolution solution (19)F NMR spectra of fluorine-labeled rhodopsin mutants in detergent micelles. Single cysteine substitution mutants in the cytoplasmic face of rhodopsin were labeled by attachment of the trifluoroethylthio (TET), CF(3)-CH(2)-S, group through a disulfide linkage. TET-labeled cysteine mutants at amino acid positions 67, 140, 245, 248, 311, and 316 in rhodopsin were thus prepared. Purified mutant rhodopsins (6-10 mg), in dodecylmaltoside, were analyzed at 20 degrees C by solution (19)F NMR spectroscopy. The spectra recorded in the dark showed the following chemical shifts relative to trifluoroacetate: Cys-67, 9.8 ppm; Cys-140, 10.6 ppm; Cys-245, 9.9 ppm; Cys-248, 9.5 ppm; Cys-311, 9.9 ppm; and Cys-316, 10.0 ppm. Thus, all mutants showed chemical shifts downfield that of free TET (6.5 ppm). On illumination to form metarhodopsin II, upfield changes in chemical shift were observed for (19)F labels at positions 67 (-0.2 ppm) and 140 (-0.4 ppm) and downfield changes for positions 248 (+0.1 ppm) and 316 (+0.1 ppm) whereas little or no change was observed at positions 311 and 245. On decay of metarhodopsin II, the chemical shifts reverted largely to those originally observed in the dark. The results demonstrate the applicability of solution (19)F NMR spectroscopy to studies of the tertiary structures in the cytoplasmic face of intact rhodopsin in the dark and on light activation.