Dysfunctional interactions of metal ions, especially Cu, Zn, and Fe, with the amyloid-beta (A beta) peptide are hypothesized to play an important role in the etiology of Alzheimer's disease (AD). In addition to direct effects on A beta aggregation, both Cu and Fe catalyze the generation of reactive oxygen species (ROS) in the brain further contributing to neurodegeneration. Disruption of these aberrant metal-peptide interactions via chelation therapy holds considerable promise as a therapeutic strategy to combat this presently incurable disease. To this end, we developed two multifunctional carbohydrate-containing compounds N,N'-bis[(5-beta-D-glucopyranosyloxy-2-hydroxy)benzyl]-N,N'-dimethyl-ethane-1,2-diamine (H2GL1) and N,N'-bis[(5-beta-D-glucopyranosyloxy-3-tert-butyl-2-hydroxy)benzyl]-N,N'-dimethyl-ethane-1,2-diamine (H2GL2) for brain-directed metal chelation and redistribution. Acidity constants were determined by potentiometry aided by UV-vis and 1H NMR measurements to identify the protonation sites of H2GL1,2. Intramolecular H bonding between the amine nitrogen atoms and the H atoms of the hydroxyl groups was determined to have an important stabilizing effect in solution for the H2GL1 and H2GL2 species. Both H2GL1 and H2GL2 were found to have significant antioxidant capacity on the basis of an in vitro antioxidant assay. The neutral metal complexes CuGL1, NiGL1, CuGL2, and NiGL2 were synthesized and fully characterized. A square-planar arrangement of the tetradentate ligand around CuGL2 and NiGL2 was determined by X-ray crystallography with the sugar moieties remaining pendant. The coordination properties of H2GL1,2 were also investigated by potentiometry, and as expected, both ligands displayed a higher affinity for Cu2+ over Zn2+ with H2GL1 displaying better coordinating ability at physiological pH. Both H2GL1 and H2GL2 were found to reduce Zn2+- and Cu2+- induced Abeta1-40 aggregation in vitro, further demonstrating the potential of these multifunctional agents as AD therapeutics.

Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor g (PPARg) contribute to hypoxiainduced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxiaexposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O 2 ) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARg, and transforming growth factor (TGF)b1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O 2 ) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARg and TGF-b1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H 2 O 2 release, proliferation, and TGF-b1 expression and blunted reductions in PPARg in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-b1 and reductions in PPARg expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxiainduced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.Keywords: rosiglitazone; PPARg; TGF-b; pulmonary hypertension Pulmonary hypertension (PH) is a progressive disorder associated with significant morbidity and mortality. Although recent therapeutic advances have improved survival for patients with PH, the prognosis remains poor (1). The pathobiology of PH is complex, and factors that contribute to endothelial dysfunction have been implicated in pathogenesis (2, 3). Among these factors, NADP reduced (NADPH) oxidase enzymes that produce reactive oxygen species (ROS) contribute to the development of a variety of vascular diseases, such as atherosclerosis (4) and systemic (5) and pulmonary hypertension (6). NADPH oxidases catalyze the reduction of molecular oxygen to generate superoxide (O 2 .2 ), hydrogen peroxide (H 2 O 2 ), or secondary oxidants (7). Seven isoforms of the catalytic moiety of the nonphagocytic NADPH oxidase enzyme have been described (Nox1-5, Duox1-2). These subunits are homologous to the catalytic moiety of the prototype phagocytic NADPH oxidase Nox2 (or gp91 phox ) but differ from each other regarding cellular localization, tissue distribution, regulation, activation, and expression (7,8). For example, although both Nox1 and Nox4 are expressed in vascular smooth muscle cells (VSMCs), they are targeted to discreet intracellular locations, are differe...

Two of the biochemical features of Alzheimers disease (AD) that contribute to neurodegeneration are intracellular oxidative stress and elevated levels of trace metal ions, especially Fe III , Cu II , and Zn II . [1] Both are factors involved in formation of the histological features in the brain used typically for postmortem diagnosis of AD, namely b-amyloid (Ab) plaques and neurofibrillary tangles. Therapeutic interventions under current investigation elsewhere include clioquinol [2] and desferrioxamine, [3] which are metal chelators that target elevated trace-metal ions in the brain, although neither are intended to affect oxidative stress directly and nor are they targeted to the brain. Antioxidant supplements have been studied separately as palliative-only measures for alleviation of the symptoms of AD.[4]Herein, we present for the first time a trifunctional approach to AD therapy. Modified and functionalized bidentate hydroxypyridinone pro-ligands (Scheme 1) address both the metal-ion and the oxidative imbalances inherent in AD while incorporating a glucose-receptor targeting feature.These prodrugs are designed to cross the blood-brain barrier (BBB), lose the pendant carbohydrate by enzymatic cleavage, passivate excess metal ions in the brain, and also protect neuronal cells against reactive oxygen species (ROS). Each of these functionalities has been demonstrated, thereby establishing the trifunctional principle as a valid goal in AD therapy. The prodrug strategy solves the potential problem of premature metal binding by using carbohydrates as both masking and directing substituents. In the context of increasing empirical support for re-establishing normal metal-ion homeostasis in neurodegenerative diseases, including AD, the trifunctional approach permits selective, tissue-dependent metal binding as a tailor-made, biologically compatible therapy.To demonstrate the utility of this approach, a series of assays on prototype compounds have been undertaken, including both in vitro and in vivo studies. This strategy is aimed at reducing neurodegeneration from oxidative stress; by passivating the pro-oxidant metal ions Fe III and Cu II , the production of ROS can be expected to be lower. By changing the R group on the pyridinone ring, the aqueous solubility, lipophilicity, and BBB permeability can be modified. Prodrug hydroxy (OH) groups have been elaborated by glycosylation (Scheme 1 b) such that, after enzymatic deprotection, the free ligands will have ring OH groups available that can either efficiently trap radicals or bind metal complexes (Scheme 1 a). Removing metal ions that promote Ab aggregation, such as Cu II and Zn II, also serves to prevent or reverse Scheme 1. Hydroxypyridinones: a) nonglycosylated pro-ligands and b) their glycosylated prodrug forms designed for metal passivation in the brain as a therapeutic intervention in Alzheimer's disease (AD).

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