Alzheimer's disease (AD) affects 10% of people older than 65 and is characterized by a progressive loss of cognitive function with an abnormal accumulation of amyloid β (Aβ ) plaques and neurofibrillary tangles (NFT) in the brain. Efforts to reduce brain Aβ plaques continue to be investigated as a therapeutic approach for AD. We report here development of dual targeting agents with affinity for Aβ plaque/P-glycoprotein (Pgp) and Aβ plaque/α4β 2* nicotinic acetylcholine receptors (nAChR). These novel dual agents may be able to efflux Aβ plaques via the paravascular (glymphatic) pathways. Ferulic acid (FA), ferulic acid ethyl ester (FAEE), and curcumin (CUR) were used for Aβ plaques, fexofenadine (FEX) was used as substrate for Pgp and nifrolidine (NIF) was used for α4β 2* nAChRs. Aβ plaque/α4β 2* nAChR dual agent, FA-NIF (GKS-007) exhibited IC₅₀ = 3-6 nM for α4β 2* nAChRs in [³H]cytisine-radiolabeled thalamus and frontal cortex in rat brain slices. In postmortem human AD frontal cortex, Aβ plaques labeled with [³H]PIB, FEX-CUR showed a 35% reduction in gray matter (GM)/white matter (WM) [³H]PIB binding, while CUR alone showed a 50% reduction. In vivo biodistribution studies are required of the Aβ-Pgp and Aβ-α4β 2* nAChRs dual targeting agents in order to evaluate their potential as therapeutic approaches for reducing brain Aβ plaques.

Nicotinic acetylcholine α4β2∗ receptors (nAChRs) are implicated in various neurodegenerative diseases and smoking addiction. Imaging of brain high-affinity α4β2∗ nAChRs at the cellular and subcellular levels would greatly enhance our understanding of their functional role. Since better resolution could be achieved with fluorescent probes, using our previously developed positron emission tomography (PET) imaging agent [18F]nifrolidine, we report here design, synthesis and evaluation of two fluorescent probes, nifrodansyl and nifrofam for imaging α4β2∗ nAChRs. The nifrodansyl and nifrofam exhibited nanomolar affinities for the α4β2∗ nAChRs in [3H]cytisine-radiolabeled rat brain slices. Nifrofam labeling was observed in α4β2∗ nAChR-expressing HEK cells and was upregulated by nicotine exposure. Nifrofam co-labeled cell-surface α4β2∗ nAChRs, labeled with antibodies specific for a β2 subunit extracellular epitope indicating that nifrofam labels α4β2∗ nAChR high-affinity binding sites. Mouse brain slices exhibited discrete binding of nifrofam in the auditory cortex showing promise for examining cellular distribution of α4β2∗ nAChRs in brain regions.

Nicotinic acetylcholinergic receptors (nAChR's) have been implicated in several brain disorders, including addiction, Parkinson's disease, Alzheimer's disease and schizophrenia. Here we report in vitro selectivity and functional properties, toxicity in rats, in vivo evaluation in humans, and comparison across species of [¹⁸ F]Nifene, a fast acting PET imaging agent for α4β2* nAChRs. Nifene had subnanomolar affinities for hα2β2 (0.34 nM), hα3β2 (0.80 nM) and hα4β2 (0.83 nM) nAChR but weaker (27-219 nM) for hβ4 nAChR subtypes and 169 nM for hα7 nAChR. In functional assays, Nifene (100 μM) exhibited 14% agonist and >50% antagonist characteristics. In 14-day acute toxicity in rats, the maximum tolerated dose (MTD) and the no observed adverse effect level (NOAEL) were estimated to exceed 40 μg/kg/day (278 μg/m² /day). In human PET studies, [¹⁸ F]Nifene (185 MBq; <0.10 μg) was well tolerated with no adverse effects. Distribution volume ratios (DVR) of [¹⁸ F]Nifene in white matter thalamic radiations were ∼1.6 (anterior) and ∼1.5 (superior longitudinal fasciculus). Habenula known to contain α3β2 nAChR exhibited low levels of [¹⁸ F]Nifene binding while the red nucleus with α2β2 nAChR had DVR ∼1.6-1.7. Females had higher [¹⁸ F]Nifene binding in all brain regions, with thalamus showing >15% than males. No significant aging effect was observed in [¹⁸ F]Nifene binding over 5 decades. In all species (mice, rats, monkeys, and humans) thalamus showed highest [¹⁸ F]Nifene binding with reference region ratios >2 compared to extrathalamic regions. Our findings suggest that [¹⁸ F]Nifene PET may be used to study α4β2* nAChRs in various CNS disorders and for translational research.