Nicotinic acetylcholine receptors (nAChRs) are involved in various central nervous system functions and have also been implicated in several neurodegenerative disorders. The heteromeric α4β2* and homomeric α7 are two major nAChR subtypes which have been studied in the brain using positron emission tomography (PET). Our comparative autoradiographic studies of the two receptor types in the mouse and rat brains show major differences in the thalamus (α4β2* >> α7), hippocampus (α7 >> α4β2*), and subiculum (α4β2* >> α7). A relatively newer heteromeric α7β2 nAChR subtype has been identified in the brain which may have a greater role in neurodegeneration. We report the development of KS7 (3-(2-(S)-azetidinylmethoxy)-5-(1,4-diaza-bicyclo[3.2.2]nonane)pyridine) which incorporates structural features of Nifzetidine (high affinity for α4β2* nAChR) and ASEM (high affinity for α7 nAChR) in an effort to target α7 and β2 subunits in α7β2 nAChR. KS7 exhibited higher affinities (IC50 = 50 to 172 nM) for [3H]cytisine radiolabeled sites and weaker affinities (IC50 = 10 μM) for [125I]-α-bungarotoxin radiolabeled rat brain sites in several brain regions. The weaker affinity of KS7 to α7 nAChR may suggest lack of binding at the α7 subunit of α7β2 nAChR. A radiolabeled derivative of KS7 may be required to identify any specific binding to brain regions suggested to contain α7β2 nAChR.

Alpha-klotho is a single-pass membrane protein primarily expressed by the kidneys. Klotho deficiency in chronic kidney disease contributes to an accelerated aging phenotype. We report here development of [89Zr]DFO-anti-klotho positron emission tomography (PET) imaging as a novel non-invasive method for assessing whole-body alpha-klotho distribution. Rat monoclonal anti-mouse klotho antibody was reacted with SCN-Bn-deferoxamine (DFO) and was radiolabeled using Zirconium-89. In vitro testing of [89Zr]DFO-anti-mKlotho was done in a distal convoluted tubule kidney cell line and with 40-micron whole kidney sections from C57BL/6J mice. Competitive binding was assessed in co-incubation studies with unlabeled anti-mKlotho antibody. For in vivo testing, C57BL/6J mice were injected retro-orbitally with [89Zr]DFO-anti-mKlotho and were scanned using Inveon PET/CT. Autoradiographs of kidney sections were obtained post-imaging on select animals. Radiochemical yield of [89Zr]DFO-anti-mKlotho was >70% and radiochemical purity was confirmed by iTLC. Specific binding in the kidney cell line was reduced by 60% in the presence of unlabeled anti-mKlotho. In the PET/CT scans, initial uptake of [89Zr]DFO-anti-mKlotho was observed in the intestines and liver. Selective retention of radioactivity was observed in the kidneys in the subsequent 24, 48, and 72 hrs scans with cortical binding of [89Zr]DFO-anti-mKlotho clearly visualized. Sites of lower alpha-klotho expression were not visualized. In summary, we have successfully synthesized [89Zr]DFO-anti-mKlotho and our initial in vitro and in vivo studies in mice demonstrate selective binding in the kidney cortex, which is known to express high levels of alpha-klotho. PET imaging promises to be a novel tool for in vivo evaluation of alpha-klotho distribution.