The Chemogenetic Receptor Ligand Clozapine N-Oxide Induces in vivo Neuroreceptor Occupancy and Reduces Striatal Glutamate Levels

Chemogenetic studies with the ligand clozapine N-oxide (CNO) are predicated upon the assumption that CNO is devoid of actions at natural neuroreceptors. However, recent evidence shows that CNO may be converted back to clozapine (CLZ) in vivo, which could yield plasma concentrations that may be sufficient to occupy inter alia dopamine D2/3 and serotonin 5HT2A receptors in living brain. To test this phenomenon, we measured striatal dopamine D2/3 receptor occupancy with [18F]fallypride PET and serotonin 5HT2A occupancy ex vivo using [18F]MH.MZ. We found a CNO dose-dependent effect on the availability of both neuroreceptor sites. In parallel MR spectroscopy experiments, we found that CNO reduced creatine + phosphcreatine (Cr+PCr) and increased N-acetylaspartate + N-acetylaspartylglutamate (NAA+NAAG) signals in the prefrontal cortex, and also reduced the glutamate signal in dorsal striatum, with peak effect at 2 mg/kg. Thus, our findings suggest that conversion of CNO to CLZ in living rats imparts significant occupancy at endogenous neuroreceptors and significant changes to neurometabolite levels

In vitro and in vivo characterization of dibenzothiophene derivatives [¹²⁵I]iodo-ASEM and [¹⁸F]ASEM as radiotracers of homo- And heteromeric α7 nicotinic acetylcholine receptors

The α7 nicotinic acetylcholine receptor (α7 nAChR) is involved in several cognitive and physiologic processes; its expression levels and patterns change in neurologic and psychiatric diseases, such as schizophrenia and Alzheimer’s disease, which makes it a relevant drug target. Development of selective radioligands is important for defining binding properties and occupancy of novel molecules targeting the receptor. We tested the in vitro binding properties of [125I]Iodo-ASEM [(3-(1,4-diazabycyclo[3.2.2]nonan-4-yl)-6-(125I-iododibenzo[b,d]thiopentene 5,5-dioxide)] in the mouse, rat and pig brain using autoradiography. The in vivo binding properties of [18F]ASEM were investigated using positron emission tomography (PET) in the pig brain. [125I]Iodo-ASEM showed specific and displaceable high affinity (~1 nM) binding in mouse, rat, and pig brain. Binding pattern overlapped with [125I]α-bungarotoxin, specific binding was absent in α7 nAChR gene-deficient mice and binding was blocked by a range of α7 nAChR orthosteric modulators in an affinity-dependent order in the pig brain. Interestingly, relative to the wild-type, binding in β2 nAChR gene-deficient mice was lower for [125I]Iodo-ASEM (58% ± 2.7%) than [125I]α-bungarotoxin (23% ± 0.2%), potentially indicating different binding properties to heteromeric α7β2 nAChR. [18F]ASEM PET in the pig showed high brain uptake and reversible tracer kinetics with a similar spatial distribution as previously reported for α7 nAChR. Blocking with SSR-180,711 resulted in a significant decrease in [18F]ASEM binding. Our findings indicate that [125I]Iodo-ASEM allows sensitive and selective imaging of α7 nAChR in vitro, with better signal-to-noise ratio than previous tracers. Preliminary data of [18F]ASEM in the pig brain demonstrated principal suitable kinetic properties for in vivo quantification of α7 nAChR, comparable to previously published data