Combined In Silico, Ex Vivo, and In Vivo Assessment of L-17, a Thiadiazine Derivative with Putative Neuro- and Cardioprotective and Antidepressant Effects.

Depression associated with poor general medical condition, such as post-stroke (PSD) or post-myocardial infarction (PMID) depression, is characterized by resistance to classical antidepressants. Special treatment strategies should thus be developed for these conditions. Our study aims to investigate the mechanism of action of 2-morpholino-5-phenyl-6H-1,3,4-thiadiazine, hydrobromide (L-17), a recently designed thiadiazine derivative with putative neuro- and cardioprotective and antidepressant-like effects, using combined in silico (for prediction of the molecular binding mechanisms), ex vivo (for assessment of the neural excitability using c-Fos immunocytochemistry), and in vivo (for direct examination of the neuronal excitability) methodological approaches. We found that the predicted binding affinities of L-17 to serotonin (5-HT) transporter (SERT) and 5-HT3 and 5-HT1A receptors are compatible with selective 5-HT serotonin reuptake inhibitors (SSRIs) and antagonists of 5-HT3 and 5-HT1A receptors, respectively. L-17 robustly increased c-Fos immunoreactivity in the amygdala and decreased it in the hippocampus. L-17 dose-dependently inhibited 5-HT neurons of the dorsal raphe nucleus; this inhibition was partially reversed by the 5-HT1A antagonist WAY100135. We suggest that L-17 is a potent 5-HT reuptake inhibitor and partial antagonist of 5-HT3 and 5-HT1A receptors; the effects of L-17 on amygdaloid and hippocampal excitability might be mediated via 5-HT, and putatively mediate the antidepressant-like effects of this drug. Since L-17 also possesses neuro- and cardioprotective properties, it can be beneficial in PSD and PMID. Combined in silico predictions with ex vivo neurochemical and in vivo electrophysiological assessments might be a useful strategy for early assessment of the efficacy and neural mechanism of action of novel CNS drugs.

Measuring drug occupancy in the absence of a reference region: the Lassen plot re-visited.

Quantitative estimation of neuroreceptor occupancy by exogenous drugs using positron emission tomography is based on the reduction in the total volume of distribution (V(T)) of site-specific radioligands after drug administration. An estimate of the distribution volume of free and nonspecifically bound radioligand (V(ND)) is also required to distinguish specific from total binding. However, a true reference region, devoid of specific binding, is often not available. We present a transformation of a graphical method, originally introduced by Lassen, using regional estimates of V(T) alone to determine occupancy, together with an extension that does not require baseline data.

Experiment assessment of mass effects in the rat: implications for small animal PET imaging.

In vivo imaging using positron emission tomography (PET) is important in the development of new radiopharmaceuticals in rodent animal models for use as biochemical probes, diagnostic agents, or in drug development. We have shown mathematically that, if small animal imaging studies in rodents are to have the same "quality" as human PET studies, the same number of coincidence events must be detected from a typical rodent imaging "voxel" as from the human imaging voxel. To achieve this using the same specific activity preparation, we show that roughly the same total amount of radiopharmaceutical must be given to a rodent as to a human subject. At high specific activities, the mass associated with human doses, when administered to a rodent, may not decrease the uptake of radioactivity at non saturable sites or sites where an enzyme has a high capacity for a substrate. However, in the case of binding sites of low density such as receptors, the increased mass injected could saturate the receptor and lead to physiologic effects and non-linear kinetics. Because of the importance of the mass injected for small animal PET imaging, we experimentally compared high and low mass preparations using ex vivo biodistribution and phosphorimaging of three compounds: 2-fluoro-2-deoxyglucose (FDG), 6-fluoro-L-metatyrosine (FMT) and one receptor-directed compound, the serotonin 5HT1A receptor ligand, trans-4-fluoro-N-[2-[4-(2-methoxylphenyl) piperazino]ethyl]-N-(2-pyridyl) cyclohexane- carboxamide (FCWAY). Changes in the mass injected per rat did not affect the distribution of FDG, FMT, and FCWAY in the range of 0.6-1.9 nmol per rat. Changes in the target to nontarget ratio were observed for injected masses of FCWAY in the range of approximately 5-50 nmol per rat. If the specific activity of such compounds and/or the sensitivity of small animal scanners are not increased relative to human studies, small animal PET imaging will not correctly portray the "true" tracer distribution. These difficulties will only be exacerbated in animals smaller than the rat, e.g., mice.