Compound A and Compound B Fig were found in our
Compound A and Compound B (Fig. 4) were found in our in vitro screening assays and have similar CRF1 receptor in vitro profiles (Table 1). When binding was investigated using [125I]-CRF in ex vivo assay, Compound A inhibited binding of [125I]-CRF both in the frontal cortex and the pituitary; however, Compound B antagonized [125I]-CRF only in the pituitary and not in the frontal cortex, maybe due to its low bms-708163 receptor permeability (Fig. 5). The results of Compound A and Compound B on the activation of HPA axis by peripheral CRF challenge indicate that HPA axis regulation can be accomplished by antagonizing CRF1 receptors in the pituitary (Fig. 6A). In the comparison of i.v. and i.c.v. injected CRF on HPA axis, CRF was injected under conscious state and it was the same condition for behavioral analysis. In the evaluation of compounds on HPA axis, i.v. administration of CRF was conducted under anesthesia  because the physiological stress, holding in the apparatus for i.v. injection under conscious state, may be involved in the activation of CRF signaling in the CNS  and, with this condition, the effect of compounds on the peripheral CRF signaling may not be evaluated. From the results of behavioral analysis, antagonizing central CRF1 receptors seems to be essential to suppress increase in locomotion and anxiety behaviors because Compound B did not reduce locomotion induced by central CRF challenges (Fig. 6B). Compound A decreased locomotion (Fig. 6B) and anxiety behavior (Fig. 7) induced by the central CRF challenge. The independent regulation of anxiolytic effect and HPA axis by a compound has been reported by Philbert et al. using CRF1 antagonist SSR125543A, and it attenuated long-term cognitive deficit induced by acute inescapable stress even under the condition in which HPA axis was blunted using dexamethasone . In addition, Compound A inhibited c-fos expression in the cortex and the PVN induced by central CRF challenge (Fig. 8). Because c-fos plays an important role in signal transmission , it is used as a marker of neural activation . Recently, Takahashi et al. reported that CRF1 receptor antagonist antalarmin inhibited the c-fos expression induced by central challenge of CRF in the PVN, but not in the central nucleus of the amygdala (CeA) . Consistent with their report, Compound A inhibited c-fos expression in the PVN but not in the amygdala, although we did not segment the amygdala. One cannot exclude the possibility of subtle change in the subregion of amygdala, or the possibility of the different time course of c-fos expression changes in the amygdala. In contrast, the regulation of c-fos expression may not be the same between the central CRF challenge and acute stress, because antalarmin reduced the number of c-fos-immunopositive cells in the CeA under acute stress . Therefore, the evaluation of CRF1 receptor antagonists on biochemical changes induced by stress is also important to enable an understanding of their roles under these physiological conditions. Although there are many reports that suggest efficacy of CRF1 receptor selective antagonists on HPA axis activity, anxiety, and other behaviors in preclinical studies , , , successful results have not been reported in the clinical studies from R121919, the first CRF1 receptor selective antagonist that entered into a clinical study , to Pexacerfont . Further investigation will be needed regarding the compensation system in CRF signaling, pathophysiology of anxiety disorders, and others, to understand the discrepancy between non-clinical and clinical results. One of the key experiments for this point may be translational research to ensure target engagement such as receptor occupancy in the brain. In view of the specific distribution patterns of CRF1 receptors in the brain areas related to anxiety and stress, such as PVN, cortex, and limbic systems, it may be possible to analyze receptor occupancy by positron emission tomography (PET). There are a few reports about a radio-labeled ligand for CRF1 receptor , . In the PET studies in baboons, [11C]SN003 penetrated the BBB; however, regional variation in total binding could not be observed due to the rapid metabolism or the small number of CRF1 receptors . Further efforts have been continuously made to identify reasonable PET ligands ,  and target occupancy of CRF1 receptor antagonists will enable us to predict active doses for clinical studies. So far, because ideal radiotracers for clinical use have not been obtained, effects of CRF1 receptor antagonist R317573 on regional cerebral glucose metabolism were studied using [18F]fluoro-2-deoxy-d-glucose (FDG) PET in healthy subjects . The results showed dose-dependent increase in cerebral glucose metabolism by acute dosing in frontal cortical regions and decrease in the putamen and right amygdala, indicating the pharmacologically active doses in humans. Thus, preclinical studies for receptor occupancy or neural modulation in the brain by a compound would provide key information for the selection of a clinical candidate in addition to efficacy on central CRF-induced and stress-induced biochemical and behavioral changes.