No abstract

The histamine H 3 receptor is implicated in the pathophysiology of several central nervous system disorders. N-methyl-6-(3-cyclobutyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-yloxy)-nicotamide (GSK189254) is a highly potent, selective, and brain-penetrant H 3 receptor antagonist. Previous studies in the pig using PET have shown that 11 C-GSK189254 uptake in H 3 -rich regions of the brain can be blocked by the selective H 3 antagonist ciproxifan. The purpose of the present study was to evaluate 11 C-GSK189254 as a PET radioligand for human studies and to determine the dose-receptor occupancy relationship of GSK189254 in the human brain. Methods: Dynamic PET scans were obtained in healthy subjects over 90 min after intravenous administration of approximately 370 MBq of 11 C-GSK189254. Blood samples were taken throughout the scans to derive the arterial plasma parent input function. Each subject was scanned twice, either with tracer alone (test-retest) or before and after a single oral dose of GSK189254 (10-100 mg). Data were analyzed by compartmental analysis, and regional receptoroccupancy estimates were obtained by graphical analysis of changes in the total volumes of distribution (V T ) of the radioligand. Results: 11 C-GSK189254 readily entered the brain; its regional brain distribution reflected the known distribution of H 3 receptors, with high binding in the caudate and putamen, intermediate binding in cortical regions, and low binding in the cerebellum. GSK189254 displayed a high receptor affinity, and a marked reduction in V T was apparent at all the doses tested. The oral dose equaling 50% occupancy of the available receptor sites (ED 50 ) was estimated as 4.33 mg. Additional data on plasma pharmacokinetics after oral dosing and the plasma free fraction gave a corresponding estimate of the free concentration of GSK189254 required to occupy 50% of the available receptor sites (EC 50 ) (0.011 nM). The test-retest data showed reductions in regional V T on the second scan in all subjects. A nonlinear compartmental analysis of this effect demonstrated that this reduction was consistent with carryover of a tracer mass dose effect with an estimated in vivo apparent dissociation constant of 0.010 nM, close to the independent estimate of the plasma EC 50 . Conclusion: 11 C-GSK189254 can be used to quantify H 3 receptor availability in humans in vivo using PET but requires high specific activity; the possibility of tracer mass dose effects should be carefully analyzed.

The introduction of two-photon microscopy, along with the development of new fluorescent probes and innovative computer software, has advanced the study of intracellular and intercellular processes in the tissues of living organisms. Researchers can now determine the distribution, behavior, and interactions of labeled chemical probes and proteins in live kidney tissue in real time without fixation artifacts. Chemical probes, such as fluorescently labeled dextrans, have extended our understanding of dynamic events with subcellular resolution. To accomplish expression of specific proteins in vivo, cDNAs of fluorescently labeled proteins have been cloned into adenovirus vectors and infused by micropuncture to induce proximal tubule cell infection and protein expression. The localization and intensity of the expressed fluorescent proteins can be observed repeatedly at different time points allowing for enhanced quantitative analysis while limiting animal use. Optical sections of images acquired with the two-photon microscope can be 3-D reconstructed and quantified with Metamorph, Voxx, and Amira software programs.