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J Pharmacol Toxicol Methods. 2012 May-Jun;65(3):115-21. doi: 10.1016/j.vascn.2012.03.003. Epub 2012 Apr 1.

In vivo receptor occupancy assay of histamine H₃ receptor antagonist in rats using non-radiolabeled tracer.

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Pharmacokinetics and Drug Metabolism, Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road-5, Avenue-7, Banjara Hills, Hyderabad 500034, India.



Rapid and reliable preclinical receptor occupancy measurement at the target organ in relevant species is critical in accelerating the drug hunting process. The aim of this study was to develop in vivo receptor occupancy assay for histamine H₃ receptors (H₃R) using the non-radiolabeled GSK189254 as a tracer and to correlate the occupancy-exposure relationship for H₃R antagonists in the rats.


In vivo tracer characterization studies like brain regional distribution, dose and time dependent uptake were carried out for GSK189254 in the male Wistar rats after intravenous administration. The tracer specificity was validated by pretreatment with H₃ antagonists like ciproxifan, thioperamide, and GSK334429. The brain regional tracer levels and H₃R antagonist concentrations in plasma and brain were quantified using liquid chromatography tandem mass spectrometry. Receptor occupancy was calculated using the ratio of total binding (striatum or frontal cortex) to the nonspecific binding (cerebellum) of the tracer in animals pretreated with H₃R antagonist.


High degree of selective distribution of GSK189254 was found in striatum, frontal cortex, and low level in the cerebellum. Regional distribution of GSK189254 in the rat brain was consistent to that of H₃R distribution mapped using ³H or ¹¹C-GSK189254 in human, porcine, and rat. The calculated occupancy ED₅₀ values in the frontal cortex were 0.14, 1.58, and 0.14 mg/kg for ciproxifan, thioperamide, and GSK334429, respectively. The plasma EC₅₀ values (ng/mL) were found to be 2.33, 292.2, and 3.54 for ciproxifan, thioperamide and GSK334429, respectively.


Results from mass spectroscopy based approach to determine H₃R occupancy in rat brain is comparable with reported radiolabeled method by scintillation spectroscopy. In conclusion, non-radiolabeled GSK189254 was successfully employed as a tracer for assessing the H₃R occupancy in rats and it can be used as a preclinical tool for evaluation of novel H₃R ligands in the drug discovery.

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