Nirogi Ramakrishna, Kandikere Vishwottam, Bhyrapuneni Gopinadh, Muddana Nageswararao, Saralaya Ramanatha, Ponnamaneni Ranjith Kumar, Manoharan Arun Kumar
Pharmacokinetics and Drug Metabolism, Discovery Research, Suven Life Sciences Ltd, Serene Chambers, Road-5, Avenue-7, Banjara Hills, Hyderabad 500034, India.
J Pharmacol Toxicol Methods. 2012 May-Jun;65(3):115-21. doi: 10.1016/j.vascn.2012.03.003. Epub 2012 Apr 1.
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.
在相关物种的靶器官进行快速可靠的临床前受体占有率测量对于加速药物研发进程至关重要。本研究的目的是开发一种使用非放射性标记的GSK189254作为示踪剂的组胺H₃受体（H₃R）体内受体占有率测定法，并关联大鼠体内H₃R拮抗剂的占有率-暴露关系。
对雄性Wistar大鼠静脉注射GSK189254后，进行了诸如脑区分布、剂量和时间依赖性摄取等体内示踪剂特性研究。通过用环匹泮、硫代哌酰胺和GSK334429等H₃拮抗剂预处理来验证示踪剂的特异性。使用液相色谱串联质谱法定量血浆和脑中的脑区示踪剂水平和H₃R拮抗剂浓度。通过用H₃R拮抗剂预处理的动物中示踪剂的总结合（纹状体或额叶皮质）与非特异性结合（小脑）的比率来计算受体占有率。
发现GSK189254在纹状体、额叶皮质中高度选择性分布，在小脑中分布水平较低。GSK189254在大鼠脑中的区域分布与使用³H或¹¹C-GSK189254绘制的人、猪和大鼠中H₃R的分布一致。环匹泮、硫代哌酰胺和GSK334429在额叶皮质中的计算占有率ED₅₀值分别为0.14、1.58和0.14 mg/kg。环匹泮、硫代哌酰胺和GSK334429的血浆EC₅₀值（ng/mL）分别为2.33、292.2和3.54。
基于质谱法测定大鼠脑中H₃R占有率的结果与通过闪烁光谱法报道的放射性标记方法相当。总之，非放射性标记的GSK18沈189254成功用作评估大鼠中H₃R占有率的示踪剂，并且可作为药物发现中评估新型H₃R配体的临床前工具。
