Correlation of apparent affinity values from H3-receptor binding assays with apparent affinity (pKapp) and intrinsic activity (α) from functional bioassays

Background and purpose: Agonist apparent affinities (pKI′) in histamine H3-receptor binding assays were higher than expected from apparent affinity values (pKapp) estimated in bioassay. Here, we investigate whether the degree of pKI′ overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pKI′ of ligands with varying intrinsic activity. Experimental approach: In the guinea-pig ileum bioassay, intrinsic activity (α) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pKapp values were estimated using the method of Furchgott. The pKL of [3H]clobenpropit in guinea-pig cerebral cortex was estimated by saturation analysis in 20 mM HEPES-NaOH buffer (buffer B(0,0,0)), or buffer B(0,0,0) containing 70 mM CaCl2, 100 mM NaCl and 100 mM KCl (buffer B(0.07,0.1,0.1)). PKI values were determined in competition studies in both buffers. Key results: [3H]clobenpropit saturation isotherms had nH values of unity in both buffers. In buffer B(0.07,0.1,0.1), agonist pKI′ values were closer to pKapp values than in buffer B(0,0,0) but were associated with nH values <1. A two-site analysis of agonist data in buffer B(0.07, 0.1, 0.1) provided a better fit than a one-site fit and low affinity values (pKIL) were comparable to pKapp. Differences between the pKI′ in buffer B(0,0,0) and pKIL values in buffer B(0.07,0.1,0.1) (ΔpK) were correlated with α. Conclusions and implications: H3-receptor binding assays conducted in buffer B(0,0,0) and buffer B(0.07,0.1,0.1) can provide a measure of ligand affinity (pKapp) and intrinsic efficacy. The assay predicts that some ligands previously classified as H3-receptor antagonists may possess residual intrinsic efficacy. British Journal of Pharmacology (2007) 151, 109–124. doi:10.1038/sj.bjp.0707174