Tactics for preclinical validation of receptor-binding radiotracers

Introduction Aspects of radiopharmaceutical development are illustrated through preclinical studies of [¹²⁵I]-(E)-1-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-4-(iodoallyl)piperazine ([¹²⁵I]-E-IA-BF-PE-PIPZE), a radioligand for sigma-1 (σ₁) receptors, coupled with examples from the recent literature. Findings are compared to those previously observed for [¹²⁵I]-(E)-1-(2-(2,3-dimethoxy-5-yl)ethyl)-4-(iodoallyl)piperazine ([¹²⁵I]-E-IA-DM-PE-PIPZE). Methods Syntheses of E-IA-BF-PE-PIPZE and [¹²⁵I]-E-IA-BF-PE-PIPZE were accomplished by standard methods. In vitro receptor binding studies and autoradiography were performed, and binding potential was predicted. Measurements of lipophilicity and protein binding were obtained. In vivo studies were conducted in mice to evaluate radioligand stability, as well as specific binding to σ₁ sites in brain, brain regions and peripheral organs in the presence and absence of potential blockers. Results E-IA-BF-PE-PIPZE exhibited high affinity and selectivity for σ₁ receptors (K_i = 0.43 ± 0.03 nM, σ₂/σ₁ = 173). [¹²⁵I]-E-IA-BF-PE-PIPZE was prepared in good yield and purity, with high specific activity. Radioligand binding provided dissociation (k_off) and association (k_on) rate constants, along with a measured K_d of 0.24 ± 0.01 nM and B_max of 472 ± 13 fmol/mg protein. The radioligand proved suitable for quantitative autoradiography in vitro using brain sections. Moderate lipophilicity, Log D_7.4 2.69 ± 0.28, was determined, and protein binding was 71 ± 0.3%. In vivo, high initial whole brain uptake, >6% injected dose/g, cleared slowly over 24 h. Specific binding represented 75% to 93% of total binding from 15 min to 24 h. Findings were confirmed and extended by regional brain biodistribution. Radiometabolites were not observed in brain (1%). Conclusions Substitution of dihydrobenzofuranylethyl for dimethoxyphenethyl increased radioligand affinity for σ₁ receptors by 16-fold. While high specific binding to σ₁ receptors was observed for both radioligands in vivo, [¹²⁵I]-E-IA-BF-PE-PIPZE displayed much slower clearance kinetics than [¹²⁵I]-E-IA-DM-PE-PIPZE. Thus, minor structural modifications of σ₁ receptor radioligands lead to major differences in binding properties in vitro and in vivo.