[3H]4-(dimethylamino)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl) butyl)benzamide: a selective radioligand for dopamine D(3) receptors. II. Quantitative analysis of dopamine D(3) and D(2) receptor density ratio in the caudate-putamen

Abstract

4-(Dimethylamino)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)benzamide (WC-10), a N-phenyl piperazine analog, displays high affinity and moderate selectivity for dopamine D(3) receptors versus dopamine D(2) receptors (Chu et al. [2005] Bioorg Med Chem 13:77-87). In this study, WC-10 was radiolabeled with tritium (specific activity = 80 Ci/mmol), and quantitative autoradiography studies were conducted using rhesus monkey and Sprague-Dawley rat brain sections. K(d) values for the binding of [3H]WC-10 to D(3) receptors obtained from quantitative autoradiography with rhesus monkey and rat brain sections are in agreement with K(d) values obtained from cloned human and rat receptors (Xu et al. [2009] Synapse 63:717-728). The D(2) selective antagonist [3H]raclopride binds with 11-fold higher affinity to human HEK D(2L) (K(d) = 1.6 nM) than HEK D(3) (K(d) = 18 nM) receptors; [3H]raclopride binds to rat Sf9 rD(2L) receptors with a K(d) of 6.79 nM, a value that is 4-fold lower than binding to human HEK D(2L) receptors and 2.5-fold higher than binding to rat Sf9 rD(3) receptors. In vitro quantitative autoradiography studies with [3H]WC-10 and [3H]raclopride were conducted on adult rat and rhesus monkey brain sections. A mathematical model for calculating the absolute densities of dopamine D(2) and D(3) receptors based on the in vitro receptor binding data of [3H]WC-10 and [3H]raclopride was developed.

Fig. 1

Chemical structure of [³H]WC-10. Detailed synthesis scheme was reported previously (Xu et al., 2009).

Fig. 2

Saturation analysis of the binding of [³H]raclopride to cloned D₂-like dopamine receptors. Direct binding analysis was performed to determine the equilibrium binding affinity of [³H]raclopride for human hD₃ and hD₂ (A, B) or rat rD₃ and rD₂ (C, D) receptors. Human receptors were expressed in HEK 293 cells and the rat receptors were expressed in Sf9 cells. Scatchard plots were used to determine the dissociation constants (K_d values). The inset graphs are the Hill plots for determining the Hill coefficient (n_H values). K_d and n_H are presented as mean values ± SEM for n = 3.

Fig. 3

Saturation analysis of the binding of [³H]WC-10 to rhesus monkey and rat brain. Direct binding analysis was performed to determine the equilibrium binding affinity of [³H]WC-10 binding sites in the rhesus monkey caudate (A), putamen (B), and rat striatum (C). Nonspecific binding was determined from samples which contained 1 μM S(−)-Eticlopride. Scatchard plots were used to determine the dissociation constants (K_d values). The inset graphs are the Hill plots for determining the Hill coefficient (n_H values). K_d and n_H are presented as mean values ± SEM for n = 3.

Fig. 4

Quantitative autoradiographic analysis of the binding of [³H]WC-10 and [³H]raclopride to rat and rhesus monkey brain. Autoradiograms show neuroanatomical localization of [³H]WC-10 and [³H]raclopride binding sites in rhesus monkey (A, B) and Sprague-Dawley rat (C, D) brain sections. For this study [³H]WC-10 was used at a concentration of 4 nM (A, C) and [³H]raclopride was used at a concentration of 10 nM (B, D). The numbers 1 through 4 in panels (A) and (C) designate the following CNS anatomical regions: 1, cortex; 2, primate caudate; 3, primate putamen; and 4, rat striatum. (E) Autoradiographic image of a [³H]Microscale, which was counted for 24 h along with the brain sections for the purpose of quantitative analysis.

Fig. 5

Quantitative calibration of in-vitro autoradiography. Calibrated autoradiography typical standard curve obtained by counting a series of tritium standards of a [³H]Microscale, digitalized image was used to analyze the ROI. This curve was used to convert cpm/mm² to nCi/mg to quantify autoradiography.

Fig. 6

Film autoradiography of the binding of [³H]WC-10 to rat brain. Autoradiograms obtained with the traditional film exposure techniques with different exposure time, (A) 1 month; (B) 3 months. The numbers 1 and 2 designate the following CNS anatomical regions: 1, cortex; 2, rat striatum. For this study [³H]WC-10 was used at a concentration of 4 nM.