[D-Pen², 4'-¹²⁵I-Phe⁴, D-Pen⁵]Enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity¹

[D-Pen², 4'-¹²⁵I-Phe⁴, D-Pen⁵]enkephalin ([¹²⁵I]DPDPE) is a highly selective radioligand for the δ opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. [¹²⁵I]DPDPE binds to a single site in rate brain membranes with an equilibrium dissociation constant (K(d)) value of 421 ± 67 pM and a receptor density (B(max)) value of 36.4 ± 2.7 fmol/mg protein. The high affinity of this site for δ opioid receptor ligands and its low affinity for μ or k receptor-selective ligands are consistent with its being a δ opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of δ opioid receptors. Association and dissociation binding kinetics of 1.0 nM [¹²⁵I] DPDPE are monophasic at 25°C. The association rate (k₊1= 5.80 ± 0.88 x 10⁷ M^-1 min^-1) is about 20- and 7-fold greater than that measured for 1.0 nM [³H]DPDPE and 0.8 nM [³H][D-Pen², 4'-Cl-Phe⁴, D-Pen⁵]enkephalin, respectively. The dissociation rate of [¹²⁵I]DPDPE (0.917 ± 0.117 x 10^-2 min^-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of [¹²⁵I]DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes [¹²⁵I]DPDPE a valuable new radioligand for studies of δ opioid receptors.