Na+-dependent high affinity binding of [3H]LY515300, a 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine opioid receptor inverse agonist

Abstract Analogues of 3,4-dimethyl-4-(3-hydroxyphenyl)piperidines are high affinity inverse agonists for μ-, δ- and κ-opioid receptors. To characterize inverse agonist binding, we synthesized a high specific activity radioligand from this series, [ 3 H]LY515300 (3-[1-((3-cyclohexyl-[3,4- 3 H 2 ])-3( R , S )-hydroxypropyl)-3( R ),4( R )-dimethylpiperidin-4-yl]phenol). In membranes expressing cloned human opioid receptors, [ 3 H]LY515300 binding was saturable and exhibited low nonspecific binding. [ 3 H]LY515300 bound with high affinity to the μ- ( K d =0.07 nM), δ- ( K d =0.92 nM) and κ-( K d =0.45 nM) opioid receptors. High affinity [ 3 H]LY515300 binding to all opioid receptors was Na + -dependent, a characteristic of inverse agonists. Displacement by standard opioid compounds yielded K i values consistent with their known opioid receptor affinities. Autoradiographic localization of specific [ 3 H]LY515300 binding in rat and guinea pig brain was high in areas known to express high levels of opioid (particularly μ-opioid receptor) binding sites including the caudate, nucleus accumbens, and nucleus tractus solitarius. Thus, [ 3 H]LY515300 is the first radiolabeled opioid receptor inverse agonist useful for the study of opioid receptors in cell lines and native tissues.