EVALUATION OF A SERIES OF N-ALKYL BENZOMORPHANS IN CELL-LINES EXPRESSING TRANSFECTED DELTA-OPIOID AND MU-OPIOID RECEPTORS

Transfection of individual opioid receptors in Chinese hamster ovary ( CHO) cells provides a pure, homogeneous population of receptors for sc reening drug candidates, and an alternative to the use of selective li gands. To evaluate the potential of this system, we chose a series of (-)-5,9 imethyl-2-hydroxy-N-substituted-6,7-benzomorphans, for which t he receptor selectivity and in vivo activity had been characterized re cently, and tested them in CHO cells stably transfected with either th e rat delta-opioid receptor or the mouse mu-opioid receptor. [H-3]Dipr enorphine was used to measure opioid receptors in P-2 membrane prepara tions. A B-max of 7.58 +/- 0.8 pmol/mg protein and a K-d of 0.42 +/- 0 .04 nM was obtained in the mu-opioid receptor expressing cell line use d in these studies. In addition, [H-3]naltrindole was used to confirm the delta-specificity of the cloned receptor. Both compounds gave a B- max of 1.2 pmol/mg in the CHO cells expressing the rat delta-opioid re ceptor. Displacement assays were performed with eleven (-)-N-alkyl-ben zomorphans in the absence and presence of 150 mM NaCl, as well as know n delta- and mu-selective agonists. Sodium reduced agonist affinity in the transfected cell lines. The benzomorphan compounds displayed a ra nge of affinities in the mu- and delta-opioid receptor expressing cell lines. Good correlations were found between their affinities at the c loned mu- and delta-opioid receptors and those in rat brain and monkey cortex (r(2) from 0.73 to 0.89, P < 0.001). Comparative analysis of K -i values with in vivo potency in the mouse tail flick test indicated a high degree of correlation between antinociception and affinity in t he mu-opioid receptor cell line (r(2) = 0.83, p < 0.0001). Lesser corr elations were found between antinociception in the mouse and affinity at the rat mu-opioid receptor (r(2) = 0.6610) and at the monkey mu-opi oid receptor (r(2) = 0.695). In sum, these studies indicate that the c ell lines expressing the cloned mu and delta-opioid receptors are appr opriate models for determining the binding affinities of this class of opioid compounds. The diminishing correlations found between species when comparing in vitro and in vivo activity suggest that caution shou ld be taken when extrapolating binding data to pharmacological activit y among species.