DIHYDROCODEINONE-HYDRAZONE, DIHYDROCODEINONE-OXIME, NALOXONE-3-OME-OXIME, AND CLOCINNAMOX FAIL TO IRREVERSIBLY INHIBIT OPIOID KAPPA-RECEPTOR-BINDING

Previous work from our lab identified two subtypes of the opioid kappa receptor. Whereas the kappa, receptor can be labeled by [H-3]U69,593 (5 alpha,7 alpha,8 beta-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1- oxaspiro (4,5)dec-8-yl]-phenyl-benzeneacetamide), the kappa(2) receptor can be labeled by [I-125]IOXY (6 -(125)iodo-3,14-dihydroxy-17-cyclopropylmeth yl-4,5 alpha-epoxymorphinan). Other data demonstrate that [I-125]IOXY, like [H-3]bremazocine, labels two populations of kappa2 receptors in guinea pig brain: kappa(2a) and kappa(2b) binding sites. In the presen t study, we tested the hypothesis that certain dihydrocodeinone and ox icodone derivatives, which have been shown to irreversibly block low a ffinity [H-3]naloxone binding sites, would also bind irreversibly to o pioid kappa receptor subtypes. We also tested the novel irreversible m u receptor antagonist, clocinnamox (14 hlorocinnamoylamino)-7,8-dihydr o-N-cyclopropylmeth mesylate). Wash-resistant inhibition (WRI) assays were conducted to detect apparent irreversible inhibition. The proport ion of WRI attribuable to inhibition of receptor binding, termed recep tor inhibition (RI), was calculated by the equation: RI = WRI (wash-re sistant inhibition) - SI (supernatant inhibition or inhibition attribu table to residual drug.) Dihydrocodeinone-hydrazone, dihydrocodeinone- oxime and naloxone-3-OMeoxime failed to produce any wash-resistant inh ibition of kappa receptor binding. In contrast, preincubating guinea p ig membranes with 1 mu M clocinnamox produced a substantial degree of wash-resistant inhibition (greater than 90%) at kappa(1) and kappa(2) binding sites. However, as indicated by supernatant inhibition values of 70% to 90%, there was a large amount of residual clocinnamox which remained despite the use of an extensive washing procedure. Thus, it i s apparent that clocinnamox has essentially no irreversible effect on kappa binding sites. Moreover, these results clearly demonstrate the r equirement to determine supernatant inhibition when testing putative i rreversible ligands. The apparent inactivity of dihydrocodeinone-hydra zone, dihydrocodeinone-oxime or naloxone-3-OMe-oxime as irreversible i nhibitors of kappa receptors suggests that the low affinity [H-3]nalox one binding site eliminated by these agents may not be a kappa binding site.