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The peripheral δ opioid receptor (DOR) is an attractive target for analgesic drug development. There is evidence that DOR can form heteromers with the κ-opioid receptor (KOR). As drug targets, heteromeric receptors offer an additional level of selectivity and, because of allosteric interactions between protomers, functionality. Here we report that selective KOR antagonists differentially altered the potency and/or efficacy of DOR agonists in primary cultures of adult rat peripheral sensory neurons and in a rat behavioral model of thermal allodynia. In vitro, the KOR antagonist nor-binaltorphimine (nor-BNI) enhanced the potency of [D-Pen2,5]-enkephalin (DPDPE), decreased the potency of [D-Ala2,D-Leu5]-enkephalin (DADLE), and decreased the potency and efficacy of 4-[(R)-[(2S,5R)-4-allyl-2,5-dimethylpiperazin-1-yl](3-methoxyphenyl)methyl]-N,N-diethylbenzamide (SNC80) to inhibit prostaglandin E2(PGE2)-stimulated adenylyl cyclase activity. In vivo, nor-BNI enhanced the effect of DPDPE and decreased the effect of SNC80 to inhibit PGE2-stimulated thermal allodynia. In contrast to nor-BNI, the KOR antagonist 5'-guanidinonaltrindole (5'-GNTI) reduced the response of DPDPE both in cultured neurons and in vivo. Evidence for DOR-KOR heteromers in peripheral sensory neurons included coimmunoprecipitation of DOR with KOR, a DOR-KOR heteromer selective antibody augmented the antinociceptive effect of DPDPE in vivo, and the DOR-KOR heteromer agonist 6'-GNTI inhibited adenylyl cyclase activity in vitro as well as PGE2-stimulated thermal allodynia in vivo. Taken together, these data suggest that DOR-KOR heteromers exist in rat primary sensory neurons and that KOR antagonists can act as modulators of DOR agonist responses most likely through allosteric interactions between the protomers of the DOR-KOR heteromer.

The existence of heterodimeric opioid receptors has introduced greater complexity to the in vivo characterization of pharmacological selectivity of agonists by antagonists. Because of the possibility of cooperativity between receptors organized as heterodimers, it is conceivable that selective antagonists may antagonize an agonist bound to a neighboring, allosterically coupled receptor. As a consequence, the in vivo selectivity of an opioid antagonist may depend on the organizational state of receptors that mediate analgesia. In this regard, phenotypic delta- and kappa-opioid receptors have been proposed to arise from different organizational states that include oligomeric delta-kappa heterodimers and homomeric delta and kappa receptors. In view of the evidence for analgesia mediated by delta-kappa heterodimers in the spinal cord, but not the brain, we have investigated the selectivity of pharmacologically selective delta and kappa antagonists in mice by both i.t. and i.c.v. routes of administration to evaluate changes in selectivity. Using pharmacologically selective delta (benzylidenenaltrexone, naltrindole, and naltriben) and kappa (norbinaltorphimine) antagonists versus delta ([d-Pen(2),d-Pen(5)]-enkephalin and deltorphin II) and kappa [3,4-dichloro-N-methyl-N-[(1R,2R)-2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (U50488) and bremazocine] agonists, the delta-1/delta-2 selectivity ratios were found to be dependent on the route of administration (i.t. versus i.c.v.). The data from different routes of administration suggest that differences in molecular recognition between spinal delta-kappa heterodimers and supraspinal homomeric delta and kappa receptors may contribute to the divergent selectivity ratios of selective antagonists. In view of the observed tissue-dependent selectivity, we suggest that multiple opioid antagonists be employed routinely in establishing agonist selectivity in vivo.

Previous studies have demonstrated that peptidic and nonpeptidic delta-opioid receptor agonists have different effects depending on the measure. For example, nonpeptidic delta-opioid agonists, but not peptidic agonists, produce convulsions in rats, and in vitro studies suggested that peptidic and nonpeptidic delta-opioid agonists might have differential mechanisms of receptor downregulation. The present study evaluated potential differences between peptidic and nonpeptidic delta-opioid agonists in their ability to activate G proteins using guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) autoradiography experiments in rat brain slices. The peptidic agonist [d-Pen(2),d-Pen(5)]-enkephalin and the nonpeptidic agonist (+)BW373U86 [(+)-4-[alpha(R)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide] demonstrated concentration-dependent increases in [(35)S]GTPgammaS binding that were attenuated by the delta-opioid antagonist naltrindole. (+)BW373U86 was more potent and efficacious than the peptidic agonist, and this difference remained consistent across brain regions where significant stimulation was observed. In addition, multiple delta-opioid compounds were evaluated for their agonist activity in this assay. These data suggested that differences between peptidic and nonpeptidic delta-opioid agonists in behavioral studies were most likely caused by differences in agonist efficacy. Finally, these data also revealed that [(35)S]GTPgammaS autoradiography could be used to compare efficacy differences among agonists across various brain regions in rat brain slices.

Three prostaglandin endoperoxide analogs that possess the 2,3-diazabicyclo[2.2.1]heptane skeleton were synthesized and evaluated for activity in human platelets and in the rat fundus. All three compounds were inactive in inhibiting or stimulating platelet aggregation. One compound possessed weak contracting activity on the rat fundus.

The absolute configuration of certain (−)-N-[2-(phenalkylmethylamino)propyl]-propionanilides has been determined by relating them to D-(−)-alanine. The compounds corresponding to the L-configuration have greater analgesic potency than those with the D-configuration. An interpretation of the significance of the relationship of absolute configuration to analgesic activity is discussed in terms of an analgesic receptor surface.

A series of alkyl esters of (b) dl-α-(2-piperidyl)-phenylacetic acid have been prepared and the kinetics of the hydroxyl ion-catalyzed hydrolysis at 80° and the hydronium ion-catalyzed hydrolysis at 95° of six of these esters were studied. In the base-catalyzed reaction, the following order of reactivity was found: 2-chloroethyl > 2-methoxyethyl > methyl > ethyl >n-propyl >n-butyl. The apparent free energy reaction constant for the base-catalyzed hydrolysis of this series was determined to be + 1.372 and, from the substituent constants reported in the literature, the relative rates of the isopropyl, isobutyl, and sec-butyl esters were calculated. The relative central stimulating activities of this series of esters reveals a decrease in activity with an increase of alkyl chain length.