Pharmacological activities of optically pure enantiomers of the κ opioid agonist, U50,488, and its cis diastereomer: evidence for three κ receptor subtypes

doi:10.1016/0014-2999(89)90443-3

European Journal of Pharmacology

Volume 167, Issue 3, 29 August 1989, Pages 345-353

https://doi.org/10.1016/0014-2999(89)90443-3 Get rights and content

Abstract

De Costa et al. (FEBS Lett. 223, 335; 1987) recently described the synthesis of optically pure enantiomers of (±)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50,488). In the present study we examined the in vitro opioid receptor selectivity of (−)-(1S,2S)-U50,488, (+)-(1R,2R)-U50,488 and (±)-cis-3,4-di-chloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide (the cis diasteromers of U50,488), as well as their pharmacological activities in rhesus monkeys. Using [³H]5α,7α,8β-(−)-N-methyl-N-[7-1-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yl]-phenyl-benzeneacetamide([³H]U69,593) to label κ binding sites of guinea pig membranes, the apparent dissociation constants of the enantiomers hadU50,488 were 0.89 and 299 nM, for the (S,S) and (R,R) enantiomers, respectively. The (−)-cis and (+)-cis diastereomers had apparent K_ds of 167 and 2715 nM, respectively. Binding surface analysis of the interaction of (−)-(1S,2S)-U50,488 with κ binding sites labeled by [³H]bremazocine resolved two binding sites at which (−)-(1S,2S)-U50,488 had K_ds of 30 and 10485 nM, respectively. The (±)-cis, (−)-cis and (+)-cis diastereomers of U50,488 (1 μM) did not inhibit [³H]bremazocine binding. Rhesus monkeys were trained to discriminate ethylketocyclazocine (EKC) and saline. All compounds tested substituted completely for EKC. The order of potency was (−)-(1S,2S)-U50,488 > (±)-U50,488 > (±)-cisdiastereomerofU50,488 > (+)-(1R,2R)-U50,488. In tests of analgesia, (−)-(1S,2S)-U50,488 was 2–4 times more potent than (±)-U50,488, while (±)-cis diastereomer of U50,488 and (+)-(1R,2R)-U50,488 were inactive at the highest doses tested (32 mg/kg). Taken collectively, these data indicate that the pharmacologically active enantiomer of U50,488 is (−)-(1S,2S)-U50,488, and provide preliminary evidence for three subtypes of κ binding sites in guinea pig brain.

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Most studies investigating U-50488 have used the racemate, often because of the lack of knowledge regarding its enantiomers or their capacity for producing different effects (Ariens, 1987). For example, the enantiomers of (±)-U-50488 have different effects in drug discrimination and antinociception procedures as well as in vivo binding studies (DeCosta et al., 1987; Rothman et al., 1989). In our discrimination studies, (−)-U-50488 produced effects on (±)-U-50488-lever responding and response rate that were more potent than, or similar to, the racemate, respectively; whereas (+)-U-50488 did not increase (±)-U-50488-lever responding or decrease response rate up to doses of 32 mg/kg.
The adverse effects of mu opioid agonists have spurred a renewed interest in using kappa opioid receptor (KOR) agonists as analgesics. KOR agonists also have potential for development as diuretics for the treatment of edema and hypertension. Here, we evaluated the discriminative stimulus, antinociceptive, and diuretic effects of the kappa agonist (±)-trans-U-50488 and its stereoisomers (−)-(1S,2S)-U-50488 or (+)-(1R,2R)-U-50488) alone and in combination with the cannabinoid agonist (−)-CP 55,940. To establish (±)-U-50488 as a discriminative stimulus, rats (n = 12) were trained to discriminate intraperitoneal (i.p.) administration of 5.6 mg/kg of (±)-trans-U-50488 from saline under a fixed-ratio 20 (FR-20) schedule of food reinforcement. Then, antinociception was assessed using two procedures: warm water tail withdrawal and von Frey paw withdrawal. Diuretic effects were assessed in separate rats (n = 6/group). Doses of (±)-U-50488 and (−)-U-50488 that served as discriminative stimuli produced significant increases in urine output, but at lower doses than those that produced antinociception. In contrast, (+)-U-50488 alone had no discriminative stimulus or diuretic effects at the doses tested, but did produce antinociception in the von Frey assay. When three cannabinoids and morphine were tested in the (±)-U-50488 discrimination procedure to determine the similarity of these drugs' discriminative stimulus effects to those for (±)-U-50488, the rank order similarity was (−)-CP 55,940 > (−)-trans-THC > (+)-WIN 55,212–2 ≥ morphine. (−)-CP 55,940 alone (0.056 mg/kg) partially substituted for the discriminative stimulus effects of (±)-U-50488 and produced significant diuretic and antinociceptive effects. (−)-CP 55,940 in combination with (±)-U-50488 also produced a two-fold leftward shift in the discriminative stimulus curve for (±)-U-50488, and near-additive antinociception with (±)-U-50488 and (+)-U-50488. Further, the diuretic effect of (−)-CP 55,940 was enhanced by a dose of (+)-U50488, which itself did not alter urine output. These data together indicate that a combination of cannabinoid and kappa opioid agonists can enhance diuresis, but may have limited potential for serving as opioid-sparing pharmacotherapeutics for treatment of pain.
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Opioid receptor, a member of the superfamily of membrane proteins that transduces signals via the heterotrimeric G proteins, has myriad of roles upon activation by exogenous alkaloid and endogenous peptide ligands. The most important of these functions is the modulation of pain perception. The massive side effects associated with opioid receptor activation, especially those that can lead to addiction to the exogenous alkaloid ligands, mandate the investigation into the mechanism of opioid receptor action. In this article, we examine some of the opioid receptor signaling complexity.
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In contrast to μ- and δOP receptors, κOP receptors are largely expressed on presynaptic dopaminergic membranes and inhibit presynaptic neurotransmitter release on activation. Three κOP receptor variants have been characterized (κ1-3OP receptors), though only one (κ1OP receptor) has been cloned (Rothman et al., 1989; Mansson et al., 1994). The κOP receptors are broadly expressed throughout the CNS, including the mesolimbic system, hypothalamus, amygdala, spinal cord, and hypothalamic-pituitary axis (Mansour et al., 1995; Peng et al., 2012; Butelman and Kreek, 2015).
Prolonged treatment of chronic severe pain with opioid analgesics is frought with problematic adverse effects including tolerance, dependence, and life-threatening respiratory depression. Though these effects are mediated predominately through preferential activation of μ opioid peptide (μOP) receptors, there is an emerging appreciation that actions at κOP and δOP receptors contribute to the observed pharmacologic and behavioral profile of μOP receptor agonists and may be targeted simultaneously to afford improved analgesic effects. Recent developments have also identified the related nociceptin opioid peptide (NOP) receptor as a key modulator of the effects of μOP receptor signaling. We review here the available literature describing OP neurotransmitter systems and highlight recent drug and probe design strategies.
This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.
Evidence for multiple mechanisms of kappa opioid tolerance in mesencephalic cultures
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This eliminates variability due to differences in cell numbers between wells. U69,593 is a synthetic, potent opioid that selectively activates kappa opioid receptors [12]. In our previous work, U69,593 showed dose–response inhibition of K+-stimulated dopamine release [5].
Opioid tolerance limits the effectiveness of chronic opioid therapy. Kappa opioid tolerance in mesencephalic cultures has been established as a model system for evaluating neuronal mechanisms of tolerance. Our studies were carried out in primary cultures derived from the mesencephalon of rat embryos. [³H]Dopamine release assays were used to evaluate the kinetics of onset and recovery from tolerance to the kappa opioid receptor agonist (U69,593) which were measured with and without (1) modulators of cAMP-dependent kinase or (2) cycloheximide, an inhibitor of protein synthesis. Results were compared to the kinetics of recovery from kappa receptor alkylation by β-chlornaltrexamine. The results showed that 8-bromo-cAMP and forskolin significantly accelerated the development of tolerance to U69,593. Rp-cAMP significantly slowed the onset of tolerance. Rates of recovery of cultures after 18 h of U69,593 were not significantly different regardless of whether cultures were incubated with U69,593 alone or in combination with 8-bromo-cAMP, forskolin or Rp-cAMP. Cycloheximide significantly slowed the recovery of cultures after 7 days of exposure to U69,593 but not after 18 h of exposure to U69,593. Recovery of cultures after alkylation of kappa receptors by β-chlornaltrexamine was significantly slower than the recovery of cultures after 7 days of U69,593 exposure indicating that receptor synthesis is unlikely to be the major mechanism of recovery after prolonged opioid exposure. It is suggested that multiple mechanisms are responsible for tolerance to U69,593 in primary neuronal cultures. There are long lasting mechanisms of tolerance that do not involve destruction or permanent inactivation of cellular components.
Opioid antagonist profile of SC nor-binaltorphimine in the formalin paw assay
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The antinociceptive effects of μ and κ agonists were examined after the systemic administration of the opioid antagonists nor-binaltorphimine (nor-BNI) and naloxone in the late response or tonic nociceptive phase of the mouse formalin assay. Initially, SC morphine (ED₅₀, 0.97 mg/kg), racemic U-50488H (ED₅₀, 0.79 mg/kg), (−)U-50488 (ED₅₀, 0.41 mg/kg), and another agonist PD 117,302 (ED₅₀, 0.28 mg/kg) were found to produce graded increases in the level of antinociception as measured by this procedure; naloxone, administered immediately before morphine and U-50488H, antagonized their antinociceptive actions. The effects of morphine and U-50488H then were evaluated 10 min to 96 h after the administration of nor-BNI. Subcutaneous nor-BNI at 30.0 mg/kg, but not at 3.0 or 10.0 mg/kg, attenuated the antinociceptive effects of morphine and U-50488H when the interval separating nor-BNI and the agonists was kept constant at 1 h. Time-course analysis of the effects of combinations of nor-BNI with morphine led to irregular findings: 10.0 mg/kg of nor-BNI lessened the effects of morphine (2.0 mg/kg) if the dosing interval was 10 min, whereas 30.0 mg/kg of nor-BNI attenuated the effects of morphine (2.0 mg/kg) if the dosing interval was 1 or 4 h; 10.0 mg/kg of nor-BNI also diminished the antinociceptive effects of U-50488H (1.7 mg/kg) only if the interval spacing the two drugs was 24 h. In comparison, a threefold higher dose of nor-BNI (30.0 mg/kg) reduced the effects of U-50488H (1.7 mg/kg) if the interval was 1 h or more. In these latter experiments, the antagonist effects of SC nor-BNI (30.0 mg/kg) were evident up to 96 h posttreatment. These results show that the μ opioid antagonist activity of nor-BNI is variable and that the κ opioid antagonist selectivity of nor-BNI is a function of dose and treatment interval and is long-lasting even after systemic administration.
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The distribution of the κ opioid receptor mRNA in adult mouse brain has been determined using the technique of in situ hybridization histochemistry. The mRNA for the κ opioid receptor was expressed in distinct areas throughout the brain. The telencephalon showed high levels of expression in the deeper layers of the parietal and temporal cortex, olfactory tubercle, nucleus accumbens, claustrum, endopiriform nucleus, nucleus of the vertical and horizontal limb of the diagonal band, and medial and central nuclei of the amygdala. In the diencephalon, κ opioid receptor mRNA was present in multiple medial thalamic nuclei including the centromedial, paraventricular, parafasicular, central, and peritenial nuclei, as well as in most hypothalamic nuclei including the ventromedial, periventricular, supraoptic, arcuate, and dorsomedial nuclei. The mesencephalon showed highest levels of κ receptor mRNA in the substantia nigra pars compacta, ventral tegmental area, zona incerta, interpeduncular nucleus, superior colliculus, inferior colliculus, central grey, and the raphe nucleus. In the metencephalon, κ opioid receptor mRNA was expressed in the parabrachial nuclei, locus coeruleus, dorsal and ventral tegmental nuclei, and the raphe pontine nuclei. The distribution of the κ receptor mRNA closely coincides with the localization of binding sites in rat brain for [³H]U-69,593, a specific κ₁ opioid receptor ligand. The mRNA distribution also correlates with neuroanatomical sites of actions of κ agontists and distribution of the endogenous κ receptor ligand dynorphin.

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Pharmacological activities of optically pure enantiomers of the κ opioid agonist, U50,488, and its cis diastereomer: evidence for three κ receptor subtypes

Abstract

FEBS Lett.

Brain Res.

European J. Pharmacol.

J. Biol. Chem.

European J. Pharmacol.

Life Sci.

Life Sci.

Neuropeptides

Neuropeptides

Neuropeptides

Neuropeptides

Stereochemistry: a source of problems in medicinal chemistry

Med. Res. Rev.

Stereochemistry in the analysis of drug-action. Part II

Med. Res. Rev.

A procedure for the rapid evaluation of the discriminative stimulus effects of drugs

J. Pharmacol. Meth.