P-7521--a new irreversible opioid ligand

In the receptor binding assay, P-7521 was a potent opioid ligand which acted mainly on mu receptor. The relative affinity ratio at mu, delta and kappa sites was 66:8:1. The inhibitory effects of P-7521 were 1868 and 6060 times more potent than morphine on the electrically evoked contractions in guinea pig ileum and mouse vas deferens, respectively and were readily antagonized by naloxone and Mr2266. These results indicate that P-7521 acted on mu receptor in guinea pig ileum and mouse vas deferens. In rabbit vas deferens, the compound had no agonist activity, but could antagonize the inhibitory effect of U-50488 H, a kappa agonist, showing the antagonistic characterization was on kappa receptor. The dissociation of P-7521 binding to opioid receptor were very difficult in mu binding assay and bioassays.     

Agonist and antagonist actions of buprenorphine on three types of opioid receptor in isolated preparations

Both agonist and antagonist actions of buprenorphine on isolated preparations were studied. The Ke (equilibrium dissociation constant) values of both naloxone and Mr 2266 [(-)-2-(3-furylmethyl)-5, 9-diethyl-2'-hydroxy-6,7-benzomorphan] against buprenorphine and the ratio of IC50 (concentration of the drug to produce 50% inhibition of the twitch) value of buprenorphine after to before exposure of mouse vas deferens to beta-FNA (beta-fumarate methyl ester derivatives of naltrexone), an irreversible mu antagonist, suggest that buprenorphine acts as both a mu and kappa agonist on mouse vas deferens. The agonist effect of buprenorphine at relatively high doses on guinea-pig ileum and mouse vas deferens and the negative agonist effect on both rat and rabbit vas deferens indicate that buprenorphine acts as a partial agonist on isolated preparations. The Ke values of buprenorphine show that buprenorphine has about equal antagonist effectiveness against a mu and kappa agonist with approximately five-fold lower effectiveness against a delta agonist. The possible mechanisms for the several characteristic actions of buprenorphine on guinea-pig ileum such as the slow onset of action, the increased magnitude of inhibition after washing the tissue, the negative elimination of the inhibition by either washing the tissue or the naloxone administration, and the negative elimination of the antagonist action by washing the tissue were discussed.     

The choice of opiate receptor subtype by neo-endorphins

The choice of opiate receptor subtype by alpha- and beta-neo-endorphin was studied in isolated preparations. Neo-endorphins had significant inhibitory actions on the electrically evoked contractions of guinea-pig ileum, mouse vas deferens and rabbit ileum as well as on the rabbit vas deferens which had been shown to contain kappa-receptors exclusively. Mr 2266, a relatively specific kappa-receptor antagonist, was more effective than naloxone, a relatively mu-receptor antagonist, to antagonize the agonist actions of neo-endorphins in either the guinea-pig and rabbit ileum or in the rabbit vas deferens. By contrast, in the mouse vas deferens, the effectiveness of Mr 2266 to antagonize the agonist actions of neo-endorphins was low and similar to that of naloxone. The potencies of neo-endorphins relative to that of ethylketocyclazocine, a representative kappa-receptor agonist, in the guinea-pig ileum were similar to those in the rabbit ileum but were significant different from those in the mouse vas deferens. The data indicate that neo-endorphins act as kappa-receptor agonists in either the guinea-pig and rabbit ileum or in the rabbit vas deferens while in the mouse vas deferens they act on opiate receptor subtypes other than kappa- and mu-receptors.     

3H]U-69593 a highly selective ligand for the opioid kappa receptor

The selective kappa agonist U-50488 was recently discovered and characterized. In this study, the receptor binding properties of [3H]U-69593, an analog of U-50488, were characterized. [3H]U-69593 binds with high affinity (3 nM) to membranes prepared from guinea pig, mouse and rat brain. The number of kappa binding sites comprise only 13%, 9% and 4% of the total opioid sites, respectively. The benzmorphans, dynorphin, and compounds structurally related to U-50488 have high affinity for this kappa site.     

Opioid kappa receptors correlate with the development of conditioned suppression of motility in mice

Mice exhibited a marked suppression of motility (conditioned suppression) when placed in the same environment in which they had previously received an electric footshock. The conditioned suppression of motility was potentiated by U-69,593, a selective kappa (non-mu) agonist. This effect of U-69,593 was antagonized by MR-2266, a kappa antagonist. Additionally, in contrast to U-69,593, MR-2266 alone attenuated the conditioned suppression of motility. These results suggest that the activation of kappa receptors may be responsible for the development of conditioned suppression of motility.     

HS-599: a novel long acting opioid analgesic does not induce place-preference in rats

1. When administered subcutaneously HS-599, a new didehydroderivative of buprenorphine (18,19-dehydrobuprenorphine), produced a long-lasting antinociceptive response in rats. Its potency exceeded twice that of buprenorphine. In the tail-flick test it acted as a full agonist but in the plantar test only as a partial agonist. Whereas the mu-opioid antagonists naloxone and naltrexone antagonized HS-599 antinociception the delta-opioid antagonist naltrindole and the kappa-opioid antagonist nor-binaltorphimine did not. 2. Unlike buprenorphine and morphine, HS-599 never induced conditioned place-preference in rats. 3. In radioligand binding assays, compared with buprenorphine HS-599 had 3 fold higher mu-opioid receptor affinity but lower delta- and kappa-opioid receptor affinity. 4. In isolated guinea-pig ileum preparations, HS-599 only partially inhibited the electrically-stimulated contraction, acting as a partial opioid agonist. When tested against the mu-opioid receptor agonist dermorphin, it behaved as a non-equilibrium antagonist. Conversely, in mouse vas deferens (rich in delta-opioid receptors) and rabbit vas deferens preparations (rich in kappa-opioid receptors) HS-599 acted as a pure equilibrium antagonist, shifting the log-concentration-response curves of the delta-opioid agonist deltorphin I and the kappa-opioid agonist U-69593 to the right. 5. In conclusion, HS-599 is a novel buprenorphine derivative with higher affinity, selectivity and potency than the parent compound, for mu-opioid receptors. It produces intense and long-lasting antinociception and does not induce place-preference in rats.     

Ethylketocyclazocine and bremazocine analgesia in neonatal rats

In three experiments we examined the analgesic potency of kappa opioid receptor agonists in 2- and 16-day-old rats. Ethylketocyclazocine (1–50 mg/kg) produced similar dose- and time-dependent increases in the latency to retract a hind paw from a noxious thermal stimulus in rats of both ages. Bremazocine (0.001–10 mg/kg), a kappa agonist with reported antagonist activity at mu receptors, was also effective in producing analgesia in 2-day-old rats. The dose-effect relationship for bremazocine was nonmonotonic. Bremazocine analgesia (0.1 mg/kg) was reversed by both naltrexone and MR2266, a putative kappa opioid antagonist. These results are discussed in terms of the functional integrity of a kappa analgesic system in the developing rat.     

Centrally administered opioid antagonists, nor-binaltorphimine, 16-methyl cyprenorphine and MR2266, suppress intake of a sweet solution

Three opioid antagonists (MR2266, 16-methyl cyprenorphine and nor-binaltorphimine) were tested independently for their ability to suppress the intake of a highly palatable saccharin and glucose (S/G) solution after central administration. MR2266 is an equally potent antagonist at kappa (kappa) and mu (mu) opioid receptors. Nor-binaltorphimine (N-BNI) and 16-methyl cyprenorphine (M80) are two recently developed opioid antagonists that were chosen based upon their ability to act more selectively than naloxone at kappa and delta (delta) opioid receptor types, respectively. Prior research has demonstrated that when dissolved in acid and administered centrally, MR2266 (20 micrograms) fails to suppress S/G intake. Because all three antagonists are rather insoluble in water, they were dissolved in dimethyl sulfoxide (DMSO). Rats with chronic ventricular cannula were allowed to consume S/G for a 0.5 hr bout. They received a single intracerebroventricular (ICV) injection of antagonist (MR2266: 0, 10, 20 and 40 micrograms; M80: 0, 5, 10, 20 and 40 micrograms or N-BNI: 0, 1, 3, and 10 micrograms) 10 min prior to the start of the drinking bout. Administration of DMSO alone failed to alter drinking relative to saline, whereas each antagonist significantly attenuated S/G intake. We conclude that, when dissolved in DMSO, these antagonists suppress drinking by blockade of opioid receptors.     

选择性κ阿片激动剂K-Ⅱ与U-50488的药理作用比较

K-Ⅱ系k阿片激动剂U-50488的同类物。通过部分离体和整体实验比较了K-Ⅱ与U-50488的药理作用。实验发现,K-Ⅱ抑制电刺激兔输精管收缩的IC₅₀值为0.42 nmol/L,U-50488为26.5 nmol/L;K-Ⅱ抑制小鼠运动功能(横筛法)的ED₅₀值为1.7 mg/g,U-50488为15.3 mg/kg;K-Ⅱ的小鼠LD₅₀值为152.5 mg/kg,U-50488为118.4 mg/g;K-Ⅱ明显降低小鼠自发活动的作用比U-50488强5倍。结果表明,K-Ⅱ是一个药理作用较U-50488强的k受体激动剂。     

CJ-15,208, a novel kappa opioid receptor antagonist from a fungus,Ctenomyces serratus ATCC15502

A novel kappa opioid receptor binding inhibitor CJ-15,208 (I) was isolated from the fermentation broth of a fungus, Ctenomyces serratus ATCC15502. The structure of I was determined to be a cyclic tetrapeptide consisting of one tryptophan, one D-proline, and two L-phenylalanine. Compound I was a selective binding inhibitor for the kappa opioid receptor: 47 nM (IC50) for kappa, 260 nM for mu, and 2,600 nM for delta. In the electrically-stimulated twitch response assay of rabbit vas deferens I recovered the suppression by a kappa agonist asimadoline with an ED50 of 1.3 microM, indicating that it is a kappa antagonist.     

Pharmacological profiles of fentanyl analogs at mu, delta and kappa opiate receptors.

Receptor binding assays using [3H]DAGO ([D-Ala2,MePhe4-Gly5-ol]enkephalin) (mu), [3H]DPDPE ([D-Pen2,D-Pen5]enkephalin) (delta) and [3H]U-69593 (kappa) were done in guinea pig whole brain membranes. Agonist activity was determined in norbinaltorphimine or beta-funaltrexamine (beta-FNA) treated guinea pig ileum (mu and kappa, respectively) and beta-FNA-treated mouse vas deferens (delta). The compounds with highest affinity were the most potent at the mu-receptor. The selectivity observed in the binding affinities was also found in in vitro activity. No correlation was found between mu-affinity and selectivity; the highest affinity analog, lofentanil, was found to be among the least selective, while another high affinity analog, R30490, was the most mu-selective. The results show that not all fentanyls are highly mu-selective, and could produce actions through delta- and kappa-opiate receptors.     

Inhibition of elevated arginine vasopressin secretion in response to osmotic stimulation and acute haemorrhage by U-62066E, a kappa-opioid receptor agonist.

1. The effect of kappa (kappa) opioid receptor activation on the increase in arginine vasopressin (AVP) secretion evoked by two acute and quite different stimuli (i.e., haemorrhage and osmotic stimulus due to hypertonic saline infusion) were evaluated in conscious Long-Evans rats, by use of U-62066E, a highly selective kappa-opioid receptor agonist, and MR2266, an opioid receptor antagonist with some selectivity for kappa-receptors. 2. An acute haemorrhage, which reduced the mean blood pressure by approximately 50%, resulted in a large increase in the plasma AVP (pAVP) levels of control rats. However, the administration of U-62066E (0.2 mg kg-1 or 2.0 mg kg-1) reduced the increase due to haemorrhage in a dose-dependent manner. In contrast, concomitant administration of 2.0 mg kg-1 of MR2266 with U-62066E significantly attenuated the inhibition of pAVP levels produced by U-62066E 2.0 mg kg-1. 3. Hypertonic saline infusion (5% hypertonic saline solution at a rate of 0.24 ml kg-1 min-1 for 10 min) caused the elevation of plasma osmolality (pOsm) from 294.0 +/- 1.6 mosmol kg-1 to 304.4 +/- 1.9 mosmol kg-1, simultaneously resulting in a significant increase in pAVP levels from 2.34 +/- 0.28 pg ml-1 to 4.54 +/- 0.51 pg ml-1. However, the administration of U-62066E (0.05 mg kg-1 or 0.2 mg kg-1) reduced the osmotically induced increase in pAVP in a dose-dependent manner although pOsm showed the same degree of increase as in controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of dynorphin-(1-8) in isolated preparations by three peptidases

Inactivation of dynorphin-(1-8) in three in vitro isolated preparations, guinea-pig ileum, mouse vas deferens and rabbit vas deferens, was estimated by employing the relatively specific inhibitors of enkephalin-hydrolyzing enzymes. All three enzyme inhibitors, amastatin, captopril and phosphoramidon, significantly enhanced the inhibitory potency of dynorphin-(1-8) in the three isolated preparations. The magnitude of the enhancement of the dynorphin potency by captopril was significantly higher than that by either amastatin or phosphoramidon in guinea-pig ileum; that by amastatin was significantly higher than that by either captopril or phosphoramidon in rabbit vas deferens; and that by amastatin was similar to that by captopril, but significantly higher than that by phosphoramidon in mouse vas deferens. The Ke values of three antagonists, naloxone, Mr 2266 and ICI 154129, against dynorphin-(1-8) in the presence of the three peptidase inhibitors indicated that dynorphin-(1-8) acted on kappa receptors in guinea-pig ileum and on both kappa and delta receptors in mouse vas deferens. Since amastatin, captopril and phosphoramidon produced the naloxone-reversible inhibition of contractions of guinea-pig ileum in the presence of dynorphin-(1-8), all three dynorphin-inactivating enzymes were indicated to be located very close to kappa receptors.     

The hallucinogen derived from Salvia divinorum, salvinorin A, has kappa-opioid agonist discriminative stimulus effects in rats

Data from clinical and preclinical studies converge implicating the plant-derived hallucinogen salvinorin A as an important pharmacologic tool; this psychoactive compound may expand scientific understandings on mammalian kappa-opioid receptor systems. Human salvinorin A effects, consistent with kappa-opioid receptor agonism, include antinociception, sedation, dysphoria and distorted perceptions. The experiments reported here measured salvinorin A (1-3mg/kg, i.p.) discriminative stimulus properties in male Sprague-Dawley rats conditioned to recognize the discriminative stimulus cue generated by the well characterized kappa-opioid agonist U-69593 (0.56 mg/kg, i.p.). At three distinct active doses, salvinorin A fully substituted for U-69593 without altering response rates. The lever choice pattern in U-69593 trained animals reverted to vehicle lever responding when a kappa selective antagonist compound, nor-BNI (4.5 nM, i.c.v.) was administered 1h prior to salvinorin A, yet nor-BNI alone failed to impact the rate or pattern of subject responses. These findings confirm and extend results published after similar drug discrimination tests were performed in rhesus monkeys. The discussion section of this article highlights public concern over salvinorin A misuse and emphasizes several potential pharmacotherapeutic applications for salvinorin A or analogue compounds.     

An analysis of precipitated withdrawal in rats acutely dependent on morphine

Acute dependence on a single dose of morphine was assessed in Wistar rats by observing the frequencies of occurrence of several signs of withdrawal precipitated by naloxone, diprenorphine, Mr2097, Mr1452 and Mr2266. Naloxone significantly precipitated urination, paw shakes, head shakes and chewing. Diprenorphine significantly precipitated urination and chewing. Mr2097 precipitated urination, head shakes, teeth chattering and chewing. The selective kappa antagonists Mr1452 and Mr2266 significantly precipitated only urination and teeth chattering. Signs of the precipitated withdrawal by Mr2097 were mediated by stereoselective opioid receptors, as the other diastereoisomer, Mr2097, did not precipitate them. Stereospecific opioid receptors were also involved in the induction of acute dependence, as naloxone precipitated withdrawal only in I-methadone-treated rats, but not in d-methadone treated rats. All the opioid antagonists produced at least some degree of "abstinoid" signs in morphine-free rats which might be caused by the blockade of endogenous opioids acting on mu and/or kappa receptors. The signs of withdrawal precipitated by naloxone and Mr2097 might be primarily mediated by mu receptors, those of diprenorphine by both mu and kappa receptors, and those by Mr1452 and Mr2266 were likely to be selectively mediated by kappa receptors. The latter aspect was further supported by experiments showing that the novel kappa agonist U-50488H did not precipitate withdrawal. A low degree of precipitation of withdrawal by Mr1452 and Mr2266 and the absence of precipitation of abstinence by U-50488H might be related to either a lack or an existence of a low proportion of kappa receptors in rat brain. Further experiments using selective agonists and antagonists are needed to evaluate these findings.     

Characterization of rabbit ear artery opioid receptors using a delta-selective agonist and antagonist

In order to characterize pre-junctional delta opioid receptors in the rabbit ear artery, the delta-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), and delta-selective antagonist, ICI 174,864, were used. The selectivity of these compounds for the delta receptor was confirmed using a standard opioid sensitive preparation, the mouse was deferens. Relative potencies of a series of opioid agonists were similar in the rabbit ear artery and the mouse vas deferens. Furthermore, Ke values for IC 174,864 were not different in the two tissues. These findings demonstrate that delta opioid receptors in the rabbit ear artery and mouse vas deferens have similar properties.     

Stereospecific inhibition of gastrointestinal transit by kappa opioid agonists in mice

Systemic administration of highly selective kappa opiate agonists, U-50488H and U-69593 (3, 10 and 30 mg/kg i.p.) produced significant inhibition of the gastrointestinal transit in mice as assessed by charcoal meal test. In contrast, the (+) stereoisomer of U-50488H, U-53455E did not inhibit the gastrointestinal motility. Furthermore, the kappa-selective antagonist, Mr 2266 (3 mg/kg) when administered along with the agonists, reversed the effects of the agonists. These results suggest that stereospecific kappa opiate receptors are involved in inhibition of gut motility in mice.     

In vitro characterization of J-113397, a non-peptide nociceptin/orphanin FQ receptor antagonist

The lack of availability of a selective, highly potent, competitive antagonist for the nociceptin receptor (OP4) devoid of residual agonistic activity has hampered studies in this area. We report here the in vitro pharmacological properties of the novel non-peptide OP4 antagonist, J-113397, which was recently discovered by Banyu Pharmaceutical investigators. The compound was synthesized as a racemic mixture in our laboratories. J-113397 was shown to antagonize (pA2 7.52) the nociceptin-induced inhibition of cAMP formation in cells expressing the recombinant human OP4 receptor (CHOhOP4) and to displace [125I]Tyr14nociceptin from CHOhOP4 membranes with a pKi of 8.56. It also competitively antagonized the contractile actions of nociceptin in the mouse colon (pA2 8.07) and the inhibitory effect of nociceptin in electrically stimulated preparations such as the mouse vas deferens (pA2 7.85), the guinea pig ileum (7.75), and the rat vas deferens (7.77). At high concentrations (10 microM), the compound was devoid of agonist activity in the mouse vas deferens and CHOhOP4, while it contracted the mouse colon and increased the twitch response of the rat vas deferens, and produced a naloxone-sensitive inhibition of the electrically evoked twitches in the guinea pig ileum. pA2 values for the new antagonist against deltorphin I in the mouse vas deferens (OP1 receptors), or against dermorphin in the guinea pig ileum (OP3 receptors), etorphine in the rat vas deferens (OP receptors), U69593 in the rabbit vas deferens (OP2 receptors) and endomorphin 1 in the mouse colon (OP3 receptors) were lower than 6. Taken together, these data indicate that J-113397 is a high-affinity, selective and competitive antagonist of the OP4 receptor; this novel pharmacological tool will be of great value in studies directed at evaluating the physiological roles of the nociceptin/OP4 system.     

Evidence that dynorphin-(1-13) acts as an agonist on opioid kappa-receptors

The study concerned the opioid-receptor subtype on which dynorphin-(1-13) acts in in vitro isolated preparations. The potency of dynorphin-(1-13) relative to that of ethylketocyclazocine (Mr 2266), a representative kappa-receptor agonist, in inhibiting the electrically evoked contractions of the guinea-pig ileum was found to be similar to that found with either mouse was deferens or rabbit ileum. Moreover, Mr 2266 was found to be several-fold more effective than naloxone to antagonize the agonist actions of both kappa-receptor agonists such as ethylketocyclazocine, ketocyclazocine and bremazocine, and dynorphin-(1-13) either in the guinea-pig ileum, mouse vas deferens, or in rabbit ileum. Additionally, dynorphin-(1-13) was found to have a significant inhibitory action on the rabbit vas deferens which had been shown to contain kappa-receptors exclusively. The data indicate that dynorphin-(1-13) acts as an endogenous agonist on kappa-receptors.