Regulation of opioid peptides on the release of arginine vasotocin in the hen

Arginine vasotocin (AVT), an avian neurohypophysial hormone, is released during osmotic stimulation and oviposition. In the present study, the role of opioid peptides on AVT release was studied by examining the effects of an opioid agonist and antagonist on osmotic- and oviposition-induced secretion of AVT. The administration of hypertonic saline (1.5 M NaCl) induced an increase in the plasma levels of AVT. The simultaneous administration of morphine, an opioid receptor agonist, inhibited the osmotically induced increase in plasma levels of AVT in a dose-dependent manner. On the other hand, the co-administration of morphine with naloxone, an opioid receptor antagonist, attenuated the inhibitory effect of morphine. Moreover, injection of naloxone alone enhanced the osmotically induced increase in plasma levels of AVT. However, the administration of morphine did not inhibit the oviposition-induced increase in plasma levels of AVT. These results suggest that osmotic-induced release of AVT may be under opioid regulation, while oviposition-induced release of AVT may be controlled by a different mechanism. J. Exp. Zool. 286:481-486, 2000.     

The pharmacological profile of delta opioid receptor ligands, (+) and (-) TAN-67 on pain modulation

We designed the nonpeptidic highly selective delta opioid receptor agonist on the basis of message address concept and the accessory site theory and synthesized (+/-) TAN-67. In spite of highly potent agonistic activity in in vitro assay, (+/-) TAN-67 (racemate) afforded a weak antinociceptive effect in the mouse tail-flick test. This result led us to separate (+/-) TAN-67 to optical pure compounds, (+) and (-) TAN-67. An i.t.-treatment with (-) TAN-67 produced profound antinociceptive effects through specifically acting on delta1 receptors. Unlike (-) TAN-67, i.t.-administered (+) TAN-67 displayed dose-related nociceptive behaviors such as scratching, biting and licking. The effect of (+) TAN-67 was blocked by i.t.-treatment with NTI (delta receptor antagonist) and (-) TAN-67 (delta1 receptor agonist), but not by morphine (mu receptor agonist). The mechanisms involved in spinal pain modulation induced by (+) and (-) TAN-67 were also described.     

Syntheses of 4,6'-epoxymorphinan derivatives and their pharmacologies

A modification of the message site in the skeleton of naltrexone was carried out to improve the potency and selectivity of the compound for an opioid receptor subtype. In the course of conversion, we synthesized 7-membered ring ether derivatives, which had an inserted OCH(2) group between 4- and 6-positions of morphinan skeleton. One of the 7-membered ring ether derivatives possessed more potent antagonistic activity than naltrexone for the mu opioid receptor. Another compound possessing 17-methyl group derived from noroxycodone may be a mu opioid receptor partial agonist and showed analgesic activity. We are currently examining the subtype selectivity of these compounds.     

Effects of interferon-alpha on cloned opioid receptors expressed in Xenopus oocytes

Interferon-α (IFNα) affects the opioid system. However, the direct action of IFNα on cloned opioid receptors remains unknown. Taking advantage of the functional coupling of cloned opioid receptors to G protein-activated inwardly rectifying K⁺ (GIRK) channels in a Xenopus oocyte expression system, we investigated the effects of recombinant IFNα on cloned μ-, δ- and κ-opioid receptors. In oocytes co-injected with mRNAs for either the δ- or κ-opioid receptor and for GIRK channel subunits, IFNα at high concentrations induced small GIRK currents that were abolished by naloxone, an opioid-receptor antagonist, compared with the control responses to each selective opioid agonist. Additionally, IFNα induced no significant current response in oocytes injected with mRNA(s) for either opioid receptor alone or GIRK channels. In oocytes expressing the μ-opioid receptor and GIRK channels, IFNα had little or no effect. Moreover, in oocytes expressing each opioid receptor and GIRK channels, GIRK current responses to each selective opioid agonist were not affected by the presence of IFNα, indicating no significant antagonism of IFNα toward the opioid receptors. Furthermore, IFNα had little or no effect on the μ/δ-, δ/κ- or μ/κ-opioid receptors expressed together with GIRK channels in oocytes. Our results suggest that IFNα weakly activates the δ and κ-opioid receptors. The direct activation of the δ- and κ-opioid receptors by IFNα may partly contribute to some of the IFNα effects under its high-dose medication.     

Structure selectivity relationship studies of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl)carboxamido]morphinan derivatives toward the development of the mu opioid receptor antagonists

Mu opioid receptor antagonists have been applied to target a variety of diseases clinically. The current study is designed to explore the structure selectivity relationship (SSR) of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl)carboxamido]morphinan (NAP), a lead compound identified as a selective mu opioid receptor antagonist based on the previous study. Among a series of NAP derivatives synthesized, compounds 6 (NMP) and 9 (NGP) maintained comparable binding affinity, selectivity and efficacy to the lead compound. Particularly, the mu opioid receptor selectivity over kappa opioid receptor of NGP was considerably enhanced compared to that of NAP. Overall, the preliminary SSR supported our original hypothesis that an alternate 'address' domain may exist in the mu opioid receptor, which favors the ligands carrying a hydrogen bond acceptor and an aromatic system to selectively recognize the mu opioid receptor.     

Drug design and synthesis of epsilon opioid receptor agonist: 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821) inducing antinociception mediated by putative epsilon opioid receptor

Here we report the new drug design and synthesis of a series of 6,14-endoethenomorphinan-7-carboxamide derivatives as a putative epsilon opioid receptor agonist. One of these compounds, 17-(cyclopropylmethyl)-4,5alpha-epoxy-3,6beta-dihydroxy-6,14-endoethenomorphinan-7alpha-(N-methyl-N-phenethyl)carboxamide (TAN-821), showed agonistic activity for a putative epsilon opioid receptor (IC(50) = 71.71nM) in the rat vas deferens (RVD) preparations. TAN-821 stimulated the binding of the nonhydrolyzable guanosine 5'-triphosphate analog, guanosine 5'-(gamma-thio)-triphosphate (GTPgammaS), to the mouse pons/medulla membrane via the activation of putative epsilon opioid receptor. Moreover, TAN-821 given intracerebroventricularly (i.c.v.) produced a marked antinociception in the tail-flick test (ED(50) = 1.73 microg) and the hot-plate test (ED(50) = 2.05 microg) in a dose-dependent manner. The antinociception induced by TAN-821 administered i.c.v. was blocked by the i.c.v.-pretreatment with a putative epsilon opioid receptor partial agonist beta-endorphin [1-27], but not a mu opioid receptor antagonist beta-FNA, a delta opioid receptor antagonist NTI, or a kappa opioid receptor antagonist nor-BNI. The present results suggest that TAN-821 may be a useful tool for the investigation on the pharmacological properties of the putative epsilon opioid receptor.     

Opioid agonist/antagonist effect of naloxone in modulating rabbit jejunum contractility in vitro.

Opioid peptides are the most effective drugs in controlling pain; their action is elicited by binding to specific membrane receptors. The gastrointestinal tract represents, after the nervous system, the site in which the opioid receptors are expressed at high levels. The opioid agonist morphine has a significant inhibitory effect on intestinal motility, this action is blocked by naloxone an opioid antagonist mainly active at mu and kappa receptors. In this study the presence of mu opioid receptor on rabbit jejunum was investigated by western blot. The effects of beta-endorphin, the endogenous opioid peptide with the highest affinity to the mu opioid receptor and those of naloxone on spontaneous rabbit jejunum contractions were evaluated. Beta-endorphin (10(-6) M) showed a relaxant effect on jejunum contractility while naloxone showed a dual effect inducing an increase of spontaneous contractility at low concentrations (10(-6) M, 10(-7) M, 10(-8) M) and a decrease when high concentrations (10(-3) M, 10(-4) M, 10(-5) M) were utilized. The obtained results demonstrate that mu opioid receptor is expressed in rabbit jejunum and suggest that this receptor may be involved in mediating the effects of both opioid agonist and antagonist on jejunum contractions.     

长时间给予高选择性δ阿片受体激动剂抑制δ受体mRNA表达

我们采用反转录－聚合酶链反应（ＲＴ－ＰＣＲ）方法,观察了δ阿片受体肽类激动剂「Ｄ－Ｐｅｎ＾２．５」ｅｎｋｅｐｈａｌｉｎ（ＤＰＤＰＥ）及非肽类激动剂ＢＷ３７３Ｕ８６对δ受体ｍＲＮＡ表达的影响,并比较了两者作用的差异性。结果如下：（１）ＤＰＤＰＥ作用２４及４８ｈ可使δ受体ｍＲＮＡ表达水平显著降低,则ＢＷ３７３Ｕ８６只在２４ｈ有显著抑制作用;（２）ＤＰＤＰＥ在１０＾－６ｍｏｌ／Ｌ时即可使δ受体的ｍＲＮＡ     

Optically pure (-)-4-[(N-allyl-3-methyl-4-piperidinyl)phenyl-amino]-N,N-diethylbenzami de displays selective binding and full agonist activity for the delta opioid receptor

The optical isomers of 4-[(N-allyl-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzamide+ ++ (3) have been prepared and tested in both binding and functional assays. The data show that (-)-3 is responsible for the delta opioid activity demonstrated by the racemic material. This compound displays a binding affinity of 5.5 nM for the delta opioid receptor as well as a 470-fold delta versus mu selectivity. Importantly, (-)-3 is a full agonist at the delta receptor in comparison with SNC-80 (2). Taken together, the data suggest that (-)-3 behaves more like the prototypical delta agonists, BW373U86 or SNC-80, and less like the peptidomimetic compound SL-3111 (5).     

Absolute configuration and conformation of the pure opioid antagonist (+)-2,9 alpha-dimethyl-5-(m-hydroxyphenyl)morphan.

(+)-2,9 alpha-Dimethyl-5-(m-hydroxyphenyl)morphan is the only phenylmorphan analog whose affinity for opioid kappa-receptors is greater than its affinity for opioid mu-receptors. Pharmacologically, the compound is a pure opioid antagonist devoid of agonist activity in in vivo assays of antinociception. The absolute configuration of the compound has been determined to be (1R,5S,9R) from an X-ray crystallographic study of the chloride salt. Thus, the absolute configuration corresponds to that of the atypical opioid agonist (-)-phenylmorphan while the weak atypical agonist (-)-2,9 alpha-dimethyl-5-(m- hydroxyphenyl)morphan corresponds to the potent morphine-like (+)-phenylmorphan. The preferred orientations of the phenyl ring for the two stereoisomers were determined using the molecular mechanics program MM2-87 and found to vary from that of the two parent compounds. The atypical properties of the two 9 alpha-methyl analogs is consistent with an opioid ligand model which proposes that morphine-like properties require a particular range of phenyl orientations. There was good agreement between the structure obtained from X-ray crystallography and computed with the MM2-87 program.     

Nociceptin in rVLM mediates electroacupuncture inhibition of cardiovascular reflex excitatory response in rats.

Electroacupuncture (EA) at Neiguan-Jianshi acupoints through an opioid mechanism inhibits the cardiovascular pressor response induced by mechanical stimulation of the stomach. Because nociceptin also may regulate cardiovascular activity through its action in the brain stem, we hypothesized that this neuromodulator serves a role in the EA-related inhibitory effect. Blood pressure in ventilated male Sprague-Dawley rats (400-600 g) anesthetized by ketamine and alpha-chloralose was measured during balloon inflation of the stomach. Gastric distension with 6-8 ml of air induced consistent pressor reflexes of 26 +/- 1 mmHg that could be repeated every 10 min for 100 min. When nociceptin (10 nM) was microinjected into the rostral ventrolateral medulla (rVLM), the pressor response induced by gastric distension was inhibited by 68 +/- 6%. Thirty minutes of EA also decreased the reflex response by 75 +/- 11%; microinjection of saline into the rVLM did not alter the inhibitory effect of EA. In contrast, microinjection of a nociceptin receptor antagonist into the rVLM promptly reversed the EA response. Pretreatment with the opioid receptor antagonist naloxone did not influence the EA-like inhibitory effect of nociceptin on the distension-induced pressor reflex (22 +/- 1 to 8 +/- 2 mmHg). Furthermore, a mu-opioid receptor agonist microinjected into the rVLM after microinjection of a nociceptin receptor antagonist during EA promptly reversed the nociceptin receptor antagonist-related inhibition of the EA effect. Thus, in addition to the classical opioid system, nociceptin, through opioid receptor-like-1 receptor stimulation in the rVLM, participates in the modulatory influence of EA on reflex-induced increases in blood pressure.     

八肽胆囊收缩素对抗阿片肽的中枢性降血压作用

过去的工作已经证明八肽胆囊收缩素(CCK-8)能够对抗阿片肽的镇痛作用,本工作探讨CCK-8是否能够对抗阿片肽的心血管抑制作用。给戊巴比妥钠麻醉大鼠脊髓蛛网膜下腔(ith)注射 CCK-8可以对抗 ith 注射 mu(μ)型阿片受体激动剂[NMePhe~3,D-Pro~4]Morphiceptin(PL017)(5μg)、delta(δ)型受体激动剂[D-Ala~2,D-Leu~5]Enkephalin(DADLE)(25μg)和 Kappa(K)型受体激动剂[N-Me Tyr,N-Me Arg~7,D-Leu~8]Dynorphin 1-8 ethyla-mide(66A-078)(1μg)引起的降低血压和减慢心率作用。在 MAP 的表现上,CCK-8的拮抗作用(10μg及以下剂量)具有量-效关系,并可被 CCK 受体阻断剂丙谷胺(Proglumide)(100μg)翻转。在 HR 的表现上,上述剂量的 CCK-8也显示了一定的拮抗作用,但量-效关系不如 MAP 表现得明显。单纯将 CCK-8或 Proglumide ith 注射,可见大剂量(50μg)CCK-8可以引起明显的降血压作用和短时的降心率作用,小剂量(0.05μg)CCK-8则表现出明显的降心率作用;ith 注射 Proglumide 100μg,30 min 后也表现出减慢心率的作用。以上结果提示:在脊髓水平,一定剂量范围内的 CCK-8能够对抗阿片肽的心血管抑制效应,此对抗作用是通过 CCK 受体实现的。本工作的结果支持关于 CCK-8是一种抗阿片物质的设想。     

Activation of G-proteins in the mouse pons/medulla by beta-endorphin is mediated by the stimulation of mu- and putative epsilon-receptors

The activation of mu-, delta- and kappa1-opioid receptors by their respective agonists increases the binding of the non-hydrolyzable GTP analog guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS) to G proteins. Beta-endorphin is an endogenous opioid peptide which binds nonselectively to mu-, delta- and putative epsilon-opioid receptors. The present experiment was designed to determine which opioid receptors are involved in the stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the mouse pons/medulla. The mouse pons/medulla membranes were incubated in an assay buffer containing 50 pM [35S]GTPgammaS, 30 microM GDP and various concentrations of beta-endorphin. Beta-endorphin (0.1 nM-10 microM) increased [35S]GTPgammaS binding in a concentration-dependent manner, and 10 microM beta-endorphin produced a maximal stimulation of approximately 260% over baseline. This stimulation of [35S]GTPgammaS binding by beta-endorphin was partially attenuated by the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA), but not by the delta-opioid receptor antagonist naltrindole (NTI) or the kappa1-opioid receptor antagonist nor-binaltorphimine (nor-BNI). Beta-endorphin stimulated [35S]GTPgammaS binding by about 80% in the presence of 10 microM beta-FNA, 30 nM NTI and 100 nM nor-BNI. The same concentrations of these antagonists completely blocked the stimulation of [35S]GTPgammaS binding induced by 10 microM [D-Ala2,NHPhe4,Gly-ol]enkephalin, [D-Pen(2,5)]enkephalin and U50,488H, respectively. Moreover, the residual stimulation of [35S]GTPgammaS binding induced by beta-endorphin in the presence of the three opioid receptor antagonists was significantly attenuated by 100 nM of the putative epsilon-opioid receptor partial agonist beta-endorphin (1-27). These results indicate that the stimulation of [35S]GTPgammaS binding induced by beta-endorphin is mediated by the stimulation of both mu- and putative epsilon-opioid receptors in the mouse pons/medulla.     

3-Aryl pyridone derivatives. Potent and selective kappa opioid receptor agonists.

A new series of 3-aryl pyridone based kappa opioid receptor agonists was designed and synthesised, based on an understanding of the classical kappa opioid receptor pharmacophore. The most potent of the new compounds were comparable to U-69,593 in receptor affinity, selectivity and functional agonist effect at the cloned human kappa opioid receptor.     

Synthesis and evaluation of novel peripherally restricted kappa-opioid receptor agonists

A series of 3-substituted analogs (3) of the parent kappa agonist, 1, were prepared to limit access to the central nervous system. With the exception of compound 3j, all other compounds bound to the human kappa opioid receptor with high affinity (K(i)=0.31-9.5 nM) and were selective for kappa over mu and delta opioid receptors. Compounds 3c, d, and 3g-i produced potent antinociceptive activity in the rat formalin assay (i.paw) and the mouse acetic acid-induced writhing assay (s.c.), with weak activity in the mouse platform sedation test. The peripheral restriction indices of 3c, d, 3g, and 3i were improved 2- to 7-fold compared to the parent compound 1, and these compounds were approximately 2- to 5-fold more potent than the peripheral kappa agonist ICI 204448.     

Synthesis of a novel universal opioid receptor agonist with the 1,3,5-trioxazatriquinane skeleton and its pharmacologies

We designed and synthesized of 1,3,5-trioxazatriquinanes with o- or p-hydroxyphenyl rings as analogs of the κ opioid receptor agonist SYK-146 with m-hydroxyphenyl groups. Although almost all tested compounds did not bind to the opioid receptors, only 17b (SYK-524) with two o-hydroxyphenyl rings showed moderate or potent binding affinities and exhibited agonistic activities for the three opioid receptor types. Because the basicity of the nitrogen atom in the 1,3,5-trioxazatriquinane structure was predicted to be very low due to the electron withdrawing effect of the three oxygen atoms, SYK-524 was a novel non-morphinan and nonpeptidic opioid universal agonist lacking a basic nitrogen atom.     

Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis

The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex. The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa.     

Opioid and melanocortin receptors: do they have overlapping pharmacophores?

We have identified compound 1 as a novel ligand for opioid and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the delta opioid receptor, 2,6-dimethyltyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and a small molecule for the MC receptor, Tic-DPhe(p-Cl)-piperidin-4-yl-N-phenyl-propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the delta opioid receptor (Ki = 0.38 nM in binding assay, EC(50) = 0.48 nM in GTP-gamma-S binding assay, IC(50) = 74 nM in MVD) as an agonist instead of an antagonist and showed selective binding affinity (IC(50) = 2.3 muM) at the MC-3 receptor rather than at the MC-5 receptor. A study of the structure-activity relationships demonstrated that the residues in positions 2, 3, and the C-terminus act as a pharmacophore for the MC receptors, and the residues in positions 1 and 2 act as a pharmacophore for the opioid receptors. Thus, this structural construct can be used to prepare chimeric structures with adjacent or overlapping pharmacophores for opioid and MC receptors.     

Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu,delta, and kappa opioid receptors

In an effort to improve biphalin’s potency and efficacy at the µ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1–5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate that C-terminal flexible linkers play an important role in KOR activity compared to that of the other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki?=?0.27, 0.46, and 0.87?nM; EC₅₀?=?3.47, 1.45, and 13.5?nM at MOR, DOR, and KOR, respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on relieving pain and further studies including in vivo tests are worthwhile.