Analgesic, receptor binding, and peripheral opioid activities of synthetic dermorphin-dynorphin hybrid peptides

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin-dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin1-5 sequences caused a remarkable increase in analgesic potency, and a 3-6 fold increase in potency of binding against [3H]-dihydromorphine. The potency of receptor binding against [3H]-EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100 mM), the effect of enhancing binding against [3H]-EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid mu-receptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decreased in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that the N-terminal dermorphin moiety, but not the C-terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.     

Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3-4-5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by micro selective antagonists (naloxone and/or CTOP) but not by the delta selective antagonist naltrindole, thus indicating that the compound acted as a micro opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the micro-receptor.     

Mu and delta receptors: their role in analgesia in the differential effects of opioid peptides on analgesia

Utilizing the mouse tail-flick assay, the rank order of analgesic potency for various opioids (i.c.v.) is beta h-endorphin greater than D-Ala2-D-Leu5-enkephalin greater than morphine greater than D-Ala2-met-enkephalinamide much greater than met-enkephalin much greater than leu-enkephalin. Assuming mu receptor mediation of analgesia, there is an affinity and analgesic potency (ie: D-Ala2-Leu5-enkephalin has 1/7 the affinity of morphine for the mu receptor but is 18X more potent as an analgesic). Additionally, sub-analgesic doses of various opioid peptides have opposite effects on analgesic responses. Leu-enkephalin, D-Ala2-D-Leu5-enkephalin or beta h-endorphin potentiate morphine or D-Ala2-met-enkephalinamide analgesia whereas met-enkephalin or D-Ala2-met-enkephalinamide antagonize opioid-induced analgesia. Using the enkephalins as the prototypic delta ligands (100 fold selective) and based on their effects on analgesia, we suggest that Leu-enkephalin-like peptides interact with the delta receptor as an "agonist" to facilitate and met-enkephalin-like peptides as an "antagonist" to attenuate analgesia. Given the biochemical evidence of a coupling between mu and delta receptors, we suggest that the mechanism of facilitation or attenuation of analgesia by the enkephalins is a direct in vivo consequence of this coupling. Further, the analgesic potencies of various opioid ligands can be better correlated to the combination of their simultaneous occupancy of mu and delta receptors.     

Analgesic activity and selectivity of isothiocyanate derivatives of fentanyl analogs for opioid receptors

The analgesic activity and opioid receptor binding characteristics were studied for the isothiocyanate ohmefentanyl (OMFIT), and isothiocyanate carfentanil (CarFIT), isothiocyanate 4-methoxymethylfentanyl (MethoFIT), isothiocyanate 3-methylfentanyl (superFIT) and their amide analogs. Antinociceptive activity was evaluated using the mouse hot plate test; selectivity for opioid receptor was determined in bioassay and binding assay. SuperFIT, CarFIT, OMFIT and MethoFIT exhibited an analgesic ED50 lower than those of their parent compounds without isothiocyanate (SCN) group. Furthermore these compounds exhibited potent inhibitory actions on the electrically evoked contractions of mouse vas deferens, which could be antagonized by naloxone, but their actions were weaker than those of their parent compounds without SC N-group. The inhibitory actions of these compounds on binding of [3H]OMF to mouse brain membrane was weaker than those of their parent compounds without SCN-group. CarFIT and MethoFIT showed weaker inhibitory actions on the binding of [3H] DADLE than their parent compounds without SCN-group, but SuperFIT and OMFIT stronger than their parent compounds, 3-methylfentanyl and ohmefentanyl. The selectivity of these isothiocyanate derivatives for delta opioid receptors increased. In conclusion, introducing isothiocyanato-group into 1-position of phenyl ring of ohmefentanyl and other fentanyl analogs would enhance the selectivity of these compounds for delta-opioid receptors, but decrease their analgesic activity.     

delta EPhe4-enkephalin analogs. Delta receptors in rat brain are different from those in mouse vas deferens

Conformationally restricted enkephalin analogs containing E-cyclopropylphenylalanine (delta EPhe), [D-Ala2, (2R,3S)-delta EPhe4,Leu5]enkephalin and its (2S,3R) isomer, were evaluated in receptor-binding assays using rat brain and in assays using muscle preparations. The (2S,3R) isomer was almost completely inactive in all assays. In contrast, the (2R,3S) isomer showed a very high affinity for the delta and a very weak affinity for the mu receptors in rat brain. The extent of delta affinity and the selectivity of this isomer were almost equal to those of [D-Pen2,D-Pen5]enkephalin. However, the (2R,3S) isomer was inactive in both the mouse vas deferens and guinea pig ileum assays, and showed no antagonistic activity in these tissues. These results indicate that the (2R,3S) isomer interacts with the delta receptors in rat brain, but not with those in the mouse vas deferens, and they suggest that the delta receptors in the central and peripheral nervous systems are different from each other.     

Potentiation of opioid analgesia by cocaine: the role of spinal and supraspinal receptors.

These studies examined the effect of cocaine on the analgesia produced by systemically and centrally administered opioid agonists. Cocaine (50 mg/kg, s.c.) increased the analgesic potency of systemic, ICV and IT morphine; and the ICV and IT analgesic effects of the delta selective peptide, [D-Pen2,D-Pen5]enkephalin (DPDPE). Cocaine also increased the analgesic potency of the mu selective ligand [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) administered ICV. However, cocaine did not alter the ED50 for IT DAGO. GC-MS studies indicated that brain cocaine concentration was approximately 3.0 micrograms/g wet weight 45 min following s.c. administration. These results suggest that cocaine-induced increases in opioid analgesic potency are mediated at brain mu and delta receptors and spinal mu receptors. Furthermore, there might be functional differences between spinal and supraspinal sites at which DAGO produces analgesia.     

Kentsin: tetrapeptide from hamster embryos produces naloxone-sensitive effects without binding to opioid receptors

The opioid nature of kentsin (Thr-Pro-Arg-Lys) and its ability to alter pain perception and intestinal transit were examined. Kentsin (30,000 nM) did not inhibit electrically stimulated contractions of the guinea pig ileum (GPI) or mouse vas deferens (MVD), nor did it cause a rightward displacement of the inhibitory concentration-response curves of the mu-selective opioid agonist PL017 in the GPI or the delta-selective agonist DPDPE in the MVD. Kentsin (10,000 nM) did not displace [3H] naloxone from rat brain homogenates. These results indicate that kentsin lacks opioid agonist and mu and delta opioid antagonist properties and does not bind to opioid receptors. In vivo, kentsin produced dose-dependent analgesia in both the hotplate and abdominal stretch tests when administered intracerebroventricularly (ICV) and intrathecally but not intravenously. The central analgesic effect of kentsin was partially antagonized by the opioid antagonist naloxone. Kentsin inhibited intestinal transit in a dose-dependent manner after ICV administration only. The intestinal antitransit effect of kentsin was not blocked by pretreatment with naloxone. These results suggest that kentsin acts centrally to produce both opioid and non-opioid effects. Further, the opioid-mediated analgesic effects of kentsin involve mechanisms other than direct interaction with opioid receptors.     

Opioid activity of C8813, a novel and potent opioid analgesic

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.     

Beta-endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic beta-endorphin (beta-EP) analogs containing dermorphin or dynorphin-A-(1-13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2-terminal 1-7 segment of camel beta-EP [beta c-EP-(1-7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1-7)]-beta c-EP was 120 times more potent than beta c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2-terminal 1-13 segment of human beta-EP [beta h-EP-(1-13)] with dynorphin-A-(1-13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1-13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that beta c-EP-(8-31) facilitates the dermorphin moiety to act on opiate mu and delta receptors but not on the epsilon receptor, while beta h-(14-31) reduces the action of dynorphin on mu, delta and kappa receptors.     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

Comparative study on the effect of morphine and the opioid-like peptides in the vas deferens of rodents: species and strain differences, evidence for multiple opiate receptors

The effect of an opiate alkaloid and an opioid-like peptide was studied on the electrically evoked twitching of the vas deferens of 3 common laboratory rodents. Normorphine and the synthetic opioid peptide D-Alanine₂ methionine enkephalinamide (D-Ala₂) produced dose dependent inhibitions of the twitching response in the mouse vas deferens. In the rat vas, while β-endorphin (β-EP) caused an inhibitory effect in three strains of rats to a similar degree, morphine produced a dose related enhancement of the twitching. In the guinea pig, both morphine and β-EP caused an increased in the muscular twitch. The results are interpreted in terms of an heterogenous mixture of opiate receptors present in the vas deferens from these rodents. The mouse appears to contain mainly δ receptors while the rat has mostly ε receptors characterized by their specificity and sensitivity to the action of β-EP. The guinea pig vas deferens has apparently lost the sensitivity to the inhibitory influence of the opioids, suggesting the absence of μ or δ opiate receptors in this tissue.     

Bremazocine: a potent, long-acting opiate kappa-agonist

The benzomorphan analogue bremazocine is a potent, centrally-acting analgesic with a long duration of action. In animal models it is free of physical and psychological dependence liability, produces no respiratory depression, and has a variety of other properties which justify its classification as a putative opiate kappa-receptor agonist.Binding studies with tritiated (?)-bremazocine on rat brain membrane preparations show that this molecule differs in its binding properties from previously investigated exogenous or endogenous opioids. Studies on isolated guinea-pig ileum and mouse vas deferens indicate a preference for opiate kappa-receptors.In mice (hot plate, tail flick) and rhesus monkeys (shock titration), bremazocine is a potent analgesic with a long duration of action. Here also, the actions of the antagonists naloxone and Mr 2266 suggest a preference for opiate kappa-receptors.Bremazocine differs from morphine in the non-production of mydriasis and the Straub tail phenomenon in mice, in its lack of effects on respiration in rats, in that it is not self-administered by rhesus monkeys, and in that programmed administration in the same species does not lead to a morphine-like withdrawal syndrome upon cessation of drug treatment or upon naloxone challenge. Prolonged treatment of animals with bremazocine leads to tolerance to its analgesic effects; morphine treatment of such tolerant animals causes analgesia. Conversely, treatment of morphine-tolerant animals with bremazocine does not cause analgesia; these findings suggest that morphine and bremazocine interact with different subpopulations of opiate receptors.     

TENA, a selective kappa opioid receptor antagonist

A number of opioid antagonists (TENA, naloxone, Mr 2266, WIN 44441) were evaluated for their selectivity in antagonizing the effect of mu, kappa, and delta agonists in the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Among these four antagonists, TENA was the most potent and the only ligand which was selective for kappa receptors. In this regard TENA was approximately 27-times more effective in antagonizing the kappa agonist, U-50488H, relative to the mu agonist, morphine, and it was about 5-times more effective against ethylketazocine (EK) relative to morphine. At the same concentration (20 nM) TENA did not significantly antagonize the delta agonist, [D-Ala2,D-Ala5]enkephalin (DADLE), in the MVD. Also, TENA was more effective than naloxone, EK, or U-50488H in protecting kappa receptors from irreversible blockage by beta-CNA. The results of this study indicate that TENA is the most selective kappa antagonist yet reported.     

Effects of stress and beta-funal trexamine pretreatment on morphine analgesia and opioid binding in rats

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.     

Single residue modifications of the delta opioid receptor selective peptide, [D-Pen2,D-Pen5]-enkephalin (DPDPE). Correlation of pharmacological effects with structural and conformational features

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [D-Pen2,D-Pen5]enkephalin, Tyr-D-Pen-Gly-Phe-D-PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by 1H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine1-DPDPE ([DAT1]DPDPE) and phenylglycine4-DPDPE ([Pgl4]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced mu receptor binding affinity of the carboxamide terminal DPDPE-NH2 appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the alpha-aminoisobutyric acid substituted analog [Aib3]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the delta opioid receptor.     

Effect of beta-FNA on opiate delta receptor binding

beta-FNA, the beta-fumaramate methyl ester of naltrexone, has been shown to antagonize irreversibly the actions of morphine on the guinea pig ileum and mouse vas deferens bioassays but does not affect the actions of delta-receptor ligands on the mouse vas deferens bioassay, suggesting that the compound does not irreversibly bind to the delta receptor. In this paper we examine the effect of beta-FNA on the binding of the prototypic delta agonists, Leu-enkephalin and D-Ala2-D-Leu5-enkephalin, its metabolically stable analogue, and show that treatment of membranes with beta-FNA does lead to alterations in the in vitro properties of delta receptors.     

Opioid peptides derived from food proteins. The exorphins.

Peptides with opioid activity are found in pepsin hydrolysates of wheat gluten and alpha-casein. The opioid activity of these peptides was demonstrated by use of the following bioassays: 1) naloxone-reversible inhibition of adenylate cyclase in homogenates of neuroblastoma X-glioma hybrid cells; 2) naloxone-reversible inhibition of electrically stimulated contractions of the mouse vas deferens; 3) displacement of [3H]dihydromorphine and [3H-Tyr, dAla2]met-enkephalin amide from rat brain membranes. Substances which stimulate adenylate cyclase and increase the contractions of the mouse vas deferens but do not bind to opiate receptors are also isolated from gluten hydrolysates. It is suggested that peptides derived from some food proteins may be of physiological importance.     

Antinociceptive interactions of opioid delta receptor agonists with morphine in mice: supra- and sub-additivity.

In this study, the antinociceptive interactions of fixed ratio combinations of intracerebroventricularly (i.c.v.) given morphine and subantinociceptive doses of the delta agonists, [D-Pen2, D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin (DELT) or [Met5]enkephalin (MET) were examined using the mouse warm water tail flick test. When morphine was coadministered with DPDPE or DELT in a 4:1 and 9:1 mixture, respectively, a synergistic antinociceptive effect was observed. In contrast, when morphine was coadministered with MET in a 1:2 fixed ratio mixture, a subadditive interaction occurred. These results demonstrate both positive and negative modulatory interactions of delta agonists with morphine in an antinociceptive endpoint and that these interactions can be either supra- or subadditive. The data support the concept of a functional interaction between opioid mu and delta receptors and a potential regulatory role for the endogenous ligands of the opioid delta receptor.     

High and low affinity opioid binding sites: relationship to mu and delta sites

Binding and pharmacological studies suggest a common opiate and enkephalin binding site in addition to their previously reported selective sites. This common high affinity site has tentatively been named mu1, distinguishing it from the morphine-selective site (mu2) and enkephalin-selective site (delta). The existence of this additional common high affinity site and its association with opiate and opioid peptide analgesia may help explain some pharmacological observations, such as the cross tolerance between morphine and enkephalin analgesia and the lack of cross tolerance between them in the guinea pig ileum and mouse vas deferens bioassays.     

In vitro pharmacological profile of peptide III-BTD: a novel ligand for nociceptin/orphanin FQ and opioid receptors

Peptide III-BTD has been recently identified as a non-selective nociceptin/orphanin FQ receptor ligand by screening of a synthetic peptide combinatorial library. In the present study we evaluated the pharmacological profile of peptide III-BTD in isolated tissues (mouse and rat vas deferens, guinea pig ileum) sensitive to both nociceptin and opioid peptides. In the mouse vas deferens and guinea pig ileum, III-BTD concentration dependently inhibited the electrically induced twitch (pEC50 5.91 and 6.18, respectively; Emax 94 +/- 1% and 94 +/- 2%) and this effect was blocked by naloxone (1 microM). In the rat vas deferens, III-BTD was inactive in most of the tissues, while in few others it elicited a slight inhibition only at the highest concentration tested (10 microM). In the presence of 1 microM naloxone, 1 microM III-BTD shifted to the right the concentration response curve of nociceptin in a parallel manner, showing pKB values in the range 6.6-6.9. These data confirm on native nociceptin receptors the pharmacological profile of peptide III-BTD which behaved as a mixed nociceptin receptor antagonist/opioid receptor agonist in the [35S]GTPyS binding assay performed on cells expressing the recombinant human receptors.