Design of novel bicyclic analogues derived from a potent oxytocin antagonist.

Eleven new analogues were synthesized by modification of the potent oxytocin antagonist (OTA) [(S)Pmp(1), D-Trp(2), Pen(6), Arg(8)]-Oxytocin, or PA (parent antagonist), in which (S)Pmp = beta,beta-(3-thiapentamethylene)-beta-mercapto-propionic acid. By internal acylation of Lys, Orn, L-1,4-diaminobutyric acid (Dab), L-1,3-diaminopropionic acid (Dap) at position 4 with the C-terminal Gly of the peptide tail, we prepared cyclo-(4-9)-[Lys(4), Gly(9)]-PA (pA(2) = 8.77 +/- 0.27), 1, and cyclo-(4-9)-[Orn(4), Gly(9)]-PA (pA(2) = 8.81 +/- 0.25), 3, which are equipotent with PA (pA(2) = 8.68 +/- 0.18) in the rat uterotonic assay and cyclo-(4-9)-[Dab(4), Gly(9)]-PA, 4, cyclo-(4-9)-[Dap(4), Gly(9)]-PA, 5, and cyclo-(4-9)-[Pmp(1), Lys(4), Gly(9)]-PA, 2, which were weaker OTAs. Neither 1 nor 3 had activity as agonists or antagonists in the antidiuretic assay. In the pressor assay, both analogues 1 and 3, with pA(2) = 7.05 +/- 0.10 and pA(2) = 6.77 +/- 0.12, respectively, are somewhat weaker antagonists than PA (pA(2) = 7.47 +/- 0.35) showing significant gain in specificity. The [desamido(9)] PA-ethylenediamine monoamide, 6, and the dimer ([desamido(9)]-PA)(2) ethylenediamine diamide, 7, had lower potency in the uterotonic assay than PA. Additionally, we synthesized cyclo-(1-5)-[(HN)Pmp(1), Asp(5)]-PA, 8, inactive in all tests, which suggests that the intact Asn(5) side chain may be critical in the interaction of the OTAs with the oxytocin (OT) receptor. Similarly, cyclo-(5-9)-[Dap(5), Gly(9)]-PA, 9, had very low uterotonic potency. Two derivatives of PA truncated from the C-terminus were internally cyclized to Lys(4), giving rise to cyclo-(4-8)-desGly-NH(2)(9)[Lys(4), Arg(8)]-PA, 10 (pA(2) = 8.35 +/- 0.20), which maintains the high potency of PA and has no activity in the rat antidiuretic assay, and in the rat pressor assay it is about ten times weaker (pA2 = 6.41 +/- 0.15) than PA (pA2 = 7.47 +/- 0.35), thus showing gains in specificity, and to cyclo-(4-7)-desArg-Gly-(NH)(2)(8-9)[Lys(4), Pro(7))-PA, 11, which has much weaker potency than PA. Synthesis of cyclo-(4-6)-desPro-Arg-Gly-(NH)(2)(7-9)[Lys(4)]-PA failed.     

Analogues of oxytocin antagonists bearing a ureido group in the amino acid side chain at position 4 or 5.

Substitution of the side chain carboxamido group at position 4 in the potent oxytocin antagonist (OTA) [ThiaPmp(1), D-Trp(2), Cys(6), Arg(8)]-OT, PA, in which ThiaPmp = beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, led to [Orn(Car)(4)]-PA, ([Cit(4)]-PA), which had uterotonic antagonistic activity equal to that of PA. The same modification at position 5, leading to [Cit(5)]-PA, resulted in antagonistic potency more than 10 times lower than that of PA. This paper also describes the same substitutions introduced in the highly potent OTA [Pen(6)]-PA (antioxytocic in vitro pA(2) = 8.72). Analogues of the general formula [U(4)-X(5)-Pen(6)]-PA, in which U = Lys, Orn, Dab, Dap or X = Orn, Dab or Dap, were synthesized by SPPS. Each of these analogues was carbamoylated by treatment with KCNO in DMF-H(2)O, yielding the corresponding U(Car)(4) or X(Car)(5) derivatives. In the uterotonic assay, the substitution with the ureido group at Gln(4) results in retention of high antagonistic potency, albeit somewhat lower than that of PA, e.g. [Orn(Car)(4), Pen(6)]-PA and [Dab(Car)(4), Pen(6)]-PA having pA(2) = 8.52 and pA(2) = 8.42 respectively. In the pressor assay, [Lys(Car)(4), Pen(6)]-PA and [Dab(Car)(4), Pen(6)]-PA were somewhat weaker antagonists of arginine vasopressin than [Pen(6)]-PA; [Dap(Car)(4), Pen(6)]-PA showed only a faint trace of pressor agonistic activity. The substitution with the ureido group at position 5 leads to a significant loss of OTA potency in the in vitro uterotonic assay. The [Orn(Car)(5), Pen(6)]-PA was the most potent of the series (pA(2) = 8.05). An interesting finding is that [Dap(Car)(5), Pen(6)]-PA is equipotent with its precursor [Dap(5), Pen(6)]-PA (potency in the uterotonic test in vitro, pA(2) = 7.71 and pA(2) = 7.68, respectively). Furthermore, neither [Dap(5), Pen(6)]-PA nor [Dap(5), Pen(6), Gly(9)]-PA exhibited activity in the antidiuretic or pressor assays. Although these last two analogues show some decrease in antioxytocin potency, they behave as pure oxytocin antagonists, which makes them attractive candidates for further studies on the development of potent and specific OTAs.     

Synthesis,characterization and nociceptive screening of new VV-hemorphin-5 analogues

In the present study, some new analogues of VV-hemorphin-5, modified at position 1 and 7 by the non-proteinogenic and/or natural amino acids followed the structures Xxx-Val-Val-Tyr-Pro-Trp-Thr-Gln-NH₂ and Val-Val-Tyr-Pro-Trp-Thr-Yyy-NH₂, where Xxx is Ile or Aib and Yyy is Lys/Orn/Dap/Dab were synthesized to investigate their potential antinociceptive activities. We report also the redox potentials and the acid/base properties as pKa values of these peptide analogues which were compared toward electrochemical behaviour of tryptophan containing peptides. All analogues showed a short lasting initial antinociceptive effect, however H2 hemorphin analogue is characterized with prolong and strong antinociceptive effect, while the other peptide analogues exerted more variable effects on the visceral nociception depending on the dose or time after the intracerebral injection.     

Synthesis and biological activity of new series of N-modified analogues of the N/OFQ(1–13)NH2 with aminophosphonate moiety

New series of N-modified analogues of the N/OFQ(1-13)NH(2) with aminophosphonate moiety have been synthesized and investigated for biological activity. These peptides were prepared by solid-phase peptide synthesis-Fmoc-strategy. The N/OFQ(1-13)NH(2) analogues were tested for agonistic activity in vitro on electrically stimulated rat vas deferens smooth-muscle preparations isolated from Wistar albino rats. Our study has shown that the selectivity of the peptides containing 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids to the N-side of Phe is not changed-they remain selective agonists of NOP receptors. The derivative with the largest ring (NOC-6) demonstrated efficacy similar to that of N/OFQ(1-13)NH(2), but in a 10-fold higher concentration. The agonistic activity of newly synthesized N-modified analogues of N/OFQ(1-13)NH(2) with aminophosphonate moiety was investigated for the first time.     

Potent side-chain to side-chain cyclized dermorphin analogues containing a carbonyl bridge.

A new family of cyclic opioid peptide analogues related to the 1-4 sequence of dermorphin/deltorphin (Tyr-D-Aaa2-Phe-Aaa4-NH2) has been synthesized. The synthesis of the linear precursor peptides was accomplished by the solid-phase method and ring formation was achieved via a ureido group incorporating the side chain amino functions of D-Aaa2 (D-Lys, D-Orn) and Aaa4 (Lys, Orn, Dab, Dap). The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Most showed very high agonist potency in the GPI assay. The peptide containing D-Lys in position 2 and Dab in position 4 was 210 times more active than enkephalin, and that containing Orn and Dab, respectively, was 150 times more active than enkephalin. The latter peptide was also very active in the MVD assay, and showed an IC50 MVD/GPI ratio of 0.816. NMR spectra of selected peptides were recorded, and structural parameters were determined. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. With the help of the NMR spectra each peptide was described as an ensemble of conformations. The conformations have been interpreted with regard to the opioid activities, and comparisons have been made with a model proposed earlier for enkephalin analogues.     

Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation

Nociceptin/orphanin FQ (N/OFQ) produces several biological actions by activating the N/OFQ peptide receptor (NOP). It has been previously shown that N/OFQ stimulates leukocyte chemotaxis both in vitro and in vivo. In the present study we investigated the ability of N/OFQ, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human neutrophil and monocyte chemotaxis and the release of lysozyme and superoxide anion (O2-) production from neutrophils. fMLP stimulated all the leukocyte functions examined. N/OFQ stimulated monocyte (pEC50 12.15) but not neutrophil chemotaxis. The production of O2- from neutrophils was not affected by N/OFQ while the release of lysozyme was increased in a concentration dependent manner (pEC50 11.00) although the maximal effects evoked by N/OFQ were about half of those of fMLP. The NOP ligands [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, Ro 64-6198, UFP-101 and the opioid antagonist naloxone were used for pharmacologically characterizing the receptor involved in the monocyte chemoattractant action of N/OFQ. [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, and Ro 64-6198 mimicked the action of N/OFQ showing similar maximal effects and the following order of potency: [Arg14, Lys15]N/OFQ (pEC50 13.22)>Ro 64-6198 (pEC50 12.96)>N/OFQ(1-13)NH2 (pEC50 12.67)>N/OFQ (pEC50 12.15). Moreover, the monocyte chemoattractant action of N/OFQ was not modified by naloxone 1 microM while antagonized by UFP-101 10 microM (pA2 7.00). Thus, the order of potency of agonists and the antagonist selectivity demonstrated that N/OFQ stimulates human monocyte chemotaxis via NOP receptor activation.     

Analogues of a potent oxytocin antagonist with truncated C-terminus or shorter amino acid side chain of the basic amino acid at position 8.

Twelve analogues were synthesized, their structure derived from modifications of [(S)Pmp1, D-Trp2, Pen6, Arg8]oxytocin, PA, in which (S)Pmp = beta,beta-(3-thiapentamethylene-beta-mercaptopropionic acid). PA is a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 8.86) and in the baboon. Truncated analogues of PA from the C-terminus were systematically prepared ending in either the free acid or the amide, i.e. PA1-9 acid, PA1-8 acid, PA1-7 acid, PA1-6 acid, PA1-8 amide, PA1-7 amide and PA1-6 amide. PA1-8 amide was roughly as potent as PA in the rat uterotonic assay in vitro, and the shorter amides were only somewhat weaker antagonists. All four acid analogues were weaker antagonists than PA but still maintained rather high antagonistic potency. These findings suggest that, if these truncated acids form as metabolites in vivo, they may contribute to the overall biological effect of PA and their contribution should be taken into account. Furthermore, using these analogues, the radioimmunoassay measurements of PA may be standardized, as they may cross react with PA antibodies and interfere with the determination. In addition, five analogues were made by substituting Arg8 of PA with Lys, Orn8, Dab8, Dap8 and Cit8. All of these analogues maintained high potency as OTAs in the uterotonic assay, although their activity was only about 1.5-3 times lower than PA. The most potent analogue in the uterotonic assay, [Dap8]PA, pA2 = 8.53, had weak pressor activity (pA2 = 6.90) and no antidiuretic effect. The pressor activity was lower for all tested acids, and for PA1-6 acid it was even below the detection limit. Additionally, PA1-9 acid, PA1-7 acid and PA1-6 acid showed no antidiuretic activity. Hence, the PA1-6 acid is a potent OTA with pA2 = 8.27 and no measurable effect in the pressor or antidiuretic tests and thus it is a pure oxytocin antagonist. This fact makes it an attractive candidate for further studies on inhibition of OT biological effects and on preterm labour.     

Toward understanding the cationicity of defensins. Arg and Lys versus their noncoded analogs

Human defensins are a family of small antimicrobial proteins found predominantly in leukocytes and epithelial cells that play important roles in the innate and adaptive immune defense against microbial infection. The most distinct molecular feature of defensins is cationicity, manifested by abundant Arg and/or Lys residues in their sequences. Sequence analysis indicates that Arg is strongly selected over Lys in alpha-defensins but not in beta-defensins. To understand this Arg/Lys disparity in defensins, we chemically synthesized human alpha-defensin 1 (HNP1) and several HNP1 analogs where three Arg residues were replaced by each of the following six alpha-amino acids: Lys, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), N,N-dimethyl-Lys ((diMe)Lys), and homo-Arg ((homo)Arg). In addition, we prepared human beta-defensin 1 (hBD1) and (Lys-->Arg)hBD1 in which all four Lys residues were substituted for Arg. Bactericidal activity assays revealed the following. 1) Arg-containing HNP1 and (Lys-->Arg)hBD1 are functionally better than Lys-HNP1 and hBD1, respectively; the difference between Arg and Lys is more evident in the alpha-defensin than in the beta-defensin and is more evident at low salt concentrations than at high salt concentrations. 2) For HNP1, the Arg/Lys disparity is much more pronounced with Staphylococcus aureus than with Escherichia coli, and the Arg-rich HNP1 kills bacteria faster than its Lys-rich analog. 3) Arg and Lys appear to have optimal chain lengths for bacterial killing as shortening Lys or lengthening Arg in HNP1 invariably becomes functionally deleterious. Our findings provide insights into the Arg/Lys disparity in defensins, and shed light on the cationicity of defensins with respect to their antimicrobial activity and specificity.     

Rational design, conformational studies and bioactivity of highly potent conformationally constrained calcitonin analogues.

Calcitonin is known for its hypocalcaemic effect and the inhibition of bone resorption, and is used therapeutically for the treatment of osteoporosis and Paget's disease. Our studies on the conformational features of human calcitonin (hCt) bioactivity have led to the conformationally constrained hCt analogue cyclo17,21-[Asp17, Lys21]hCt (1), which had a 5-10 times higher in vivo hypocalcaemic potency than hCt [Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem. 38, 836-847]. We hypothesized that a stabilized, possibly type I beta turn/beta sheet conformation between residues 17 and 21 could play a crucial role in hCt bioactivity. Here, we designed, synthesized and studied the conformation and bioactivity of 19-member to 17-member ring-size analogues of 1 with the structure cyclo17,21-[Asp17,XX21]hCt with XX = Orn (2), Dab (3) and Dap (4), of the control peptide [Asp17,Orn21]hCt (5), and of the 19-member cyclo17,21-[Glu17,Dab21]hCt (6). Analyses of the far-UV CD spectra indicated increased type I beta turn and antiparallel beta sheet content in the bicyclic analogues compared with hCt. In the in vivo hypocalcaemic assay, cyclo17,21-[Asp17,Orn21]hCt (2) was found to have a 400-fold higher potency than hCt and was fourfold more potent than salmon calcitonin (sCt), which has been the most potent known Ct. Analogue 3 had a 30-fold higher potency than hCt, whereas the highly constrained analogue 4 was as potent as hCt. Bioactivity was not enhanced for the nonbridged compound [Asp17, Orn21]hCt (5), whereas cyclo17,21-[Glu17,Dab21]hCt (6) showed the same bioactivity as 1. This study identifies 2 as exhibiting the highest in vivo potency among currently known Cts, while it differs in only one amino acid residue from hCt, strongly suggesting that the introduced constraint may have served in 'freezing' hCt in a bioactive conformation. Our findings provide evidence for the first time that a beta turn/beta sheet conformation in region 17-21 of hCt and the topological features of the side chain of Asn17 are strongly associated with in vivo bioactivity, and offer a novel lead structure for a hCt-based drug for the treatment of osteoporosis and other bone-disorder-related diseases.     

Nonpeptide/peptide chimeric ligands for the nociceptin/orphanin FQ receptor: design, synthesis and in vitro pharmacological activity.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the G-protein coupled receptor referred to as N/OFQ peptide (NOP) receptor. NOP receptor activation by N/OFQ modulates several biological functions both at central and peripheral level. Structure activity relationship (SAR) studies demonstrated that the N/OFQ sequence can be divided into a N-terminal tetrapeptide 'message' crucial for receptor activation and a C-terminal 'address' important for receptor binding. On the basis of this message/address concept we synthesized some chimeric compounds in which we substituted the natural message domain with the nonselective nonpeptide NOP ligand (8-Naphthalen-1-yl-methyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4,5]dec-3-yl)-aceticacid methyl ester (NNC 63-0532) and used as address domain the peptide sequences Thr-NH2, N/OFQ(5-9)-NH2, N/OFQ(5-13)-NH2 and N/OFQ(5-17)-NH2. All the compounds were pharmacologically evaluated in the electrically stimulated guinea-pig ileum. NNC 63-0532 produced a concentration-dependent inhibition of the electrically induced twitches showing, in comparison with N/OFQ, lower potency and higher maximal effects. In addition, contrary to N/OFQ, the effects of NNC 63-0532 were insensitive to the NOP selective antagonist [Nphe1, Arg14, Lys15]N/OFQ-NH2 (UFP-101) while prevented by naloxone. Similar results were obtained with NNC 63-0532/Thr-NH2 and NNC 63-0532/N/OFQ(1-9)-NH2. On the contrary, the inhibitory effects of NNC 63-0532/N/OFQ(5-13)-NH2 and NNC 63-0532/N/OFQ(5-17)-NH2 were slightly antagonized by UFP-101 while naloxone prevented the effects of the high but not of the low concentrations of the two ligands. These data indicate that it is possible to functionalize with the N/OFQ address sequence a nonpeptide NOP ligand for increasing its binding to the NOP receptor. Moreover, these results corroborate the idea that the 5-13 sequence represents the crucial core of the N/OFQ address domain.     

Nociceptin/orphanin FQ and related peptides reduce the increase in plasma corticosterone elicited in mice by an intracerebroventricular injection

Nociceptin/orphanin FQ (=N/OFQ), the endogenous ligand of ORL1 receptor (=NOP), has been reported to induce, in rodents, after intracerebroventricular (i.c.v.) administration, anti-stress and anxiolytic effects. We have observed that the handling of mice followed by an i.c.v. injection of saline, induced a marked increase in the plasma corticosterone level (+250%) measured 30 minutes later. When N/OFQ was injected intracerebroventricularly, using a 1 microg dose, the increase in plasma corticosterone was significantly lower than in saline injected mice. N/OFQ(1-13)NH(2), known as a NOP receptor agonist, at the same 1 microg dose, also induced a lesser increase in plasma corticosterone level than a saline i.c.v. injection. The pseudopeptide [Phe(1)-psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2), defined either as an agonist or an antagonist of NOP receptor, at the 0.1 microg dose, behaved in a similar manner as N/OFQ, by decreasing the plasma corticosterone level. Finally, [Nphe(1)]N/OFQ(1-13)NH(2), although presumed to be a selective NOP receptor antagonist, also decreased the corticosterone level at the 0.1 microg dose. These observations suggest the implication of N/OFQ in the regulation of response to stress, through an action on the hypothalamo-pituitary-adrenocortical axis. Moreover, they evidence a similar effect of N/OFQ and N/OFQ(1-13)NH(2), but also of two other related peptides displaying antagonist properties on NOP receptors. These data suggest that several subtypes of N/OFQ receptors could exist.     

Gastrointestinal effects of intracerebroventricularly injected nociceptin/orphaninFQ in rats

Nociceptin/orphanin FQ/(N/OFQ), a novel heptadecapeptide recently isolated from porcine and rat brain, is the endogenous ligand of the N/OFQ peptide receptor (NOP, previously known as ORL-1). In this study we examined the effects of intracerebroventricularly (icv) injected N/OFQ on gastric emptying, gastrointestinal transit, colonic propulsion and gastric acid secretion in rats. N/OFQ (0.01-10 nmol/rat) significantly delayed gastric emptying of a phenol red meal, inhibited transit of a non-absorbable charcoal marker through the small intestine and increased the mean colonic bead expulsion time. These N/OFQ-motor effects were abolished by the NOP receptor selective antagonist [NPhe(1)]N/OFQ(1-13)-NH(2) (50 nmol/rat), but were unaltered by the classical opioid receptor antagonist, naloxone (9.2 micromol/kg). Icv injected N/OFQ (10 nmol/rat) decreased gastric acid secretion in 2-h pylorus ligated rats in a naloxone sensitive manner. [NPhe(1)]N/OFQ(1-13)-NH(2) (100 nmol/rat) icv administered alone stimulated gastric acid secretion. These results indicate that N/OFQ activates via NOP receptor stimulation a central inhibitory pathway modulating gastrointestinal propulsive activity and gastric acid secretion in rats.     

Nociceptin/orphanin FQ increases ANP secretion in neonatal cardiac myocytes

Nociceptin (N/OFQ) is a novel heptadecapeptide with an amino acid sequence similar to that of endogenous opioid peptide dynorphin A. Dynorphin have been reported to increase the secretion of atrial natriuretic peptide (ANP) via selective activation of kappa-opioid receptor in cultured atrial cardiocytes. The present study was designed to investigate the direct effect of N/OFQ on the ANP secretion in cultured neonatal rat cardiac myocytes via N/OFQ receptor (NOP) activation. The secretion of ANP from cultured neonatal cardiac myocytes was increased in terms of incubation time. N/OFQ, at a dose of 0.3, 1, 3, and 10 microM, caused increases in ANP secretion in a dose-dependent manner. The N/OFQ-induced ANP secretion was completely antagonized by antagonists of NOP, 1 microM each of [Phe1 (CH2-NH) Gly2] nociceptin (1-13)-NH2 ([FG]N/OFQ(1-13)NH2) or naloxone benzoylhydrazone. In contrast, naloxone (1 microM), the non-selective opioid receptor antagonist, did not alter ANP response to N/OFQ. N/OFQ at 3 microM inhibited basal and forskolin-stimulated cAMP production, which was partially antagonized with the pretreatment of [FG]N/OFQ(1-13)NH2. An increase in ANP secretion by N/OFQ was also partially blocked by the pretreatment of forskolin. Homologous competition studies in neonatal cardiomyocyte membranes revealed the presence of two distinct sites. The high affinity site (10.9 +/- 1.6 nM) was far less abundant than the low affinity site. Therefore, these results suggest that N/OFQ causes an increase in ANP secretion in cultured neonatal cardiac myocytes by decreasing cAMP through its binding sites.     

Lack of involvement of dopamine and serotonin during the orphanin FQ/Nociceptin (OFQ/N)-induced prolactin secretory response

The purpose of these studies was to examine possible mechanisms of Orphanin FQ/Nociceptin (OFQ/N)-induced prolactin release. We investigated the involvement of the dopaminergic neurons by quantifying DOPAC:DA levels in the median eminence and neurointermediate lobe following central administration of OFQ/N to female Sprague-Dawley rats. To specifically determine the involvement of the tuberoinfundibular dopaminergic neurons, immunocytochemical studies were conducted to visualize c-fos protein expression in the arcuate nucleus following central administration of OFQ/N. In addition, the role of serotonergic activation was examined in dose response studies using the selective serotonin antagonist ritansarin and the nonselective antagonist metergoline. Finally, the pharmacological specificity of the prolactin response was examined by pretreating animals with [Nphe1] NC (1-13)NH2, a drug reported to antagonize OFQ/N effects. The results of these studies indicate that the increase in prolactin release following central administration of OFQ/N does not inhibit tuberoinfundibular, tuberohypophyseal or periventricular hypophysial dopaminergic neuronal activity at 10 min after drug administration, a time when prolactin levels were significantly elevated. Furthermore, serotonergic activation is not involved since pharmacological blockade of serotonergic receptors did not alter the prolactin secretory response to OFQ/N. NC (1-13)NH2 did not antagonize the stimulatory effects of OFQ/N on prolactin secretion. The neural effects of OFQ/N on dopaminergic neuronal activity may occur following a different time course than that of the prolactin increase.     

Novel potent agonist [(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 and antagonist [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 of nociceptin/orphanin FQ receptor

Two novel ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe4,Aib7, Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-1) and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-2), have been generated by combining different modifications of N/OFQ sequence. In the present study, we investigated the actions of two analogues and compared them with those of N/OFQ in four assays. Peptide-1 mimicked N/OFQ effects in mouse vas deferens and mouse colon and showed similar maximal effects but higher potency relative to N/OFQ. The effects of peptide-1 were sensitive to NOP receptor selective antagonist ([Nphe1]N/OFQ(1-13)-NH2) but not to naloxone in vitro. Peptide-1 (25 pmol, i.c.v.) mimicked the pronociceptive action of N/OFQ (2.5 nmol, i.c.v.) in mouse tail withdrawal assay, displaying higher potency and longer lasting effects. In anesthetized rats, peptide-1 (1 nmol/kg, i.v.) produced a marked decrease in mean arterial pressure, which was comparable to that evoked by i.v. N/OFQ (100 nmol/kg). Peptide-2 did not produce any effect per se but antagonized N/OFQ actions in mouse vas deferens and mouse colon assays. Peptide-2 is active in vivo where it prevented the pronociceptive effect induced by 2.5 nmol N/OFQ i.c.v. in the mouse tail withdrawal assay. Furthermore, peptide-2 at 5 nmol produced alone a robust and long lasting antinociceptive effect. Moreover, peptide-2 (10 and 40 nmol/kg i.v.) didn't produce any effect per se but antagonized hypotensive actions produced by i.v. administration of N/OFQ. Collectively, these findings demonstrate that [(pF)Phe4,Aib7,Aib11, Arg14,Lys15]N/OFQ-NH2 behaves as a highly potent NOP receptor agonist which produces long lasting effects in vivo and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 acts as a pure and competitive antagonist of the NOP receptor.     

The effects of [Arg14, Lys15] nociceptin/orphanin FQ, a highly potent agonist of the NOP receptor, on in vitro and in vivo gastrointestinal functions

Nociceptin/orphanin FQ (N/OFQ) administered into the lateral left cerebral ventricle of rats has been reported to inhibit in vivo gut motor and secretory functions. Recently, a novel N/OFQ analog, [Arg14, Lys15] N/OFQ, was synthesized and demonstrated to behave as a highly potent agonist at the human recombinant N/OFQ peptide (NOP) receptors and to produce long-lasting effects in vivo in mice compared with the natural ligand N/OFQ. In the present study, the pharmacological profile of [Arg14, Lys15] N/OFQ was further evaluated and compared with that of N/OFQ in vitro on guinea pig exocrine pancreas and in vivo on gastric emptying, colonic propulsion and gastric acid secretion in rats. [Arg14, Lys15] N/OFQ and N/OFQ significantly decreased the KCl-evoked amylase secretion from isolated pancreatic lobules of the guinea pig. In in vivo experiments, [Arg14, Lys15] N/OFQ mimicked the effects of N/OFQ, inducing, after intracerebroventricular injection, a delay (up to 70%) in the gastric emptying of a phenol red meal, an increase (about 40 times) of the mean bead colonic expulsion time and a decrease (up to 90%) of gastric acid secretion in water loaded rats after 90 min pylorus ligature. In all these assays, [Arg14, Lys15] N/OFQ was more effective than N/OFQ, and its effective doses were at least 10-fold lower than N/OFQ effective doses. The highly selective NOP receptor antagonist, UFP-101, decreased the efficacy of [Arg14, Lys15] N/OFQ in in vitro and in vivo assays above reported. These findings: (a) show that pancreatic NOP receptors mediate an in vitro inhibitory effect on stimulated guinea pig amylase secretion; (b) confirm that the stimulation of central NOP receptors exerts an inhibitory control on gastric emptying, colonic motility and gastric secretion in rats and (c) put in evidence that [Arg14, Lys15] N/OFQ, being more potent and effective than the natural ligand N/OFQ, represents a new pharmacological tool for the study of the physiological and pharmacological roles mediated by the N/OFQ-NOP receptor system.     

Study in vitro and in vivo of nociceptin/orphanin FQ(1-13)NH2 analogues substituting N-Me-Gly for Gly2 or Gly3

In the present study, two analogues containing N-Me-Gly (Sarcosine, Sar) were synthesized to further investigate the structural-activity relationships of orphanin FQ/nociceptin (OFQ/NC, NC). The replacement of Gly(2) or Gly(3) with Sar increased the flexibility and decreased the hydrophobicity of the N-terminal tetrapeptide. The activity of the analogues was investigated in a series of assays in vivo and in vitro. [Sar(2)]NC(1-13)NH(2) was found to (1) produce dose-dependent inhibition of the electrically induced contraction in MVD assay (pEC(50) = 6.14); (2) produce significant hyperalgesia effects in a dose-dependent manner when intracerebroventricularly (i.c.v.) injected in mice. The inhibitive effects of [Sar(2)]NC(1-13)NH(2) in MVD assay could be significantly antagonized by [Nphe(1)]NC(1-13)NH(2), and partially antagonized by naloxone; the hyperalgesic effect of [Sar(2)]NC(1-13)NH(2) could be significantly antagonized by naloxone, and partially antagonized by [Nphe(1)]NC(1-13)NH(2). On the contrary, [Sar(3)]NC(1-13)NH(2) showed no effects in these assays. All the findings suggest that the flexibility of the peptide bond between Phe(1) and Gly(2) and between Gly(2) and Gly(3) play an important role in NC-OP(4) receptor interaction, and the hydrophobicity of the N-terminal tetrapeptide showed no significant effect on this interaction. The present work also helps to provide a novel method to elucidate structural and conformational requirements of the opioid peptide-receptor interaction.     

Nociceptin/orphanin FQ inhibits electrically induced contractions of the human bronchus via NOP receptor activation

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit neurogenic contractions in various tissues, including guinea pig airways. In the present study, we investigated the ability of N/OFQ to affect cholinergic contractions of human bronchi elicited by electrical field stimulation (EFS). Tissues were obtained from 23 patients undergoing surgery for lung cancer. EFS (20 Hz, 320 mA, 1.5 ms, 10 s) was applied five times every 20 min. Contractions induced by EFS were abolished by either TTX (1 microM) or atropine (1 microM) and concentration-dependently (10 nM-1 microM) inhibited by N/OFQ (Emax, 11.5+/-1.8% inhibition). The inhibitory effects of N/OFQ were mimicked by the N/OFQ receptor (NOP) ligand [Arg14, Lys15]N/OFQ which displayed however, higher significant maximal effects (17.7+/-2.9% inhibition, P<0.05). The actions of N/OFQ and [Arg14, Lys15]N/OFQ were not affected by naloxone (1 microM) while prevented by the selective NOP receptor antagonist UFP-101 (10 microM). Moreover, the inhibitory effects of NOP agonists were no longer evident in tissues treated with tertiapin (10 microM), an inhibitor of inward-rectifier potassium channels. In conclusion, the present data demonstrate that N/OFQ inhibited acetylcholine (ACh) release in the human bronchi via NOP receptor activation. This effect may involve stimulation of potassium currents.     

Effects of structural analogues of nociceptin(1-13)NH₂ on brain antioxidant status in kainic acid-treated rats

The in vivo effects of nociceptin (N/OFQ(1–13)NH₂) and its structural analogues ([Dab⁹]N/OFQ(1–13)NH₂, [Dap⁹]N/OFQ(1–13)NH₂ and [Cav⁹]N/OFQ(1–13)NH₂) on the levels of lipid peroxidation and cell antioxidants (enzyme and non‐enzyme) in brain of control and kainic acid (KA)‐treated rats were studied. In control animals, [Dab⁹]N/OFQ(1–13)NH₂ and [Dap⁹]N/OFQ(1–13)NH₂, unlike N/OFQ(1–13)NH₂ and [Cav⁹]N/OFQ(1–13)NH₂, slightly increased the brain lipid peroxidation; the rest of the parameters were unchanged by all neuropeptides tested. KA (0.25 µg in 0.5 µl, i.c.v) increased the lipid peroxidation (4 and 24 h after KA‐injection) and decreased the glutathione level (1 h after KA‐administration). One hour after KA‐administration, the neuropeptides (2 µg in 0.5 µl, injected 30 min before KA) showed the following effects: a slight decrease in the KA‐induced lipid peroxidation by all nociceptin analogues and an enhancement of the KA‐decreased GSH level, but by [Cav⁹]N/OFQ(1–13)NH₂ only. The brain antioxidant enzyme activities were unchanged in all used experimental groups. In addition, the nociceptin analogues, especially [Can⁹]N/OFQ(1–13)NH₂, showed a good antioxidant capacity in chemical systems, generating reactive oxygen species. In conclusion, the substitution of lysin (Lys) in N/OFQ(1–13)NH₂ molecule with other amino acids might contribute to changes in its antioxidant properties. Copyright © 2011 John Wiley & Sons, Ltd.     

Possible involvement of endogenous nociceptin/orphanin FQ in the pain-related behavioral responses induced by its own metabolite, nociceptin/orphanin FQ(14-17)

Nociceptin/orphanin FQ(14-17) (N/OFQ(14-17)) is one of the major fragments that are released from N/OFQ, an endogenous ligand for the opioid receptor like-1 (ORL-1) receptor by endopeptidase 24.11. In the present study, we determined the pharmacological profiles of N/OFQ(14-17) on pain-related behavioral responses in the mouse. Intrathecal (i.t.) administration of N/OFQ(14-17) (5-160 pmol) evoked pain-related behaviors, and these behavioral responses were reduced by i.t. co-administration of an ORL-1 receptor antagonist, [Nphe(1)]N/OFQ(1-13)NH2 (4 pmol). However, in the ligand-binding receptor assay, N/OFQ(14-17) had no affinity for the ORL-1 receptor. Furthermore, i.t. pretreatment with an antiserum against N/OFQ (1:50) diminished the N/OFQ(14-17)-induced pain-related behaviors, suggesting that endogenous N/OFQ is involved in their expression. Therefore, N/OFQ(14-17)-induced pain-related behaviors may be mediated through the release of endogenous N/OFQ in the mouse spinal cord.