Hyperactive cyclic motor activity in the distal colon after colonic surgery as defined by high-resolution colonic manometry.

BACKGROUND: Recovery after colonic surgery is invariably delayed by disturbed gut motility. It is commonly assumed that colonic motility becomes quiescent after surgery, but this hypothesis has not been evaluated rigorously. This study quantified colonic motility through the early postoperative period using high-resolution colonic manometry. METHODS: Fibre-optic colonic manometry was performed continuously before, during and after surgery in the left colon and rectum of patients undergoing right hemicolectomy, and in healthy controls. Motor events were characterized by pattern, frequency, direction, velocity, amplitude and distance propagated. RESULTS: Eight patients undergoing hemicolectomy and nine healthy controls were included in the study. Colonic motility became markedly hyperactive in all operated patients, consistently dominated by cyclic motor patterns. Onset of cyclic motor patterns began to a minor extent before operation, occurring with increasing intensity nearer the time of surgery; the mean(s.d.) active duration was 12(7) per cent over 3 h before operation and 43(17) per cent within 1 h before surgery (P = 0.024); in fasted controls it was 2(4) per cent (P < 0·001). After surgery, cyclic motor patterns increased markedly in extent and intensity, becoming nearly continuous (active duration 94(13) per cent; P < 0·001), with peak frequency 2-4 cycles per min in the sigmoid colon. This postoperative cyclic pattern was substantially more prominent than in non-operative controls, including in the fed state (active duration 27(20) per cent; P < 0·001), and also showed higher antegrade velocity (P < 0·001). CONCLUSION: Distal gut motility becomes markedly hyperactive with colonic surgery, dominated by cyclic motor patterns. This hyperactivity likely represents a novel pathophysiological aspect of the surgical stress response. Hyperactive motility may contribute to gut dysfunction after surgery, potentially offering a new therapeutic target to enhance recovery.

In vitro and in vivo characterization of the bifunctional µ and δ opioid receptor ligand UFP-505.

BACKGROUND AND PURPOSE: Targeting more than one opioid receptor type simultaneously may have analgesic advantages in reducing side-effects. We have evaluated the mixed µ opioid receptor agonist/ δ opioid receptor antagonist UFP-505 in vitro and in vivo. EXPERIMENTAL APPROACH: We measured receptor density and function in single µ, δ and µ /δ receptor double expression systems. GTPγ35 S binding, cAMP formation and arrestin recruitment were measured. Antinociceptive activity was measured in vivo using tail withdrawal and paw pressure tests following acute and chronic treatment. In some experiments, we collected tissues to measure receptor densities. KEY RESULTS: UFP-505 bound to µ receptors with full agonist activity and to δ receptors as a low efficacy partial agonist At µ, but not δ receptors, UFP-505 binding recruited arrestin. Unlike morphine, UFP-505 treatment internalized µ receptors and there was some evidence for internalization of δ receptors. Similar data were obtained in a µ /δ receptor double expression system. In rats, acute UFP-505 or morphine, injected intrathecally, was antinociceptive. In tissues harvested from these experiments, µ and δ receptor density was decreased after UFP-505 but not morphine treatment, in agreement with in vitro data. Both morphine and UFP-505 induced significant tolerance. CONCLUSIONS AND IMPLICATIONS: In this study, UFP-505 behaved as a full agonist at µ receptors with variable activity at δ receptors. This bifunctional compound was antinociceptive in rats after intrathecal administration. In this model, dual targeting provided no advantages in terms of tolerance liability. LINKED ARTICLES: This article is part of a themed section on Emerging Areas of Opioid Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.14/issuetoc.

Limited evidence of abnormal intra-colonic pressure profiles in diverticular disease - a systematic review.

AIM: Abnormal colonic pressure profiles and high intraluminal pressures are postulated to contribute to the formation of sigmoid colon diverticulosis and the pathophysiology of diverticular disease. This study aimed to review evidence for abnormal colonic pressure profiles in diverticulosis. METHOD: All published studies investigating colonic pressure in patients with diverticulosis were searched in three databases (Medline, Embase, Scopus). No language restrictions were applied. Any manometry studies in which patients with diverticulosis were compared with controls were included. The Newcastle-Ottawa Quality Assessment Scale (NOS) for case-control studies was used as a measure of risk of bias. A cut-off of five or more points on the NOS (fair quality in terms of risk of bias) was chosen for inclusion in the meta-analysis. RESULTS: Ten studies (published 1962-2005) met the inclusion criteria. The studies followed a wide variety of protocols and all used low-resolution manometry (sensor spacing range 7.5-15 cm). Six studies compared intra-sigmoid pressure, with five of six showing higher pressure in diverticulosis vs controls, but only two reached statistical significance. A meta-analysis was not performed as only two studies were above the cut-off and these did not have comparable outcomes. CONCLUSION: This systematic review of manometry data shows that evidence for abnormal pressure in the sigmoid colon in patients with diverticulosis is weak. Existing studies utilized inconsistent methodology, showed heterogeneous results and are of limited quality. Higher quality studies using modern manometric techniques and standardized reporting methods are needed to clarify the role of colonic pressure in diverticulosis.

Restoration of normal colonic motor patterns and meal responses after distal colorectal resection.

BACKGROUND: Colorectal resections alter colonic motility, including disruption of control by neural or bioelectrical cell networks. The long-term impact of surgical resections and anastomoses on colonic motor patterns has, however, never been assessed accurately. Fibreoptic high-resolution colonic manometry was employed to define motility in patients who had undergone distal colorectal resection. METHODS: Recruited patients had undergone distal colorectal resections more than 12 months previously, and had normal bowel function. Manometry was performed in the distal colon (36 sensors; 1-cm intervals), with 2-h recordings taken before and after a meal, with comparison to controls. Analysis quantified all propagating events and frequencies (cyclical, short single, and long single motor patterns), including across anastomoses. RESULTS: Fifteen patients and 12 controls were recruited into the study. Coordinated propagating events directly traversed the healed anastomoses in nine of 12 patients with available data, including antegrade and retrograde cyclical, short single and long single patterns. Dominant frequencies in the distal colon were similar in patients and controls (2-3 cycles/min) (antegrade P = 0·482; retrograde P = 0·178). Compared with values before the meal, the mean(s.d.) number of dominant cyclical retrograde motor patterns increased in patients after the meal (2·1(2·7) versus 32·6(31·8) in 2 h respectively; P < 0·001), similar to controls (P = 0·178), although the extent of propagation was 41 per cent shorter in patients, by a mean of 3·4 cm (P = 0·003). Short and long single propagating motor patterns were comparable between groups in terms of frequency, velocity, extent and amplitude. CONCLUSION: Motility patterns and meal responses are restored after distal colorectal resection in patients with normal bowel function. Coordinated propagation across healed anastomoses may indicate regeneration of underlying cellular networks.

Development and characterisation of novel fentanyl-delta opioid receptor antagonist based bivalent ligands.

BACKGROUND: Opioid tolerance is a limiting factor in chronic pain. Delta opioid peptide (DOP)(δ) receptor antagonism has been shown to reduce tolerance. Here, the common clinical mu opioid peptide (MOP)(µ) receptor agonist fentanyl has been linked to the DOP antagonist Dmt-Tic (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydrisoquinoline-3-carboxylic acid) to create new bivalent compounds. METHODS: Binding affinities of bivalents(#9, #10, #11, #12 and #13) were measured in Chinese hamster ovary (CHO) cells expressing recombinant human MOP, DOP, Kappa opioid peptide (KOP)(κ) and nociceptin/orphanin FQ opioid peptide (NOP) receptors. Functional studies, measuring GTPγ[(35)S] or ß-arrestin recruitment, were performed in membranes or whole cells respectively expressing MOP and DOP. RESULTS: The new bivalents bound to MOP (pKi : #9:7.31; #10:7.58; #11:7.91; #12:7.94; #13:8.03) and DOP (#9:8.03; #10:8.16; #11:8.17; #12:9.67; #13:9.71). In GTPγ[(35)S] functional assays, compounds #9(pEC50:6.74; intrinsic activity:0.05) #10(7.13;0.34) and #11(7.52;0.27) showed weak partial agonist activity at MOP. Compounds #12 and #13, with longer linkers, showed no functional activity at MOP. In antagonist assays at MOP, compounds #9 (pKb:6.87), #10(7.55) #11(7.81) #12(6.91) and #13(7.05) all reversed the effects of fentanyl. At DOP, all compounds showed antagonist affinity (#9:6.85; #10:8.06; #11:8.11; #12:9.42; #13:9.00), reversing the effects of DPDPE ([D-Pen(2,5)]enkephalin). In ß-arrestin assays, compared with fentanyl (with response at maximum concentration (RMC):13.62), all compounds showed reduced ability to activate ß-arrestin (#9 RMC:1.58; #10:2.72; #11:2.40; #12:1.29; #13:1.58). Compared with fentanyl, the intrinsic activity was: #9:0.12; #10:0.20; #11:0.18; #12:0.09 and #13:0.12. CONCLUSIONS: The addition of a linker between fentanyl and Dmt-Tic did not alter the ability to bind to MOP and DOP, however a substantial loss in MOP functional activity was apparent. This highlights the difficulty in multifunctional opioid development.

In vitro and in vivo pharmacological characterization of nociceptin/orphanin FQ tetrabranched derivatives.

BACKGROUND AND PURPOSE: An innovative chemical approach, named peptide welding technology (PWT), allows the synthesis of multibranched peptides with extraordinary high yield, purity and reproducibility. With this approach, three different tetrabranched derivatives of nociceptin/orphanin FQ (N/OFQ) have been synthesized and named PWT1-N/OFQ, PWT2-N/OFQ and PWT3-N/OFQ. In the present study we investigated the in vitro and in vivo pharmacological profile of PWT N/OFQ derivatives and compared their actions with those of the naturally occurring peptide. EXPERIMENTAL APPROACH: The following in vitro assays were used: receptor and [(35)S]-GTPγS binding, calcium mobilization in cells expressing the human N/OFQ peptide (NOP) receptor, or classical opioid receptors and chimeric G proteins, electrically stimulated mouse vas deferens bioassay. In vivo experiments were performed; locomotor activity was measured in normal mice and in animals with the NOP receptor gene knocked out [NOP(-/-)]. KEY RESULTS: In vitro PWT derivatives of N/OFQ behaved as high affinity potent and rather selective full agonists at human recombinant and animal native NOP receptors. In vivo PWT derivatives mimicked the inhibitory effects exerted by the natural peptide on locomotor activity showing 40-fold higher potency and extremely longer lasting action. The effects of PWT2-N/OFQ were no longer evident in NOP(-/-) mice. CONCLUSIONS AND IMPLICATIONS: The results showed that the PWT can be successfully applied to the peptide sequence of N/OFQ to generate tetrabranched derivatives characterized by a pharmacological profile similar to the native peptide and associated with a higher potency and marked prolongation of action in vivo.

EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk.

In an effort to identify novel biallelically inactivated tumor suppressor genes (TSGs) in sporadic invasive and preinvasive non-small-cell lung cancer (NSCLC) genomes, we applied a comprehensive integrated multiple 'omics' approach to investigate patient-matched, paired NSCLC tumor and non-malignant parenchymal tissues. By surveying lung tumor genomes for genes concomitantly inactivated within individual tumors by multiple mechanisms, and by the frequency of disruption in tumors across multiple cohorts, we have identified a putative lung cancer TSG, Eyes Absent 4 (EYA4). EYA4 is frequently and concomitantly deleted, hypermethylated and underexpressed in multiple independent lung tumor data sets, in both major NSCLC subtypes and in the earliest stages of lung cancer. We found that decreased EYA4 expression is not only associated with poor survival in sporadic lung cancers but also that EYA4 single-nucleotide polymorphisms are associated with increased familial cancer risk, consistent with EYA4s proximity to the previously reported lung cancer susceptibility locus on 6q. Functionally, we found that EYA4 displays TSG-like properties with a role in modulating apoptosis and DNA repair. Cross-examination of EYA4 expression across multiple tumor types suggests a cell-type-specific tumorigenic role for EYA4, consistent with a tumor suppressor function in cancers of epithelial origin. This work shows a clear role for EYA4 as a putative TSG in NSCLC.

Pharmacological characterization of the bifunctional opioid ligand H-Dmt-Tic-Gly-NH-Bzl (UFP-505).

BACKGROUND: While producing good-quality analgesia, µ-opioid (MOP) receptor activation produces a number of side-effects including tolerance. Simultaneous blockade of δ-opioid (DOP) receptors has been shown to reduce tolerance to morphine. Here, we characterize a prototype bifunctional opioid H-Dmt-Tic-Gly-NH-Bzl (UFP-505). METHODS: We measured receptor binding affinity in Chinese hamster ovary (CHO) cells expressing recombinant human MOP, DOP, k-opioid (KOP), nociceptin/orphanin (NOP) receptors. For activation, we measured the binding of GTPγ(35)S to membranes from CHO(hMOP), CHO(hDOP), rat cerebrocortex, and rat spinal cord. In addition, we assessed 'end organ' responses in the guinea pig ileum and mouse vas deferens. RESULTS: UFP-505 bound to CHO(hMOP) and CHO(hDOP) with (binding affinity) pK(i) values of 7.79 and 9.82, respectively. There was a weak interaction at KOP and NOP (pK(i) 6.29 and 5.86). At CHO(hMOP), UFP-505 stimulated GTPγ(35)S binding with potency (pEC(50)) of 6.37 and in CHO(hDOP) reversed the effects of a DOP agonist with affinity (pK(b)) of 9.81 (in agreement with pK(i) at DOP). UFP-505 also stimulated GTPγ(35)S binding in rat cerebrocortex and spinal cord with pEC(50) values of 6.11-6.53. In the guinea pig ileum (MOP-rich preparation), UFP-505 inhibited contractility with pEC(50) of 7.50 and in the vas deferens (DOP-rich preparation) reversed the effects of a DOP agonist with an affinity (pA(2)) of 9.15. CONCLUSIONS: We have shown in a range of preparations and assays that UFP-505 behaves as a potent MOP agonist and DOP antagonist; a MOP/DOP bifunctional opioid. Further studies in dual expression systems and whole animals with this prototype are warranted.

National Institute of Academic Anaesthesia research priority setting exercise.

BACKGROUND: Formal research priority setting is a recognized way of identifying important clinical research questions and promoting these as topics for commissioned research. This paper describes a research priority setting exercise conducted by the National Institute of Academic Anaesthesia (NIAA). METHODS: Possible research questions were identified from a questionnaire sent to holders of the Final Fellowship in Anaesthesia in Great Britain and Ireland and to lay representatives. The responses to the first questionnaire were collated to produce a list of potential research questions which were then sent to the same constituency for scoring. The results of this scoring process were considered by an expert panel and statements of research need generated for selected questions. The questions from the first round were also reviewed with the help of representatives of NIHR Evaluation, Trials and Studies Coordinating Centre (NETSCC). RESULTS: For the first questionnaire, 308 responses with 447 suggestions for research were received. A total of 15 questions were included in the second questionnaire, for which 2226 responses were received. The expert panel identified five questions for prioritization. A further nine were identified from discussions with representatives of NETSCC. CONCLUSIONS: A total of 14 research priorities were identified by the exercise, two of which have been submitted to the NIHR Health Technology Assessment (HTA) programme as statements of research need. Potential funding streams for the remaining questions are being sought. We discuss some implications of this exercise for research strategy in the speciality.

Opioid receptor subtypes: fact or artifact?

There is a vast amount of pharmacological evidence favouring the existence of multiple subtypes of opioid receptors. In addition to the primary classification of µ (mu: MOP), δ (delta: DOP), κ (kappa: KOP) receptors, and the nociceptin/orphanin FQ peptide receptor (NOP), various groups have further classified the pharmacological µ into µ(1-3), the δ into δ(1-2)/δ(complexed/non-complexed), and the κ into κ(1-3). From an anaesthetic perspective, the suggestions that µ(1) produced analgesia and µ(2) produced respiratory depression are particularly important. However, subsequent to the formal identification of the primary opioid receptors (MOP/DOP/KOP/NOP) by cloning and the use of this information to produce knockout animals, evidence for these additional subtypes is lacking. Indeed, knockout of a single gene (and hence receptor) results in a loss of all function associated with that receptor. In the case of MOP knockout, analgesia and respiratory depression is lost. This suggests that further sub-classification of the primary types is unwise. So how can the wealth of pharmacological data be reconciled with new molecular information? In addition to some simple misclassification (κ(3) is probably NOP), there are several possibilities which include: (i) alternate splicing of a common gene product, (ii) receptor dimerization, (iii) interaction of a common gene product with other receptors/signalling molecules, or (iv) a combination of (i)-(iii). Assigning variations in ligand activity (pharmacological subtypes) to one or more of these molecular suggestions represents an interesting challenge for future opioid research.

Persistent postoperative pain: where are we now?

There has been considerable interest and controversy around persistent postoperative pain for several years. Most of the available data arise from studies with methodological problems (especially its definition in terms of duration, severity, and effect on quality of life and function); however, more recent investigations have begun to address these issues. Although the quoted incidence varies considerably, analysis of the most conservative data shows that there is no doubt that persistent postoperative pain is a significant clinical problem and a burden to those who suffer from it. There is a wealth of literature describing factors associated with increased likelihood of persistent postoperative pain. Although it is difficult to be precise, it is clear that psychosocial factors probably play a role in some situations and that significant preoperative pain, severe immediate postoperative pain, and nerve damage are often good predictors. There are some data indicating that the incidence and severity of persistent postoperative pain can be reduced by special perioperative interventions; however, as yet, the evidence is not compelling and consistent. A reliable prevention strategy is not yet emerging from the published literature and considerably more work is required to deliver this.

Simultaneous targeting of multiple opioid receptors: a strategy to improve side-effect profile.

Opioid receptors are currently classified as mu (mu: mOP), delta (delta: dOP), kappa (kappa: kOP) with a fourth related non-classical opioid receptor for nociceptin/orphainin FQ, NOP. Morphine is the current gold standard analgesic acting at MOP receptors but produces a range of variably troublesome side-effects, in particular tolerance. There is now good laboratory evidence to suggest that blocking DOP while activating MOP produces analgesia (or antinociception) without the development of tolerance. Simultaneous targeting of MOP and DOP can be accomplished by: (i) co-administering two selective drugs, (ii) administering one non-selective drug, or (iii) designing a single drug that specifically targets both receptors; a bivalent ligand. Bivalent ligands generally contain two active centres or pharmacophores that are variably separated by a chemical spacer and there are several interesting examples in the literature. For example linking the MOP agonist oxymorphone to the DOP antagonist naltrindole produces a MOP/DOP bivalent ligand that should produce analgesia with reduced tolerance. The type of response/selectivity produced depends on the pharmacophore combination (e.g. oxymorphone and naltrindole as above) and the space between them. Production and evaluation of bivalent ligands is an emerging field in drug design and for anaesthesia, analgesics that are designed not to be highly selective morphine-like (MOP) ligands represents a new avenue for the production of useful drugs for chronic (and in particular cancer) pain.

Nociceptin and urotensin-II concentrations in critically ill patients with sepsis.

BACKGROUND: The systemic inflammatory response to infection (sepsis) involves widespread organ dysfunction, including changes in immune modulation, cardiovascular derangements, and neural activation. Two neuropeptide/receptor systems, nociceptin/orphanin FQ (N/OFQ) which acts at the non-classical opioid receptor NOP and urotensin-II (U-II) which acts at the urotensin receptor (UT), have been implicated in neural, immune, and cardiovascular system function. In this study, we make measurements of these peptides in critically ill patients. METHODS: Plasma samples from 21 critically ill patients with sepsis were collected over four consecutive days. Plasma N/OFQ and U-II concentrations were determined by radioimmunoassay and compared with biochemical and clinical markers of illness severity, including serum creatinine, bilirubin, platelet and white cell counts, admission APACHE II and serial SOFA scores. RESULTS: Median (inter-quartile range) admission plasma N/OFQ concentrations in sepsis were higher in patients who died within 30 days (n=4) compared with survivors (n=17); 3.0 (2.5-5.0) vs 1.0 (1.0-2.5) pg ml(-1) (P=0.028). Plasma N/OFQ concentrations were increased in a subgroup of five patients who had undergone major gastrointestinal surgery. There were no significant changes in plasma U-II concentrations. There were no correlations between plasma U-II and N/OFQ concentrations and markers of illness severity and organ system dysfunction. CONCLUSIONS: Plasma N/OFQ concentrations were increased in critically ill patients with sepsis who had undergone major gastrointestinal surgery and in patients who subsequently died. Further work is required to clarify the significance of plasma N/OFQ concentrations in sepsis.

Effects of receptor density on Nociceptin/OrphaninFQ peptide receptor desensitisation: studies using the ecdysone inducible expression system.

Pretreatment of the G-protein coupled nociceptin receptor (NOP) with nociceptin/orphaninFQ (N/OFQ) produces desensitisation. The influences of receptor expression and genomic effects are largely unknown. We have used an ecdysone-inducible NOP expression system in a CHO line (CHO INDhNOP) to examine the effects of N/OFQ pretreatment upon receptor density, GTPgamma[35S] binding, cAMP formation and NOP-mRNA. CHO(INDhNOP) induced with 5 and 10 microM PonasteroneA (PonA) for 20 h produced NOP densities (Bmax) of 194 and 473 fmol. mg(-1) protein, respectively. This was accompanied by decreased NOP mRNA. The lower Bmax is typical of the central nervous system. Pretreatment with 1 microM N/OFQ significantly (p < 0.05) reduced Bmax at 5 and 10 microM PonA to 100 and 196 fmol. mg(-1) protein, respectively. There was no change in binding affinity. Along with the reduction in Bmax), potency and efficacy for N/OFQ-stimulated GTPgamma[35S] binding were also reduced (5 microM PonA: pEC50-control = 8.55 +/- 0.06, pretreated = 7.88 +/- 0.07; Emax-control = 3.52 +/- 0.43, pretreated = 2.48 +/- 0.10; 10 microM PonA: pEC50-control = 8.41 +/- 0.18, pretreated = 7.76 +/- 0.03; Emax-control = 5.07 +/- 0.17, pretreated = 3.38 +/- 0.19). For inhibition of cAMP formation, there was a reduction in potency (5 microM PonA: pEC50-control = 9.78 +/- 0.08, pretreated = 8.92 +/- 0.13; 10 microM PonA: pEC50-control = 9.99 +/- 0.07, pretreated = 9.04 +/- 0.14), but there was no reduction in efficacy. In addition, there were 39 and 31% reductions in NOP mRNA at 5 and 10 microM PonA, respectively, but these measurements were made following concurrent N/OFQ challenge and PonA induction. In CHO INDhNOP, we have shown a reduction in cell surface receptor numbers and a reduction in functional coupling after N/OFQ pretreatment. This was observed at pseudo-physiological and supraphysiological receptor densities. Moreover, we also report a reduction in NOP mRNA, but further studies are needed which include 'pulsing' PonA and desensitizing following wash-out.

In vitro pharmacological characterisation of a novel cyclic nociceptin/orphanin FQ analogue c[Cys(7,10)]N/OFQ(1-13)NH (2).

Nociceptin/orphanin FQ (N/OFQ) is the endogenous 17 amino acid peptide ligand for the G(i)-protein-coupled N/OFQ receptor (NOP). In an attempt to improve the metabolic stability of N/OFQ, we have produced a truncated cyclic analogue with cysteine residues at positions 7 and 10, c[Cys(7,10)]N/OFQ(1-13)NH(2) (c[Cys(7,10)]). c[Cys(7,10)], the template N/OFQ(1-13)NH(2) and N/OFQ displaced the binding of [(3)H]N/OFQ to Chinese hamster ovary cells expressing recombinant human NOP (CHO(hNOP)) with pK ( i ) values of 9.98, 9.83 and 9.18, respectively. In addition, c[Cys(7,10)], N/OFQ(1-13)NH(2) and N/OFQ stimulated the binding of guanosine triphosphate gamma [(35)S] to CHO(hNOP) cells with pEC(50)/E (max) (stimulation factor) of 9.16/5.5, 9.11/4.9 and 8.35/5.5, respectively. c[Cys(7,10)], N/OFQ(1-13)NH(2) and N/OFQ inhibited forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation with pEC(50) values of 10.08, 10.11 and 9.78, respectively. All ligands produced complete inhibition of cAMP formation. In both functional assays, c[Cys(7,10)] was a full agonist. In a series of metabolism experiments, incubation of 1 nM c[Cys(7,10)], N/OFQ(1-13)NH(2) and N/OFQ with a rat brain homogenate produced a time-dependent loss of peptide that was greatest for the native peptide N/OFQ. Amidation in N/OFQ(1-13)NH(2) produced some metabolic protection, but this was not significantly improved by further inclusion of c[Cys(7,10)]. In summary, c[Cys(7,10)] is a high-affinity, high-potency full agonist of the NOP receptor. However, we were unable to demonstrate clear metabolic protection.