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The use of α-amino-γ lactam of Freidinger (Agl) may serve as an impressive method to increase the biological stability of peptides and an appropriate tool to elucidate their structure-activity relationships. The endomorphin-2 (EM-2) and [D-Ala 2 , des-Leu 5 ] enkephalin amide (DAPEA) are two linear opioid tetrapeptides agonists of MOR and MOR/DOR respectively. Herein, we investigated the influence of the incorporation of (R/S)-Agl in position 2 and 3 on the biological profile of the aforementioned products in vitro and in vivo. Receptor radiolabeled displacement and functional assays were used to measure in vitro the binding affinity and receptors activation of the novel analogues. The mouse tail flick and formalin tests allowed to observe their antinociceptive effect in vivo. Data revealed that peptide A2D was able to selectively bind and activate MOR with a potent antinociceptive effect after intracerebroventricular (i.c.v.) administration, performing better than the parent compounds EM-2 and DAPEA. Molecular docking calculations helped us to understand the key role exerted by the Freidinger Agl moiety in A2D for the interaction with the MOR binding pocket., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background/aim: Physical exercise is a state of physiological stress that requires adaptation of the organism to physical activity. Glycogen is an important and essential energy source for muscle contraction. Skeletal muscle and liver are two important glycogen stores, and the energy required to maintain exercise in rodents are provided by destruction of this glycogen depot. In this study, the effects of endogenous opioid peptide antagonism at the central nervous system level on tissue glycogen content after exhaustive exercise were investigated., Materials and Methods: Rats had intracerebroventricularly (icv) received nonspecific opioid peptide receptor antagonist, naloxone (50 μg/10 μL in saline) and δ-opioid receptor-selective antagonist naltrindole (50 μg/10 μL in saline) and then exercised till exhaustion. After exhaustion, skeletal muscle, heart, and liver were excised immediately., Results: Both opioid peptide antagonists decreased glycogen levels in skeletal muscle. Although, in soleus muscle, this decrease was not statistically significant (p > 0.05), in gastrocnemius muscle, it was significant in the icv naloxone administered group compared with control (p < 0.05). Heart glycogen levels increased significantly in both naloxone and naltrindole groups compared to control and sham-operated groups (p < 0.05). Heart glycogen levels were higher in the naloxone group than naltrindole (p < 0.05). Liver glycogen levels were elevated significantly with icv naloxone administration compared with the control group (p < 0.05). Glycogen levels in the naloxone group was also significantly higher than the naltrindole group (p < 0.05)., Conclusion: Our findings indicate that icv administered opioid peptide antagonists may play a role in glycogen metabolism in peripheral tissues such as skeletal muscle, heart, and liver., (This work is licensed under a Creative Commons Attribution 4.0 International License.)

The current study has determined the ability of exendin-4 to augment the antidiabetic benefits of the recently characterised GIP/xenin hybrid, (DAla 2 )GIP/xenin-8-Gln. As such, combined activation of metabolic pathways linked to various gut derived hormones has been shown to exert complementary beneficial metabolic effects in diabetes. (DAla 2 )GIP/xenin-8-Gln and exendin-4 were administered twice daily to high fat fed (HFF) or db/db mice for 28 days and antidiabetic benefits assessed. Persistence of beneficial metabolic effects in HFF mice was also examined. Twice-daily injection of (DAla 2 )GIP/xenin-8-Gln for 28 days in HFF mice significantly reduced energy intake, body weight, circulating glucose, HbA 1c and improved glucose tolerance and insulin sensitivity. Overall pancreatic islet, alpha- and beta-cell areas were reduced, with concurrent reduction in alpha- and beta-cell proliferation that was more apparent in the combined treatment group. Addition of exendin-4 to (DAla 2 )GIP/xenin-8-Gln therapy did not significantly improve metabolic control. Remarkably, beneficial effects were still evident 14 days following complete cessation of peptide administration. Thus, circulating glucose and insulin, HbA 1c concentrations and glucose tolerance were still significantly improved when compared to control HFF mice on day 42, with minimal changes to pancreatic islet architecture. In contrast to HFF mice, combined treatment of db/db mice with (DAla 2 )GIP/xenin-8-Gln plus exendin-4 was required to induce beneficial effects on key metabolic parameters, which were not observed with either treatment alone. This included improvements in glucose tolerance and insulin sensitivity, but no effect on pancreatic architecture. These studies highlight the clear, and persistent, metabolic advantages of sustained activation of GLP-1 receptors, alongside concurrent activation of related GIP and xenin cell signalling pathways, in diabetes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Among androgen-regulated genes, soluble guanylyl cyclase α1 (sGCα1) is significant in promoting the survival and growth of prostate cancer cells and does so independent of nitric oxide (NO) signaling. Peptides were designed targeting sGCα1 to block its pro-cancer functions and one peptide is discussed here. Peptide B-8R killed both androgen-dependent and androgen-independent prostate cancer cells that expressed sGCα1, but not cells that do not express this gene. Peptide B-8R induced apoptosis of prostate cancer cells. Importantly, Peptide B-8R does not affect nor its cytotoxicity depend on NO signaling, despite the fact that it associates with sGCα1, which dimerizes with sGCβ1 to form the sGC enzyme. Just as with a previously studied Peptide A-8R, Peptide B-8R induced elevated levels of reactive oxygen species (ROS) in prostate cancer cells, but using a ROS-sequestering agent showed that ROS was not responsible the cytotoxic activity of Peptide B-8R. Interestingly, Peptide B-8R induced elevated levels of p53 and phosphorylated p38, but neither of these changes is the cause of the peptide's cytotoxicity. Additional drugs were used to alter levels of iron levels in cells and these studies showed that Peptide B-8R activity does not depend on Ferroptosis. Thus, future work will be directed at defining the mechanism of cytotoxic action of Peptide B-8R against prostate cancer cells.

The intergeniculate leaflet (IGL) is a flat thalamic nucleus implicated in the modulation of circadian rhythmicity. In rat, two main GABAergic subpopulations can be distinguished in the IGL: neurons synthesizing neuropeptide Y (NPY), which directly innervates the suprachiasmatic nuclei, and enkephalinergic cells, which connect contralaterally located leaflets. The aim of this study was to evaluate possible effects of inner IGL neurotransmitters on the spontaneous and synaptic activity of IGL neurons. The data presented in this article provide evidence that enkephalin, and not NPY, could act upon the majority of IGL neurons. Moreover, we investigated the type of opioid receptor activated by enkephalin and showed that the μ-receptor is functionally predominant in the IGL. The application of met-enkephalin not only robustly hyperpolarized IGL neurons (both putatively NPY-synthesizing and putatively enkephalinergic neurons), but it also was able to inhibit GABAergic and glutamatergic synaptic transmission. Based on this and previous studies, we hypothesize that IGL enkephalinergic neurons may act as powerful interneurons that inhibit themselves and NPY-synthesizing neurons, also in the contralaterally located IGL., (Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neuropathic pain is relatively less responsive to opioids than other types of pain, which is possibly due to a disrupted opioid system partially caused by the profound microglial cell activation that underlines neuroinflammation. We demonstrated that intrathecally injected biphalin, a dimeric enkephalin analog, diminished symptoms of neuropathy in a preclinical model of neuropathic pain in rats (CCI, chronic constriction injury of the sciatic nerve) at day 12 postinjury. Using primary microglial cell cultures, we revealed that biphalin did not influence cell viability but diminished NO production and expression of Iba1 in LPS-stimulated cells. Biphalin also diminished MOP receptor level, as well as pronociceptive mediators (iNOS, IL-1 β , and IL-18) in an opioid receptor-dependent manner, and it was correlated with diminished p-NF- κ B, p-I κ B, p-p38MAPK, and TRIF levels. Biphalin reduced IL-6, IL-10, TNF α , p-STAT3, and p-ERK1/2 and upregulated SOCS3, TLR4, and MyD88; however, this effect was not reversed by naloxone pretreatment. Our study provides evidence that biphalin diminishes neuropathy symptoms, which might be partially related to reduced pronociceptive mediators released by activated microglia. Biphalin may be a putative drug for future pain therapy, especially for the treatment of neuropathic pain, when the lower analgesic effects of morphine are correlated with profound microglial cell activation.

The dorsal horn (DH) of the spinal cord contains a heterogenous population of neurons that process incoming sensory signals before information ascends to the brain. We have recently characterized calretinin-expressing (CR+) neurons in the DH and shown that they can be divided into excitatory and inhibitory subpopulations. The excitatory population receives high-frequency excitatory synaptic input and expresses delayed firing action potential discharge, whereas the inhibitory population receives weak excitatory drive and exhibits tonic or initial bursting discharge. Here, we characterize inhibitory synaptic input and neuromodulation in the two CR+ populations, in order to determine how each is regulated. We show that excitatory CR+ neurons receive mixed inhibition from GABAergic and glycinergic sources, whereas inhibitory CR+ neurons receive inhibition, which is dominated by glycine. Noradrenaline and serotonin produced robust outward currents in excitatory CR+ neurons, predicting an inhibitory action on these neurons, but neither neuromodulator produced a response in CR+ inhibitory neurons. In contrast, enkephalin (along with selective mu and delta opioid receptor agonists) produced outward currents in inhibitory CR+ neurons, consistent with an inhibitory action but did not affect the excitatory CR+ population. Our findings show that the pharmacology of inhibitory inputs and neuromodulator actions on CR+ cells, along with their excitatory inputs can define these two subpopulations further, and this could be exploited to modulate discrete aspects of sensory processing selectively in the DH., (Copyright © 2016. Published by Elsevier Ltd.)

Background: Endogenous opioid systems may be engaged in the control of arterial pressure (MAP), however, given the risk of addiction, opioid receptor agonists are not used in antihypertensive therapy. We examined cardiovascular effects of biphalin, a potentially non-addictive dimeric enkephalin analog, an agonist of opioid μ and δ receptors., Methods: Biphalin was infused iv at 150μg/kg/h to anesthetized spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Along with MAP and heart rate (HR), renal blood flow (RBF) and iliac blood flow (IBF, a measure of hind limb perfusion) were measured using Transonic probes on renal and iliac artery, respectively. The effects of biphalin were compared with those of intravenous morphine (1.5mg/kg/h)., Results: In two SHR groups biphalin decreased MAP from 143±2 to 130±2 and from 177±4 to 167±3mmHg (p<0.001) while HR did not change or modestly decreased. The renal blood flow (RBF) increased modestly and both renal and hind limb vascular resistances decreased significantly (p<0.001). The responses were blocked by inhibition of peripheral opioid receptors with naloxone methiodide. Unlike in SHR, in WKY rats biphalin did not change MAP or vascular resistances. Morphine infusion decreased MAP in SHR from 169±6 to 150±6mmHg (less decrease in WKY) and significantly decreased RBF and IBF., Conclusion: Since biphalin, a non-addictive synthetic opioid, lowers MAP in SHR, a model of hypertension with pronounced neurogenic component, such analogs might find therapeutic application in human stress-induced hypertensive states. Biphalin's advantage is no associated reduction of renal perfusion., (Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

MrgC receptors are selectively expressed on peripheral and central terminals of small calibre nociceptive fibres. Peptide agonists of the MrgC receptor were reported to modulate nociceptive transmission exerting either pro- or antinociceptive effects depending on site of action and pain model used. Here, we investigated the effect of intraplantar and intrathecal administration of the selective MrgC receptor agonist BAM6-22 on mechanically and electrically evoked nociceptive reflex activity as a uniform readout measure in naïve, monoarthritic and mononeuropathic rats. In naïve rats, intraplantar BAM6-22 enhanced, whereas intrathecal BAM6-22 did not modulate mechanically-evoked nociceptive reflex activity. In monoarthritic rats, intraplantar BAM6-22 had no effect, whereas intrathecal BAM6-22 inhibited mechanically evoked nociceptive reflex activity. In mononeuropathic rats, BAM6-22 reduced mechanically evoked nociceptive reflex activity after both intraplantar and intrathecal administration. BAM6-22 did not modulate electrically evoked nociceptive reflex activity in any condition. Thus, the results of the present investigation confirm and add to previous studies demonstrating that site of action, (patho)-physiological state and stimulus modality determine the effect quality of MrgC receptor agonists. It still needs to be explored how concurrent activation of peripheral and spinal MrgC receptors modulates nociceptive processing under conditions of both acute and chronic pain to evaluate the therapeutic potential of putative small molecule MrgC receptor agonists as innovative analgesics., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We describe the design and synthesis of novel bivalent ligands based on the conjugation of 4-anilidopiperidine derivatives with enkephalin analogues. The design of non-peptide analogues is explored with 5-amino substituted (tetrahydronaphthalen-2yl) methyl containing 4-anilidopiperidine derivatives, while non-peptide-peptide ligands are explored by conjugating the C-terminus of enkephalin analogues (H-Xxx-DAla-Gly-Phe-OH) to the amino group of 4-anilidopiperidine small molecule derivatives with and without a linker. These novel bivalent ligands are evaluated for biological activities at μ and δ opioid receptors. They exhibit very good affinities at μ and δ opioid receptors, and potent agonist activities in MVD and GPI assays. Among these the lead bivalent ligand 17 showed excellent binding affinities (0.1 nM and 0.5 nM) at μ and δ opioid receptors respectively, and was found to have very potent agonist activities in MVD (56 ± 5.9 nM) and GPI (4.6 ± 1.9 nM) assays. In vivo the lead bivalent ligand 17 exhibited a short duration of action (<15 min) comparable to 4-anilidopiperidine derivatives, and moderate analgesic activity. The ligand 17 has limited application against acute pain but may have utility in settings where a highly reversible analgesic is required., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Eribis Peptide 94 (EP94) is an enkephalin analog with cardioprotective properties in ischemia and reperfusion. The aim of the present study was to define the optimal timing and dosing of the administration of EP94 during ischemia and reperfusion in a rat model. 172 anesthetized and mechanically ventilated male Sprague-Dawley rats were randomly assigned to different administration protocols of EP94 and subjected to 30 or 40 min of coronary artery occlusion followed by 2h of reperfusion. EP94 was administered intravenously at different doses and time intervals. Area at risk (AAR) and infarct size (IS) were determined by staining with Evans Blue (EB) and Triphenyl tetrazolium chloride (TTC), respectively. EP94 reduced IS/AAR when administered as a double bolus (0.5 µg/kg per dose), whereas single (1 μg/kg) or triple boluses (0.5 μg/kg per dose) did not confer any protection. Reduction of IS/AAR was of highest magnitude if EP94 was administered 5 and 0 min before the 30 min ischemic period (47% reduction, P<0.05), with declining cardioprotective effect with later administration during ischemia. When EP94 was administered after 15 and 20 min of a 40-min ischemic period, reduction of IS/AAR was of the same magnitude as when given after 5 and 10 min of a 30-min ischemic period. It is concluded that EP94 confers cardioprotection after double bolus administration. The effects are highly dependent on the timing of administration in relation to ischemia and reperfusion. Time of reperfusion from drug administration seems to be more critical than the total duration of ischemia., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: To study the synergism between neuropeptides and lithium ions., Material and Methods: An experimental model of stroke (chronic bilateral occlusion of the common carotid arteries in rats), neuronal culture studies, histomorphological analyses, determination of micronutrient profile of brain substrates were used., Results: A complex of experimental studies revealed that the effect of cerebrolysin is influenced by the synergism between lithium ions and the neuropeptide contentof this drug. Pharmacokinetic synergism promotes the accumulation of lithium in brain tissues during cerebrolysin treatment. The existence of the pharmacokinetic synergism is evident from the potentiation of neuroprotective effects of the drug under the action of lithium ions established in the model of stroke., Conclusion: Lithium ions potentiate neuroprotective effects of cerebrolysin.

Enkephalins are active in regulation of nociception in the body and are key in development of new synthetic peptide analogs that target centrally located opioid receptors. In this study, we investigated the in vivo blood-brain barrier (BBB) penetration behavior and antinociceptive activity of two cyclic enkephalin analogs with a thiourea (CycS) or a N-methyl-guanidine bridge (CycNMe), and their linear counterparts (LinS and LinNMe) in mice, as well as their in vitro metabolic stability. (125)I-LinS had the highest blood-brain clearance (K1=3.46μL/gmin), followed by (125)I-LinNMe, (125)I-CycNMe, and (125)I-CycS (K1=1.64, 0.31, and 0.11μL/gmin, respectively). Also, these peptides had a high metabolic stability (t1/2>1h) in mouse serum and brain homogenate, and half-inhibition constant (Ki) values in the nanomolar range with predominantly μ-opioid receptor selectivity. The positively charged NMe-enkephalins showed a higher antinociceptive activity (LinNMe: 298% and CycNMe: 205%), expressed as molar-dose normalized area under the curve (AUC) relative to morphine, than the neutral S-enkephalins (CycS: 122% and LinS: 130%)., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Biphalin, a mixed MOP/DOP agonist, displays a potent antinociceptive activity in numerous animal models of pain. The aim of the study was to characterize the anti-inflammatory and antinociceptive action of biphalin in the mouse models of colitis. The anti-inflammatory effect of biphalin (5mg/kg, twice daily, i.c. and i.p.) was characterized in a semi-chronic mouse model of colitis, induced by i.c. injection of trinitrobenzenesulfonic acid (TNBS). The antinociceptive action of biphalin (5mg/kg, i.p. and i.c.) in inflamed mice was assessed in mustard oil-induced model of visceral pain and in the hot plate test. In the semi-chronic mouse model of colitis, biphalin i.c. (5mg/kg), but not i.p. improved colitis macroscopic score (2.88±0.19 and 4.99±0.80 units for biphalin and vehicle treated animals, respectively). Biphalin injected i.p. and i.c. (5mg/kg) displayed a potent antinociceptive action in the mustard oil-induced pain test. In the hot plate test, biphalin (5mg/kg, i.p.) produced a potent antinociceptive activity in inflamed mice, suggesting central site of action. Our data suggest that biphalin may become a novel opioid-based analgesic agent in IBD therapy and warrant further investigation of its pharmacological profile., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The aim of this study was to test the efficacy of a single direct injection of viral vector encoding for encephalin to induce a widespread expression of the transgene and potential analgesic effect in trigeminal behavioral pain models in mice. After direct injection of herpes simplex virus type 1 based vectors encoding for human preproenkephalin (SHPE) or the lacZ reporter gene (SHZ.1, control virus) into the trigeminal ganglia in mice, we performed an orofacial formalin test and assessed the cumulative nociceptive behavior at different time points after injection of the viral vectors. We observed an analgesic effect on nociceptive behavior that lasted up to 8 weeks after a single injection of SHPE into the trigeminal ganglia. Control virus-injected animals showed nociceptive behavior similar to naive mice. The analgesic effect of SHPE injection was reversed/attenuated by subcutaneous naloxone injections, a μ-opioid receptor antagonist. SHPE-injected mice also showed normalization in withdrawal latencies upon thermal noxious stimulation of inflamed ears after subdermal complete Freund's adjuvant injection, indicating widespread expression of the transgene. Quantitative immunohistochemistry of trigeminal ganglia showed expression of human preproenkephalin after SHPE injection. Direct injection of viral vectors proved to be useful for exploring the distinct pathophysiology of the trigeminal system and could also be an interesting addition to the pain therapists' armamentarium.

The influence of regulatory peptide type of AFP on the course of experimental ulcer was investigated. It has been shown that activation of apoptosis enhances local necrotic inflammatory reaction, on the one hand, and enables the development of adhesions with on the other.

Objective: Previous in vitro studies have shown that the degradation of [Leu5]enkephalin during incubation with cerebral membrane preparations is almost completely prevented by a mixture of three peptidase inhibitors such as amastatin, captopril, and phosphoramidon. The present in vivo study was performed to examine the antinociceptive effect of [Leu5]enkephalin administered intrathecally under pretreatment with these three peptidase inhibitors., Methods: A tail-flick test was used to determine the nociceptive threshold after administration of [Leu5]enkephalin. The time-course profiles of the latency to flick the tail and the area under the time response curve were evaluated for the antinociceptive action of the drug., Results: The antinociceptive effect of [Leu5]enkephalin administered intrathecally on the tail-flick test was increased more than 100-fold under i.t. pretreatment with three peptidase inhibitors. The antinociceptive effect produced by [Leu5]enkephalin in rats pretreated with any single peptidase inhibitor increased antinociception compared to that with saline. The antinociceptive potency of [Leu5]enkephalin under pretreatment with any combination of two peptidase inhibitors was smaller than that in rats pretreated with three peptidase inhibitors together. These results indicate that any residual single peptidase inactivates significant amounts of [Leu5]enkephalin., Conclusion: The present data, together with those of earlier studies, clearly demonstrate that amastatin-, captopril-, and phosphoramidon-sensitive enzymes play an important role in the inactivation of [Leu5]enkephalin administered intrathecally in rat.

Biphalin is an opioid linear octapeptide, which displays a broad affinity for all opioid receptors (μ, δ and κ), as well as exceptionally high antinociceptive activity. AM 94 is a biphalin analog and a selective agonist at μ and δ opioid receptors. This study investigated the antinociceptive profile of AM 94. All antinociception evaluations were made in adult male rats using the hot-plate test. AM 94 proved to induce greater and longer antinociception compared to biphalin following intracebroventricular (1 nmol/kg) and intravenous administration (1200 nmol/kg) as evaluated by % maximum possible effect (M.P.E.), when administered intracerebroventricularly and intravenously and sustained analgesia up to 210 min. The antinociceptive activities of biphalin and AM 94 were antagonized by naloxone (10mg/kg intraperitoneally). Our data suggest that AM 94 could be regarded as a novel pharmacologically active opioid compound for eliciting potent and sustained analgesia after central and peripheral administration., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The oral use of neuropeptides to treat brain disease is currently not possible because of a combination of poor oral absorption, short plasma half-lives and the blood-brain barrier. Here we demonstrate a strategy for neuropeptide brain delivery via the (a) oral and (b) intravenous routes. The strategy is exemplified by a palmitic ester prodrug of the model drug leucine(5)-enkephalin, encapsulated within chitosan amphiphile nanoparticles. Via the oral route the nanoparticle-prodrug formulation increased the brain drug levels by 67% and significantly increased leucine(5)-enkephalin's antinociceptive activity. The nanoparticles facilitate oral absorption and the prodrug prevents plasma degradation, enabling brain delivery. Via the intravenous route, the nanoparticle-prodrug increases the peptide brain levels by 50% and confers antinociceptive activity on leucine(5)-enkephalin. The nanoparticle-prodrug enables brain delivery by stabilizing the peptide in the plasma although the chitosan amphiphile particles are not transported across the blood-brain barrier per se, and are excreted in the urine.

Eribis peptide 94 (EP 94) is a novel enkephalin derivative that binds with high potency to μ and δ opioid receptors with less affinity for the κ opioid receptor. This compound has recently been shown to produce an acute reduction in myocardial infarct size in the anesthetized pig and rat partially via an endothelial nitric oxide synthase and KATP channel-dependent mechanism. EP 94 also was found to produce a chronic reduction in infarct size 24 hours postdrug administration via the upregulation of inducible nitric oxide synthase in rats. Despite these findings, no data have emerged in which the opioid receptor subtype responsible for cardioprotection has been identified and the site of action, heart, other peripheral organs, or the central nervous system, has not been addressed. In the current study, EP 94, was administered in 2 divided doses (0.5 μg/kg, intravenously) at 5 and 10 minutes into the ischemic period, and the opioid antagonists were administered 10 minutes before the onset of the 30-minute ischemic period. The selective antagonists used were the μ receptor antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2), the δ receptor antagonists naltrindole and BNTX (7-benzylidenenaltrexone), and the κ receptor antagonist nor-BNI (norbinaltorphimine). Surprisingly, only CTOP completely blocked the cardioprotective effect of EP 94, whereas naltrindole, BNTX, and nor-BNI had modest but nonsignificant effects. Because there is controversial evidence suggesting that μ receptors may be absent in the adult rat myocardium, it was hypothesized that the protective effect of EP 94 may be mediated by an action outside the heart, perhaps in the central nervous system. To test this hypothesis, rats were pretreated with the nonselective opioid antagonist, naloxone hydrochloride, which penetrates the blood-brain barrier or naloxone methiodide, the quaternary salt of naloxone hydrochloride, which does not penetrate the blood-brain barrier before EP 94 administration. In support of a central nervous system site of action for EP 94, naloxone hydrochloride completely blocked its cardioprotective effect, whereas naloxone methiodide had no effect. These results suggest that EP 94 reduces infarct size (expressed as a percent of the area at risk) in the rat primarily via activation of central μ opioid receptors.

Eribis peptide 94 (EP 94) is a novel enkephalin analog, thought to interact with the μ- and δ-opioid receptors. The purpose of the present study was to examine the cardioprotective potential of EP 94 in two clinically relevant porcine models of myocardial ischaemia and reperfusion, and to investigate if such an effect is associated with an increased expression of endothelial nitric oxide synthase (eNOS). Forty-one anesthetized pigs underwent 40min of coronary occlusion followed by 4h of reperfusion. In Protocol I, balloon occlusion of the left anterior descending artery was performed with concurrent intravenous administration of (A) vehicle (n=7), (B) EP 94 (1ug/kg) after 5, 12, 19 and 26min of ischaemia (n=4) or (C) EP 94 (1ug/kg) after 26, 33, 40min of ischaemia (n=6). In Protocol II, open-chest pigs were administered (D) vehicle (n=6) or (E) 0.2ug/kg/min of EP 94 (n=6) through an intracoronary infusion into the jeopardized myocardium, started after 30min of ischaemia and maintained for 15min. The hearts were stained and the protein content of eNOS measured. EP 94 reduces infarct size when administered both early and late during ischaemia compared with vehicle (infarct size group A 61.6±2%, group B 50.2±3% and group C 49.2±2%, respectively, P<0.05), as well as when infused intracoronary (infarct size group D 82.2±3.9% and group E 61.2±2.5% respectively, P<0.01). Phosphorylated eNOS Ser(1177) in relation to total eNOS was significantly increased in the group administered EP 94, indicating activation of nitric oxide production., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Many peptides with the potential of therapeutic action for brain disorders are not in clinical use because they are unable to cross the blood-brain barrier (BBB) following peripheral administration. We have developed two potential strategies for the delivery of peptides to the brain and demonstrated their feasibility with enkephalins. In the first approach, designated induced reversible lipophilization, Leu/Met Enkephalins were converted to 9-fluorenylmethoxycarbonyl (Fmoc) derived lipophilic prodrug analogues, which undergo slow, spontaneous hydrolysis under physiological conditions, generating the native agonists. In contrast to Enkephalin, Fmoc-Met-Enkephalin was found to facilitate an analgesic effect following intraperitoneal administration in mice. Fmoc-Leu-Enkephalin was not analgesic. In the second approach, Enkephalin was linked to BBB transport vectors through an Fmoc based linker spacer, forming conjugates that slowly release Enkephalin under physiological conditions. A pronounced antinociceptive response was thus obtained following intraperitoneal administration of either cationized-human serum albumin-Fmoc-Enkephalin or polyethylene glycol(5)-Fmoc-Enkephalin. Derivatives of Enkephalin covalently linked to the same BBB-transport vectors through a stable (nonreversible) chemical bond were not analgesic. In summary, we have demonstrated that lipophilicity can be conferred to hydrophilic peptides to a degree permitting the permeation of the BBB by passive diffusion, without the drawback of agonist inactivation, which is often caused by irreversible derivatization. Similarly, in the second strategy, the conjugation to BBB-permeable vectors overcomes the obstacle of peptide inactivation by releasing the active form in the central nervous system.

Biphalin is an opioid peptide analogue that currently is under clinical development as a new type of site-directed analgesic. In rats, the intrathecal (i.t.) analgesic potency of biphalin is 1000-fold greater than morphine. Such a high activity may reflect this compound's activation of three types of opioid receptors (mu, delta and kappa). NMDA receptors also play an important role in nociceptive processing. Therefore, we investigated in rats whether an NMDA antagonist may influence biphalin-induced antinociception. In the present study, ketamine was chosen because of the widespread safe use of this drug in clinical practice. I.t. application of ketamine alone had relatively little analgesic effect and its excitatory effects limited possible doses of the drug. Co-administration of ketamine with biphalin or morphine produced markedly greater antinociception than biphalin or morphine alone in acute, thermal tail flick testing. These results suggest that NMDA antagonists may be useful potentiators of biphalin analgesia. Thus, to obtain the same spinal antinociceptive effect, lower doses of biphalin or morphine are required when ketamine is co-administered.

This study assessed enkephalin expression induced by intra-articular application of recombinant, enkephalin-encoding herpes virus (HSV-1) and the impact of expression on nociceptive behaviours and synovial lining inflammation in arthritic rats. Replication-conditional HSV-1 recombinant vectors with cDNA encoding preproenkephalin (HSV-ENK), or control transgene beta-galactosidase cDNA (HSV-beta-gal; control) were injected into knee joints with complete Freund's adjuvant (CFA). Joint temperatures, circumferences and nociceptive behaviours were monitored on days 0, 7, 14 and 21 post CFA and vector treatments. Lumbar (L4-6) dorsal root ganglia (DRG) and spinal cords were immunostained for met-enkephalin (met-ENK), beta-gal, HSV-1 proteins and Fos. Joint tissues were immunostained for met-ENK, HSV-1 proteins, and inflammatory mediators Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) and cyclo-oxygenase-2, or stained with haematoxylin and eosin for histopathology. Compared to exuberant synovial hypertrophy and inflammatory cell infiltration seen in arthritic rats treated with CFA only or CFA and HSV-beta-gal, the CFA- and HSV-ENK-treated arthritic rats had: (i) striking preservation of synovial membrane cytoarchitecture with minimal inflammatory cell infiltrates; (ii) significantly improved nociceptive behavioural responses to mechanical and thermal stimuli; (iii) normalized Fos staining in lumbar dorsal horn; and (iv) significantly increased met-ENK staining in ipsilateral synovial tissue, lumbar DRG and spinal cord. The HSV-1 and transgene product expression were confined to ipsilateral lumbar DRG (HSV-1, met-ENK, beta-gal). Only transgene product (met-ENK and beta-gal) was seen in lumbar spinal cord sections. Targeted delivery of enkephalin-encoding HSV-1 vector generated safe, sustained opioid-induced analgesia with protective anti-inflammatory blunting in rat inflammatory arthritis.

Opioid receptors in the spinal cord produce strong analgesia, but the mechanisms controlling their activation by endogenous opioids remain unclear. We have previously shown in spinal cord slices that peptidases preclude mu-opioid receptor (MOR) internalization by opioids. Our present goals were to investigate whether enkephalin-induced analgesia is also precluded by peptidases, and whether it is mediated by MORs or delta-opioid receptors (DORs). Tail-flick analgesia and MOR internalization were measured in rats injected intrathecally with Leu-enkephalin and peptidase inhibitors. Without peptidase inhibitors, Leu-enkephalin produced neither analgesia nor MOR internalization at doses up to 100 nmol, whereas with peptidase inhibitors it produced analgesia at 0.3 nmol and MOR internalization at 1 nmol. Leu-enkephalin was 10 times more potent to produce analgesia than to produce MOR internalization, suggesting that DORs were involved. Selective MOR or DOR antagonists completely blocked the analgesia elicited by 0.3 nmol Leu-enkephalin (a dose that produced little MOR internalization), indicating that it involved these two receptors, possibly by an additive or synergistic interaction. The selective MOR agonist endomorphin-2 produced analgesia even in the presence of a DOR antagonist, but at doses substantially higher than Leu-enkephalin. Unlike Leu-enkephalin, endomorphin-2 had the same potencies to induce analgesia and MOR internalization. We concluded that low doses of enkephalins produce analgesia by activating both MORs and DORs. Analgesia can also be produced exclusively by MORs at higher agonist doses. Since peptidases prevent the activation of spinal opioid receptors by enkephalins, the coincident release of opioids and endogenous peptidase inhibitors may be required for analgesia.

Systemically active, nonpeptidic delta opioid receptor agonists have been shown to produce antidepressant and anxiolytic effects in animal models in rodents. In addition, delta agonists have been shown to increase expression of brain-derived neurotrophic factor (BDNF) mRNA, an effect of some antidepressants, which may be important for the clinical efficacy of antidepressant drugs. The present study examined whether a variety of peptidic delta agonists, DPDPE, JOM-13, a systemically active derivative of DPDPE, deltorphin II, and H-Dmt-Tic-NH-CH2-Bid could produce convulsions and antidepressant-like effects in the forced swim test. In addition, some of these compounds were examined for their influence on BDNF mRNA expression. All four agonists dose-dependently decreased immobility in the forced swim test, indicating an antidepressant-like effect. Only JOM-13 produced convulsions at doses required for antidepressant-like effects. In addition, DPDPE increased BDNF mRNA expression, as measured by in situ hybridization, in the frontal cortex. The antidepressant-like effect of the agonists in the forced swim test and the increase in BDNF mRNA expression produced by DPDPE were blocked by the delta antagonist naltrindole. Therefore, activation of the delta receptor by centrally administered peptidic agonists and intravenously administered JOM-13 produces behavioral antidepressant-like effects without producing convulsions, and some peptidic agonists can increase BDNF mRNA expression, however, not as consistently as the systemically active nonpeptidic agonists.

The effects of bovine adrenal medulla 22 (BAM22), a cleaved product of proenkephalin A, were investigated on the noxious stimulus-evoked expressions of spinal c-fos-like immunoreactivity (FLI). Heat (51 degrees C) applied to the tail evoked FLI predominantly in laminae I-II of the sacral spinal cord. Intrathecal (i.t.) BAM22 at a dose of 7 nmol decreased the expressions of the heat-evoked FLI by 68%, 64% and 56% in laminae I-II, III-IV and V-VI, respectively, and the decrease pattern was comparable to that induced by i.t. morphine (10 mug). Naloxone (1 mg/kg, i.p.) significantly enhanced the heat-evoked FLI in laminae III-VI, prevented the morphine-induced inhibition, and decreased the potencies of BAM22 in laminae I-II and V-VI by 23-40%. Higher dose of naloxone (10 mg/kg, i.p.) also partially reduced the BAM22-induced suppression. Following intraplantar injection of formalin (2.5%), FLI neurons were preferentially distributed not only in laminae I-II but also in laminae III-IV and V-VI of segments L4-L5. Pretreatment with BAM22 (7 nmol, i.t.) reduced the formalin-evoked FLI neurons by 72%, 61% and 58%, in laminae I-II, III-IV and V-VI, respectively. Naloxone (1 mg/kg. i.p.) enhanced the formalin-evoked expressions of FLI in laminae III-VI and decreased the potencies of BAM22 by 22-38% in laminae I-II and V-VI. The present study provided evidence at a cellular level showing that opioid and non-opioid effects of BAM22 on nociceptive processing in acute and persistent pain models were associated with modulation of noxious stimulus-evoked activity of the spinal dorsal horn neurons.

The aim of this study was to investigate the importance of the amino acidic sequence at N-terminal end of certain minimum structure enkephalin-like peptides for the analgesic activity. Different groups of mice or rats were treated with 1) L-tyrosine (i.p. 200 mg/kg), 2) Tyr-Phe (i.t. 0.5 mg/rat), 3) Tyr-Pro-Phe (i.t. 0.5 mg/rat), 4) Gly-Tyr (i.t. 0.5 mg/rat), 5) Tyr-Gly-Gly (i.t. 0.5 mg/rat). Different tests were utilized to evaluate the antinociceptive effect of the substances tested: thermal nociception (hot plate test, plantar test), mechanical nociception (analgesymeter test). Tyr-Pro-Phe, Tyr-Gly-Gly, Tyr-Phe, but not Gly-Tyr, elicited analgesic activity. So, the presumption made in the case of atypical opioid peptides that opioid-like activity in case of peptides presumes a tyrosine residue at the N-terminal sequence, applies for shorter peptides. It appears also that minimal structure brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opioids, can provide better affinity for the opioid receptors and stronger analgesic activity. The inhibition of their analgesic effect by previous administration of naloxone proves that this effect is mediated through the endogenous opioid system.

The present study was carried out in five cats which did not attack the rats spontaneously. Predatory attack on an anaesthetized rat was elicited by electrical stimulation of lateral hypothalamus at a mean current strength of 650 microA. The attack was accompanied by minimal affective display and culminated in neck biting. Microinfusions of DAME (delta-alanine methionine enkephaline) in 500 ng dose in substantia nigra facilitated the predatory attack and there was a significant reduction in the threshold current strength for affective display as well as somatomotor components. Microinfusions of naloxone, an opioid antagonist in 1.0 microg dose when DAME effect was at its peak reversed the facilitatory effects and the threshold returned to the control levels within 10 minutes of naloxone infusion at the same locus. Microinfusions of naloxone alone in similar dosage completely blocked the predatory attack response as indicated by an increase in the threshold current strength for somatomotor as well as affective display components. The somatomotor were completely inhibited and could not be elicited even when the current strength was increased to 1000 microA. Control injections of saline in similar volumes (0.5 microl) failed to produce any response Microinfusions of naloxone in lower dose (250 ng) failed to produce any blocking effect. These findings indicate that hypothalamically elicited predatory attack is facilitated by enkephalinergic mechanisms operating at the midbrain level.

Intraspinal drug delivery, based on the concept of controlling pain by delivering drug to a nociceptive target rich in opioid and other relevant receptors is increasingly used clinically. The therapeutic ratio for opioids or other centrally acting agents is potentially greater if they are administered intrathecally (i.t.) than outside the central nervous system (CNS). The present study was designed with the ultimate goal of formulating a controlled release system for intrathecal analgesia characterized by effectiveness, rapid onset and few side effects for chronic pain control. A biodegradable copolymer poly(L-lactide-co-glycolide) (PLGA) was used to prepare a rod-shaped drug delivery system containing hydromorphone (HM), bupivacaine (BP), both HM and BP, or biphalin (BI). In vitro drug release kinetics of these systems showed a zero-order release rate for HM and BP from PLGA (85:15) rods. Drug-loaded rods were implanted i.t. Control groups received only placebo implants. Measurement of analgesic efficacy was carried out with tail flick and paw-withdrawal tests. In vivo studies showed potent, prolonged analgesia in comparison to controls for all active treatments. Analgesic synergy was observed with HM and BP. With further refinements of drug release rate, these rods may offer a clinically relevant alternative for intrathecal analgesia.

Rheumatoid arthritis is characterized by erosive inflammation of the joints, new bone proliferation, and ankylosis, leading to severely reduced locomotion and intense chronic pain. In a model of this disease, adjuvant-induced polyarthritis in the rat, neurons involved in pain transmission and control undergo plastic changes, especially at the spinal level. These changes affect notably neurons that contain opioids, such as enkephalins deriving from preproenkephalin A (PA) precursor protein. Using recombinant herpes simplex virus containing rat PA cDNA, we enhanced enkephalin synthesis in sensory neurons of polyarthritic rats. This treatment markedly improved locomotion and reduced hyperalgesia. Furthermore, the progression of bone destruction slowed down, which is the most difficult target to reach in the treatment of patients suffering from arthritis. These data demonstrate the therapeutic efficacy of enkephalin overproduction in a model of systemic inflammatory and painful chronic disorder.

We compared the physical dependence liability of biphalin, a dimeric enkephalin analogue that possesses high antinociceptive activity, with that of morphine in equipotent intravenous doses. Naloxone challenge produced severe withdrawal signs after a 5-day infusion of morphine but only minor withdrawal signs after a 5-day biphalin infusion. In a cross-dependence study, biphalin did not suppress body weight loss after morphine withdrawal, but successfully suppressed weight loss after pentazocine withdrawal. These data support consideration of biphalin as a new analgesic with a novel pharmacological profile and minimum dependence liability.

This study evaluated the contribution of supraspinal opioid receptors to gastric mucosal protection in the rat. Intracerebroventricular (i.c.v.) and intracisternal (i.c.) injections of selective delta- [[D-Ala(2),D-Leu(5)]-enkephalin (DADLE), [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE), deltorphin II], selective mu- [[D-Ala(2),Phe(4),Gly(5)-ol]-enkephalin (DAGO)] opioid receptor agonists and beta-endorphin (ligand of both receptor types) produced a dose-dependent inhibition of acidified ethanol-induced gastric mucosal damage. The ED(50) values for beta-endorphin, DAGO, DADLE, deltorphin II, and DPDPE were 3.5, 6.8, 75, 120, and 1100 pmol/rat, respectively, following i.c.v. and 0.8, 9.0, 45, 0.25, and 7 pmol/rat following i.c. injection. The gastroprotective effect of DADLE, deltorphin II, and DPDPE, but not that of DAGO, was inhibited by naltrindole, the selective delta-receptor antagonist. Since the delta(2)-receptor agonist deltorphin II was more potent than the delta(1)-receptor agonist DPDPE, the dominant role of central delta(2)-receptors in gastroprotection might be raised. The site of action for delta-receptor agonists is likely to be the brain stem since the peptides were more potent following i.c. than following i.c.v. administration. The gastroprotective effect was reduced following acute bilateral cervical vagotomy. Moreover, both the nitric-oxide synthase inhibitor N(G)-nitro-L-arginine (3 mg/kg i.v.) and the prostaglandin synthesis inhibitor indomethacin (20 mg/kg p.o.) decreased the protective effect of opioid peptides. The results indicate that 1) activation of supraspinal delta- and mu-opioid receptors induces gastric mucosal protection, 2) integrity of vagal nerve is necessary for the gastroprotective action of opioids, and 3) mucosal nitric oxide and prostaglandins may be involved in the opioid-induced gastroprotection.

A major challenge in the development of sustained-release formulations for protein and peptide drugs is to achieve high drug loading sufficient for prolonged therapeutic effect coupled with a high recovery of the protein/peptide. This challenge has been successfully met in the formulation of several peptide and protein drugs using the DepoFoam, multivesicular lipid-based drug delivery system. DepoFoam technology consists of novel multivesicular liposomes characterized by their unique structure of multiple non-concentric aqueous chambers surrounded by a network of lipid membranes. The objective of this paper is to demonstrate that DepoFoam technology can be used to develop sustained-release formulations of therapeutic proteins and peptides with high loading. DepoFoam formulations of a protein such as insulin, and peptides such as leuprolide, enkephalin and octreotide have been developed and characterized. The data show that these formulations have high drug loading, high encapsulation efficiency, low content of free drug in the suspension, little chemical change in the drug caused by the formulation process, narrow particle size distribution, and spherical particle morphology. Drug release assays conducted in vitro in biological suspending media such as human plasma indicate that these formulations provide sustained release of encapsulated drug over a period from a few days to several weeks, and that the rate of release can be modulated. In vivo pharmacodynamic studies in rats also show a sustained therapeutic effect over a prolonged period. These results demonstrate that the DepoFoam system is capable of efficiently encapsulating therapeutic proteins and peptides and effectively providing controlled delivery of these biologically active macromolecules.

The effects of opioid peptides on the osmotic release of neurohypophyseal hormones, arginine vasotocin (AVT) and mesotocin (MT), were determined in 2-day-old chicks. Experiment 1 examined the effect of a variety of doses of naloxone, an opioid antagonist, on chicks administered isotonic or hypertonic solution. Plasma osmolality in chicks administered hypertonic solution was significantly higher than that in groups administered isotonic solution. None of the doses of naloxone affected plasma osmolality in response to isotonic and hypertonic solution. Plasma levels of AVT increased in hypertonic solution and this response was further enhanced by naloxone injection as the doses increased. The hypertonic solution alone did not affect plasma levels of MT, but additional treatment with naloxone slightly increased plasma levels of MT. Experiment 2 examined the effect of DAMGO ([d-Ala(2), N-Me-Phe(4),Gly-ol]-enkepha lin), a specific mu receptor agonist. Relatively high plasma osmolality caused by hypertonic solution was not affected by additional treatment with DAMGO. Plasma levels of AVT in response to hypertonic solution and to additional treatment with naloxone were reduced by higher doses of DAMGO. Experiment 3 examined the effect of naloxone on chicks administered different concentrations of NaCl. Administration of hypertonic solution resulted in an increase in plasma osmolality and plasma levels of AVT. Naloxone administration enhanced the increase in plasma AVT levels in response to hypertonic solution. Experiment 4 examined the effect of naloxone on different kinds of hypertonic solution, 0.15 M NaCl, 1.5 M NaCl, 2.55 M urea, and 1.95 M sucrose. The increases in plasma osmolality resulting from the administration of the urea and sucrose solutions were the same as those in the chicks injected with 1.5 M NaCl. In sucrose-treated chicks, plasma levels of AVT increased in chicks administered naloxone but not in chicks injected with normal saline. In contrast, no significant changes in plasma levels of AVT were observed in urea treatment with or without naloxone. In Experiments 3 and 4, plasma levels of MT after administration of hypertonic solutions did not change. However, naloxone administration enhanced plasma levels of MT in osmotically stimulated chicks. The results of the present study suggest that opioid peptides attenuate the increase in plasma AVT and MT in hypertonic states., (Copyright 1999 Academic Press.)

In addition to its central actions, morphine has important peripheral effects. To examine peripheral analgesic mechanisms, we developed a topical opioid paradigm in which the tail was immersed in a dimethyl sulfoxide (DMSO) solution containing various drugs. Alone, DMSO was inactive in the tail-flick assay in mice. DMSO solutions containing morphine and peptides such as [D-Ala2,MePhe4, Gly(ol)5]enkephalin (DAMGO) produced a potent, dose-dependent analgesia with the radiant heat tail-flick assay. The actions of the drugs were local. Analgesia was observed only in regions of the tail exposed to the solution and not in more proximal unexposed portions of the tail. Immersion of the tail in a solution containing either 125I-labeled morphine or 125I-labeled DAMGO revealed no detectable uptake of radioactivity into the brain, spinal cord, or blood. In the tail, radioactivity was limited only to the regions actually immersed in the solutions. The topical drugs potentiated systemic agents, similar to the previously established synergy between peripheral and central sites of action. Local tolerance was rapidly produced by repeated daily exposure of the tail to morphine. Topical morphine tolerance was effectively blocked by the N-methyl-D-aspartate (NMDA) antagonist MK801 given either systemically or topically but not intrathecally. The ability of a topical NMDA antagonist to block local morphine tolerance suggests that peripheral NMDA receptors mediate topical morphine tolerance. Morphine was cross-tolerant to DAMGO, but not to morphine-6beta-glucuronide, implying different mechanisms of action. These observations are significant in the design and use of opioids clinically.

The participation of central cholecystokinin-8 (CCK-8) receptors in the modulatory effect of D-Ala2, N-Me-Phe4, Gly5-ol enkephalin (DAGO), a selective mu-opioid receptor agonist, on the spike burst activity of the gastrointestinal tract (rumen, reticulum, antrum, duodenum, colon and caecum) in sheep was investigated. DAGO was infused intracerebroventricularly (i.c.v.) at doses of 0.1-1 microg/kg body weight (BW). It was shown that DAGO significantly inhibited myoelectrical activity of the wall of the forestomachs, abomasum and colon but stimulated this activity in the duodenum (rate of myoelectrical migrant complex-MMC). The effects of DAGO were prevented by CCK-8 antagonists (L-364.718 and L-365.260) previously infused at doses of 5-20 microg/kg BW. The results of this present study indicate that central receptors of CCK-8 participated in the modulatory action of an opioid on myoelectrical activity of the gastrointestinal tract in sheep. Furthermore, this result suggests that CCK-8 is released in response to mu-receptor stimulation, because CCK-8 antagonists (L-364.718 and L-365.260) prevented the modulatory action of DAGO on the gastrointestinal motility in sheep.

Previous work indicates that the antianalgesic action of pentobarbital and neurotensin administered intracerebroventricularly in mice arises from activation of a descending system to release cholecystokinin (CCK) in the spinal cord where CCK is known to antagonize morphine analgesia. Spinal dynorphin, like CCK, has an antianalgesic action against intrathecally administered morphine. This dynorphin action is indirect; even though it is initiated in the spinal cord, it requires the involvement of an ascending pathway to the brain and a descending pathway to the spinal cord where an antianalgesic mediator works. The aim of the present investigation was to determine if the antianalgesic action of intrathecal dynorphin A involved spinal CCK. All drugs were administered intrathecally to mice in the tail flick test. Morphine analgesia was inhibited by dynorphin as shown by a rightward shift of the morphine dose-response curve. The effect of dynorphin was eliminated by administration of the CCK receptor antagonists lorglumide and PD135 158. One hour pretreatment with CCK antiserum also eliminated the action of dynorphin. On the other hand, the antianalgesic action of CCK was not affected by dynorphin antiserum. Thus, CCK did not release dynorphin. Both CCK and dynorphin were antianalgesic against DSLET but not DPDPE, delta 2 and delta 1 opioid receptor peptide agonists, respectively. The results suggest that the antianalgesic action of dynorphin occurred through an indirect mechanism ultimately dependent on the action of spinal CCK.

The analgesic effect of three different opioid agonists, DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin), U-50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidynyl)cyclohexyl] benzene-aceramide methane sulphonate), and [D,Pen2-D,Pen5]-enkephalin, which act upon mu, delta and kappa opioid receptors, respectively, was compared in the presence and absence of salmon-calcitonin (s-CT). The analgesic test used was the writhing test in mice. The analgesic effect of the opioids was significantly enhanced by pretreatment of the animals with s-CT intraperitoneally (i.p.) administered. This effect was more evident for the delta and kappa-agonists. The present result suggests that the joint administration of s-CT and opioids may be a useful and interesting alternative in the treatment of painful diseases resistant to other treatments.

The role of mu-opioid receptor in gastrointestinal transit was investigated using mu-opioid receptor knockout mice (MOR-KO). Our result establishes unequivocally that inhibition of GI transit by morphine is a mu-opioid receptor mediated function. In addition, we show that neither delta nor kappa receptor agonist given supraspinally or peripherally are able to inhibit GI transit in MOR-KO animals. It was interesting to observe that basal GI motility was lower in MOR-KO (-/-) compared to heterozygous (+/-) and wild type (+/+) animals., (Copyright 1998 Elsevier Science B.V.)

Morphine has been shown to decrease proliferative responses of rat T-lymphocytes via central opioid receptors, however, the specific receptor subtype(s) mediating this effect have not been established. To determine the potential role of central mu opioid receptors in morphine-mediated suppression of T-lymphocyte proliferation, 20 nmol/2 microliters of either morphine sulfate or DAMGO (mu-selective agonist) were administered into the lateral ventricle of freely moving Sprague-Dawley rats. Lymphocyte proliferative response to the polyclonal T cell mitogen concanavalin A (ConA), changes in splenic natural killer cell (NK) cytolytic activity, activation of the hypothalamic-pituitary-adrenal (HPA) axis and antinociception (tail-flick latency) were examined. Results indicated that like morphine, DAMGO decreased blood lymphocyte proliferative responses by 80% and elevated both tail-flick latency and plasma corticosterone when compared to saline-treated animals. The proliferation response of lymphocytes from the spleen or thymus and splenic NK cell activity were not significantly altered by either morphine or DAMGO treatment. The effects of DAMGO were determined to be dose-dependent and completely antagonized by naltrexone pretreatment. Central administration of DPDPE (delta-selective agonist) and U-50488 (kappa-selective agonist) produced between 40-50% suppression of blood lymphocyte proliferation responses only at a dose five times greater (100 nmol) than DAMGO treatment, without altering antinociception or activation of the HPA axis. To determine the central opioid receptor subtype(s) involved in the effects of morphine, selective opioid antagonists were microinjected into the lateral ventricle prior to morphine treatment (6 mg/kg, s.c.). CTOP (mu-selective antagonist, 5 micrograms/2 microliters) completely blocked the effects of morphine on all parameters measured, however, naltrindole (delta-selective antagonist, 2 micrograms/2 microliters) or nor-binaltorphimine (kappa-selective antagonist, 73.5 micrograms/2 microliters) failed to block the effects of morphine. Collectively, these results provide evidence that morphine acts primarily through central mu receptors to modulate peripheral blood lymphocyte proliferation responses. Further, the antinociception and blood lymphocyte effects show greater sensitivity to opioids than either natural killer cell cytolytic activity or activation of the HPA axis.

Objective: Our purpose was to assess the effects of an intravenous dose of a highly selective mu-(DALDA) and delta-(DPDPE) opioid peptide to determine which class of peptide has the best clinical potential., Study Design: Chronically instrumented pregnant ewes received a 0.3 mg/kg intravenous bolus of each peptide with and without opioid receptor blockade by means of a randomized prospective design., Results: Intravenous DALDA produced only mild hypertension and a loss of heart rate variability, whereas DPDPE produced respiratory depression, maternal hypertension, and a fall in heart rate in both mother and fetus. Uterine blood flow, oxygen uptake, and glucose uptake were unchanged with both drugs. The effects of DALDA but not DPDPE were reversed by opioid receptor blockade., Conclusion: The delta-selective agonist had multiple nonopioid adverse effects, whereas the mu-selective agonist was well tolerated by the pregnant ewe, suggesting that mu-selective agonists have better potential for clinical use as an analgesic in pregnancy.

The effects of acute and chronic administration of cocaine on the antinociception and tolerance to the antinociceptive actions of mu-(morphine), kappa-(U-50,488H), and delta-([D-Pen2,D-Pen5]enkephalin; DPDPE), opioid receptor agonists were determined in male Swiss-Webster mice. Intraperitoneal injection of 40 mg/kg of cocaine by itself produced weak antinociceptive response as measured by the tail-fick test but the lower doses were ineffective. Administration of morphine (10 mg/kg, SC), U-50,488H (25 mg/kg, IP) or DPDPE (10 microg/mouse, ICV) produced antinociception in mice. Cocaine (20 mg/kg) potentiated the antinociceptive action of morphine and DPDPE but had no effect on U-50,488H-induced antinociception. Administration of morphine (20 mg/kg, SC), U-50,488H (25 mg/kg, IP) or DPDPE (20 microg/mouse, ICV) twice a day for 4 days resulted in the development of tolerance to their antinociceptive actions. Tolerance to the antinociceptive actions of morphine and U-50,488H was inhibited by concurrent treatment with 20 or 40 mg/kg doses of cocaine; however, tolerance to the antinociceptive action of DPDPE was not modified by cocaine. It is concluded that cocaine selectively potentiates the antinociceptive action of mu- and delta- but not of the kappa-opioid receptor agonist. On the other hand, cocaine inhibits the development of tolerance to the antinociceptive actions of mu- and kappa- but not of delta-opioid receptor agonists in mice.

Background: Hypothermic cardioplegia provides adequate myocellular protection, yet stunning and dysfunction remain significant problems. Interestingly, the subcellular changes of hibernation parallel the altered biology of induced cardiac ischemia, but are well tolerated by hibernating mammalian myocardium. Hibernation induction trigger (HIT) from winter-hibernating animal serum induces hibernation in active animals. Hibernation induction trigger is opiate in nature and is similar to the delta 2 opioids., Methods: To determine whether HIT could improve myocardial recovery following global ischemia, we gave 37 isolated rabbit hearts either standard cardioplegia or cardioplegia containing summer-active woodchuck, hibernating woodchuck, or black bear HIT serum or a delta 2 opioid, D-Ala2-Leu5-enkephalin, before 2 hours of global ischemia., Results: Hibernation induction trigger appeared not to have an active mechanism during ischemia, as all hearts had equal recovery. In contrast, when examining for a preischemia mechanism, 23 additional rabbits received 3 days pretreatment with summer-active woodchuck or HIT hibernating woodchuck or black bear serum, or were preperfused with D-Ala2-Leu5-enkephalin or D-pen2,5-enkephalin, a-delta 1 opioid, again before 2 hours of global ischemia. Postischemic ventricular function, coronary flows, myocardial oxygen consumption, and ultrastructural preservation were all significantly improved with HIT and D-Ala2-Leu5-enkephalin pretreatment., Conclusion: "Natural" HIT protection is superior to standard cardioplegia alone and may have clinical application.

We determined the dose-dependent effects of central mu-opioid receptor stimulation on rates of tissue protein synthesis. Chronically catheterized conscious rats received an intracerebroventricular injection of [D-Ala2, N-Me-Phe4,Gly5-ol]enkephalin (DAGO, 0.5, 2, or 8 nmol/rat) or water (5 microliters) 45 min before determination of protein synthesis by the flooding dose technique. DAGO produced a significant decrease in tissue protein synthesis in liver (57%), spleen (54%), gut mucosa (36%), gut serosa (23%), kidney (48%), gastrocnemius (33%), and plantaris muscle (27%), but it did not alter rates of protein synthesis in the brain, heart, and soleus muscle. DAGO produced an acute dose-dependent respiratory depression 30 min after intracerebroventricular injection; this depression resulted in acidosis, hypoxia, and hypercapnia (pH 7.19 +/- 0.04, arterial partial O2, pressure 44.2 +/- 3.4 Torr, arterial O2 saturation 65.3 +/- 5.5%, and PCO2 66.3 +/- 4.4 Torr). Intracerebroventricular DAGO increased circulating levels of catecholamines, corticosterone, and growth hormone but did not alter those of insulin and insulin-like growth factor I. Significant positive correlations between protein synthesis and pH were observed in the tissues studied (i.e., liver protein synthesis vs. pH, P < 0.0001, r = 0.902; gastrocnemius protein synthesis vs. pH, P < 0.0001, r = 0.830). Our results indicate that mu-receptor stimulation inhibits tissue protein synthesis, and this effect appears to be secondary to respiratory depression and the resulting acidosis and/or hypoxia. Furthermore, our findings suggest differential sensitivity in tissue response to alterations in pH, hypoxia, and stress hormone elevation.

We previously reported that beta-endorphin and morphine administered supraspinally produce antinociception by activating different descending pain-inhibitory systems. To determine the role of spinal calcium channels, calmodulin and calcium/calmodulin-dependent protein kinase II in the production of antinociception induced by morphine, [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO) or beta-endorphin administered supraspinally, the effects of nimodipine (an L-type calcium channel blocker), omega-conotoxin GVIA (an N-type voltage-dependent calcium channel blocker), calmidazolium (a calmodulin antagonist) or KN-62 (a calcium/calmodulin-dependent protein kinase II inhibitor) injected intrathecally (i.t.) on the antinociception induced by morphine, DAMGO or beta-endorphin administered intracerebroventricularly (i.c.v.) were examined in the present study. Antinociception was assessed by the mouse tail-flick test. The i.t. injection of nimodipine (from 0.024 to 2.4 pmol), omega-conotoxin GVIA (from 0.0033 to 0.33 pmol), calmidazolium (from 0.0015 to 0.15 pmol) or KN-62 (from 0.0014 to 0.14 pmol) alone did not affect the basal tail-flick latencies. The i.t. pretreatment of mice with nimodipine, omega-conotoxin GVIA, calmidazolium or KN-62 dose dependently attenuated the inhibition of the tail-flick response induced by beta-endorphin administered i.c.v. However, the inhibition of the tail-flick response induced by morphine or DAMGO administered i.c.v. was not changed by i.t. pretreatment with nimodipine, omega-conotoxin GVIA, calmidazolium or KN-62. The results suggest that spinally located L- and N-type calcium channels, calmodulin and calcium/calmodulin-dependent protein kinase II may be involved in the modulation of antinociception induced by beta-endorphin, but not morphine and DAMGO, administered supraspinally.

The role of dopamine neurotransmission in opioid reward was investigated using a rate-independent measure for determining brain stimulation reward (BSR) thresholds. Intra-accumbens infusions of the mu- and delta-specific peptides, D-Ala2, N-Me-Phe4, Gly-ol5-Enkephalin and D-Pen2, D-Pen5-Enkephalin caused significant lowering of BSR thresholds. The dopamine D1/D2 antagonist, cis-flupenthixol, blocked these effects at a dose that did not significantly alter thresholds when given alone. These data suggest both mu- and delta-opioid potentiation of BSR is dopamine dependent.

Previous results using an amphibian model showed that systemic and spinal administration of opioids selective for mu, delta and kappa-opioid receptors produce analgesia. It is not known whether non-mammalian vertebrates also contain supraspinal sites mediating opioid analgesia. Thus, opioid agonists selective for mu (morphine; fentanyl), delta (DADLE, [D-Ala2, D-Leu5]-enkephalin; DPDPE, [D-Pen2, D-Pen5]-enkephalin) and kappa (U50488, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methanesulfonate; CI977, (5R)-(544alpha,744alpha,845beta)-N-methyl-N-[7-(1-p yrr olidinyl)-1-oxaspiro[4,5]dec-8yl]-4-benzofuranaceta mide++ + monohydrochloride) opioid receptors were tested for analgesia following i.c.v. administration in the Northern grass frog, Rana pipiens. Morphine, administered at 0.3, 1, 3 and 10 nmol/frog, produced a dose-dependent and long-lasting analgesic effect. Concurrent naltrexone (10 nmol) significantly blocked analgesia produced by i.c.v. morphine (10 nmol). ED50 values for the six opioids ranged from 2.0 for morphine to 63.9 nmol for U50488. The rank order of analgesic potency was morphine > DADLE > DPDPE > CI977 > fentanyl > U50488. These results show that supraspinal sites mediate opioid analgesia in amphibians and suggest that mechanisms of supraspinal opioid analgesia may be common to all vertebrates.

The changes in the latency for tail withdrawal in response to noxious heating of the skin induced by microinjection of opioid or serotonergic agonists into the anterior pretectal nucleus (APtN) was studied in rats. The mu-opioid agonist DAMGO (78 and 156 picomol), but not the delta-opioid agonist DADLE (70 and 140 pmol), the kappa-opioid agonist bremazocine (0.24 and 0.48 nanomol) or the sigma-opioid agonist N-allylnormetazocine (0.54 nanomol), produced a dose-dependent antinociceptive effect. The 5-HT1 agonist 5-carboxamidotryptamine (19 and 38 nanomol) and the 5-HT1B agonist, CGS 12066B (1.12 and 2.24 nanomol), but not the non-selective 5-HT agonist m-CPP (41 to 164 nanomol), 5-HT2 agonist alpha-methylserotonin (36 and 72 nanomol) and 5-HT3 agonist 2-methylserotonin (36 and 72 nanomol), produced a dose-dependent antinociceptive effect. These results indicate that the antinociceptive effects of opioid or serotonergic agonists microinjected into the APtN depend on drug interaction with local mu or 5-HT1B receptors, respectively.