Selective MOR activity of DAPEA and Endomorphin-2 analogues containing a (R)-γ-Freidinger lactam in position two.

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-Ala2, des-Leu5] 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.

Effects of intracerebroventricularly administered opioid peptide antagonists on tissue glycogen levels in rats after exercise

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.

A GIP/xenin hybrid in combination with exendin-4 improves metabolic status in db/db diabetic mice and promotes enduring antidiabetic benefits in high fat fed mice.

The current study has determined the ability of exendin-4 to augment the antidiabetic benefits of the recently characterised GIP/xenin hybrid, (DAla2)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. (DAla2)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 (DAla2)GIP/xenin-8-Gln for 28 days in HFF mice significantly reduced energy intake, body weight, circulating glucose, HbA1c 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 (DAla2)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, HbA1c 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 (DAla2)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.

Peptide B targets soluble guanylyl cyclase α1 and kills prostate cancer cells.

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 sGCß1 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.

Biphalin, a Dimeric Enkephalin, Alleviates LPS-Induced Activation in Rat Primary Microglial Cultures in Opioid Receptor-Dependent and Receptor-Independent Manners.

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-1ß, and IL-18) in an opioid receptor-dependent manner, and it was correlated with diminished p-NF-κB, p-IκB, p-p38MAPK, and TRIF levels. Biphalin reduced IL-6, IL-10, TNFα, 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.

Enkephalin and neuropeptide-Y interaction in the intergeniculate leaflet network, a part of the mammalian biological clock.

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.

Distinct forms of synaptic inhibition and neuromodulation regulate calretinin-positive neuron excitability in the spinal cord dorsal horn.

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.

An antihypertensive opioid: Biphalin, a synthetic non-addictive enkephalin analog decreases blood pressure in spontaneously hypertensive rats.

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.

Effects of the mas-related gene (Mrg) C receptor agonist BAM6-22 on nociceptive reflex activity in naive, monoarthritic and mononeuropathic rats after intraplantar and intrathecal administration.

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.

Design and synthesis of novel bivalent ligands (MOR and DOR) by conjugation of enkephalin analogues with 4-anilidopiperidine derivatives.

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.

[Pharmacokinetic and pharmacodynamic synergism between neuropeptides and lithium in the neurotrophic and neuroprotective action of cerebrolysin].

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.

Cardioprotection of the enkephalin analog Eribis peptide 94 in a rat model of ischemia and reperfusion is highly dependent on dosing regimen and timing of administration.

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.

Blood-brain transfer and antinociception of linear and cyclic N-methyl-guanidine and thiourea-enkephalins.

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%).

Anti-inflammatory and antinociceptive action of the dimeric enkephalin peptide biphalin in the mouse model of colitis: new potential treatment of abdominal pain associated with inflammatory bowel diseases.

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.

Gene therapy for trigeminal pain in mice.

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.

[Lymphocyte apoptosis enhancement by the synthetic peptide E in experimental ulcer].

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.

Increase in antinociceptive effect of [leu5]enkephalin after intrathecal administration of mixture of three peptidase inhibitors.

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.

A prodrug nanoparticle approach for the oral delivery of a hydrophilic peptide, leucine(5)-enkephalin, to the brain.

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.

The analgesic activity of biphalin and its analog AM 94 in rats.

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.

Eribis peptide 94 reduces infarct size in rat hearts via activation of centrally located µ opioid receptors.

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.