deltorphin and Pain

We recently found that PKCε was required for spinal analgesic synergy between two GPCRs, δ opioid receptors and α2 A adrenoceptors, co-located in the same cellular subpopulation. We sought to determine if co-delivery of μ and δ opioid receptor agonists would similarly result in synergy requiring PKCε.. Combinations of μ and δ opioid receptor agonists were co-administered intrathecally by direct lumbar puncture to PKCε-wild-type (PKCε-WT) and -knockout (PKCε-KO) mice. Antinociception was assessed using the hot-water tail-flick assay. Drug interactions were evaluated by isobolographic analysis.. All agonists produced comparable antinociception in both PKCε-WT and PKCε-KO mice. Of 19 agonist combinations that produced analgesic synergy, only 3 required PKCε for a synergistic interaction. In these three combinations, one of the agonists was morphine, although not all combinations involving morphine required PKCε. Morphine + deltorphin II and morphine + deltorphin I required PKCε for synergy, whereas a similar combination, morphine + deltorphin, did not. Additionally, morphine + oxymorphindole required PKCε for synergy, whereas a similar combination, morphine + oxycodindole, did not.. We discovered biased agonism for a specific signalling pathway at the level of spinally co-delivered opioid agonists. As the bias is only revealed by an appropriate ligand combination and cannot be accounted for by a single drug, it is likely that the receptors these agonists act on are interacting with each other. Our results support the existence of μ and δ opioid receptor heteromers at the spinal level in vivo.. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Drug Therapy, Combination; Female; Hot Temperature; Ligands; Male; Mice, Knockout; Morphine; Morpholines; Oligopeptides; Pain; Protein Kinase C-epsilon; Protein Multimerization; Receptors, Opioid, delta; Receptors, Opioid, mu; Spinal Cord

To further understand the relationship between melatonin (MT) and deltorphins (Dels) in pain modulation, two chimeric peptides (Del I-5-methoxytryptamine and Del II-5-methoxytryptamine) both containing 5-methoxytryptamine at the carboxyl-terminal of Dels mimicking MT were designed, synthesized and characterized by tail-flick assay in mice. Results showed that intracerebroventricular (i.c.v.) administration of Del I-5-methoxytryptamine (YaFDVVG-X, X is 5-methoxytryptamine, 5, 50 nmol/kg) or Del II-5-methoxytryptamine (YaFEVVG-X, X is 5-methoxytryptamine, 5, 50 nmol/kg) produced stronger analgesia than deltorphins (Del I or Del II alone), and acting even longer and stronger than cocktails containing Del I or Del II (50 nmol/kg) and MT (50 nmol/kg). Naloxone (i.p., 100 nmol/kg) could totally block the analgesic effects induced by the chimeric peptides, while luzindole (specific antagonist of melatonin receptor, i.p., 250 nmol/kg) could only partially inhibit the effects down to that induced by Dels alone. Interestingly, Del I-5-methoxytryptamine and Del II-5-methoxytryptamine act weaker with δ receptor than Dels in vitro but could induce much longer analgesia through co-activating δ opioid receptor and melatonin receptor.

Topics: 5-Methoxytryptamine; Analgesics; Animals; Male; Melatonin; Mice; Oligopeptides; Opioid Peptides; Pain

Enhancement of peripheral opioid analgesia following tissue injury or inflammation in animal models is well-documented, but clinical results of peripheral opioid therapy remain inconsistent. Previous studies in the central nervous system have shown that co-administration of μ- and δ-opioid receptor agonists can enhance analgesic outcomes; however, less is known about the functional consequences of opioid receptor interactions in the periphery. The present study examines the effects of intraplantar injection of the μ- and δ-opioid receptor agonists, morphine and deltorphin, alone and in combination on behavioral tests of nociception in naïve rats and on potassium-evoked release of CGRP from sciatic nerves of naïve rats. Neither drug alone affected nociceptive behaviors or CGRP release. Two separate measures of mechanical nociceptive sensitivity remained unchanged after co-administration of the two drugs. In contrast, when deltorphin was co-injected with morphine, dose-dependent and peripherally restricted increases in paw withdrawal latencies to radiant heat were observed. Similarly, concentration-dependent inhibition of CGRP release was observed when deltorphin and morphine were administered in sequence prior to potassium stimulation. However, no inhibition was observed when morphine was administered prior to deltorphin. All combined opioid effects were blocked by co-application of antagonists. Deltorphin exposure also enhanced the in vivo and in vitro effects of another μ-opioid receptor agonist, DAMGO. Together, these results suggest that under normal conditions, δ-opioid receptor agonists enhance the effect of μ-opioid receptor agonists in the periphery, and local co-administration of δ- and μ-opioid receptor agonists may improve results of peripheral opioid therapy for the treatment of pain.

Topics: Analgesics, Opioid; Analysis of Variance; Animals; Calcitonin Gene-Related Peptide; Dose-Response Relationship, Drug; Drug Therapy, Combination; Male; Morphine; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu; Sciatic Nerve

The peptide nociceptin/orphanin FQ (N/OFQ) and its receptor ORL-1, also designated opioid receptor 4 (OP(4)) are involved in the modulation of nociception. Using OP(4)-knockout mice, we have studied their response following opioid receptor stimulation and under neuropathic conditions.In vas deferens from wild-type and OP(4)-knockout mice, DAMGO (mu/OP(3) agonist), deltorphine II (delta/OP(1) agonist) and (-)-U-50488 (kappa/OP(2) agonist) induced similar concentration-dependent inhibition of electrically-evoked contractions. Naloxone and naltrindole (delta/OP(1) antagonists) shifted the curves of DAMGO (pA(2)=8.6) and deltorphine II (pA(2)=10.2) to the right, in each group. In the hot-plate assay, N/OFQ (10 nmol per mouse, i.t.) increased baseline latencies two-fold in wild-type mice while morphine (10mg/kg, s.c.), deltorphine II (10 nmol per mouse, i.c.v.) and dynorphin A (20 nmol per mouse, i.c.v.) increased hot-plate latencies by about four- to five-fold with no difference observed between wild-type and knockout mice. Furthermore, no change was evident in the development of the neuropathic condition due to chronic constriction injury (CCI) of the sciatic nerve, after both thermal and mechanical stimulation. Altogether these results suggest that the presence of OP(4) receptor is not crucial for (1) the development of either acute or neuropathic nociceptive responses, and for (2) the regulation of full receptor-mediated responses to opioid agonists, even though compensatory mechanisms could not be excluded.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Dynorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Female; Male; Mice; Mice, Knockout; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Nociceptin; Nociceptin Receptor; Oligopeptides; Opioid Peptides; Pain; Receptors, Opioid; Time Factors; Vas Deferens

1. When administered subcutaneously HS-599, a new didehydroderivative of buprenorphine (18,19-dehydrobuprenorphine), produced a long-lasting antinociceptive response in rats. Its potency exceeded twice that of buprenorphine. In the tail-flick test it acted as a full agonist but in the plantar test only as a partial agonist. Whereas the mu-opioid antagonists naloxone and naltrexone antagonized HS-599 antinociception the delta-opioid antagonist naltrindole and the kappa-opioid antagonist nor-binaltorphimine did not. 2. Unlike buprenorphine and morphine, HS-599 never induced conditioned place-preference in rats. 3. In radioligand binding assays, compared with buprenorphine HS-599 had 3 fold higher mu-opioid receptor affinity but lower delta- and kappa-opioid receptor affinity. 4. In isolated guinea-pig ileum preparations, HS-599 only partially inhibited the electrically-stimulated contraction, acting as a partial opioid agonist. When tested against the mu-opioid receptor agonist dermorphin, it behaved as a non-equilibrium antagonist. Conversely, in mouse vas deferens (rich in delta-opioid receptors) and rabbit vas deferens preparations (rich in kappa-opioid receptors) HS-599 acted as a pure equilibrium antagonist, shifting the log-concentration-response curves of the delta-opioid agonist deltorphin I and the kappa-opioid agonist U-69593 to the right. 5. In conclusion, HS-599 is a novel buprenorphine derivative with higher affinity, selectivity and potency than the parent compound, for mu-opioid receptors. It produces intense and long-lasting antinociception and does not induce place-preference in rats.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Benzeneacetamides; Binding, Competitive; Buprenorphine; Dose-Response Relationship, Drug; Guinea Pigs; Ileum; Male; Membranes; Mice; Morphine; Oligopeptides; Pain; Pyrrolidines; Rabbits; Rats; Rats, Wistar; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Vas Deferens

kappa-opioid receptor agonists (kappa-ORAs) have been shown to modulate visceral nociception through an interaction with a peripheral, possibly novel, kappa-opioid-like receptor. We used in the present experiments an antisense strategy to further explore the hypothesis that kappa-ORA effects in the colon are produced at a site different from the cloned kappa-opioid receptor (KOR). An antisense oligodeoxynucleotide (ODN) to the cloned rat KOR was administered intrathecally (12.5 microg, twice daily for 4 d) to specifically knock-down the cloned KOR. Efficacy of the KOR antisense ODN treatment was behaviorally evaluated by assessing the antinociceptive effects of peripherally administered kappa- (EMD 61, 753 and U 69,593), mu- (DAMGO) and delta- (deltorphin) ORAs in the formalin test. Intrathecal antisense, but not mismatch ODN blocked the actions of EMD 61,753 and U 69,593 without affecting the actions of DAMGO or deltorphin; a complete recovery of antinociceptive actions of the kappa-ORA EMD 61,753 was observed 10 d after the termination of antisense ODN treatment. In contrast, the ability of EMD 61,753 to dose-dependently attenuate responses of pelvic nerve afferent fibers to noxious colonic distension was unaffected in the same rats in which the antisense ODN effectively knocked-down the KOR as assessed in the formalin test. Additionally, Western blot analysis demonstrated a significant downregulation of KOR protein in the L4-S1 dorsal root ganglia of antisense, but not mismatch ODN-treated rats. The present results support the existence of a non-kappa-opioid receptor site of action localized in the colon.

Topics: Acetamides; Analgesics, Opioid; Animals; Colon; Disinfectants; Electrophysiology; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Formaldehyde; Ganglia, Spinal; Male; Nociceptors; Oligonucleotides, Antisense; Oligopeptides; Pain; Physical Stimulation; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, kappa; Rectum; Visceral Afferents

The solid phase procedure, based on the Fmoc chemistry, was used to prepare some opioid deltorphin (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, DEL C) and dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DER) analogues in which a D-glucopyranosyl moiety is beta-O-glycosidically linked to a Thr4 or Thr7 side chain. Their activities were determined in binding studies based on displacement of mu- and delta-receptor selective radiolabels from rat brain membrane synaptosomes, in guinea pig ileum and rabbit jejenum bioassays, and, in vivo, by a mouse tail-flick test after intracerebroventricular (icv) and subcutaneous (sc) administrations. The glyco analogues modified at position 4 displayed low opioid properties, while Thr7-glycosylated peptides retained high delta- or mu-selectivity and remarkable activity in vivo. In particular, as systemic antinociceptive agents, the latter glucoside-bearing compounds were more potent than the parent unglycosylated peptide counterparts, showing a high blood to brain rate of influx which may be due to the glucose transporter GLUT-1.

Topics: Amino Acid Sequence; Analgesics, Opioid; Animals; Binding, Competitive; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Glycopeptides; Guinea Pigs; Ileum; In Vitro Techniques; Indicators and Reagents; Jejunum; Kinetics; Male; Mice; Muscle Contraction; Muscle, Smooth; Oligopeptides; Opioid Peptides; Pain; Rabbits; Rats; Rats, Sprague-Dawley; Receptors, Opioid; Structure-Activity Relationship; Synaptosomes

A series of deltorphin analogues was prepared by solid-phase peptide synthesis. Their opioid activity was evaluated in rat opiatic assay and their conformation was determined by two-dimension Nuclear Magnetic Resonance Spectroscopy. The analogues containing D-alanine acid at position 2 were much more potent in the assay than their corresponding isomers containing L-alanine acid at this position. The conformational analysis on NMR study in DMSO showed that C-terminal tetrapeptides of both deltorphin II and its L-alanine analog might form a 3(10) helix, which confirms that the substitution of D-amino acid at position 2 decreased the opioid activity.

Topics: Analgesics; Animals; Chromatography, High Pressure Liquid; Dimethyl Sulfoxide; Indicators and Reagents; Magnetic Resonance Spectroscopy; Male; Models, Molecular; Oligopeptides; Pain; Protein Conformation; Rats; Rats, Wistar; Spectrometry, Mass, Fast Atom Bombardment; Structure-Activity Relationship

Microinjection of [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO) into either the periaqueductal gray (PAG) or the rostral ventral medulla (RVM) elicits analgesia in the tailflick assay in the rat. Co-administration of DAMGO into both regions together results in a profound synergistic interaction similar to that we previously reported with morphine. U50,488H and DPDPE are inactive when given into either region. [D-Ala2,Glu4]Deltorphin (deltorphin), on the other hand, elicits an analgesic response, although the maximal response is less than than mu agonists. Co-administration of DAMGO into one region with deltorphin in the other also results in a significant synergy. However, co-administration of DAMGO and deltorphin together in the same region gives only additive effects. These results confirm the existence of mu/mu synergy between the PAG and RVM. kappa 1 and delta 1 agents are inactive, but the delta 2 agonist deltorphin is active in both regions. Our results indicate the presence of mu/delta 2 synergy between the PAG and RVM which appears to involve interactions of pathways rather than receptor interactions at the cellular level.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Amino Acid Sequence; Analgesics; Animals; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Male; Medulla Oblongata; Molecular Sequence Data; Oligopeptides; Pain; Periaqueductal Gray; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Receptors, Opioid, mu

Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 is an extraordinarily potent and highly mu-selective opioid heptapeptide isolated from amphibian skin. It is unique among peptides synthesized by animal cells in having an amino acid residue in the D-configuration. At least two different preprodermorphin cDNAs were cloned from skin of Phylomedusa sauvagei; their predicted amino acid sequences contained four to five homologous repeats of 35 amino acids, each repeat including one copy of the dermorphin progenitor sequence. Tyr-Ala-Phe-Gly-Tyr-Pro-Ser-Gly, flanked by Lys-Arg at the amino end and by Glu-Ala-Lys-Lys at the carboxyl end [Science (Wash. D. C.) 238:200-202 (1987)]. The D-Ala in position 2 in dermorphin is encoded by a usual Ala codon in the precursor sequence. Of the two prodermorphin molecules, one has a dermorphin copy replaced with a distinct heptapeptide same processing signals. Assuming the same pathway as for the release of dermorphin, processing of this precursor may yield, beside dermorphin, a copy of a new peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2. We have synthetized this peptide together with its (L-Met2)-counterpart and evaluated their respective opioid receptor selectivity in the mouse vas deferens and guinea pig ileum assays and in rat brain membrane binding assays. Overall, the data collected demonstrate that the putative prodermorphin product Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 named dermenkephalin, behaves as a potent delta opioid agonist exhibiting high affinity and high selectivity for the delta opioid receptor. Prodermorphin, thus, offers a surprising example of an opioid biosynthetic precursor that might simultaneously generate highly potent and fully selective agonists for the mu- (morphine) and the delta (enkephalin) opioid receptors, respectively. In addition, because dermenkephalin has no structural features in common with the sequence of all the hitherto known opioid peptides, it should be a useful tool for identifying conformational determinants for high affinity and selective binding of opioids to the delta receptor.

Topics: Amino Acid Sequence; Analgesia; Animals; Binding, Competitive; Brain; Cell Membrane; Cerebral Ventricles; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; Injections, Intraventricular; Kinetics; Male; Mice; Oligopeptides; Opioid Peptides; Pain; Rats; Receptors, Opioid; Receptors, Opioid, delta