beta-funaltrexamine and dermorphin

A series of potent electrophilic affinity labels (IC(50) = 0.1-5 nM) containing either a bromoacetamide or isothiocyanate based on the mu opioid receptor (MOR) selective peptide dermorphin were prepared. All four analogues exhibited wash resistant inhibition of [(3)H]DAMGO binding at subnanomolar to nanomolar concentrations, suggesting that these analogues bind covalently to MOR. To our knowledge, these peptides are the highest affinity peptide-based affinity labels for MOR reported to date.

Topics: Affinity Labels; Amino Acid Sequence; Animals; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Drug Discovery; Opioid Peptides; Receptors, Opioid, mu

The dermorphin-derived peptide [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2)), labels mu-opioid receptors with high affinity and selectivity in receptor binding assays. In mouse, radiant heat tail-flick assay [Dmt(1)]DALDA produced profound spinal and supraspinal analgesia, being approximately 5000- and 100-fold more potent than morphine on a molar basis, respectively. When administered systemically, [Dmt(1)]DALDA was over 200-fold more potent than morphine. Pharmacologically, [Dmt(1)]DALDA was distinct from morphine. [Dmt(1)]DALDA displayed no cross-tolerance to morphine in the model used and it retained supraspinal analgesic activity in morphine-insensitive CXBK mice. Supraspinally, it also differed from morphine in its lack of sensitivity towards naloxonazine. Finally, in antisense mapping studies, [Dmt(1)]DALDA was insensitive to MOR-1 exon probes that reduced morphine analgesia, implying a distinct receptor mechanism of action. Thus, [Dmt(1)]DALDA is an interesting and extraordinarily potent, systemically active peptide analgesic, raising the possibility of novel approaches in the design of clinically useful drugs.

Topics: Analgesics; Animals; Drug Tolerance; Humans; Mice; Morphine; Naloxone; Naltrexone; Narcotic Antagonists; Oligodeoxyribonucleotides, Antisense; Oligopeptides; Opioid Peptides; Pain Measurement; Receptors, Opioid, mu; Time Factors

Neurons in the rostroventromedial medulla (RVM) project to spinal loci where the neurons inhibit or facilitate pain transmission. Abnormal activity of facilitatory processes may thus represent a mechanism of chronic pain. This possibility and the phenotype of RVM cells that might underlie experimental neuropathic pain were investigated. Cells expressing mu-opioid receptors were targeted with a single microinjection of saporin conjugated to the mu-opioid agonist dermorphin; unconjugated saporin and dermorphin were used as controls. RVM dermorphin-saporin, but not dermorphin or saporin, significantly decreased cells expressing mu-opioid receptor transcript. RVM dermorphin, saporin, or dermorphin-saporin did not change baseline hindpaw sensitivity to non-noxious or noxious stimuli. Spinal nerve ligation (SNL) injury in rats pretreated with RVM dermorphin-saporin failed to elicit the expected increase in sensitivity to non-noxious mechanical or noxious thermal stimuli applied to the paw. RVM dermorphin or saporin did not alter SNL-induced experimental pain, and no pretreatment affected the responses of sham-operated groups. This protective effect of dermorphin-saporin against SNL-induced pain was blocked by beta-funaltrexamine, a selective mu-opioid receptor antagonist, indicating specific interaction of dermorphin-saporin with the mu-opioid receptor. RVM microinjection of dermorphin-saporin, but not of dermorphin or saporin, in animals previously undergoing SNL showed a time-related reversal of the SNL-induced experimental pain to preinjury baseline levels. Thus, loss of RVM mu receptor-expressing cells both prevents and reverses experimental neuropathic pain. The data support the hypothesis that inappropriate tonic-descending facilitation may underlie some chronic pain states and offer new possibilities for the design of therapeutic strategies.

Topics: Animals; Behavior, Animal; Brain Stem; Disease Models, Animal; Immunotoxins; Ligation; Male; Medulla Oblongata; Microinjections; N-Glycosyl Hydrolases; Naltrexone; Neuralgia; Neurons; Oligopeptides; Opioid Peptides; Pain Measurement; Physical Stimulation; Plant Proteins; Radioligand Assay; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, Opioid, mu; Recombinant Fusion Proteins; Ribosome Inactivating Proteins, Type 1; Saporins; Spinal Nerves

To examine the role of mu-opioid receptor subtypes, we assessed the antinociceptive effect of H-Tyr-D-Arg-Phe-beta-Ala-OH (TAPA), an analogue of dermorphin N-terminal peptide in mice, using the tail-flick test. Intracerebroventricularly (i.c.v.) or intrathecally (i.t.) injected TAPA produced potent antinociception with tail-flick as a thermal noxious stimulus. The selective mu(1)-opioid receptor antagonist, naloxonazine (35 mg/kg, s.c.), or the selective mu-opioid receptor antagonist, beta-funaltrexamine, 24 h before testing antagonized the antinociceptive effect of i.t. or i.c.v. TAPA on the response to noxious stimuli. Pretreatment with beta-funaltrexamine completely antagonized the antinociception by both i.c.v. and i.t. administered TAPA and [D-Ala(2), Me-Phe(4), Gly(ol)(5)]enkephalin (DAMGO). Especially in the tail-flick test, pretreatment with naloxonazine produced a marked rightward displacement of the i.t. TAPA dose-response curve for antinociception. Though DAMGO is a highly selective mu-opioid receptor agonist, pretreatment with naloxonazine partially blocked the antinociceptive response to DAMGO after i.c.v., but not after i. t. injection. These results indicate that TAPA can act as a highly selective mu(1)-opioid receptor agonist (notable naloxonazine-sensitive receptor agonist) at not only the supraspinal level, but also the spinal level. These data also reveal different antinociceptive mechanisms for DAMGO and for TAPA.

Topics: Analgesics; Analgesics, Opioid; Animals; Drug Antagonism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Injections, Intraventricular; Male; Mice; Naloxone; Naltrexone; Narcotic Antagonists; Oligopeptides; Opioid Peptides; Pain Measurement; Receptors, Opioid, mu; Time Factors

1. The effects of intracerebroventricular administration of Tyr-D-Arg-Phe-beta-Ala-NH2 (TAPA), a novel dermorphin analog, on plus-maze learning and spontaneous alternation performance were investigated in mice. 2. The pre- or posttraining or preretention administration of TAPA (0.3-3.0 ng) alone failed to affect transfer latency of plus-maze learning, whereas TAPA (3 ng) produced a significant decrease in percent alternation without affecting total arm entries. 3. beta-Funaltrexamine (5 micrograms) almost completely reversed the TAPA (3 ng)-induced decrease in percentage of alternation. 4. These results suggest that stimulation of mu-opioid receptors disrupts spontaneous alternation performance associated with spatial working memory.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Exploratory Behavior; Injections, Intraventricular; Male; Maze Learning; Memory; Mice; Mice, Inbred Strains; Naltrexone; Narcotic Antagonists; Oligopeptides; Opioid Peptides

The involvement of mu-opioid receptors in memory retrieval was examined in mice by using Tyr-D-Arg-Phe-beta-Ala-NH2 (TAPA), a novel dermorphin analog with high affinity for mu-opioid receptors, and passive avoidance learning. TAPA was intracerebroventricularly administered to mice before retention tests of passive avoidance learning. A 0.3-ng dose of TAPA markedly shortened step-down latency of passive avoidance learning, and the shortening of step-down latency was reversed by treatment with beta-funaltrexamine (5 micrograms), a mu-opioid receptor antagonist, indicating that TAPA (0.3 ng) attenuates memory retrieval. Although the attenuating dose (0.3 ng) of TAPA failed to affect horizontal or vertical locomotor activity, a 3-ng dose of TAPA showed a tendency to decrease vertical locomotor activity. A 30-ng dose of TAPA produced a significant increase in horizontal locomotor activity accompanied by a marked reduction of vertical locomotor activity. TAPA (3 ng) produced a significant increase in step-down latency of passive avoidance learning with lower intensity of electroshock or without electroshock during training. These results suggest that the activation of mu-opioid receptors impairs memory retrieval.

Topics: Amino Acid Sequence; Analgesics, Opioid; Analysis of Variance; Animals; Avoidance Learning; Binding, Competitive; Drug Interactions; Injections, Intraventricular; Male; Mice; Molecular Sequence Data; Motor Activity; Naltrexone; Narcotic Antagonists; Oligopeptides; Opioid Peptides; Receptors, Opioid, mu