Antidepressant Effect of Intracerebroventricularly Administered Deltorphin Analogs in the Mouse Tail Suspension Test.

Several studies have proposed δ opioid receptors as influential targets for developing novel antidepressants. Deltorphin (DLT) and deltorphin II (DLT-II) have high affinity and selectivity for δ opioid receptors; thus, it is likely that DLT analogs possess antidepressant-like effects. Based on this, we evaluated the effects of four DLT analogs (DLT-related synthetic peptides) on immobility behavior in the tail suspension test. Intracerebroventricular administration of DLT or [N-isobutyl-Gly6]DLT in mice significantly decreased immobile behavior. However, administration of DLT did not affect locomotor activity, whereas that of [N-isobutyl-Gly6]DLT significantly increased locomotion in mice. The effect of the shortened immobility time following DLT administration was counteracted by the administration of the selective δ1 opioid receptor antagonist 7-benzylidenenaltrexone, but not by the selective δ2 opioid receptor antagonist naltriben. These findings suggest that DLT has an antidepressant-like effect by activating the central δ1 opioid receptor in mice.

Intracerebroventricular Administration of Dermorphin-Dynorphin Analogs Producing Antidepressant-Like Effects through Activation of µ1- and κ-Opioid Receptors in Mice.

The opioid system in the central nervous system regulates depressive-like behavior in animals. Opioid receptors and their endogenous ligands have been focused on as novel therapeutic targets for depression. We synthesized dermorphin (DRM)-dynorphin (DYN) analogs (DRM-DYN001-004) using the message-address concept concerning interactions with opioid receptors. It has previously been reported that DRM-DYN001, 003, and 004 have shown high affinities for µ- and κ-opioid receptors, whereas all analogs had a lower affinity for the δ-opioid receptor than for other receptors using a receptor binding assay. However, it remains unknown whether these analogs show antidepressant-like effects in mice. We examined the effects of DRM-DYN analogs on the duration of immobile behavior in a tail suspension test. Intracerebroventricular administration of DRM-DYN001 in mice shortened the duration of immobile behavior, but did not affect locomotion. The DRM-DYN001-induced antidepressant-like effect was inhibited by co-administration of naloxone (non-selective opioid receptor antagonist), naloxonazine (selective µ1-opioid receptor antagonist), or nor-BNI (κ-opioid receptor antagonist), but not naltrindole (δ-opioid receptor antagonist). These data suggest that DRM-DYN001 exerts an antidepressant-like effect via activation of the central µ1- and κ-opioid receptors in mice and may represent a new lead peptide for further investigation for the development of novel therapeutic approaches for depression.

Endomorphin analogues with mixed µ-opioid (MOP) receptor agonism/δ-opioid (DOP) receptor antagonism and lacking ß-arrestin2 recruitment activity.

Analogues of endomorphin (Dmt-Pro-Xaa-Xaa-NH2) modified at position 4 or at positions 4 and 3, and tripeptides (Dmt-Pro-Xaa-NH2) modified at position 3, with various phenylalanine analogues (Xaa=Trp, 1-Nal, 2-Nal, Tmp, Dmp, Dmt) were synthesized and their effects on in vitro opioid activity were investigated. Most of the peptides exhibited high µ-opioid (MOP) receptor binding affinity (KiMOP=0.13-0.81nM), modest MOP-selectivity (Kiδ-opioid (DOP)/KiMOP=3.5-316), and potent functional MOP agonism (GPI, IC50=0.274-249nM) without DOP and κ-opioid (KOP) receptor agonism. Among them, compounds 7 (Dmt-Pro-Tmp-Tmp-NH2) and 9 (Dmt-Pro-1-Nal-NH2) were opioids with potent mixed MOP receptor agonism/DOP receptor antagonism and devoid of ß-arrestin2 recruitment activity. They may offer a unique template for the discovery of potent analgesics that produce less respiratory depression, less gastrointestinal dysfunction and that have a lower propensity to induce tolerance and dependence compared with morphine.

Relationship between structure and P-glycoprotein inhibitory activity of dimeric peptides related to the Dmt-Tic pharmacophore.

To develop novel inhibitors of P-glycoprotein (P-gp), dimeric peptides related to an opioid peptide containing the Dmt-Tic pharmacophore were synthesized and their P-gp inhibitory activities were analyzed. Of the 30 analogs synthesized, N(α),N(ε)-[(CH(3))(2)Mle-Tic](2)Lys-NH(2) and its D-Lys analog were found to exhibit potent P-gp inhibitory activity, twice that of verapamil, in doxorubicin-resistant K562 cells. Structure-activity studies indicated that the correct hydrophobicity and spacer length between two aromatic rings are important structural elements in this series of analogs for inhibition of P-gp.

Role of 2',6'-dimethyl-l-tyrosine (Dmt) in some opioid lead compounds.

Here we evaluated how the interchange of the amino acids 2',6'-dimethyl-L-tyrosine (Dmt), 2',6'-difluoro-L-tyrosine (Dft), and tyrosine in position 1 can affect the pharmacological characterization of some reference opioid peptides and pseudopeptides. Generally, Dft and Tyr provide analogues with a similar pharmacological profile, despite different pK(a) values. Dmt/Tyr(Dft) replacement gives activity changes depending on the reference opioid in which the modification was made. Whereas, H-Dmt-Tic-Asp *-Bid is a potent and selective delta agonist (MVD, IC(50)=0.12nM); H-Dft-Tic-Asp *-Bid and H-Tyr-Tic-Asp *-Bid are potent and selective delta antagonists (pA(2)=8.95 and 8.85, respectively). When these amino acids are employed in the synthesis of deltorphin B and its Dmt(1) and Dft(1) analogues, the three compounds maintain a very similar delta agonism (MVD, IC(50) 0.32-0.53 nM) with a decrease in selectivity relative to the Dmt(1) analogue. In the less selective H-Dmt-Tic-Gly *-Bid the replacement of Dmt with Dft and Tyr retains the delta agonism but with a decrease in potency. Antagonists containing the Dmt-Tic pharmacophore do not support the exchange of Dmt with Dft or Tyr.

Synthesis of a potent and selective (18)F-labeled delta-opioid receptor antagonist derived from the Dmt-Tic pharmacophore for positron emission tomography imaging.

Identification and pharmacological characterization of two new selective delta-opioid receptor antagonists, derived from the Dmt-Tic pharmacophore, of potential utility in positron emission tomography (PET) imaging are described. On the basis of its high delta selectivity, H-Dmt-Tic--Lys(Z)-OH (reference compound 1) is a useful starting point for the synthesis of (18)F-labeled compounds prepared by the coupling of N-succinimidyl 4-[ (18)F]fluorobenzoate ([(18)F]SFB) with Boc-Dmt-Tic--Lys(Z)-OH under slightly basic conditions at 37 degrees C for 15 min, deprotection with TFA, and HPLC purification. The total synthesis time was 120 min, and the decay-corrected radiochemical yield of [(18)F]- 1 was about 25-30% ( n = 5) starting from [(18)F]SFB ( n = 5) with an effective specific activity about 46 GBq/micromol. In vitro autoradiography studies showed prominent uptake of [ (18)F]- 1 in the striatum and cortex with significant blocking by 1 and UFP-501 (selective delta-opioid receptor antagonist), suggesting high specific binding of [(18)F]- 1 to delta-opioid receptors. Noninvasive microPET imaging studies revealed the absence of [(18)F]- 1 in rat brain, since it fails to cross the blood-brain barrier. This study demonstrates the suitability of [ (18)F]- 1 for imaging peripheral delta-opioid receptors.

Role of benzimidazole (Bid) in the delta-opioid agonist pseudopeptide H-Dmt-Tic-NH-CH(2)-Bid (UFP-502).

H-Dmt-Tic-NH-CH(2)-Bid (UFP-502) was the first delta-opioid agonist prepared from the Dmt-Tic pharmacophore. It showed interesting pharmacological properties, such as stimulation of mRNA BDNF expression and antidepression. To evaluate the importance of 1H-benzimidazol-2-yl (Bid) in the induction of delta-agonism, it was substituted by similar heterocycles: The substitution of NH(1) by O or S transforms the reference delta-agonist into delta-antagonists. Phenyl ring of benzimidazole is not important for delta-agonism; in fact 1H-imidazole-2-yl retains delta-agonist activity.

Bifunctional [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues substituted at position 3 with alkylated phenylalanine derivatives yield potent mixed mu-agonist/delta-antagonist and dual mu-agonist/delta-agonist opioid ligands.

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) and [Dmt1]EM-2 (Dmt = 2',6'-dimethyl-l-tyrosine) analogues, containing alkylated Phe3 derivatives, 2'-monomethyl (2, 2'), 3',5'- and 2',6'-dimethyl (3, 3', and 4', respectively), 2',4',6'-trimethyl (6, 6'), 2'-ethyl-6'-methyl (7, 7'), and 2'-isopropyl-6'-methyl (8, 8') groups or Dmt (5, 5'), had the following characteristics: (i) [Xaa3]EM-2 analogues exhibited improved mu- and delta-opioid receptor affinities. The latter, however, were inconsequential (Kidelta = 491-3451 nM). (ii) [Dmt1,Xaa3]EM-2 analogues enhanced mu- and delta-opioid receptor affinities (Kimu = 0.069-0.32 nM; Kidelta = 1.83-99.8 nM) without kappa-opioid receptor interaction. (iii) There were elevated mu-bioactivity (IC50 = 0.12-14.4 nM) and abolished delta-agonism (IC50 > 10 muM in 2', 3', 4', 5', 6'), although 4' and 6' demonstrated a potent mixed mu-agonism/delta-antagonism (for 4', IC50mu = 0.12 and pA2 = 8.15; for 6', IC50mu = 0.21 nM and pA2 = 9.05) and 7' was a dual mu-agonist/delta-agonist (IC50mu = 0.17 nM; IC50delta = 0.51 nM).

Synthesis of opioidmimetics, 3-[H-Dmt-NH(CH(2))(m)]-6-[H-Dmt-NH(CH(2))(n)]-2(1H)-pyrazinones, and studies on structure-activity relationships.

Opioidmimetics containing 3-[H-Dmt-NH-(CH(2))(m)]-6-[H-Dmt-NH-(CH(2))(n)]-2(1H)-pyrazinone symmetric (m = n, 1-4) (1 - 4) and asymmetric (m, n = 1 - 4) aliphatic chains (5 - 16) were synthesized using dipeptidyl chloromethylketone intermediates. They had high mu-affinity (K(i)mu = 0.021 - 2.94 nM), delta-affinity (K(i)delta = 1.06 - 152.6 nM), and mu selectivity (K(i)delta/K(i)mu = 14 - 3,126). The opioidmimetics (1 - 16) exhibited mu agonism in proportion to their mu-receptor affinity. delta-Agonism was essentially lacking in the compounds except (4) and (16), and (1) and (2) indicated weak delta antagonism (pA(2) = 6.47 and 6.56, respectively). The data verify that a specific length of aliphatic linker is required between the Dmt pharmacophore and the pyrazinone ring to produce unique mu-opioid receptor ligands.

Effect of lysine at C-terminus of the Dmt-Tic opioid pharmacophore.

Substitution of Gly with side-chain-protected or unprotected Lys in lead compounds containing the opioid pharmacophore Dmt-Tic [H-Dmt-Tic-Gly-NH-CH(2)-Ph, mu agonist/delta antagonist; H-Dmt-Tic-Gly-NH-Ph, mu agonist/delta agonist; and H-Dmt-Tic-NH-CH(2)-Bid, delta agonist (Bid = 1H-benzimidazole-2-yl)] yielded a new series of compounds endowed with distinct pharmacological activities. Compounds (1-10) included high delta- (Ki(delta) = 0.068-0.64 nM) and mu-opioid affinities (Ki(mu) = 0.13-5.50 nM), with a bioactivity that ranged from mu-opioid agonism {10, H-Dmt-Tic-NH-CH[(CH2)4-NH2]-Bid (IC50 GPI = 39.7 nM)} to a selective mu-opioid antagonist [3, H-Dmt-Tic-Lys-NH-CH2-Ph (pA2(mu) = 7.96)] and a selective delta-opioid antagonist [5, H-Dmt-Tic-Lys(Ac)-NH-Ph (pA2(delta) = 12.0)]. The presence of a Lys linker provides new lead compounds in the formation of opioid peptidomimetics containing the Dmt-Tic pharmacophore with distinct agonist and/or antagonist properties.

Potent in vivo antinociception and opioid receptor preference of the novel analogue [Dmt1]endomorphin-1.

[Dmt1]Endomorphin-1 is a novel analogue of the potent mu-opioid agonist endomorphin-1. Given the physiological role of endomorphin-1 in vivo, this compound was investigated to determine if the antinociception occurred through systemic, supraspinal or in a combination of both neuronal pathways. This compound exhibited a potent dose-dependent effect intracerebroventricularly in both spinal and supraspinal regions, and was blocked by opioid antagonist naloxone, which verified the involvement of opioid receptors. Specific opioid antagonists characterized the apparent receptor type: beta-funaltrexamine (mu1/mu2-irreversible antagonist) equally inhibited spinal- and central-mediated antinociception; on the other hand, naloxonazine (mu1-subtype) was ineffective in both neural pathways and naltrindole (delta-selective antagonist) partially (26%), though not significantly, blocked only the spinal-mediated antinociception. Therefore, spinal antinociception was primarily triggered by mu2-subtypes without involvement of mu1-opioid receptors; however, although a slight enhancement of antinociception by delta-receptors cannot be completely ruled out since functional bioactivity indicated mixed mu-agonism/delta-antagonism. In terms of the CNS action, [Dmt1]endomorphin-1 appears to act through mu2-opioid receptor subtypes.

Development of potent mu-opioid receptor ligands using unique tyrosine analogues of endomorphin-2.

Six analogues of tyrosine, which contained alkyl groups at positions 2', 3', and 6', either singly or in combination on the tyramine ring, were investigated for their effect on the opioid activity of [Xaa(1)]endomorphin-2 (EM-2). The opioid analogues displayed the following characteristics: (i) high mu-opioid receptor affinity [K(i)(mu) = 0.063-2.29 nM] with selectivity [K(i)(delta)/K(i)(mu)] ranging from 46 to 5347; (ii) potent functional mu-opioid agonism [GPI assay (IC(50) = 0.623-0.924 nM)] and with a correlation between delta-opioid receptor affinities and functional bioactivity using MVD; (iii) intracerebroventricular administration of [Dmt(1)]- (14) and [Det(1)]EM-2 (10) produced a dose-response antinociception in mice, with the former analogue more active than the latter; and (iv) a marked shift occurred from the trans-orientation at the Tyr(1)-Pro(2) bond to a cis-conformer compared to that observed previously with [Dmt(1)]EM-2 (14) (Okada et al. Bioorg. Med. Chem. 2003, 11, 1983-1984) except [Mmt(1)]EM-2 (7). The active profile of the [Xaa(1)]EM-2 analogues indicated that significant modifications on the tyramine ring are possible while high biological activity is maintained.

Potent Dmt-Tic pharmacophoric delta- and mu-opioid receptor antagonists.

A series of dimeric Dmt-Tic (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) analogues (8-14, 18-22) were covalently linked through diaminoalkane and symmetric or asymmetric 3,6-diaminoalkyl-2(1H)-pyrazinone moieties. All the compounds exhibited high affinity for both delta-opioid receptors [Ki(delta) = 0.06-1.53 nM] and mu-opioid receptors [Ki(mu) = 1.37-5.72 nM], resulting in moderate delta-receptor selectivity [Ki(mu)/Ki(delta) = 3-46]. Regardless of the type of linker between the Dmt-Tic pharmacophores, delta-opioid-mediated antagonism was extraordinarily high in all analogues (pA2 = 10.42-11.28), while in vitro agonism (MVD and GPI bioassays) was essentially absent (ca. 3 to >10 microM). While an unmodified N-terminus (9, 13, 18) revealed weak mu-opioid antagonism (pA2 = 6.78-6.99), N,N'-dimethylation (21, 22), which negatively impacts on mu-opioid-associated agonism (Balboni et al., Bioorg. Med. Chem. 2003, 11, 5435-5441), markedly enhanced mu-opioid antagonism (pA2 = 8.34 and 7.71 for 21 and 22, respectively) without affecting delta-opioid activity. These data are the first evidence that a single dimeric opioid ligand containing the Dmt-Tic pharmacophore exhibits highly potent delta- and mu-opioid antagonist activities.

Synthesis and antinociceptive activity of cyclic endomorphin-2 and morphiceptin analogs.

Cyclic analogs of the opioid peptides endomorphin-2 and morphiceptin of the type Tyr-X-Phe-Phe-Y-NH2 and Tyr-X-Phe-D-Pro-Y-NH2 (X = Lys or Asp and Y = Lys or Asp), respectively, were synthesized in order to test their structure-activity relationships. Antinociceptive activity of the new analogs was assessed in the hot-plate test after intracerebroventricular administration in mice. The strong analgesic effect was observed for the analogs with Asp in position 2, while the analogs with Lys in the second position were inactive. Antinociception caused by Asp2 analogs was dose-dependent and reversed by the concomitant administration of the universal opioid antagonist naloxone and by the selective kappa antagonist, nor-BNI. However, receptor binding studies revealed poor affinity of all cyclic analogs at the mu-opioid receptor and no affinity at delta- and kappa-opioid receptors. It is most likely that the new cyclic analogs produced their antinociception by the release of dynorphin A, which subsequently acted on the kappa-opioid receptor.

Differentiation of opioid receptor preference by [Dmt1]endomorphin-2-mediated antinociception in the mouse.

The potent opioid [Dmt1]endomorphin-2 (Dmt-Pro-Phe-Phe-NH2) differentiated between the opioid receptor subtypes responsible for the antinociception elicited by endomorphin-2 in mice. Antinociception, induced by the intracerebroventricular administration of [Dmt1]endomorphin-2 and inhibited by various opioid receptor antagonists [naloxone, naltrindole, beta-funaltrexamine, naloxonazine], was determined by the tail-flick (spinal effect) and hot-plate (supraspinal effect) tests. The opioid receptor subtypes involved in [Dmt1]endomorphin-2-induced antinociception differed between these in vivo model paradigms: naloxone (non-specific opioid receptor antagonist) and beta-funaltrexamine (irreversible mu1/mu2-opioid receptor antagonist) blocked antinociception in both tests, although stronger inhibition occurred in the hot-plate than the tail-flick test suggesting involvement of other opioid receptors. Consequently, we applied naloxonazine (mu1-opioid receptor antagonist) that significantly blocked the effect in the hot-plate test and naltrindole (delta-opioid receptor antagonist), which was only effective in the tail-flick test. The data indicated that [Dmt1]endomorphin-2-induced spinal antinociception was primarily mediated by both mu2- and delta-opioid receptors, while a supraspinal mechanism involved only mu1/mu2-subtypes.

Oral bioavailability of a new class of micro-opioid receptor agonists containing 3,6-bis[Dmt-NH(CH(2))(n)]-2(1H)-pyrazinone with central-mediated analgesia.

The inability of opioid peptides to be transported through epithelial membranes in the gastrointestinal tract and pass the blood-brain barrier limits their effectiveness for oral application in an antinociceptive treatment regime. To overcome this limitation, we enhanced the hydrophobicity while maintaining the aqueous solubility properties in a class of opioid-mimetic substances by inclusion of two identical N-termini consisting of Dmt (2',6'-dimethyl-l-tyrosine) coupled to a pyrazinone ring platform by means of alkyl chains to yield the class of 3,6-bis[Dmt-NH-(CH(2))(n)]-2(1H)-pyrazinones. These compounds displayed high micro-opioid receptor affinity (K(i)micro = 0.042-0.115 nM) and selectivity (K(i)delta/K(i)micro = 204-307) and functional micro-opioid receptor agonism (guinea-pig ileum, IC(50) = 1.3-1.9 nM) with little or undetectable bioactivity toward delta-opioid receptors (mouse vas deferens) and produced analgesia in mice in a naloxone reversible manner when administered centrally (intracerebroventricular, i.c.v.) or systemically (subcutaneously and orally). Furthermore, the most potent compound, 3,6-bis(3'-Dmt-aminopropyl)-5-methyl-2(1H)-pyrazinone (7'), lacked functional delta-opioid receptor bioactivity and was 50-63-fold and 18-21-fold more active than morphine by icv administration as measured analgesia using tail-flick (spinal involvement) and hot-plate (supraspinal effect) tests, respectively; the compound ranged from 16 to 63% as potent upon systemic injection. These analgesic effects are many times greater than unmodified opioid peptides. The data open new possibilities for the rational design of potential opioid-mimetic drugs that pass through the epithelium of the gastrointestinal tract and the blood-brain barrier to target brain receptors.

Development of potent bifunctional endomorphin-2 analogues with mixed mu-/delta-opioid agonist and delta-opioid antagonist properties.

The C terminus of endomorphin-2 (EM-2) analogues (Tyr-Pro-Phe-NH-X) was modified with aromatic, heteroaromatic, or aliphatic groups (X = phenethyl,benzyl, phenyl, naphthyl, pyridyl, quinolyl, isoquinolyl, tert-butyl, cyclohexyl, or adamantyl; 3-18) to study their effect on opioid activity. Only 9 (1-naphthyl), 11 (5-quinolyl), 16 (cyclohexyl), and 18 (2-adamantyl) exhibited mu-opioid receptor affinity in the nanomolar range (K(i) = 2.41-6.59 nM), which, however, was 3- to 10-fold less than the parent peptide. Replacement of Tyr(1) by Dmt (2',6'-dimethyl-l-tyrosine) (19-32) exerted profound effects: (i) acquisition of high mu-opioid receptor affinity (K(i) = 0.11-0.52 nM) except 23 (Ph); (ii) presence of potent functional mu-opioid receptor agonism (IC(50) < 1 nM) for 19 ([Dmt(1)]EM-2), 27 (1-naphthyl), 29 (5-quinolyl), and 32 (5-isolquinolyl); (iii) association of weak delta-opioid antagonist activity (pA(2) = 5.41-7.18) except 19 ([Dmt(1)]EM-2), 20 (H), 27 (1-naphthyl), and in particular 29 (5-quinolyl) with its potent delta-agonism (IC(50) = 0.62 nM, pA(2) = 5.88); (iv) production of antinociception after ic administration of 32 (5-isoquinolyl) in mice, a bioactivity absent in the corresponding Tyr(1) analogue (14); and (v) preferential cis orientation (cis/trans = 3:2 to 7:3) at the Dmt-Pro amide bond, in contrast to the Tyr-Pro amide trans orientation (cis/trans = 1:2 to 1:3). Thus, [Dmt(1)]EM-2 analogues with hydrophobic C-terminal extensions provide model compounds with potent mu-opioid receptor bioactivity and dual functional agonism.

Unique high-affinity synthetic mu-opioid receptor agonists with central- and systemic-mediated analgesia.

Unique opioid mimetic substances containing identical N-terminal aromatic residues separated by an unbranched alkyl chain containing two to eight methylene groups were developed. Regardless of the length of interposing alkyl chain, the bis-Tyr and bis-Phe compounds were inactive; however, replacement by a single Dmt (2',6'-dimethyl-L-tyrosine) residue enhanced activity by orders of magnitude. Moreover, the bis-Dmt compounds were another 10-fold more potent with an optimum intra-aromatic ring distance of about four to six methylene units. 1,4-Bis(Dmt-NH)butane (7) had high mu-opioid receptor affinity (K(i) = 0.041 nM) and functional mu-opioid agonist bioactivity (IC(50) = 5.3 nM) with in vivo central (intracerebroventricular) and systemic (subcutaneous) analgesia in mice (1.5- to 2.5-fold greater than and 10-12% relative to morphine, respectively); these activities were reversed by naloxone to the same degree. It appears that the bis-Dmt compounds indiscriminately act as both message and address domains.

2',6'-dimethylphenylalanine: a useful aromatic amino Acid surrogate for tyr or phe residue in opioid peptides.

Two aromatic amino acids, Tyr(1) and Phe(3) or Phe(4), are important structural elements in opioid peptides because they interact with opioid receptors. The usefulness of an artificial amino acid residue 2',6'-dimethylphenylalanine (Dmp) was investigated as an aromatic amino acid surrogate for several opioid peptides, including enkephalin, dermorphin, deltorphin, endomorphin, dynorphin A, and nociceptin peptides. In most peptides, substitutions of Phe(3) by a Dmp residue produced analogs with improved receptor-binding affinity and selectivity, while the same substitution of Phe(4) induced markedly reduced receptor affinity and selectivity. Interestingly, replacement of Tyr(1) by Dmp produced analogs with unexpectedly high affinity or produced only a slight drop in receptor affinity and bioactivity for most peptides. Thus, Dmp is also a useful surrogate for the N-terminal Tyr residue in opioid peptides despite the lack of a phenolic hydroxyl group, which is considered necessary for opioid activity. The Dmp(1)-substituted analogs are superior to 2',6'-dimethyltyrosine (Dmt)(1)-substituted analogs for high receptor selectivity since the latter generally have poor receptor selectivity. Thus, Dmp is very useful as an aromatic amino acid surrogate in opioid peptides and may be useful for developing other novel peptide mimetics with high receptor specificity.

Possible involvement of ß-endorphin in docosahexaenoic acid-induced antinociception.

We have previously demonstrated that the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) has an antinociceptive effect on various pain stimuli in a naloxone-reversible manner. In the present study, the role of the endogenous opioid peptide ß-endorphin in DHA-induced antinociception was examined. DHA-induced antinociception was abolished when mice were pretreated with the µ-opioid receptor antagonist ß-funaltrexamine (ß-FNA) and the δ-opioid receptor antagonist naltrindole, but not by the κ-opioid receptor antagonist nor-binaltorphimine (nor-BNI) in the acetic acid-induced writhing test. In the radioligand binding assay, DHA itself did not have affinity for µ- , δ- or κ-opioid receptors. On the other hand, the pretreatment of anti-ß-endorphin antiserum inhibited DHA-induced antinociception. Furthermore, the intracerebroventricular injection of DHA dose-dependently reduced writhing behavior, and this effect was inhibited by d-Phe-Cys-Tyr-Orn-Thr-Pen-Thr-NH(2) (CTOP) and naltrindole, but not nor-BNI. ß-endorphin-induced antinociception was inhibited by the pretreatment of ß-FNA, but not naltrindole or nor-BNI, and its levels in plasma were increased by DHA treatment. These findings suggest that the induction of antinociception by DHA may partially involve the µ-opioid receptor via the release of ß-endorphin.