Endomorphin analogues with mixed μ-opioid (MOP) receptor agonism/δ-opioid (DOP) receptor antagonism and lacking β-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.

DOR activation inhibits anoxic/ischemic Na+ influx through Na+ channels via PKC mechanisms in the cortex.

Activation of delta-opioid receptors (DOR) is neuroprotective against hypoxic/ischemic injury in the cortex, which is at least partially related to its action against hypoxic/ischemic disruption of ionic homeostasis that triggers neuronal injury. Na(+) influx through TTX-sensitive voltage-gated Na(+) channels may be a main mechanism for hypoxia-induced disruption of K(+) homeostasis, with DOR activation attenuating the disruption of ionic homeostasis by targeting voltage-gated Na(+) channels. In the present study we examined the role of DOR in the regulation of Na(+) influx in anoxia and simulated ischemia (oxygen-glucose deprivation) as well as the effect of DOR activation on the Na(+) influx induced by a Na(+) channel opener without anoxic/ischemic stress and explored a potential PKC mechanism underlying the DOR action.

Engineering endomorphin drugs: state of the art.

Introduction: Although endomorphins-1 (EM-1; H-Tyr-Pro-Phe-Trp-NH(2)) and -2 (EM-2; H-Tyr-Pro-Phe-Phe-NH(2)) are primarily considered agonists for the μ-opioid receptor (MOR), systematic alterations to specific residues provided antagonists and ligands with mixed μ/δ-opioid properties, suitable for application to health-related topics. While the application of endomorphins as antinociceptive agents and numerous biological endpoints were experimentally delineated in laboratory animals and in vitro, clinical use is currently absent. However, structural alterations provide enhanced stability; formation of MOR antagonists or mixed and dual μ/δ-acting ligands could find considerable therapeutic potential.

Developmental potential for endomorphin opioidmimetic drugs.

Morphine, which is agonist for μ-opioid receptors, has been used as an anti-pain drug for millennia. The opiate antagonists, naloxone and naltrexone, derived from morphine, were employed for drug addiction and alcohol abuse. However, these exogenous agonists and antagonists exhibit numerous and unacceptable side effects.

Current research on opioid receptor function.

The use of opioid analgesics has a long history in clinical settings, although the comprehensive action of opioid receptors is still less understood. Nonetheless, recent studies have generated fresh insights into opioid receptor-mediated functions and their underlying mechanisms. Three major opioid receptors (μ-opioid receptor, MOR; δ-opioid receptor, DOR; and κ-opioid receptor, KOR) have been cloned in many species.

Opioid bifunctional ligands from morphine and the opioid pharmacophore Dmt-Tic.

Bifunctional ligands containing an ester linkage between morphine and the δ-selective pharmacophore Dmt-Tic were synthesized, and their binding affinity and functional bioactivity at the μ, δ and κ opioid receptors determined. Bifunctional ligands containing or not a spacer of β-alanine between the two pharmacophores lose the μ agonism deriving from morphine becoming partial μ agonists 4 or μ antagonists 5. Partial κ agonism is evidenced only for compound 4.

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.

Evolution of the Bifunctional Lead μ Agonist / δ Antagonist Containing the Dmt-Tic Opioid Pharmacophore.

Based on a renewed importance recently attributed to bi- or multifunctional opioids, we report the synthesis and pharmacological evaluation of some analogues derived from our lead μ agonist / δ antagonist, H-Dmt-Tic-Gly-NH-Bzl. Our previous studies focused on the importance of the C-teminal benzyl function in the induction of such bifunctional activity. The introduction of some substituents in the para position of the phenyl ring (-Cl, -CH(3), partially -NO(2), inactive -NH(2)) was found to give a more potent μ agonist / antagonist effect associated with a relatively unmodified δ antagonist activity (pA(2) = 8.

Novel multiple opioid ligands based on 4-aminobenzazepinone (Aba), azepinoindole (Aia) and tetrahydroisoquinoline (Tic) scaffolds.

The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced delta/mu antagonists, as determined by the functional [S(35)]GTPgammaS binding assay, the dimerization of potent Aia-based 'parent' ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds.

A novel insight into neuroprotection against hypoxic/ischemic stress.

The use of opioid analgesics has a long history in clinical settings, although the functions of opioid receptors, especially their role in the brain, are not well understood yet. Recent studies have generated abundant new data on opioid receptor-mediated functions and the underlying mechanisms. The most exciting finding in the past decade is probably the neuroprotection against hypoxic/ischemic stress mediated by delta-opioid receptors (DOR).

delta-Opioid receptors protect from anoxic disruption of Na+ homeostasis via Na+ channel regulation.

Hypoxic/ischemic disruption of ionic homeostasis is a critical trigger of neuronal injury/death in the brain. There is, however, no promising strategy against such pathophysiologic change to protect the brain from hypoxic/ischemic injury. Here, we present a novel finding that activation of delta-opioid receptors (DOR) reduced anoxic Na+ influx in the mouse cortex, which was completely blocked by DOR antagonism with naltrindole.

Influence of the side chain next to C-terminal benzimidazole in opioid pseudopeptides containing the Dmt-Tic pharmacophore.

To improve the structure-activity studies of the lead delta opioid agonist H-Dmt-Tic-Asp*-Bid, we synthesized and pharmacologically characterized a series of analogues in which the side chain next to 1H-benzimidazole-2-yl (Bid) was substituted by those endowed with different chemical properties. Interesting results were obtained: (1) only Gly, Ala, and Asp resulted in delta agonism, (2) Phe yielded delta antagonism, (3) and all other residues except Glu (devoid of any activity) gave mu agonism.

Orally administered H-Dmt-Tic-Lys-NH-CH2-Ph (MZ-2), a potent mu/delta-opioid receptor antagonist, regulates obese-related factors in mice.

Orally active dual mu-/delta-opioid receptor antagonist, H-Dmt-Tic-Lys-NH-CH(2)-Ph (MZ-2) was applied to study body weight gain, fat content, bone mineral density, serum insulin, cholesterol and glucose levels in female ob/ob (B6.V-Lep/J homozygous) and lean wild mice with or without voluntary exercise on wheels for three weeks, and during a two week post-treatment period under the same conditions. MZ-2 (10mg/kg/day, p.

Conformationally constrained opioid ligands: the Dmt-Aba and Dmt-Aia versus Dmt-Tic scaffold.

Replacement of the constrained phenylalanine analogue 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in the opioid Dmt-Tic-Gly-NH-Bn scaffold by the 4-amino-1,2,4,5-tetrahydro-indolo[2,3-c]azepin-3-one (Aia) and 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffolds has led to the discovery of novel potent mu-selective agonists (Structures 5 and 12) as well as potent and selective delta-opioid receptor antagonists (Structures 9 and 15). Both stereochemistry and N-terminal N,N-dimethylation proved to be crucial factors for opioid receptor selectivity and functional bioactivity in the investigated small peptidomimetic templates. In addition to the in vitro pharmacological evaluation, automated docking models of Dmt-Tic and Dmt-Aba analogues were constructed in order to rationalize the observed structure-activity data.

The novel micro-opioid receptor antagonist, [N-allyl-Dmt(1)]endomorphin-2, attenuates the enhancement of GABAergic neurotransmission by ethanol.

Aims: We investigated the effects of [N-allyl-Dmt(1)]endomorphin-2 (TL-319), a novel and highly potent micro-opioid receptor antagonist, on ethanol (EtOH)-induced enhancement of GABA(A) receptor-mediated synaptic activity in the hippocampus.

Methods: Evoked and spontaneous inhibitory postsynaptic currents (eIPSCs and sIPSCs) were isolated from CA1 pyramidal cells from brain slices of male rats using whole-cell patch-clamp techniques.

Results: TL-319 had no effect on the baseline amplitude of eIPSCs or the frequency of sIPSCs.

Further studies on lead compounds containing the opioid pharmacophore Dmt-Tic.

Some reference opioids containing the Dmt-Tic pharmacophore, especially the delta agonists H-Dmt-Tic-Gly-NH-Ph (1) and H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid (4) (UFP-512) were evaluated for the influence of the substitution of Gly with aspartic acid, its chirality, and the importance of the -NH-Ph and N(1)H-Bid hydrogens in the inductions of delta agonism. The results provide the following conclusions: (i) Asp increases delta selectivity by lowering the mu affinity; (ii) -NH-Ph and N(1)H-Bid nitrogens methylation transforms the delta agonists into delta antagonists; (iii) the substitution of Gly with L-Asp/D-Asp in the delta agonist H-Dmt-Tic-Gly-NH-Ph gave delta antagonists; the same substitution in the delta agonist H-Dmt-Tic-NH-CH2-Bid yielded more selective agonists, H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid and H-Dmt-Tic-NH-(R)CH(CH2-COOH)-Bid; (iv) L-Asp seems important only in functional bioactivity, not in receptor affinity; (v) H-Dmt-Tic-NH-(S)CH(CH2-COOH)-Bid(N(1)-Me) (10) evidenced analgesia similar to 4, which was reversed by naltrindole only in the tail flick. 4 and 10 had opposite behaviours in mice; 4 caused agitation, 10 gave sedation and convulsions.

Inhibition of the development of morphine tolerance by a potent dual mu-delta-opioid antagonist, H-Dmt-Tic-Lys-NH-CH2-Ph.

Three analogues of the dual mu-/delta-antagonist, H-Dmt-Tic-R-NH-CH2-Ph (R = 1, Lys-Z; 2, Lys-Ac; 3, Lys) were examined in vivo: 1 and 2 exhibited weak bioactivity, while 3 injected intracerebroventricularly was a potent dual antagonist for morphine- and deltorphin C-induced antinociception comparable to naltrindole (delta-antagonist), but 93% as effective as naloxone (nonspecific opioid receptor antagonist) and 4% as active as CTOP, a mu antagonist. Subcutaneous or oral administration of 3 antagonized morphine-induced antinociception indicating passage across epithelial and blood-brain barriers. Mice pretreated with 3 before morphine did not develop morphine tolerance indicative of a potential clinical role to inhibit development of drug tolerance.

Synthesis and in vitro evaluation of a library of modified endomorphin 1 peptides.

Endomorphin 1 (Endo-1=Tyr-Pro-Trp-Phe-NH(2)), an endogenous opioid with high affinity and selectivity for mu-opioid receptors, mediates acute and neuropathic pain in rodents. To overcome metabolic instability and poor membrane permeability, the N- and C-termini of Endo-1 were modified by lipoamino acids (Laa) and/or sugars, and 2',6'-dimethyltyrosine (Dmt) replacement of Tyr. Analogues were assessed for mu-opioid receptor affinity, inhibition of cAMP accumulation, enzymatic stability, and permeability across Caco-2 cell monolayers.

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.

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.

Anxiolytic- and antidepressant-like activities of H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512), a novel selective delta opioid receptor agonist.

Knockout and pharmacological studies have shown that delta opioid peptide (DOP) receptor signalling regulates emotional responses. In the present study, the in vitro and in vivo pharmacological profile of the DOP ligand, H-Dmt-Tic-NH-CH(CH2-COOH)-Bid (UFP-512) was investigated. In receptor binding experiments performed on membranes of CHO cells expressing the human recombinant opioid receptors, UFP-512 displayed very high affinity (pKi 10.

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.

A new opioid designed multiple ligand derived from the micro opioid agonist endomorphin-2 and the delta opioid antagonist pharmacophore Dmt-Tic.

Opioid compounds with mixed micro agonist/delta antagonist properties could be used as analgesics with low propensity to induce tolerance and dependence. Here we report the synthesis of a new designed multiple ligand deriving from the micro selective agonist endomorphin-2 and the delta selective antagonist pharmacophore Dmt-Tic. As predicted, the resulting bivalent ligand showed a micro agonist/delta antagonist profile deriving from the corresponding activities of each pharmacophore.

Design and synthesis of opioidmimetics containing 2',6'-dimethyl-L-tyrosine and a pyrazinone-ring platform.

Twelve 2',6'-dimethyl-L-tyrosine (Dmt) analogues linked to a pyrazinone platform were synthesized as 3- or 6-[H-Dmt-NH(CH(2))(n)],3- or 6-R-2(1H)-pyrazinone (n=1-4). 3-[H-Dmt-NH-(CH(2))(4)]-6-beta-phenethyl-5-methyl-2(1H)-pyrazinone 11 bound to mu-opioid receptors with high affinity (K(i)mu=0.13 nM; K(i)delta/K(i)mu=447) with mu-agonism (GPI IC(50)=15.

[N-allyl-Dmt1]-endomorphins are micro-opioid receptor antagonists lacking inverse agonist properties.

[N-allyl-Dmt1]-endomorphin-1 and -2 ([N-allyl-Dmt1]-EM-1 and -2) are new selective micro-opioid receptor antagonists obtained by N-alkylation with an allyl group on the amino terminus of 2',6'-dimethyl-L-tyrosine (Dmt) derivatives. To further characterize properties of these compounds, their intrinsic activities were assessed by functional guanosine 5'-O-(3-[35S]thiotriphosphate) binding assays and forskolin-stimulated cyclic AMP accumulation in cell membranes obtained from vehicle, morphine, and ethanol-treated SK-N-SH cells and brain membranes isolated from naive and morphine-dependent mice; their mode of action was compared with naloxone or naltrexone, which both are standard nonspecific opioid-receptor antagonists. [N-allyl-Dmt1]-EM-1 and -2 were neutral antagonists under all of the experimental conditions examined, in contrast to naloxone and naltrexone, which behave as neutral antagonists only in membranes from vehicle-treated cells and mice but act as inverse agonists in membranes from morphine- and ethanol-treated cells as well as morphine-treated mice.