Supraspinal and spinal effects of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 on nociception in the rat

A new derivative of the neuropeptide nociceptin (NC) has recently been developed. This molecule, the pseudopeptide [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was found to antagonize NC inhibitory effects in peripheral smooth muscle preparations in vitro. However, contrasting results have appeared as regards its pharmacodynamic profile in the CNS. Here, we investigated the pseudopeptide effects, in vivo, on nociceptive responses in the rat. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) (alone or in combination with NC), and tail-flick latencies (TFL) to radiant heat were assessed. I.c.v. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 (1-10 nmol/rat) caused a short-lasting decrease (5 min) of TFL and did not antagonize the threshold lowering effect of i.c.v. NC (1 nmol/rat). At the spinal level, the i.t. administration (0.2-10 nmol/rat) of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 produced a dose-dependent and long-lasting antinociceptive effect that was not modified by the administration of a high dose (30 nmol/rat i.t.) of the opioid antagonist naloxone. The i.t. co-administration of the pseudopeptide (10 nmol/rat) did not block the antinociceptive effect of i.t. NC (10 nmol/rat). These data indicate that the pseudopeptide behaves as an NC agonist at supraspinal and spinal levels in the rat tail-flick test of nociception. These different profiles in the periphery and the CNS could suggest differences between central and peripheral NC receptor/s and provide a basis for further development of antagonist molecules suitable for their characterization.     

Pseudononapeptide bombesin antagonists containing C-terminal Trp or Tpi.

Seven new antagonists of bombesin (Bn)/gastrin-releasing peptide (GRP) containing C-terminal Trp or Tpi (2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-3-carboxylic acid) in a reduced peptide bond were synthesized by solid phase methods and evaluated biologically. The reduced bond in four [Leu13 psi(CH2NH)Trp14]Bn(6-14) analogs was formed by reductive alkylation at the dipeptide stage. In the case of three [Leu13 psi(CH2N)Tpi14]Bn(6-14) analogs, the Trp dipeptide with reduced bond was reacted with formaldehyde to form the corresponding Tpi derivative. These Tpi-containing analogs have a new reduced bond which is structurally more constrained. Leu13 psi(CH2N)Tpi14 analogs inhibit [125I][Tyr4]bombesin binding to Swiss 3T3 cells with IC50 values of 2-4 nM, compared to 5-10 nM for Leu13 psi(CH2NH)Trp14 analogs. Leu13 psi(CH2N)Tpi14 analogs are also more potent than Leu13 psi(CH2NH)Trp14 analogs in growth inhibition studies using Swiss 3T3 cells. The two best bombesin antagonists of this series, [D-Trp6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3415) and [Tpi6,Leu13 psi(CH2N)Tpi14]Bn(6-14) (RC-3440), inhibited GRP-stimulated growth of Swiss 3T3 cells with IC50 values less than 1 nM. RC-3440 was also active in vivo, suppressing GRP(14-27)-stimulated serum gastrin secretion in rats. Bombesin/GRP antagonists, such as RC-3440, containing the new reduced bond (CH2N) reported herein are very potent.     

Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase N.

Bradykinin (BK) analogs such as Lys-Lys-BK, des-Arg9-BK and [Leu8]des-Arg9-BK were poor substrates for angiotensin I converting enzyme (ACE), and analogs containing D-Phe7 residues, or a pseudopeptide C-terminal bond, were completely resistant. However, many of these analogs were metabolized by carboxypeptidase N (CPN) including Lys-Lys-BK, [Tyr8(OMe)]BK and D-Phe7-containing analogs, with Km and Vmax values comparable to those for BK. The only analogs completely resistant to both ACE and CPN were the B2 agonist [Phe8 psi(CH2NH)Arg9]BK, the B2 agonist D-Arg[Hyp3,D-Phe7,Phe8 psi(CH2NH)Arg9]BK, and the B1 agonist [D-Phe8]des-Arg9-BK. These data indicate an important role for plasma CPN and vascular CPN-like activity in the metabolism of the widely used ACE-resistant/D-Phe7-containing antagonists of B2 kinin receptors.     

A gastrin-releasing peptide antagonist containing A psi (CH2O) amide bond surrogate

The [Leu26-psi(CH2O)Leu27] derivative of N-Ac-GRP20-27-peptide amide was prepared and evaluated as a gastrin-releasing peptide antagonist. This psi(CH2O) derivative was found to be a more potent inhibitor of [3H-Phe15]GRP15-24NH2 binding and N-Ac-GRP20-27NH2 induced mitogenesis in Swiss 3T3 fibroblasts than the related nitrogen analog [Leu13-psi(CH2NH)Leu14] bombesin. Possible reasons for the improved activity of the (CH2O) insert relative to the (CH2NH) group include increased hydrophobicity and a reduced tendency of the oxygen derivative to form hydrogen bonds.     

Antinociceptive effects of [D-Ala2]deltorphin II, a highly selective delta agonist in vivo

The present study has characterized the antinociceptive actions of [D-Ala2]deltorphin II following intracerebroventricular (i.c.v.) administration in the mouse tail-flick test. [D-Ala2]deltorphin II produced dose- and time-related antinociception, with maximal effects at +10 min and significant antinociception which lasted for 40-60 min. [D-Ala2]deltorphin II was 13-fold more potent than i.c.v. [D-Pen2, D-Pen5]enkephalin (DPDPE), a second highly selective delta agonist, and approximately equipotent with i.c.v. morphine in producing antinociception. The antinociceptive effects of i.c.v. [D-Ala2]deltorphin II and DPDPE, but not those of morphine, were antagonized by the selective delta antagonist, ICI 174,864. In contrast, pretreatment with the non-equilibrium mu antagonist, beta-funaltrexamine blocked morphine antinociception, but failed to antagonize [D-Ala2]deltorphin II and DPDPE antinociception. These data indicate that [D-Ala2]deltorphin II produced its antinociceptive effects at a supraspinal delta receptor. [D-Ala2]deltorphin II appears to be the most appropriate delta opioid agonist currently available for studies in vivo and support the involvement of delta receptors in supraspinal antinociception.     

BACE-1 inhibition by a series of psi[CH2NH] reduced amide isosteres

A series of beta-site amyloid precursor protein cleaving enzyme (BACE-1) inhibitors containing a psi(CH2NH) reduced amide bond were synthesized. Incorporation of this reduced amide isostere as a non-cleavable peptide surrogate afforded inhibitors possessing low nanomolar potencies in both an enzymatic and cell-based assay.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a psi (E,CH = CH) or psi (CH2CH2) isosteric replacement.

The peptide CO-NH function was replaced by a trans carbon-carbon double bond or by a CH2-CH2 isostere in enkephalin analogues of DADLE, DCDCE-NH2 or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly3-Phe4 bond was replaced by the isosteres Gly psi (E,CH = CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or Gly psi (CH2CH2)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH2-CH2 replacement. The Gly3 psi (E,CH = CH)Phe4 DCDCE-NH2 analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

In vitro and in vivo characterization of opioid activities of endomorphins analogs with novel constrained C-terminus: evidence for the important role of proper spatial disposition of the third aromatic ring

In the present study, the C-terminus of endomorphin (Tyr(1)-Pro(2)-Trp/Phe(3)-Phe(4)-NH(2), EMs) analogs [Xaa(4)-R]EMs, modified by substitution of a non-aromatic residue for Phe(4) and ending up with -NH-benzyl, were designed to generate an atypical conformationally constrained peptide set. We investigated the effects of these analogs on the opioid receptors affinity, guinea pig ileum (GPI) and mouse vas deferens (MVD) activity, system arterial pressure (SAP), heart rate (HR), antinociception and colonic motility. Analogs 5 ([D-V(4)-Bzl]EM1) and 10 ([D-V(4)-Bzl]EM2), which exhibit appropriate spatial orientations of the third aromatic ring, were about 3-4 times more potent than their parents both in vivo and in vitro. However, a drastic loss of activity was found in analogs 2 ([A(4)-Bzl]EM1) and 7 ([A(4)-Bzl]EM2), which possess improper spatial orientations of the third aromatic ring. Interestingly, analog 7 or 3 ([G(4)-Bzl]EM1), when injected intravenously (i.v.), produced significantly different changes in SAP from their parents. Surprisingly, analog 4 displayed relatively higher vasodepressor activity but significantly less potent colonic contractile activity than analog 5. This may be elicited by the differences in the spatial disposition of the third aromatic ring, which were verified by molecular modeling. Our results indicate that the proper spatial disposition of the third aromatic ring plays an important role in the regulation of pharmacological activities of EMs.     

Alpha-aminoxy acids as building blocks for the oxime and hydroxylamine pseudopeptide links. Application to the synthesis of human elastase inhibitors.

The aminoxy acids NH2-O-C(alpha)HR-CO2H are much more easily obtained in the enantiomerically pure form than the analogous hydrazino acids NH2-NH-C(alpha)HR-CO2H, and it has been shown that the isosteric amidoxy psi[CO-NH-O] and hydrazide psi[CO-NH-NH] amide surrogates Induce two quite similar gamma-like folded structures. An aminoxy acid can also be N-coupled to a peptide aldehyde to give the aldoxime psi[CH = N-O] link or to a peptide ketone to form the ketoxime psi[CR= N-O] link. The former can be further reduced into the hydroxylamine psi[CH2-NH-O] link which gives rise to reduced amidoxy peptides. The structural properties Induced by these amide surrogates were studied, using IR and NMR spectroscopy, paying particular attention to the Z/E-isomerism of the oxime link. In order to investigate their inhibitory potency, the three amide surrogates were introduced in the Pro3-Val4 and Val4-Ala5 position of Z-Ala1-Ala2-Pro3-Val4-Ala5-Ala6-NHiPr, a substrate which is cleaved in the Val4-Ala5 position by human leukocyte elastase (HLE). The [Val4psi[CO-NH-O]Ala5] analogue was still a substrate, while the [Pro3psi[CO-NH-O]Val4] and [Val4psi[CH = N-O]Ala5] pseudopeptides acted as HLE competitive inhibitors.     

Reduced peptide bond pseudopeptide analogues of substance P. A new class of substance P receptor antagonists with enhanced specificity

Each of the last 6 peptide bonds in the COOH terminus of [Leu11]substance P [( Leu11]SP) and [Nle11]spantide were replaced with [CH2NH], and each analogue was tested for SP agonist or antagonist activity by determining its ability to interact with SP receptors on dispersed acini from guinea pig pancreas. Each of the 6 spantide and 5 of the 6 SP analogues had no agonist activity, whereas [psi 9-10]SP was an agonist. For the spantide pseudopeptides, the psi 10-11 analogue (Ki,2.8 microM) was equipotent as an antagonist to spantide itself, whereas the psi 9-10, psi 8-9, psi 7-8, and psi 6-7 analogues were 2.5, 7, 5, and 3 times less potent. For the SP pseudopeptides, the psi 10-11 analogue was the most potent antagonist (Ki, 6.2 microM), whereas the psi 8-9, psi 7-8, and psi 6-7 analogues were 7-, 36-, and 39-fold less potent. There was a close correlation between the ability of each pseudopeptide to inhibit binding of 125I-Bolton-Hunter-SP and to affect amylase secretion. [psi 10-11]SP inhibited SP-stimulated amylase release in a competitive manner, and its inhibitory ability was specific for the SP receptor. Despite [psi 10-11]SP, spantide, and [psi 10-11]spantide having similar affinities for the SP receptor (Ki, 2-6 microM), for inhibition of binding of 125I-[Tyr4]bombesin, the analogues differed with [psi 10-11]SP having a 50-fold lower affinity than for the SP receptor, whereas [psi 10-11]spantide had a 4-fold lower affinity and spantide a 1.5-fold lower affinity for the SP receptor. These results demonstrate that SP pseudopeptides represent a new class of SP receptor antagonists and, in contrast to the currently described SP receptor antagonists, are more specific for SP receptors.     

Reduced peptide bond pseudopeptide analogues of secretin. A new class of secretin receptor antagonists.

The ability to assess the importance of secretin in various physiological processes is limited by the lack of specific potent antagonists. Recently, reduced peptide bond (psi) analogues of bombesin or substance P in which the -CONH- bond is replaced by -CH2NH- are reported to be receptor antagonists. To attempt to develop a new class of secretin receptor antagonists, we have adopted a similar strategy with secretin and sequentially altered the eight NH2-terminal peptide bonds, the biological active portion of secretin. In guinea pig pancreatic acini, secretin caused a 75-fold increase in cyclic AMP (cAMP). Secretin inhibited 125I-secretin binding with a half-maximal effect at 7 nM. Each of the psi analogues inhibited 125I-secretin binding. [psi 4,5]Secretin was the most potent, causing the half-maximal inhibition at 4 microM, and was 2-fold more potent than the [psi 1,2]secretin; 7-fold more than [psi 3,4]secretin, [psi 5,6]secretin, and [psi 8,9]secretin; 9-fold more than [psi 7,8]secretin; 13-fold more potent [psi 6,7]secretin, and 17-fold more than [psi 2,3]secretin. Secretin caused a half-maximal increase in cAMP at 1 nM. At concentrations up to 10 microM, [psi 2,3]secretin, [psi 4,5]secretin, and [psi 8,9]secretin did not alter cAMP whereas [psi 1,2]secretin and [psi 6,7]secretin caused a detectable increase in cAMP at 10 nM, [psi 7,8]secretin at 300 nM, [psi 5,6]secretin at 1 microM, and [psi 3,4]secretin at 10 microM. The [psi 4,5], [psi 2,3], and [psi 8,9] analogues of secretin each inhibited 1 nM secretin-stimulated cAMP as well as [psi 3,4]secretin, which functioned as a partial agonist. [psi 4,5]Secretin was the most potent, causing half-maximal inhibition at 3 microM whereas [psi 8,9]secretin was 6-fold less potent, and [psi 2,3]secretin and [psi 3,4]secretin were 17-fold less potent. [psi 4,5]Secretin inhibited secretin-stimulated cAMP and binding of 125I-secretin in a competitive manner. [psi 4,5]Secretin did not interact with cholecystokinin, bombesin, calcitonin gene-related peptide, or cholinergic receptors but did interact with receptors for vasoactive intestinal peptide, causing half-maximal inhibition at 72 microM and thus had a 18-fold higher affinity for secretin than vasoactive intestinal peptide receptors. These results indicate that reduced peptide bond analogues of the NH2 terminus of secretin represent a new class of secretin receptor antagonists. It is likely that in the future even more potent members of this class can be developed which may be useful to investigate the role of secretin in various physiological processes.     

Synthesis and antinociceptive effects of endomorphin-1 analogs with C-terminal linked by oligoarginine

Endomorphins (EMs) cannot be delivered into the central nervous system (CNS) in sufficient quantity to elicit antinociception when given systemically because they are severely restricted by the blood-brain barrier (BBB). In the present study, we investigated herein a series of EM-1 analogs with C-terminal linked by oligoarginine in order to improve the brain delivery and antinociception after systemic administration. Indeed, all these analogs decreased the opioid receptor affinity and in vitro pharmacological activity. Moreover, analogs 4, 7-9 produced a less potent antinociceptive activity after intracerebroventricular (i.c.v.) administration, with the ED₅₀ values about 11- to 13-fold lower potencies than that of EM-1. Nevertheless, our results revealed that EM-1 failed to induce any significant antinociception at a dose of 50 μmol/kg after subcutaneous (s.c.) administration, whereas equimolar dose of these four analogs produced a little low but significant antinociceptive effects. Naloxone (10 nmol/kg, i.c.v.) significantly blocked the antinociceptive effects, indicating an opioid and central mechanism. These results demonstrated that C-terminal of EM-1 linked to oligoarginine improved the brain delivery, eliciting potent antinociception following peripheral administration.     

1H and 13C n.m.r. studies of pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin

1H and 13C n.m.r. study of pseudo-peptide analogues of the C-terminal tetrapeptide of gastrin, obtained by replacing each peptide bond by a "reduced peptide bond", one at a time, e.g. Boc-Trp psi (CH2NH)Leu-Asp-Phe-NH2 2, Boc-Trp-Leu psi (CH2NH) Asp-Phe-NH2 3, Boc-Trp-Leu-Asp psi (CH2NH)Phe-NH2 4, were reported. The CH2NH bond was completely characterized. 1H and 13C spectroscopic data were reported. It appeared from the present work that the modifications produced by the replacement of a peptide bond by a CH2NH bond were localized around the CH2NH.     

125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP: iodination and binding characteristics of a vasopressin receptor ligand

A radioiodinated vasopressin antagonist, d(CH2)5[Tyr(NH2)9]AVP has been prepared. Iodination was carried out at the phenyl moiety of the tyrosylamide residue at position 9, followed by HPLC purification. Non-radiolabelled monoiodinated antagonist was used as a reference for identification. 125I-d(CH2)5[Tyr(Me)2, Tyr(NH2)9]AVP binding appeared to take place with a dissociation constant of 0.28 +/- 0.09 nM (Kd +/- SD) to V1 vasopressin receptors on rat liver membranes.     

Conformational studies on bombesin antagonists: CD and NMR characterization of [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14).

The conformational flexibility of the [Thr6, Leu13 psi(CH2NH) Met14] bombesin (6-14) nonapeptide has been studied by CD and one- and two-dimensional (1D and 2D) nmr techniques. The CD and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for the parent bombesin (BN) and [D-Phe12, Leu14]BN. A preliminary investigation on spantide is also reported. In particular, the results obtained from CD measurements indicate that there is a shift from random coil structures, in aqueous solutions, toward folded structures in apolar media (2,2,2-trifluoroethanol) and in a membrane-mimetic environment (40 mM SDS) for all three peptides, namely BN, [D-Phe12, Leu14]BN, and [Thr6, Leu13 psi(CH2NH) Met14]BN (6-14). Spantide, which also possesses some inhibitory activity against BN but very little sequence similarity, even in water, shows an ordered conformation. Nuclear magnetic resonance parameters such as backbone NH-alpha CH coupling constant values, amidic temperature coefficients, and the presence of only sequential nuclear Overhauser effects have not provided, so far, any clear evidence for a preferential ordered structure in the peptides studied, and this may be due to rapid exchange among different conformers in the nmr time scale.     

SR142801 behaves as a tachykinin NK-3 receptor agonist on a spinal nociceptive reflex in the rat

Effects of two commonly used tachykinin NK-3 receptor antagonists (SR 142801 and R820) intrathecally (i.t.) administered were assessed in the rat tail-flick test. SR142801 and its (R)-enantiomer SR142806 (1.3, 6.5 and 65 nmol) were found as potent as senktide and [MePhe7]NKB (NK-3 selective agonists) to induce transient antinociceptive effects. Naloxone (10 microg) and R820 (6.5 nmol) blocked reversibly the responses to 6.5 nmol senktide, [MePhe7]NKB, SR142801 and SR142806 when administered i.t. 15 min earlier. However, the antinociceptive responses induced by SR142801 and SR142806 were not affected by i.t. pretreatments with NK-1 (6.5 nmol SR140333) and NK-2 (6.5 nmol SR48968) receptor antagonists. In control experiments, the NK-1 and NK-2 antagonists prevented the hyperalgesic effects to NK-1 ([Sar9,Met(O2)11]SP) and NK-2 ([beta-Ala8] NKA(4-10)) receptor agonists (6.5 nmol i.t.), respectively. R820 had no direct effect on nociceptive threshold and failed to alter angiotensin II-induced antinociception. The data suggest that the antinociceptive effect of SR142801 is due to an agonist effect at NK-3 receptor in the rat spinal cord that involves a local opioid mechanism. These results can be best explained by the existence of inter-species NK-3 receptor subtypes.     

Solid phase synthesis of peptides containing the CH2NH peptide bond isostere

The CH2NH peptide bond can be directly introduced by the reductive alkylation reaction between a tert-butoxycarbonyl-amino acid aldehyde and an amine on the resin bound peptide employing sodium cyanoborohydride in acidified dimethylformamide solution and this facile method was successfully applied to the synthesis of a psi[CH2NH] pseudopeptide somatostatin octapeptide analogue.     

Structure-conformation relationships of synthetic peptide inhibitors of human renin studied by resonance energy transfer and molecular modeling

The structure-conformation relationships of a series of angiotensinogen6-13 (ANG6-13, His-Pro-Phe-His-Leu-Val-Ile-His) congeners substituted by Nin-For-Trp (Ftr), D-Ftr or Trp at the N-terminus, Tyr at the C-terminus and Phe psi[CH2NH]Phe at the P1-P'1 cleavage site (i.e. Leu10-Val11) were studied using resonance energy transfer coupled with molecular modeling of the peptide conformation using macromolecular energy refinement and dynamics simulation. Average end-to-end intramolecular distances (r) of the peptides in solution were determined by fluorescence spectroscopy. For example, Ac-Ftr-Pro-Phe-His-Phe psi[CH2NH]Phe-Val-Tyr-NH2 (U-70714E) gave an average intramolecular donor (Tyr)-acceptor (Ftr) distance of 16.3A in aqueous solution. This experimental value was consistent with a distance of 17.9 A determined by molecular modeling of U-70714E to a human renin 3-D structure (developed from known homologous aspartyl protease inhibitor X-ray crystallographic data) followed by simulation of the solution phase conformation of the peptide. An extended backbone secondary structure of U-70714E is suggested from these studies and the relationship(s) of structure-conformation to structure-activity was explored by analysis of several congeners of U-70714E, a potent (IC50 = 3.0 X 10(-9)M) inhibitor of human renin in vitro.     

Incorporation of vinylogous scaffolds in the C-terminal tripeptide of substance P.

Glycine-9 and leucine-10 of substance P (SP) are critical for (NK)-1 receptor recognition and agonist activity. Propsi(Z)-CH=CH(CH3)-CONH)Leu (or Met) and Propsi((E)-CH=CH(CH3)-CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C-terminal tripeptide, Gly-Leu-Met-NH2, of SP. Propsi((Z)-CH=C(CH2CH(CH3)2)-CONH)Met-NH2, with an isobutyl substituent to mimic the Leu side-chain, was also incorporated in place of the C-terminal tripeptide. The substituted-SP analogs were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK-1 receptor. The most potent SP analogs [Pro9psi((Z)CH=C(CH3)CONH)Leu10]SP and [Pro9psi ((E)CH=C(CH3)CONH)Leu10]SP, are about 100-fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)- or (E)-CH=C(CH3)- or (Z)-CH=C(CH2CH(CH3)2) moieties hamper the correct positioning of the C-terminal tripeptide of SP within both the NK-1M- and NK-1m-specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group.     

Synthesis and binding characteristics of [3H] H-Tyr-Ticpsi[CH2-NH] Cha-Phe-OH, a highly specific and stable delta-opioid antagonist.

Substitution of the Phe3 aromatic ring in H-Tyr-Ticpsi[CH2-NH]Phe-Phe-OH with cyclohexylalanine (Cha) has been reported to result in a compound, H-Tyr-Ticpsi[CH2-NH]Cha-Phe-OH (TICP[psi]), showing substantially increased delta-opioid antagonist potency and high delta selectivity. TICP[psi] was radiolabeled by catalytic tritiation of its precursor Tyr(3',5'-I2)1TICP[psi]. Binding characteristics of the new tritiated pseudopeptide were determined using the radioligand binding assay in rat brain membranes. On the basis of the results of saturation binding studies performed at 25 degrees C, an equilibrium dissociation constant (Kd) of 0.35 nM and a receptor density (Bmax) of 112 fmol/mg protein were calculated. This new tritiated ligand exhibits high affinity for delta-opioid receptors, whereas its binding to mu and kappa receptors is weak. A study of [H3]TICP[psi] binding displacement by various receptor-selective opioids showed the following rank order of potency: delta > kappa = mu. These receptor binding characteristics of the ligand, together with its high specific radioactivity (41.3 Ci/mmol) and stability, makes it a useful tool for labeling delta-opioid receptors, both in vitro and in vivo.