Opioid activity of C8813, a novel and potent opioid analgesic

Compound trans-4-(p-bromophenyl)-4-(dimethylamino)-1-(2-thiophen-2-yl-ethyl)-cyclohexanol (C8813), structurally unrelated to morphine, is a novel analgesic. The present study examined the antinociception, opioid receptor selectivity and in vitro activity of C8813. The antinociceptive activity was evaluated using mouse hot plate and acetic acid writhing tests. In mouse hot plate test, the antinociceptive ED(50) of C8813 was 11.5 microg/kg, being 591 times and 3.4 times more potent than morphine and fentanyl respectively. In mouse writhing test, the antinociceptive ED(50) of C8813 was 16.9 microg/kg, being 55 times and 2.3 times more active than morphine and fentanyl respectively. In the opioid receptor binding assay, C8813 showed high affinity for mu-opioid receptor (K(i) = 1.37 nM) and delta-opioid receptor (K(i) = 3.24 nM) but almost no affinity for kappa-opioid receptor (at 1 microM). In the bioassay, the inhibitory effect of C8813 in the guinea-pig ileum (GPI) was 16.5 times more potent than in the mouse vas deferens (MVD). The inhibitory effects of C8813 in the GPI and MVD could be antagonized by mu-opioid receptor antagonist naloxone and delta-opioid receptor antagonist ICI174,864 respectively. However, the inhibitory effect of C8813 in the rabbit vas deferens was very weak. These results indicated that C8813 was a potent analgesic and a high affinity agonist for the mu- and delta-opioid receptors.     

Characterization of [125I]AR-M100613, a high-affinity radioligand for delta opioid receptors

AR-M100613 ([I]-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the delta opioid receptor. Specific [125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (Kd = 0.080 +/- 0.008 nM, Bmax = 45.2 +/- 4.4 fmol/mg protein). [125I]AR-M100613 binding was displaced with high affinity by the delta opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the mu or kappa-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [125I]AR-M 100613 to mu opioid receptors is blocked by the addition of CTOP to the assay buffer. [35S]GTPgammaS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for delta opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [125I]AR-M100613 favor its use as a radiochemical probe for delta opioid receptors.     

Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3-4-5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by micro selective antagonists (naloxone and/or CTOP) but not by the delta selective antagonist naltrindole, thus indicating that the compound acted as a micro opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the micro-receptor.     

Stereospecific synthesis of (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) and its incorporation into an opioid peptide

To examine the effect of replacing the N-terminal amino group in opioid peptides with a methyl group on biological activity, a stereospecific synthesis of the tyrosine analogue (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) was performed. The enkephalin analogue (2S)-Mdp-D-Ala-Gly-Phe-Leu-NH2 turned out to be a quite potent delta opioid antagonist and a somewhat less potent mu antagonist, indicating that a positively charged N-terminal amino group is not a conditio sine qua non for the binding of opioid peptides to delta and mu receptors but may be required for signal transduction.     

Morphine tolerance in mice changes response of heroin from mu to delta opioid receptors

Heroin produced antinociception in the tail flick test through mu receptors in the brain of ICR and CD-1 mice, a response inhibited by 3-O-methylnaltrexone. Tolerance to morphine was produced by subcutaneous morphine pellet implantation. By the third day, the heroin response was produced through delta opioid receptors. The response was inhibited by simultaneous intracerebroventricular (i.c. v.) administration of naltrindole, a delta opioid receptor antagonist. More specifically, delta1 rather than delta2 receptors were involved because 7-benzylidenenaltrexone, a delta1 receptor antagonist, inhibited but naltriben, a delta2 antagonist, did not. Also, antinociception produced by i.c.v. heroin was inhibited by intrathecal administration of bicuculline and picrotoxin consistent with the concept that delta1 receptors in the brain mediated the antinociceptive response through descending neuronal pathways to the spinal cord to activate GABAA and GABAB receptors rather than spinal alpha2-adrenergic and serotonergic receptors activated originally by the mu agonist action in naive mice. The mu response of 6-monoacetylmorphine, a metabolite of heroin, was changed by morphine pellet implantation to a delta2 response (inhibited by naltriben but not 7-benzylidenenaltrexone). The agonist action of morphine in these morphine-tolerant mice remained mu. Thus, the opioid receptor selectivity of heroin and 6-monoacetylmorphine in the brain is changed by production of tolerance to morphine. Such a change explains how morphine tolerant mice are not cross-tolerant to heroin.     

Differential contribution of C-terminal regions of dermorphin and dermenkephalin to opioid-sites selection and binding potency

Dermorphin and dermenkephalin are D-aminoacid containing peptides generated from processing of the plurifonctional biosynthetic precursor pro-dermorphin. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM) and dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), are among the most selective and potent agonists described respectively for the mu- and delta-opioid receptors. In order to identify determinants of selectivity and high-affinity receptor binding of dermorphin and dermenkephalin, a series of analogs was investigated for their affinity at the mu- and delta-receptors in the brain. The tetrapeptide amino end of both DRM and DREK were found to display high affinity and selectivity towards mu-receptors. Substitution of the C-terminal tripeptide of DREK with that of DRM reversed the receptor selectivity of DREK from delta to mu. Replacement of the C-terminal tripeptide of DRM with the C-terminal counterpart of DREK shifted the selectivity of DRM from mu to delta. These data emphasize the critical contribution of the carboxy end of DREK to delta-selectivity. They further suggest that the potent mu-address lying in the N terminus of DREK is overwhelmed by the powerful delta-directing ability of the carboxy end. Unlike DREK, the C-terminus of DRM is not involved in opioid receptor sites selection but is important insofar as it serves to stabilize interactions of DRM with the mu-receptor binding site.     

Opioid receptor agonists and Ca2+ modulation in human B cell lines.

Opiates and opioid peptides have been shown to modulate lymphocyte functions; however, little attention has been given to the type of receptors or receptor signaling mechanisms that are involved. Receptor-mediated signaling via ionized free Ca2+ is an event thought to be important in the triggering of lymphocyte activities. We report use of the calcium indicator dye, indo-1, and flow cytometry to identify B lymphocyte calcium responses to physiologic concentrations of opioid peptides. The human B cell lines Nalm 6 and JY responded to the naturally occurring opioid pentapeptide methionine-enkephalin or other opiate receptor agonists with a rapid, dose-dependent rise in free cytoplasmic Ca2+. This opioid peptide effect on Ca2+ modulation was inhibited by the opiate receptor antagonist naloxone. The synthetic enkephalin analogue DAMGO with specificity for mu-type opiate receptors and the synthetic opiate receptor agonists U50,488H and U69,593 with selectivity for kappa-type sites also stimulated calcium responses when applied to the B cell lines. These studies provide evidence that human B cell lines express functional opiate receptors of the mu- and kappa-types and suggest that such receptors, coupled with Ca2+ modulation, are instrumental in the B cell response to opiates and endogenous opioid neuropeptides.     

Lateralization of opioid receptors and their putative ligands in the visual cortex of the turtle

Opioid mu-agonist morphine, delta-agonist D-Ala2,D-Leu5-enkephalin (DADL) and kappa-agonist bremazocine locally applied to the surface of turtle visual cortex inhibited the orthodromic evoked potential (EP; fast negative component N1). The lack of cross-desensitization to the inhibitory action of opioids upon EP indicates that the drugs exert their effects via different opioid receptors. Morphine and bremazocine predominantly inhibited the left cortex EP, whereas DADL was a potent inhibitor of the right cortex EP. Thus opioid receptors which modulate evoked electrical activity of the left visual cortex (LVC) apparently belong mostly to mu- and kappa-type while delta-receptors were predominantly responsible for the modulation of electrical activity in the right visual cortex (RVC). Application of LVC- and RVC-extracts to the cortex surface led to EP inhibition, which was partially (60-80%) prevented by antagonist naloxone. LVC-extract proved to be a more potent inhibitor of the left cortex EP, whereas RVC-extract was found to be more effective when applied to the right cortex. It is suggested that not only opioid receptors, but also their endogenous ligands are lateralized in turtle visual cortex.     

Alpha-2 adrenergic and opioid receptor-mediated gastroprotection.

Clonidine inhibited the development of gastric mucosal lesions induced by either acidified ethanol or indomethacin. The ED50 values were: 7.1 and 5.2 microg x kg(-1) orally, respectively. The gastroprotective effect was antagonised by the pre-synaptic alpha-2 antagonist yohimbine, the more selective alpha-2 antagonist Ch-38083 and the pre-synaptic alpha-2B antagonist prazosin. Moreover, the non-selective opioid receptor antagonist naloxone, the delta receptor selective naltrindole also reversed the clonidine-induced mucosal protective action. Clonidine was also effective following intracerebroventricular administration with the ED50 of 37 ng/rat against ethanol-induced mucosal damage. Our results suggest that: 1) the gastroprotective effect of clonidine is likely to be mediated by alpha-2B adrenoceptor subtype; 2) there is an interaction between pre-synaptic alpha-2 adrenoceptors and opioid system; and 3) clonidine can induce gastroprotection by central mechanism.     

Novel highly potent mu-opioid receptor antagonist based on endomorphin-2 structure

The mu-opioid agonists endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)) exhibit an extremely high selectivity for the mu-opioid receptor and thus represent a potential framework for modification into mu-antagonists. Here we report on the synthesis and biological evaluation of novel [d-2-Nal(4)]endomorphin-2 analogs, [Sar(2),d-2-Nal(4)]endomorphin-2 and [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 (Dmt=2'6'-dimethyltyrosine; Sar=N-methylglycine, sarcosine; d-2-Nal=3-(2-naphthyl)-d-alanine). [Dmt(1),Sar(2),d-2-Nal(4)]endomorphin-2 possessed very high affinity for the mu-opioid receptor (IC(50)=0.01+/-0.001 nM) and turned out to be a potent and extremely selective mu-opioid receptor antagonist, as judged by the in vitro aequorin luminescence-based calcium assay (pA(2)=9.19). However, in the in vivo hot plate test in mice this analog was less potent than our earlier mu-opioid receptor antagonist, [Dmt(1),d-2-Nal(4)]endomorphin-2 (antanal-2). The exceptional mu-opioid receptor in vitro activity and selectivity of [Dmt(1), Sar(2),d-2-Nal(4)]endomorphin-2 makes this analog a valuable pharmacological tool, but further modifications are needed to improve its in vivo profile.     

Discovery of two novel branched peptidomimetics containing endomorphin-2 and RF9 pharmacophores: Synthesis and neuropharmacological evaluation

It is well known that opioid analgesics produce side effects including tolerance and constipation. Since neuropeptide FF (NPFF) receptor antagonists reversed opioid-induced hyperalgesia and analgesic tolerance, the present work was performed to synthetize two branched peptidomimetics, EKR and RKE, containing the opioid peptide endomorphin-2 (EM-2) and the NPFF receptor antagonist RF9. Our data obtained from the in vitro cyclic adenosine monophosphate experiment demonstrated that EKR functioned as a mixed mu-, delta-opioid receptors agonist and NPFF₁ receptor antagonist/NPFF₂ receptor partial agonist, whereas RKE acted as a multi-functional peptidomimetic with the mu-opioid agonism and the NPFF₁ antagonism/NPFF₂ partial agonism. Furthermore, EKR and RKE completely blocked the NPFF₂ receptor-mediated neurite outgrowth of Neuro 2A cells. In vivo antinociception studies found that supraspinal administration of EKR and RKE dose-dependently produced potent antinociception via the mu-opioid receptor in the tail-flick test. In carrageenan inflammatory pain model, spinal administration of EKR and RKE induced dose-related analgesia, which was significantly reduced by the opioid antagonist naloxone and the NPFF antagonist RF9. Notably, compared with morphine, intracerebroventricular repeated administration of EKR and RKE maintained prolonged antinociceptive effectiveness. In addition, at the antinociceptive doses, these two branched peptidomimetics did not significantly inhibit gastrointestinal transit. Taken together, the present work suggest that EKR and RKE behave as multi-functional ligands with the opioid agonism and the NPFF₁ antagonism/NPFF₂ partial agonism, and produce prolonged antinociception with limited side effects. Moreover, our results imply that EKR and RKE might be interesting pharmacological tools for further investigating the biological function of the NPFF and opioid systems.     

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.     

The TIPP opioid peptide family: development of delta antagonists, delta agonists, and mixed mu agonist/delta antagonists

The discovery of the prototype delta opioid antagonists TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) in 1992 was followed by extensive structure-activity relationship studies, leading to the development of analogues that are of interest as pharmacological tools or as potential therapeutic agents. Stable TIPP-derived delta opioid antagonists with subnanomolar delta receptor binding affinity and extraordinary delta receptor selectivity include TIPP[Psi] (H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH] and TICP[Psi] (H-Tyr-TicPsi[CH(2)NH]Cha-Phe-OH); Cha: cyclohexylalanine), which are widely used in opioid research. Theoretical conformational analyses in conjunction with the pharmacological characterization of conformationally constrained TIPP analogues led to a definitive model of the receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related delta opioid antagonists, which is characterized by all-trans peptide bonds. Further structure-activity studies revealed that the delta antagonist vs delta agonist behavior of TIP(P)-derived compounds depended on very subtle structural differences in diverse locations of the molecule and suggested a delta receptor model involving a number of different inactive receptor conformations. A further outcome of these studies was the identification of a new class of potent and very selective dipeptide delta agonists of the general formula H-Tyr-Tic-NH-X (X = arylalkyl), which are of interest for drug development because of their low molecular weight and lipophilic character. Most interestingly, TIPP analogues containing a C-terminal carboxamide group displayed a mixed mu agonist/delta antagonist profile, and thus were expected to be analgesics with a low propensity to produce tolerance and physical dependence. This turned out to be the case with the TIPP-derived mu agonist/delta antagonist DIPP-NH(2)[Psi] (H-Dmt-TicPsi[CH(2)NH]Phe-Phe-NH(2)); Dmt: 2',6'- dimethyltyrosine).     

Synthesis and evaluation of novel peripherally restricted kappa-opioid receptor agonists

A series of 3-substituted analogs (3) of the parent kappa agonist, 1, were prepared to limit access to the central nervous system. With the exception of compound 3j, all other compounds bound to the human kappa opioid receptor with high affinity (K(i)=0.31-9.5 nM) and were selective for kappa over mu and delta opioid receptors. Compounds 3c, d, and 3g-i produced potent antinociceptive activity in the rat formalin assay (i.paw) and the mouse acetic acid-induced writhing assay (s.c.), with weak activity in the mouse platform sedation test. The peripheral restriction indices of 3c, d, 3g, and 3i were improved 2- to 7-fold compared to the parent compound 1, and these compounds were approximately 2- to 5-fold more potent than the peripheral kappa agonist ICI 204448.     

Kinin and opioid receptors in the paratrigeminal nucleus modulate the somatosensory reflex to rat sciatic nerve stimulation

The influence of kinin and opioid receptor blockade in the paratrigeminal nucleus (Pa5) on the somatosensory reflex (SSR) to sciatic nerve stimulation (SNS) was assessed in anaesthetized-paralyzed rats. SNS (square 1 ms pulses at 0.6 mA and 20 Hz for 10s) increased mean arterial pressure from 87+/-3 to 106+/-3 mmHg. Pressor responses to SNS were reduced 40-60% by HOE-140 and LF 16-0687 (B2 receptor antagonists; 20 and 100 pmol respectively), CTOP or nor-binaltorphimine (mu and kappa opioid receptor antagonists, respectively; 1 microg) but potentiated by naltrindole (delta opioid receptor antagonist) receptor antagonist microinjections into the contralateral (but not ipsilateral) Pa5. The SSR to sciatic nerve stimulation was not changed by B1 kinin receptor or NK1, NK2 and NK3 tachykinin receptor antagonists administered to the Pa5. Capsaicin pretreatment (40 mg/kg/day, 3 days) abolished the effects of the opioid receptor antagonists, but did not change the effect of kinin B2 receptor blockade on the SSR. Thus, the activity of B2 and opioid receptor-operated mechanisms in the Pa5 contribute to the SSR in the rat, suggesting a role for these endogenous peptides in the cardiovascular responses to SNS.     

trans-3,4-dimethyl-4-(3-carboxamidophenyl)piperidines: a novel class of micro-selective opioid antagonists

trans-3,4-Dimethyl-4-(3-carboxamidophenyl)piperidines constitute a novel class of micro opioid receptor antagonists. The CONH(2) group was found to be an effective isostere of the phenolic OH moiety. Structure-activity relationships at the piperidine nitrogen position led to the identification of several ligands displaying high affinity toward the cloned human micro opioid receptors, good selectivity micro/delta, micro/kappa, and potent in vitro antagonist activity.     

Novel ligands for the opioid receptors: synthesis and structure-activity relationships among 5'-aryl and 5'-heteroaryl 17-cyclopropylmethyl-4,5 alpha-epoxypyrido[2',3':6,7]morphinans

A series of pyridomorphinans possessing an aryl (10a-s) or heteroaryl (11a-h) substituent at the 5'-position of the pyridine ring of 17-cyclopropylmethyl-4,5 alpha-epoxypyrido[2',3':6,7]morphinan was synthesized and evaluated for binding and functional activity at the opioid delta, mu, and kappa receptors. All of these pyridomorphinans bound with higher affinity at the delta site than at mu or kappa sites. The binding data on isomeric compounds revealed that there exists greater bulk tolerance for substituents placed at the o-position of the phenyl ring than at m- or p-positions. Among the ligands examined, the 2-chlorophenyl (10l), 2-nitrophenyl (10n), 2-pyridyl (11a), and 4-quinolinyl (11g) compounds bound to the delta receptor with subnanomolar affinity. Compound 10c with the p-tolyl substituent displayed the highest mu/delta selectivity (ratio=42) whereas compound 10l with the 2-chlorophenyl substituent displayed the highest kappa/delta selectivity (ratio=23). At 10 microM concentration, the in vitro functional activity determined using [(35)S]GTP-gamma-S binding assays showed that all of the compounds were antagonists devoid of any significant agonist activity at the delta, mu, and kappa receptors. Antagonist potency determinations of three selected ligands revealed that the p-tolyl compound 10c is a potent delta selective antagonist. In the [(35)S]GTP-gamma-S assays this compound had a functional antagonist K(i) value of 0.2, 4.52, and 7.62 nM at the delta, mu, and kappa receptors, respectively. In the smooth muscle assays 10c displayed delta antagonist potency with a K(e) value of 0.88 nM. As an antagonist, it was 70-fold more potent at the delta receptors in the MVD than at the mu receptors in the GPI. The in vitro delta antagonist profile of this pyridomorphinan 10c resembles that of the widely used delta selective antagonist ligand naltrindole.     

Structure-activity relationship of rubiscolins as delta opioid peptides

To study the structure-activity relationship of rubiscolins (YPLDLF and YPLDL), delta opioid peptides derived from the spinach Rubisco, we substituted the amino acid residues and evaluated their activities by mouse vas deferens (MVD) and guinea pig ileum (GPI) assays as well as receptor affinity. Replacement of Leu(3) with Ile and Met in rubiscolin-6 potentiated the delta opioid activity by about four times in MVD assay. Asp(4) cannot be replaced by Ala, Glu or His. The original Leu(5) was optimal, while substitution of Phe(6) with Val potentiated its delta opioid activity by more than 10 times. The most potent derivative we obtained was YPMDLV, which was nearly 20 times more potent than rubiscolin-6 in MVD assay. The derivatives thus obtained showed higher delta receptor affinity and more potent antinociceptive activity than rubiscolins.     

Pharmacological activities of Vitex agnus-castus extracts in vitro.

The pharmacological effects of ethanolic Vitex agnus-castus fruit-extracts (especially Ze 440) and various extract fractions of different polarities were evaluated both by radioligand binding studies and by superfusion experiments. A relative potent binding inhibition was observed for dopamine D2 and opioid (micro and kappa subtype) receptors with IC50 values of the native extract between 20 and 70 mg/mL. Binding, neither to the histamine H1, benzodiazepine and OFQ receptor, nor to the binding-site of the serotonin (5-HT) transporter, was significantly inhibited. The lipophilic fractions contained the diterpenes rotun-difuran and 6beta,7beta-diacetoxy-13-hydroxy-labda-8,14-dien . They exhibited inhibitory actions on dopamine D2 receptor binding. While binding inhibition to mu and kappa opioid receptors was most pronounced in lipophilic fractions, binding to delta opioid receptors was inhibited mainly by a aqueous fraction. Standardised Ze 440 extracts of different batches were of constant pharmacological quality according to their potential to inhibit the binding to D2 receptors. In superfusion experiments, the aqueous fraction of a methanolic extract inhibited the release of acetylcholine in a concentration-dependent manner. In addition, the potent D2 receptor antagonist spiperone antagonised the effect of the extract suggesting a dopaminergic action mediated by D2 receptor activation. Our results indicate a dopaminergic effect of Vitex agnus-castus extracts and suggest additional pharmacological actions via opioid receptors.     

Prolonged in vivo antagonism of central mu- and delta-opioid receptor activity by beta-funal trexamine

beta-Funaltrexamine (beta-FNA) was tested in the spinal cord and supraspinally against inhibition of reflex bladder contractions produced in the anesthetized rat by the opioid-receptor selective agonists [D-Ala2, MePhe4, Gly (ol)5]enkephalin (DAGO, mu-agonist) and [D-Pen2, D-Pen5] enkephalin (DPDPE, delta-agonist). All agents were microinjected either intracerebroventricularly (i.c.v.) or intrathecally (i.t.). beta-FNA (1-8 micrograms) produced long-lasting antagonism of both DAGO and DPDPE. Complete recovery from its effects was only observed some 24-32 h later. Higher doses of beta-FNA (4 and 8 micrograms i.t.) produced short-lived agonistic activity though the selectivity of this was not determined. It was concluded that beta-FNA was a potent, long-lasting antagonist at central opioid receptors in vivo but was unselective for the mu and delta opioid receptor.