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This publication is the fourth in a series of safety evaluations performed by the Expert Panel of the Flavour and Extract Manufacturers' Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavouring substances under conditions of intended use. In this review, scientific data relevant to the safety evaluation of trans-anethole (i.e. 4-methoxypropenylbenzene) as a flavouring substance is critically evaluated by the FEMA Expert Panel. The evaluation uses a mechanism-based approach in which production of the hepatotoxic metabolite anethole epoxide (AE) is used to interpret the pathological changes observed in different species and sexes of laboratory rodents in chronic and subchronic dietary studies. Female Sprague Dawley rats metabolize more trans-anethole to AE than mice or humans and, therefore, are the most conservative model for evaluating the potential for AE-induced hepatotoxicity in humans exposed to trans-anethole from use as a flavouring substance. At low levels of exposure, trans-anethole is efficiently detoxicated in rodents and humans primarily by O-demethylation and omega-oxidation, respectively, while epoxidation is only a minor pathway. At high dose levels in rats, particularly females, a metabolic shift occurs resulting in increased epoxidation and formation of AE. Lower activity of the "fast" acting detoxication enzyme epoxide hydrolase in the female is associated with more pronounced hepatotoxicity compared to that in the male. The continuous intake of high dose levels of trans-anethole (i.e. cumulative exposure) has been shown in dietary studies to induce a continuum of cytotoxicity, cell necrosis and cell proliferation. In chronic dietary studies in rats, hepatotoxicity was observed when the estimated daily hepatic production of AE exceeded 30 mg AE/kg body weight. In female rats, chronic hepatotoxicity and a low incidence of liver tumours were reported at a dietary intake of 550 mg trans-anethole/kg body weight/day. Under these conditions, daily hepatic production of AE exceeded 120 mg/kg body weight. Additionally, neither trans-anethole nor AE show any evidence of genotoxicity. Therefore, the weight of evidence supports the conclusion that hepatocarcinogenic effects in the female rat occur via a non-genotoxic mechanism and are secondary to hepatotoxicity caused by continuous exposure to high hepatocellular concentrations of AE. trans-Anethole was reaffirmed as GRAS (GRASr) based on (1) its low level of flavour intake (54 microg/kg body weight/day); (2) its metabolic detoxication pathway in humans at levels of exposure from use as a flavouring substance; (3) the lack of mutagenic or genotoxic potential; (4) the NOAEL of 120 mg trans-anethole/kg body weight/day in the female rat reported in a 2 + -year study which produces a level of AE (i.e. 22 mg AE/kg body weight/day) at least 10,000 times the level (0.002 mg AE/kg body weight day) produced from the intake of trans-anethole from use as a flavouring substance; and (5) the conclusion that a slight increase in the incidence of hepatocellular tumours in the high dose group (550 mg trans-anethole/kg body weight/day) of female rats was the only significant neoplastic finding in a 2+ -year dietary study. This finding is concluded to be secondary to hepatotoxicity induced by high hepatocellular concentrations of AE generated under conditions of the study. Because trans-anethole undergoes efficient metabolic detoxication in humans at low levels of exposure, the neoplastic effects in rats associated with dose-dependent hepatotoxicity are not indicative of any significant risk to human health from the use of trans-anethole as a flavouring substance.

Endogneous delta and kappa opioid peptides possess a variety of immunomodulatory properties, and kappa-opioid receptor ligands recently were shown to suppress the expression of human immunodeficiency virus type 1 (HIV-1) in microglial cells, the resident macrophages of the brain. To determine whether the newly discovered endogenous mu-opioid receptor ligands endomorphin-1 and -2 would affect HIV-1 replication, these peptides were added to acutely infected brain cell cultures. Endomorphin-1 potentiated viral expression, in a bell-shaped dose-response manner with maximal enhancement approximately equal to 35% at 10(-10) M, in both mixed glial/neuronal cell and purified microglial cell cultures. Endomorphin-1's amplifying effect was blocked by pretreatment of brain cells with either the mu-opioid receptor selective antagonist beta-funaltrexamine or the G protein inhibitor pertussis toxin. However, the classical mu receptor agonists morphine and DAMGO (Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol) had no effect on viral expression or on endomorphin-1's amplifying effect. Taken together, these findings suggest that in this in vitro model of HIV-1 brain infection, endomorphin-1 potentiates viral expression via activation of an atypical mu-selective opioid receptor. They also provide evidence, for the first time, that an endogenous mu-opioid peptide has neuroimmunomodulatory activity.

A series of 7-arylidinenaltrexones (2a-m) related to the prototypical delta1-selective antagonist, 7-benzylidenenaltrexone 1 (BNTX), have been synthesized in an effort to develop more selective ligands. Testing in smooth muscle preparations revealed that members of the series exhibited varying degrees of selectively for delta receptors, with the o-methoxy (2e) and o-chloro (2j) congeners being most potent and most selective (Ke approximately 0.8 nm). Evaluation of 1, 2e, and 2f sc in mice using the tail-flick procedure indicated that they are selective delta1 opioid receptor antagonists in the lower dose range. At high doses these ligands, including BNTX, exhibited decreased delta1 selectivity due to increases in the ED50 ratios of [D-Ser2,Leu5]enkephalin-Thr6 and morphine. It is concluded that 2e and 2f possess in vivo selectivity similar to that of BNTX, but are less potent as delta1 antagonists.

On the basis of previous structure-activity studies of the highly potent and selective delta-opioid receptor antagonist naltrindole (1) and the spiroindanyl analogues 2 and 3, we have synthesized epimeric pairs of spirobenzocyclohexyl derivatives of naltrexone, oxymorphone, and hydromorphone (4-9). Pharmacologic evaluation in smooth muscle assays has revealed that the oxymorphone derivatives (6, 7) are delta-selective agonists and possess receptor binding profiles that are consistent with their agonist activity. It is proposed that the spirobenzocyclohexyl group of 6 and 7 orients its benzene moiety orthogonally with respect to the C ring of the opiate in a manner similar to that of the spiroindanyl analogue 3. It is suggested that this orthogonal orientation serves as an "address" to facilitate activation of delta receptors. The finding that the hydromorphone analogues (8, 9) were full mu agonists and exhibited only partial delta agonist activity suggests that the 14-hydroxyl group also contributes to the delta agonist activity. The naltrexone derivatives (4, 5) were mu-selective antagonists and exhibited relatively weak delta antagonist activity. However, the binding data indicated a very high-affinity delta-selective binding profile that was not consistent with the pharmacology. This study illustrates the differential contributions of the delta "address" to agonist and antagonist activity and supports the idea of different recognition sites for interaction of agonist and antagonist ligands with delta-opioid receptors.

The Expert Panel of the Flavor and Extract Manufacturers' Association (FEMA) has assessed the safety of furfural for its continued use as a flavour ingredient. The safety assessment takes into account the current scientific information on exposure, metabolism, pharmacokinetics, toxicology, carcinogenicity and genotoxicity. Furfural was reaffirmed as GRAS (GRASr) as a flavour ingredient under conditions of intended use based on: (1) its mode of metabolic detoxication in humans; (2) its low level of flavour use compared with higher intake levels as a naturally occurring component of food; (3) the safety factor calculated from results of subchronic and chronic studies, (4) the lack of reactivity with DNA; and (5) the conclusion that the only statistically significant finding in the 2-year NTP bioassays, an increased incidence of hepatocellular adenomas and carcinomas in the high-dose group of male mice, was secondary to pronounced hepatotoxicity. Taken together, these data do not indicate any risk to human health under conditions of use as a flavour ingredient. This evidence of safety is supported by the occurrence of furfural as a natural component of traditional foods, at concentrations in the diet resulting in a 'natural intake' that is at least 100 times higher than the intake of furfural from use as a flavour ingredient.

Substance P (SP) is released from primary afferent fibers in response to nociceptive stimuli. Capsaicin, which produces an initial hyperalgesic response followed by persistent antinociception, also elicits release of SP from primary afferent fibers. Capsaicin pretreatment decreases the content and release of SP from primary afferent fibers. This effect on SP has been hypothesized to mediate the antinociceptive effect of capsaicin. To test this hypothesis, mice were injected intrathecally (i.t.) with antinociceptive doses of capsaicin or SP(1–7) before superfusion of spinal cord tissue with 3 μ M capsaicin 24, 48, 96 or 168 h later. N-terminal metabolic fragments of SP that accumulate after capsaicin-induced SP release and are involved in the antinociceptive effect of capsaicin, were also tested. Like capsaicin SP(1–3), SP(1–4) and SP(1–7) were each antinociceptive when injected 24 h before nociceptive testing. However, at this time there was no decrease in capsaicin-evoked release of SP in tissue from capsaicin- and SP(1–7)-pretreated animals compared to those injected with vehicle. In contrast, capsaicin-evoked SP release decreased significantly in tissue from mice pretreated with capsaicin or SP(1–7) 48 h prior to testing. d -Substance P(1–7), which prevents antinociception, blocked capsaicin- and SP(1–7)-induced decreases in SP release, indicating that these effects are mediated by SP N-terminal activity. Total spinal cord content of SP did not differ amongst treatment groups. These data indicate that antinociception does not appear to depend on decreases in SP release or content as antinociception precedes decreases in SP release.

A series of 2',3'-disubstituted pyrrolomorphinans (5a-i) were synthesized to determine the role of steric hindrance at mu and kappa receptors in promoting delta opioid receptor antagonist selectivity. In smooth muscle preparations, five members of the series (5a-c,e,f) possessed Ke values in the range 2-15 nM and were delta selective. Since the unsubstituted analogue 4 possessed delta antagonist potency of similar magnitude, but was not delta selective, it is suggested that the 2',3'-substitution confers delta selectivity by hindering the interaction of the pharmacophore at mu and kappa receptors, while not affecting delta receptors.

A series consisting of spiroindanyl (5-7), benzospiroindanyl (8-10), and spiroperinaphthyl (11) derivatives of naltrexone and oxymorphone were synthesized in order to investigate the role of an orthogonal-oriented "address" for delta opioid receptors. All of the ligands exhibited a preference for delta receptors in vitro. The 7-benzospiroindanyl derivative 8 (BSINTX) was the most selective delta opioid receptor antagonist in vitro. In mice BSINTX antagonized the delta 1-selective agonist, [D-Pen2,D-Pen5]enkephalin without significantly affecting the antinociceptive potency of delta 2, mu, and kappa agonists. The results of this study are consistent with an orthogonally-oriented address favoring delta 1 activity.

Guinea pig ileum longitudinal muscle/myenteric plexus preparations contain functional mu- and kappa-opioid receptors. A preparation with blocked kappa receptors was obtained by pretreating guinea pigs with 0.1-10.0 mg/kg intraperitoneal doses of a new kappa-selective affinity label, DIPPA (2-(3,4-dichlorophenyl)-N-methyl-N-[1S-1-(-3-isothiocyanatophenyl)-2-(-1 -pyrrolidinyl) ethyl] acetamide). Determination of IC50 values for the mu-selective agonist DAMGO ([D-Ala2,MePhe4,Gly(ol)5]-enkephalin) and the kappa-selective agonist U 69,593 ([5alpha,7alpha,8beta]-(+)-N-methyl-N-[7-(1-pyrrolidin yl)-1-oxaspiro-(4,5)-dec-8-yl]) showed that 0.5 mg/kg DIPPA at 48 h produced reliable, near-complete blockade of kappa-opioid activity but a minimal shift for kappa-opioid agonism. This new assay should be useful for studying mixed agonists/antagonists that produce strong kappa-opioid receptor agonism, which prevents determination of mu-opioid receptor antagonism.

Bivalent ligands that contain two pharmacophores linked by a spacer are promising tools to investigate the pharmacology of opioid receptor heteromers. Evidence for occupation of neighboring protomers by two phamacophores of a single bivalent ligand (bridging) has relied mainly on pharmacological data. In the present study, we have employed an immunocytochemical correlate to support in vivo biological studies that are consistent with bridging. We show that a bivalent mu agonist/delta antagonist (MDAN-21) that is devoid of tolerance due to possible bridging of mu and delta protomers, prevents endocytosis of the heteromeric receptors in HEK-293 cells. Conversely, a bivalent ligand (MDAN-16) with a short spacer, or monovalent mu agonist give rise to robust internalization. The data suggest that the immobilization of proximal mu and delta protomers is due to bridging by MDAN-21. The finding that MDAN-21 and its shorter spacer homologue MDAN-16 possess equivalent activity in HEK-293 cells, but produce dramatically divergent internalization of mu-delta heteromer, is relevant to the role of internalization and tolerance.

For over 35 years, an independent panel of expert scientists has served as the primary body for evaluating the safety of flavour ingredients. This group, the Expert Panel of the Flavor and Extract Manufacturers' Association (FEMA), has achieved international recognition from the flavour industry, government regulatory bodies including the Food and Drug Administration, and the toxicology community for its unique contributions. To date, the Expert Panel has evaluated the safety of more than 1700 flavour ingredients and determined the vast majority to be "generally recognized as safe" (GRAS). Elements that are fundamental to the safety evaluation of flavour ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavour ingredients are evaluated individually taking into account the available scientific information on the group of structurally related substances. The elements of the GRAS assessment program as they have been applied by the Expert Panel to the group of 119 alicyclic substances used as flavour ingredients, and the relevant scientific data which provide the basis for the GRAS status of these substances, are described herein.

A novel boron tribromide-catalyzed rearrangement of ketones to allylic alcohols was discovered in the 7-phenylmorphinan-6-one system. The reaction involved the stereospecific migration of an axial 7β-phenyl (or a hydrogen) to the C-6 carbonyl carbon, followed by the elimination of the H-8 proton leading to the generation of allylic alcohols. A possible mechanistic pathway for this rearrangement is discussed.

Aspartic acid conjugates of 2-(3,4-dichlorophenyl)-N-methyl-N-[(1S)-1-(3-aminophenyl)-2-(1-pyrrol idinyl) ethyl]acetamide (5) were synthesized and evaluated in mice for antinociceptive activity by intravenous and intracerebroventricular routes of administration. The intravenously-administered alpha-conjugate of L-Asp (2), its D-Asp diastereomer (3), and the beta -conjugate of L-Asp (4) were found to be 11-, 31-, and 40-fold, respectively, less effective than the parent ligand 1 (ICI 199,441) in producing central nervous system mediated antinociception in the mouse abdominal stretch assay. In addition, iv-administered 2 and 3 were found to also produce potent antinociception in the tonic phase of the mouse formalin assay, which is a model of tonic rather than acute pain. This study suggests that the attachment of a zwitterionic moiety to a position in the molecule that exhibits bulk tolerance is a viable strategy for the design of peripherally-selective and peripherally-active opioids.

The relative importance of different opioid receptor types in mediating alcohol drinking behavior compared with the intake of other ingesta can be determined by characterizing the effects of selective opioid antagonists on the intake of various ingesta. Nonselective opioid receptor antagonists suppress the intake of many ingesta including alcohol, food, water, and sweets. Two distinct subtypes of delta-opioid receptors, delta 1 and delta 2, have recently been identified in rodent brain. We have previously reported that naltrindole (NTI), which blocks both delta 1 and delta 2 receptors, suppresses both alcohol and saccharin intake in rats selectively bred for high alcohol preference (P line). We now report that naltriben (NTB), an opioid antagonist that is selective for delta 2-opioid receptors, suppresses alcohol intake in rats of the P line and the effect appears to be both specific for alcohol and independent of alcohol palatability. NTB may reduce alcohol intake by attenuating the reinforcing pharmacological properties of alcohol.

This study demonstrates that the selective delta receptor antagonists ICI 174864 and naltrindole (NTI) attenuate alcohol intake in a dose-dependent manner, without altering water intake, in rats selectively bred for alcohol preference. ICI 174864 had a very limited duration of action, as evidenced by the fact that suppression of alcohol intake lasted for only an hour following ICI 174864 administration. NTI, when administered in a dose of 10 mg/kg, suppressed alcohol intake by 28%. Increasing the dose of NTI to 15 mg/kg produced a 44% suppression of alcohol intake, but a further increase to 20 mg/kg did not produce greater suppression than was seen with a dose of 15 mg/kg (46% versus 44%, respectively). This suggests that NTI is maximally effective in suppressing alcohol intake at a dose of 15.0 mg/kg. NTI displayed a long duration of action, as evidenced by attenuation of alcohol drinking that lasted for at least 8 h following drug treatment. Administering the maximally effective dose of NTI (15 mg/kg) in two parts, separated by 4 h, served to prolong the duration of action of NTI and produced an attenuation of alcohol intake, but not water intake, that lasted for at least 28 h. The effect of NTI on alcohol intake was not specific for alcohol, as evidenced by the fact that NTI reduced the intake of saccharin solutions with and without alcohol.

2-(3,4-Dichlorophenyl)-N-methyl-N-[(1S)-1-(3-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (1, DIPPA) has been previously reported to be an opioid receptor affinity label that produces selective and long-lasting κ opioid receptor antagonism in mice. High specific activity [ 3 H]DIPPA (39.7 Ci/mmol) was prepared by bromination and catalytic tritiation of the amino precursor of DIPPA followed by conversion to the isothiocyanate with thiophosgene

The antinociception induced by beta-endorphin given supraspinally has been previously demonstrated to be mediated by the release of [Met5]enkephalin acting on delta-opioid receptors in the spinal cord. The present study was designed to determine what type of opioid receptors in the spinal cord is involved in beta-endorphin-induced antinociception in the rat. Antinociception was induced by beta-endorphin (0.6 nmol) given into nucleus raphe obscurus and was assessed by the tail-flick test in pentobarbital-anesthesized rats. Naltriben (0.6-6.0 nmol), a selective delta 2-opioid receptor antagonist, given intrathecally dose-dependently attenuated beta-endorphin-induced inhibition of the tail-flick response. On the other hand, 7-benzylidene naltrexone (2.1-64.3 nmol), CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, 0.09-2.8 nmol), or nor-binaltorphimine (1.4-40.8 nmol), selective delta 1-, mu-, and kappa-opioid receptor antagonists, respectively, did not block beta-endorphin-induced antinociception. The results of present study in rats are consistent with previous experiments in mice indicating that spinal delta 2-, but not delta 1-, mu- or kappa-opioid receptors are involved in beta-endorphin-induced inhibition of the tail-flick response.

Coexpressed and colocalized μ- and δ-opioid receptors have been established to exist as heteromers in cultured cells and in vivo. However the biological significance of opioid receptor heteromer activation is less clear. To explore this significance, the efficacy of selective activation of opioid receptors by SNC80 was assessed in vitro in cells singly and coexpressing opioid receptors using a chimeric G-protein-mediated calcium fluorescence assay, SNC80 produced a substantially more robust response in cells expressing μ-δ heteromers than in all other cell lines. Intrathecal SNC80 administration in μ- and δ-opioid receptor knockout mice produced diminished antinociceptive activity compared with wild type. The combined in vivo and in vitro results suggest that SNC80 selectively activates μ-δ heteromers to produce maximal antinociception. These data contrast with the current view that SNC80 selectively activates δ-opioid receptor homomers to produce antinociception. Thus, the data suggest that heteromeric μ-δ receptors should be considered as a target when SNC80 is employed as a pharmacological tool in vivo.

Opioid conjugate vaccines have shown promise in animal models as a potential treatment for opioid addiction. Individual vaccines are quite specific and each targets only a limited number of structurally similar opioids. Since opioid users can switch or transition between opioids, we studied a bivalent immunization strategy of combining 2 vaccines that could target several of the most commonly abused opioids; heroin, oxycodone and their active metabolites. Morphine (M) and oxycodone (OXY) haptens were conjugated to keyhole limpet hemocyanin (KLH) through tetraglycine (Gly)(4) linkers at the C6 position. Immunization of rats with M-KLH alone produced high titers of antibodies directed against heroin, 6-monoacetylmorphine (6-MAM) and morphine. Immunization with OXY-KLH produced high titers of antibodies against oxycodone and oxymorphone. Immunization with the bivalent vaccine produced consistently high antibody titers against both immunogens. Bivalent vaccine antibody titers against the individual immunogens were higher than with the monovalent vaccines alone owing, at least in part, to cross-reactivity of the antibodies. Administration of a single concurrent intravenous dose of 6-MAM and oxycodone to rats immunized with the bivalent vaccine increased 6-MAM, morphine and oxycodone retention in serum and reduced the distribution of 6-MAM and oxycodone to brain. Vaccine efficacy correlated with serum antibody titers for both monovalent vaccines, alone or in combination. Efficacy of the individual vaccines was not compromised by their combined use. Consistent with the enhanced titers in the bivalent group, a trend toward enhanced pharmacokinetic efficacy with the bivalent vaccine was observed. These data support the possibility of co-administering two or more opioid vaccines concurrently to target multiple abusable opioids without compromising the immunogenicity or efficacy of the individual components.

Morphine and related drugs are widely employed as analgesics despite the side effects associated with their use. Although morphine is thought to mediate analgesia through mu opioid receptors, delta opioid receptors have been implicated in mediating some side effects such as tolerance and dependence. Here we present evidence in rhesus monkeys that morphine, fentanyl, and possibly methadone selectively activate mu-delta heteromers to produce antinociception that is potently antagonized by the delta opioid receptor antagonist, naltrindole (NTI). Studies with HEK293 cells expressing mu-delta heteromeric opioid receptors exhibit a similar antagonism profile of receptor activation in the presence of NTI. In mice, morphine was potently inhibited by naltrindole when administered intrathecally, but not intracerebroventricularly, suggesting the possible involvement of mu-delta heteromers in the spinal cord of rodents. Taken together, these results strongly suggest that, in primates, mu-delta heteromers are allosterically coupled and mediate the antinociceptive effects of three clinically employed opioid analgesics that have been traditionally viewed as mu-selective. Given the known involvement of delta receptors in morphine tolerance and dependence, our results implicate mu-delta heteromers in mediating both antinociception and these side effects in primates. These results open the door for further investigation in humans.

MDAN-21, 7′-{2-[(7-{2-[({(5α,6α)-4,5-Epoxy-3,14-dihydroxy-17-methylmorphin-6-yl}-aminocarbonyl)metoxy]-acetylamino}-heptylaminocarbonyl)-methoxy]-acetylamino}-naltrindole, a bivalent opioid ligand containing a mu-opioid receptor agonist (derived from oxymorphone) linked to the delta-opioid receptor antagonist (related to naltrindole) by a spacer of 21 atoms, was reported to have potent analgesic properties in mice. Tolerance, physical dependence, and conditioned place preference were not evident in that species. The finding that bivalent ligands in this series, with spacers 19 atoms or greater, were devoid of tolerance and dependence led to the proposal that MDAN-21 targets heteromeric mu-delta-opioid receptors. The present study focused on its effects in nonhuman primates (Macaca mulatta), a species with a physiology and behavioral repertoire not unlike humans. With regard to opioids, this species usually better predicts clinical outcomes. MDAN-21 substituted for morphine in morphine-dependent monkeys in the remarkably low dose range 0.006–0.032 mg/kg, subcutaneously. Although MDAN-21 failed to produce reliable thermal analgesia in the dose range 0.0032–0.032 mg/kg, intramuscularly, it was active in the same dose range and by the same route of administration, in the capsaicin-induced thermal allodynia assay. The results suggest that MDAN-21 may be useful in the treatment of opioid dependence and allodynia. The data provide additional evidence that opioid withdrawal is associated with sensitized pain.

The indolic nitrogen of the delta-opioid receptor antagonist, naltrindole (1), was derivatized with benzyl or substituted benzyl to afford a series (2-9) that retained delta-opioid receptor antagonist activity and selectivity in vitro. The two most potent members (2 and 8) of the series were evaluated in mice and were found to produce delta-selective antagonism of [D-Ser2,Leu5]enkephalin-Thr6 which lasted 5 days. N-Benzylnaltrindole (2) should be useful as a delta 2-selective antagonist for in vivo studies where prolonged action is desired.

Previous studies have shown that morphine promotes the replication of human immunodeficiency virus (HIV)-1 in peripheral blood mononuclear cell cocultures. In the present study, we tested the hypothesis that morphine would amplify HIV-1 expression in the chronically infected promonocytic clone U1 when cocultured with lipopolysaccharide-stimulated human fetal brain cells. Marked upregulation of HIV-1 expression was observed in these cocultures (quantified by measurement of HIV-1 p24 antigen levels in supernatants), and treatment of brain cells with morphine resulted in a bell-shaped dose-dependent enhancement of viral expression. The mechanism of morphine's amplifying effect appears to be opioid receptor-mediated and to involve enhanced production of tumor necrosis factor-alpha by microglial cells.

Naltrindole (1) (NTI) is a highly potent and selective delta-opioid receptor antagonist. In an effort to understand the origin of the high potency, affinity, and selectivity of NTI, we have examined the conformational role of its indolic benzene moiety through the synthesis of related naltrexone derivatives 3-8, which contain the benzene moiety in different orientations and at different attachments in the molecule. One of these naltrexone derivatives, 5, whose 7-indanyl benzene moiety is orthogonal to ring C of the morphinan system, is a potent delta-opioid receptor antagonist in vitro and in vivo. Computer-assisted molecular overlay studies of the minimized structures (2-8) revealed the importance of the position of the benzene moiety for effective interaction with delta-opioid receptors. In compounds 2, 4, and 5, the aromatic ring falls in the same region of space as that of the indolic benzene moiety of NTI, and all of these ligands possessed significant activity at delta-opioid receptors. Analogues (3 and 6-8) which were shown to have relatively weak delta-opioid receptor antagonist potency have their aromatic groups located in a space that is different from that of the more potent analogues.

The interaction of [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Ala2,Glu4]deltorphin with delta-opioid receptor subtypes was investigated. Pretreatment of mice with the delta 1-opioid receptor antagonist, [D-Ala2,Leu5,Cys6]enkephalin (DALCE), produced a virtually complete antagonism of the antinociceptive actions of DPDPE, but had no effect on those of [D-Ala2,Glu4]deltorphin. In DALCE pretreated mice (i.e., delta 1-opioid receptors blocked), DPDPE was able to significantly antagonize the antinociceptive effects of [D-Ala2,Glu4]deltorphin. Pretreatment of mice with the delta 2-opioid receptor antagonist, naltrindole-5'-isothiocyanate (5'-NTII) produced a virtually complete antagonism of the antinociceptive effects of [D-Ala2,Glu4]deltorphin, but had no effect on the antinociception produced by DPDPE. In 5'-NTII pretreated mice (i.e., delta 2-opioid receptors blocked), [D-Ala2,Glu4]deltorphin had no effect on the antinociception produced by DPDPE. These data suggest that [D-Ala2,Glu4]deltorphin is highly selective for the delta 2-opioid receptor in vivo, and that neither agonist nor antagonist actions can be demonstrated at delta 1-opioid receptors for this peptide. In contrast, under appropriate conditions, DPDPE can be shown to interact with both delta 1- and delta 2-opioid receptor subtypes; DPDPE may have limited efficacy (i.e., is a partial agonist) at the delta 2-opioid receptor.

Opioid agonists selective for mu- or delta opioid receptors inhibit adenylyl cyclase in membranes from rat caudate-putamen and nucleus accumbens. The presence of subtypes of delta opioid receptors has been suggested. In both brain regions we have found that the inhibition of adenylyl cyclase by DPDPE was more readily antagonized by 7-benzylidenenaltrexone (BNTX), than by naltriben. In contrast, the inhibitory effects of deltorphin-II and DSLET were more readily antagonized by naltriben, than by BNTX. Neither naltriben nor BNTX significantly antagonized the effect of a mu selective agonist. These results suggest that inhibition of adenylyl cyclase in caudate-putamen and nucleus accumbens is regulated by two forms of delta-opioid receptor with ligand selectivities similar to those two forms proposed to mediate analgesic effect.

Numerous G protein-coupled receptors (GPCRs) have been shown to form heteromeric receptors in cell-based assays. Among the many heteromers reported in the opioid receptor family are μ/κ, κ/δ, and μ/δ. However, the in vivo physiological and behavioral relevance for the proposed heteromers have not yet been established. Here we report a unique example of a ligand, N -naphthoyl-β-naltrexamine (NNTA) that selectively activates heteromeric μ/κ-opioid receptors in HEK-293 cells and induces potent antinociception in mice. NNTA was an exceptionally potent agonist in cells expressing μ/κ-opioid receptors. Intriguingly, it was found to be a potent antagonist in cells expressing only μ-receptors. In the mouse tail-flick assay, intrathecal (i.t.) NNTA produced antinociception that was ~100-fold greater than by intracerebroventricular (i.c.v.) administration. The κ-antagonist, norBNI, decreased the i.t. potency, and the activity was virtually abolished in μ-opioid receptor knockout mice. No tolerance was induced i.t., but marginal tolerance (3-fold) was observed via the i.c.v. route. Moreover, NNTA produced neither significant physical dependence nor place preference in the ED 50 dose range. Taken together, this work provides an important pharmacologic tool for investigating the in vivo functional relevance of heteromeric μ/κ-opioid receptors and suggests an approach to potent analgesics with fewer deleterious side effects.

This publication is the thirteenth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2600 substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of aliphatic and aromatic terpene hydrocarbons as flavoring ingredients are evaluated. The group of aliphatic and aromatic terpene hydrocarbons was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their rapid absorption, metabolic detoxication, and excretion in humans and other animals; their low level of flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic potential.

The fumaramate derivative of naltrexone, beta-funaltrexamine (beta-FNA), is a highly selective long-lasting mu opioid receptor antagonist that is active both in vitro and in vivo, presumably as a result of covalent binding to a mu receptor-based sulfhydryl group. Glutathione, which occurs in significant levels in brain and liver, was found to undergo a Michael-type reaction with beta-FNA in the test tube to give a stable conjugate 3 which occurred as an isomeric mixture. When tested in the GPI and MVD smooth muscle preparations, 3 was found to possess one-tenth the agonist activity of beta-FNA is both tissues, but showed no irreversible antagonist activity. The same result was found for the cysteine conjugate 4, except for some irreversible antagonism in the MVD. Both conjugates antagonize the antinociceptive effect of morphine in the mouse radiant heat tail-flick assay on icv administration. This antagonism persisted and actually increased over 24 h and generally paralleled the duration profile of beta-FNA. On sc administration, beta-FNA and 3 showed similar duration of antagonistic effect, while 4 exhibited only marginal activity at the early time interval. When the compounds are compared by the dose to produce equivalent antagonism, beta-FNA and 3 appeared equally effective and accessible by either route, whereas 4 showed a large difference between the two routes. It is possible that the ultra-long antagonism of the conjugates may result from their enzymatic conversion to beta-FNA in the central nervous system in view of the fact that conjugate 5, which cannot be converted to beta-FNA, did not produce antagonism of long duration in vivo. Alternatively, the protracted antagonism could arise from sequestration of 3 and 4 in tissue compartments that interface with mu opioid receptors.

Naloxone and naltrindole attenuate the locomotor response to amphetamine, implicating delta-opioid receptors in the opioid-antagonist/amphetamine interaction. To determine the role of delta-opioid receptor subtypes in this phenomenon, rats were pretreated with the following selective antagonists administered intracisternally: naltrindole, [D-Ala2,Leu5,Cys6]enkephalin (DALCE, delta 1 receptor selective), naltrindole-5'-isothiocyanate (delta 2 receptor selective). Cumulative dose-response curves to amphetamine were constructed (saline, 0.1, 0.4, 1.6 and 6.4 mg/kg s.c.), with injections every 30 min. Naltrindole was also tested against cumulative doses of cocaine (saline, 3.0, 10, 30 and 56 mg/kg i.p.). Gross and fine motor activity were recorded for 20 min, commencing 10 min postinjection. Amphetamine and cocaine dose dependently increased both gross and fine movements. Naltrindole (10 micrograms) attenuated the gross but not fine activity response to amphetamine, but 10 or 30 micrograms failed to influence the response to cocaine. Naltrindole-5'-isothiocyanate (30 micrograms) attenuated slightly but significantly the gross activity response to amphetamine, whereas DALCE (30 micrograms) was without effect. However, a combination of 10 micrograms each of DALCE and naltrindole-5'-isothiocyanate markedly attenuated the amphetamine-induced increases in gross movements without altering fine activity. These data provide further evidence for the involvement of delta-opioid receptors in the modulation of behavioral effects of amphetamine; both delta 1- and delta 2-opioid receptors appear to play a role. The differential effects of opioid antagonists on locomotor activity stimulated by amphetamine and cocaine suggests differences in the mechanism of action of these drugs not previously appreciated.

Recent reports provided evidence that at least two δ opioid receptors may mediate antinociception in mice. In this study, we studied further the involvement of δ opioid receptor subtypes in mediating antinociception at spinal sites in mice using subtype selective agonists and antagonists. The antinociceptive ED 50 values (95 % C.I.) of i.t. administered DPDPE [(D-Pen 2 , D-Pen 5 )enkephalin] ( δ 1 receptor agonist) and DELT II [(D-Ala 2 )deltorphin II] ( δ 2 receptor agonist) were 6.3 (5.2–7.6) and 6.4 (5.4–7.7) nmol/mouse, respectively. Administration of BNTX, s.c. increased the antinociceptive ED 50 value of DPDPE 5.9-fold whereas that of DELT II was not changed significantly. On the other hand administration of maltriben (NTB, the benzofuran derivative of naltrindole), s.c. increased the antinociceptive ED 50 value of DELT II 12.5-fold but did not alter that of DPDPE. Similarly administration of BNTX, i.t. increased the antinociceptive ED 50 value of DPDPE 4-fold without altering significantly that of DELT II. NTB given i.t. enlarged the antinociceptive ED 50 of DELT II 11-fold without affecting significantly that of DPDPE. BNTX, s.c. did not alter the antinociceptive ED 50 values of the μ-agonists, DAMGO [(D-Ala 2 , MePhe 4 , Gly-ol 5 ) enkephalin] and morphine or that of the κ-agonist, U50,488H [trans(±)−3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinylcyclohexyl]benzeneacetamide] These results demonstrate that BNTX is highly selective for δ 1 opioid receptors at spinal sites. Also, the present data provide for the involvement of both δ 1 and δ 2 opioid receptors in mediating antinociception at spinal sites in mice.

beta-Endorphin(1-27) (i.c.v.) has been reported to inhibit the antinociceptive activity of i.c.v. administered beta-endorphin in mice. In this study the antagonist activity of beta-endorphin(1-27) has been confirmed and the antagonism appears to be mediated at delta 1 opioid receptors. At higher doses than that used for antagonism, i.c.v. administered beta-endorphin(1-27) was a full antinociceptive agonist. The antinociceptive activity of beta-endorphin is attributed to the release of met-enkephalin in the spinal cord and is antagonized by the selective delta 2 opioid receptor antagonist, naltriben (NTB) but not by the selective delta 1 opioid receptor antagonist, 7-benzylidenenaltrexone (BNTX). In contrast, the antinociceptive activity of i.c.v. administered beta-endorphin(1-27) was not affected by either NTB or BNTX administered i.c.v. or i.t. Also, the antinociceptive activity of beta-endorphin(1-27) was unaffected by the selective mu opioid receptor antagonist, beta-funaltrexamine (beta-FNA) or the selective kappa opioid receptor antagonist, norbinaltorphimine (norBNI). Thus, beta-endorphin(1-27) appears to mediate antinociception supraspinally through the interaction of a unique receptor, i.e. a receptor that is different from mu, kappa, delta 1 or delta 2 opioid receptors. Alternatively, a non-opioid mechanism may be considered.

A novel boron tribromide-catalyzed rearrangement of ketones to allylic alcohols was discovered in the 7-phenylmorphinan-6-one system. The reaction involved the stereospecific migration of an axial 7β-phenyl (or a hydrogen) to the C-6 carbonyl carbon, followed by the elimination of the H-8 proton leading to the generation of allylic alcohols. A possible mechanistic pathway for this rearrangement is discussed.

Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named 'reporter affinity labels' because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.

Various concepts have served as the basis for the development of models to explore the relationship between molecular structure and biological activity. In this presentation I have outlined five concepts that have been useful in our investigation of opioid receptor multiplicity and in the design of selective opioid receptor antagonists. The first of these, the multiple binding modality concept, led to our application of four other concepts in the development of opioid receptor probes. Some of these probes are now standard tools in opioid research. These include the mu-selective affinity label beta-FNA (10), the kappa opioid receptor antagonist norBNI (15), and the delta opioid antagonist NTI (20). These highly selective antagonists have advantages over the universal opioid antagonists naloxone and naltrexone because they are of value in probing the interaction of endogenous opioid peptides with opioid receptor types. Additionally, they are useful in evaluating the selectivity of new opioid agonists. Also, selective opioid antagonists have potential clinical applications in the treatment of a variety of disorders where endogenous opioids play a modulatory role. These include constipation, immune function, drug addiction, and alcoholism, to name only a few.

The phenolic hydroxy group of opiate-derived ligands is of known importance for biological activity. On the basis of its putative role as a hydrogen-bonding donor in the interaction with opioid receptors, it was replaced with a sulfonamide group because of their similar pKa values. The first thebaine-derived 3-amino (8a, 8b) and subsequent sulfonamide analogues (10a, 10b) were synthesized from naltrexone (1a) and oxymorphone (1b) in a linear nine-step synthesis. The sulfonamides were tested in vitro and found inactive.

During the past decade, substantial evidence has documented that opioid receptor heterodimers form in cell lines expressing one or more opioid receptors. More recent studies have begun to investigate whether heterodimer formation also occurs in vivo. We have used opioid receptor knockout mice to determine whether the in vivo intrathecal (i.t.) pharmacological potency of delta, kappa, and bivalent kappa/delta ligands is altered in the absence of the KOR-1 and/or DOR-1 genes. We observe that both NorBNI (a kappa antagonist) and KDN-21 (a kappa/delta bivalent antagonist) specifically inhibit DPDPE but not deltorphin II i.t potency in wild-type mice but that following mutation of KOR-1, the ability of either compound to reduce DPDPE potency is lost. In contrast, knockout of KOR-1 unexpectedly slightly reduces the potency of deltorphin II (delta2) but not DPDPE (delta1). Finally, two compounds with kappa agonist activity, 6′-GNTI (a putative kappa/delta heterodimer selective agonist) and KDAN-18 (kappa agonist/delta antagonist bivalent ligand) show reduced potency in DOR-1 KO mice. These results show, genetically, that bivalent ligands with kappa agonist activity require delta receptors for maximal potency in vivo, which is consistent with the presence of opioid heterodimer/oligomer complexes in vivo, and also highlight the complexity of delta drug action even when complementary pharmacologic and genetic approaches are used.

In an effort to develop antagonists for kappa-mu opioid receptor heterodimers, a series of bivalent ligands 3-6 containing kappa- and mu-antagonist pharmacophores were designed and synthesized. Evaluation of the series in HEK-293 cells revealed 4 (KMN-21) to selectively antagonize the activation of kappa-mu heterodimers, suggesting possible bridging of receptors when the bivalent ligand spacer contains 21 atoms.

Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated 1,4-benzodiazepines, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (9) and (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (7), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin types 1 and 2 (CCK(1) and CCK(2)) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The (125)I-labeled CCK(1) receptor-selective compound 9 often revealed a substantially higher amount of CCK(1) receptor binding sites in tumors than the agonist (125)I-CCK. Conversely, the radioiodinated CCK(2) receptor-selective compound 7 showed generally weaker tumor binding than (125)I-CCK. In conclusion, compound 9 is an excellent radioiodinated nonpeptidic antagonist ligand for direct and selective labeling of CCK(1) receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK(1) receptor-expressing tumors in vivo.

Both mu opioid (MOP)† and type 2 cholecystokinin (CCK2) receptors are present in areas of the central nervous system that are involved in modulation of pain processing. We conducted bioluminescence resonance energy transfer (BRET) studies on COS cells coexpressing MOP and CCK2 receptors to determine whether receptor heterodimerization is involved in such modulation. These studies revealed the absence of constitutive or monovalent ligand-induced heterodimerization. Heterodimerization of MOP and CCK2 receptors therefore is unlikely to be responsible for the opposing effects between morphine and CCK in the CNS. However, association was induced, as indicated by a positive BRET signal, on exposure of the cells to bivalent ligands containing mu-opioid agonist and CCK2 receptor antagonist pharmacophores linked through spacers containing 16 to 22 atoms, but not with a shorter (9-atom) spacer. These studies demonstrate for the first time that an appropriately designed bivalent ligand is capable of inducing association of G protein-coupled receptors. The finding that opioid tolerance studies with these ligands in mice showed no correlation with the BRET data is consistent with the absence of association of MOP and CCK2 receptors in vivo.

A series of heterocyclic analogues 2-5 related to naltrindole (1) (NTI) and 6-arylnaltrexone derivatives 6-8 were synthesized in order to determine the role of the spacer and the address moieties in conferring delta opioid receptor antagonist activity. The benzofuran (NTB), quinoxaline, and quinoline analogues (2, 3, and 4, respectively) were delta-selective opioid antagonists in vitro and bound selectively to delta receptors. The tetrahydroindole derivative 5, while delta-selective, was considerably less potent than its indole analogue 13. The data for 2-4 indicate that heterocycles other than pyrrole can serve as a spacer for the delta address moiety. Moreover, the lower delta antagonist potency of 5 illustrates the importance of the aromatic address component. Molecular dynamics simulations of enkephalin using a zipper binding model are consistent with a delta address subsite that may accommodate the benzene moiety of NTI or the Phe4 phenyl group of leucine enkephalin. The considerably lower delta opioid receptor antagonist potencies of the 6-aryl derivatives 6-8 are consistent with the conformational mobility of the aryl group and its location in the molecule.

In this study we investigated the development of cross-tolerance among intrathecally (i.t.)- administered mu and delta opioid receptor selective peptides in β-funaltrexamine (β-FNA)-treated mice. Tolerance to the antinociceptive effect of i.t. administered DPDPE was accomplished by administration of 16 nmol/mouse of DPDPE, i.t. 3 hr before testing in β-FNA-treated mice (10 μmol/kg, s.c., 24 hr before the experiment). Cross-tolerance developed to the antinociceptive effect of i.t. administered DADLE but not to those of DSLETT or DAMGO. DSLET (0.1 nmol/mouse i.t.) administration in β-FNA-treated mice resulted in tolerance development to its antinociceptive effect. The same pretreatment resulted in a marginally significant increase in the antinociceptive ED 50 value of DPDPE. There was no cross-tolerance to the antinociceptive effect of i.t. administered DADLE or DAMGO. These results provide further evidence for the existence of delta opioid receptor subtypes where DADLE and DPDPE interact with one site and DSLET with a different one.

This publication is the 12th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2200 chemically-defined substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, toxicodynamics and toxicology. Scientific data relevant to the safety evaluation for the use of aliphatic, linear α,β-unsaturated aldehydes and structurally related substances as flavoring ingredients are evaluated. The group of substances was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their low level of flavor use; the rapid absorption and metabolism of low in vivo concentrations by well-recognized biochemical pathways; adequate metabolic detoxication at much higher levels of exposure in humans and animals; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies. While some of the compounds described here have exhibited positive in vitro genotoxicity results, evidence of in vivo genotoxicity and carcinogenicity occurs only under conditions in which animals are repeatedly and directly exposed to high irritating concentrations of the aldehyde. These conditions are not relevant to humans who consume α,β-unsaturated aldehydes as flavor ingredients at low concentrations distributed in a food or beverage matrix. © 2008 Elsevier Ltd. All rights reserved. Chemicals / CAS: 2 hexenal, 505-57-7; sorbic acid, 110-44-1, 22500-92-1; Aldehydes; Carcinogens; Flavoring Agents; Mutagens

We evaluated the effect of the kappa antagonist, nor-binaltorphimine (nor-BNI) on deprivation and opioid-induced feeding in rats. Intracerebroventricular administration of nor-BNI (100 nmol) decreased deprivation-induced feeding for as long as 24 h, albeit in a fairly weak manner (maximum decrease of approximately 28%). Nor-BNI (1, 10 and 100 nmol) decreased feeding induced by the kappa ligand U-50,488H by as much as 85% during the first hour of the study. This kappa antagonist also decreased feeding induced by the delta agonist DSLET and the mu agonist DAMGO. Based on previous studies indicating that nor-BNI is a selective kappa antagonist, we conclude that not only U-50,488H (kappa), but also DSLET (delta) and DAMGO (mu)-induced feeding are dependent upon an active kappa receptor.

A series of naltrexamine bivalent ligands, compounds with two naltrexamine pharmacophores separated by a spacer which contains a variable number of glycyl units flanking a succinyl group, were synthesized and evaluated in vivo in mice. These compounds possessed long-acting agonist and especially antagonist activities. The bivalent ligands, 2 and 3 displayed antinociceptive activity that lasted greater than 4 h. Compound 1, a bivalent ligand and 4, the monomer, antagonized the antinociceptive effect of morphine for a week after a single injection i.c.v. The long duration of action may be due to entrapment of these ligands in the central nervous system. These compounds may give future insights into the design of long-acting agonists and antagonists.

Highly selective nonpeptide ligands with potent delta opioid receptor antagonist activity have been developed using the message-address concept. This approach envisaged the delta opioid receptor to contain two major recognition subsites; a message subsite which recognizes the pharmacophore, and an address subsite that is unique for the delta receptor type and confers selectivity. The message and address components of the delta-selective enkephalins were postulated to be Tyr1 and Phe4, respectively, with Gly2-Gly3 functioning as a spacer. The message component of the target compounds in this study was derived from naltrexone and related structures. An indole system was fused to the C ring of naltrexone as a mimic of the address component. The benzene moiety of indole was viewed as the delta address component, mimicking the phenyl group of Phe4, and the pyrrole portion was used as a rigid spacer. Members of the series (1-23) were evaluated for opioid antagonist activity on the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Naltrindole (NTI, 1) was the most potent member of the series, with Ke values of approximately 0.1 nM at delta receptors. The antagonism by NTI was approximately 220- and 350-fold greater at delta than at mu and kappa opioid receptors. The binding of NTI and selected members of the series to guinea pig brain membranes was qualitatively consistent with their pharmacologic antagonist activity profiles in the MVD and GPI, but the Ki values were not in the same rank order. The selectivity of NTI arises mainly as a consequence of increased affinity at delta receptors. Thus, the Ke and Ki values of NTI were 1/530 and 1/90 that of the delta antagonist enkephalin analogue, ICI 174864. In contrast to NTI, ICI174864 derives its selectivity through greatly decreased recognition at mu and kappa receptors. The implications of the high affinity and selectivity of NTI as a consequence of its conformational rigidity are discussed. It is suggested that any attempt to model a receptor-bound conformation of an opioid peptide should consider affinity and potency at multiple receptor sites rather than selectivity alone.