An updated assessment of the translational promise of G-protein-biased kappa opioid receptor agonists to treat pain and other indications without debilitating adverse effects.

Kappa opioid receptor (κOR) agonists lack the abuse liability and respiratory depression effects of clinically used mu opioid receptor (µOR) analgesics and are hypothesized to be safer alternatives. However, κOR agonists have limiting adverse effects of their own, including aversion, sedation, and mood effects, that have hampered their clinical translation. Studies performed over the last 15 years have suggested that these adverse effects could result from activation of distinct intracellular signaling pathways that are dependent on ß-arrestin, whereas signaling downstream of G protein activation produces antinociception. This led to the hypothesis that agonists biased away from ß-arrestin signaling would have improved therapeutic windows over traditional unbiased agonists and allow for clinical development of analgesic G-protein-biased κOR agonists. Given a recent controversy regarding the benefits of G-protein-biased µOR agonists, it is timely to reassess the therapeutic promise of G-protein-biased κOR agonists. Here we review recent discoveries from preclinical κOR studies and critically evaluate the therapeutic windows of G-protein-biased κOR agonists in each of the adverse effects above. Overall, we find that G-protein-biased κOR agonists generally have improved therapeutic window relative to unbiased agonists, although frequently study design limits strong conclusions in this regard. However, a steady flow of newly developed biased κOR agonists paired with recently engineered behavioral and molecular tools puts the κOR field in a prime position to make major advances in our understanding of κOR function and fulfill the promise of translating a new generation of biased κOR agonists to the clinic.

Antinociceptive Efficacy of the µ-Opioid/Nociceptin Peptide-Based Hybrid KGNOP1 in Inflammatory Pain without Rewarding Effects in Mice: An Experimental Assessment and Molecular Docking.

Opioids are the most effective analgesics, with most clinically available opioids being agonists to the µ-opioid receptor (MOR). The MOR is also responsible for their unwanted effects, including reward and opioid misuse leading to the current public health crisis. The imperative need for safer, non-addictive pain therapies drives the search for novel leads and new treatment strategies. In this study, the recently discovered MOR/nociceptin (NOP) receptor peptide hybrid KGNOP1 (H-Dmt-D-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) was evaluated following subcutaneous administration in mouse models of acute (formalin test) and chronic inflammatory pain (Complete Freund's adjuvant-induced paw hyperalgesia), liabilities of spontaneous locomotion, conditioned place preference, and the withdrawal syndrome. KGNOP1 demonstrated dose-dependent antinociceptive effects in the formalin test, and efficacy in attenuating thermal hyperalgesia with prolonged duration of action. Antinociceptive effects of KGNOP1 were reversed by naltrexone and SB-612111, indicating the involvement of both MOR and NOP receptor agonism. In comparison with morphine, KGNOP1 was more potent and effective in mouse models of inflammatory pain. Unlike morphine, KGNOP1 displayed reduced detrimental liabilities, as no locomotor impairment nor rewarding and withdrawal effects were observed. Docking of KGNOP1 to the MOR and NOP receptors and subsequent 3D interaction pattern analyses provided valuable insights into its binding mode. The mixed MOR/NOP receptor peptide KGNOP1 holds promise in the effort to develop new analgesics for the treatment of various pain states with fewer MOR-mediated side effects, particularly abuse and dependence liabilities.

GTPγS-Autoradiography for Studies of Opioid Receptor Functionality.

The opioid receptors have been an interesting target for the drug industry for decades. These receptors were pharmacologically characterized in the 1970s and several drugs and peptides have emerged over the years. In 2012, the crystal structures were also demonstrated, with new data on the receptor sites, and thus new possibilities will appear. The role of opioids in the brain has attracted considerable interest in several diseases, especially pain and drug dependence. The opioid receptors are G-protein-coupled receptors (GPCR ) that are Gi coupled which make them suitable for studying the receptor functionality. The [35S]GTP γS autoradiography assay is a good option that has the benefit of generating both anatomical and functional data in the area of interest. It is based on the first step of the signaling mechanism of GPCRs. When a ligand binds to the receptor GTP will replace GDP on the a-subunit of the G-protein, leading to a dissociation of the ßγ-subunit. These subunits will start a cascade of second messengers and subsequently a physiological response.

Critical Assessment of G Protein-Biased Agonism at the µ-Opioid Receptor.

G protein-biased agonists of the µ-opioid receptor (MOPr) have been proposed as an improved class of opioid analgesics. Recent studies have been unable to reproduce the original experiments in the ß-arrestin2-knockout mouse that led to this proposal, and alternative genetic models do not support the G protein-biased MOPr agonist hypothesis. Furthermore, assessment of putatively biased ligands has been confounded by several factors, including assay amplification. As such, the extent to which current lead compounds represent mechanistically novel, extremely G protein-biased agonists is in question, as is the underlying assumption that ß-arrestin2 mediates deleterious opioid effects. Addressing these current challenges represents a pressing issue to successfully advance drug development at this receptor and improve upon current opioid analgesics.

Atomic-Level Characterization of the Methadone-Stabilized Active Conformation of µ-Opioid Receptor.

Methadone is a synthetic opioid agonist with notoriously unique properties, such as lower abuse liability and induced relief of withdrawal symptoms and drug cravings, despite acting on the same opioid receptors triggered by classic opioids-in particular the µ-opioid receptor (MOR). Its distinct pharmacologic properties, which have recently been attributed to the preferential activation of ß-arrestin over G proteins, make methadone a standard-of-care maintenance medication for opioid addiction. Although a recent biophysical study suggests that methadone stabilizes different MOR active conformations from those stabilized by classic opioid drugs or G protein-biased agonists, how this drug modulates the conformational equilibrium of MOR and what specific active conformation of the receptor it stabilizes are unknown. Here, we report the results of submillisecond adaptive sampling molecular dynamics simulations of a predicted methadone-bound MOR complex and compare them with analogous data obtained for the classic opioid morphine and the G protein-biased ligand TRV130. The model, which is supported by existing experimental data, is analyzed using Markov state models and transfer entropy analysis to provide testable hypotheses of methadone-specific conformational dynamics and activation kinetics of MOR. SIGNIFICANCE STATEMENT: Opioid addiction has reached epidemic proportions in both industrialized and developing countries. Although methadone maintenance treatment represents an effective therapeutic approach for opioid addiction, it is not as widely used as needed. In this study, we contribute an atomic-level understanding of how methadone exerts its unique function in pursuit of more accessible treatments for opioid addiction. In particular, we present details of a methadone-specific active conformation of the µ-opioid receptor that has thus far eluded experimental structural characterization.

Chronic aerobic exercise: Autoradiographic assessment of GABA(a) and mu-opioid receptor binding in adult rats.

Exercise programs have shown great potential for both the prevention and treatment of substance use disorder (SUD). As exercise has been shown to have potent effects on physical and psychological health, it is reasonable to examine the mechanism of how exercise can be used as an adjunct treatment for addiction. The present study examined the effects of chronic aerobic (treadmill) exercise on both GABA(a) and mu-opioid receptor levels in the brains of male and female rats. GABA(a) receptor binding, measured by [3H] Flunitrazepam, was increased in the cingulate cortex following exercise, but only in females. Mu-opioid receptor expression, measured by [3H] ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin) (DAMGO), showed no effect of exercise while showing an effect of sex, with increased [3H] DAMGO binding in the brains of sedentary males compared to that of sedentary females. Our findings support the potential role for GABA(a) signaling in the cingulate cortex as part of the mechanism of action of aerobic exercise. These data, along with prior reports, aid our understanding of the neurochemical impact and mechanism of chronic aerobic exercise on neuropsychiatric disease, particularly regarding addiction.

Assessment of structure-activity relationships and biased agonism at the Mu opioid receptor of novel synthetic opioids using a novel, stable bio-assay platform.

Fentanyl and morphine are agonists of the Mu opioid receptor (MOR), which is a member of the GPCR family. Their analgesic effects are associated with unwanted side effects. On a signaling level downstream from MOR, it has been hypothesized that analgesia may be mediated through the G protein pathway, whereas the undesirable effects of opioids have been linked to the ß-arrestin (ßarr) pathway. Despite being an increasingly debated subject, little is known about a potential 'bias' (i.e. the preferential activation of one pathway over the other) of the novel synthetic opioids (NSO) - including fentanyl analogs - that have emerged on the illegal drug market. We have therefore developed and applied a novel, robust bio-assay platform to study the activity of 21 NSO, to evaluate to what extent these MOR agonists show biased agonism and to investigate the potential correlation with their structure. In addition, we evaluated the functional selectivity of TRV130, a purported G protein-biased agonist. We applied newly established stable bio-assays in HEK293T cells, based on the principle of functional complementation of a split nanoluciferase, to assess MOR activation via recruitment of a mini-Gi protein (GTPase domain of Gαi subunit) or ßarr2. All but two of the tested NSO demonstrated a concentration-dependent response at MOR in both bio-assays. The developed bio-assays allow to gain insight into the ßarr2 or G protein recruitment potential of NSO, which may eventually help to better understand why certain opioids are associated with higher toxicity. Adding to the recent discussion about the relevance of the biased agonism concept for opioids, we did not observe a significant bias for any of the evaluated compounds, including TRV130.

Fentanyl Family at the Mu-Opioid Receptor: Uniform Assessment of Binding and Computational Analysis.

Interactions of 21 fentanyl derivatives with µ-opioid receptor (µOR) were studied using experimental and theoretical methods. Their binding to µOR was assessed with radioligand competitive binding assay. A uniform set of binding affinity data contains values for two novel and one previously uncharacterized derivative. The data confirms trends known so far and thanks to their uniformity, they facilitate further comparisons. In order to provide structural hypotheses explaining the experimental affinities, the complexes of the studied derivatives with µOR were modeled and subject to molecular dynamics simulations. Five common General Features (GFs) of fentanyls' binding modes stemmed from these simulations. They include: GF1) the ionic interaction between D147 and the ligands' piperidine NH⁺ moiety; GF2) the N-chain orientation towards the µOR interior; GF3) the other pole of ligands is directed towards the receptor outlet; GF4) the aromatic anilide ring penetrates the subpocket formed by TM3, TM4, ECL1 and ECL2; GF5) the 4-axial substituent (if present) is directed towards W318. Except for the ionic interaction with D147, the majority of fentanyl-µOR contacts is hydrophobic. Interestingly, it was possible to find nonlinear relationships between the binding affinity and the volume of the N-chain and/or anilide's aromatic ring. This kind of relationships is consistent with the apolar character of interactions involved in ligand⁻receptor binding. The affinity reaches the optimum for medium size while it decreases for both large and small substituents. Additionally, a linear correlation between the volumes and the average dihedral angles of W293 and W133 was revealed by the molecular dynamics study. This seems particularly important, as the W293 residue is involved in the activation processes. Further, the Y326 (OH) and D147 (Cγ) distance found in the simulations also depends on the ligands' size. In contrast, neither RMSF measures nor D114/Y336 hydrations show significant structure-based correlations. They also do not differentiate studied fentanyl derivatives. Eventually, none of 14 popular scoring functions yielded a significant correlation between the predicted and observed affinity data (R < 0.30, n = 28).

Kinetic and thermodynamic insights into sodium ion translocation through the µ-opioid receptor from molecular dynamics and machine learning analysis.

The differential modulation of agonist and antagonist binding to opioid receptors (ORs) by sodium (Na+) has been known for decades. To shed light on the molecular determinants, thermodynamics, and kinetics of Na+ translocation through the µ-OR (MOR), we used a multi-ensemble Markov model framework combining equilibrium and non-equilibrium atomistic molecular dynamics simulations of Na+ binding to MOR active or inactive crystal structures embedded in an explicit lipid bilayer. We identify an energetically favorable, continuous ion pathway through the MOR active conformation only, and provide, for the first time: i) estimates of the energy differences and required timescales of Na+ translocation in inactive and active MORs, ii) estimates of Na+-induced changes to agonist binding validated by radioligand measurements, and iii) testable hypotheses of molecular determinants and correlated motions involved in this translocation, which are likely to play a key role in MOR signaling.

Delta/mu opioid receptor interactions in operant conditioning assays of pain-depressed responding and drug-induced rate suppression: assessment of therapeutic index in male Sprague Dawley rats.

RATIONALE AND OBJECTIVES: Although delta/mu receptor interactions vary as a function of behavioral endpoint, there have been no assessments of these interactions using assays of pain-depressed responding. This is the first report of delta/mu interactions using an assay of pain-depressed behavior. METHODS: A mult-cycle FR10 operant schedule was utilized in the presence of (nociception) and in the absence of (rate suppression) a lactic acid inflammatory pain-like manipulation. SNC80 and methadone were used as selective/high efficacy delta and mu agonists, respectively. Both SNC80 and methadone alone produced a dose-dependent restoration of pain-depressed responding and dose-dependent response rate suppression. Three fixed ratio mixtures, based on the relative potencies of the drugs in the nociception assay, also produced dose-dependent antinociception and sedation. Isobolographic analysis indicated that all three mixtures produced supra-additive antinociceptive effects and simply additive sedation effects. CONCLUSIONS: The therapeutic index (TI) inversely varied as a function of amount of SNC80 in the mixture, such that lower amounts of SNC80 produced a higher TI, and larger amounts produced a lower TI. Compared to literature using standard pain-elicited assays, the orderly relationship between SNC80 and TI reported here may be a unique function of assessing pain-depressed behavior.

Pharmacologic and clinical assessment of kratom.

PURPOSE: This article reviews the pharmacology, clinical utility, adverse effects, and abuse potential of kratom. SUMMARY: The leaves of Mitragyna speciosa contain the biologically active alkaloids of kratom. Kratom exerts opioid and α-2 receptor agonistic effects as well as antiinflammatory and parasympathetic-impeding effects. There are no published human pharmacologic, pharmacokinetic, or drug interaction studies on kratom or mitragynine, making it virtually impossible to fully understand kratom's therapeutic potential and risks and the populations most likely to benefit or experience harm from its use. Kratom has been used to ameliorate opioid withdrawal symptoms but also induces withdrawal. Human pharmacologic, pharmacokinetic, and clinical data are of low quality, precluding any firm conclusions regarding safety and efficacy. Respiratory depression has not been commonly reported, but kratom does cause a host of adverse effects without clear guidance for how they should be treated. There are numerous assessments where people have been unable to stop using kratom therapy, and withdrawal signs and symptoms are problematic. Kratom does not appear in normal drug screens and, when taken with other substances of abuse, may not be recognized. Thirty-six deaths have been attributed to kratom, and the Food and Drug Administration issued a public health warning about the substance in November 2017. CONCLUSION: Kratom exerts opioid and α-2 receptor agonistic effects as well as antiinflammatory and parasympathetic-impeding effects. Human pharmacologic, pharmacokinetic, and clinical data are of low quality, precluding any firm conclusions regarding safety and efficacy.

Antinociceptive and respiratory effects following application of transdermal fentanyl patches and assessment of brain µ-opioid receptor mRNA expression in ball pythons.

OBJECTIVE To quantify plasma fentanyl concentrations (PFCs) and evaluate antinociceptive and respiratory effects following application of transdermal fentanyl patches (TFPs) and assess cerebrospinal µ-opioid receptor mRNA expression in ball pythons (compared with findings in turtles). ANIMALS 44 ball pythons (Python regius) and 10 turtles (Trachemys scripta elegans). PROCEDURES To administer 3 or 12 µg of fentanyl/h, a quarter or whole TFP (TFP-3 and TFP-12, respectively) was used. At intervals after TFP-12 application in snakes, PFCs were measured by reverse-phase high-pressure liquid chromatography. Infrared heat stimuli were applied to the rostroventral surface of snakes to determine thermal withdrawal latencies after treatments with no TFP (control [n = 16]) and TFP-3 (8) or TFP-12 (9). Breathing frequency was measured in unrestrained controls and TFP-12-treated snakes. µ-Opioid receptor mRNA expression in brain and spinal cord tissue samples from snakes and turtles (which are responsive to µ-opioid receptor agonist drugs) were quantified with a reverse transcription PCR assay. RESULTS Mean PFCs were 79, 238, and 111 ng/mL at 6, 24, and 48 hours after TFP-12 application, respectively. At 3 to 48 hours after TFP-3 or TFP-12 application, thermal withdrawal latencies did not differ from pretreatment values or control treatment findings. For TFP-12-treated snakes, mean breathing frequency significantly decreased from the pretreatment value by 23% and 41% at the 24- and 48-hour time points, respectively. Brain and spinal cord tissue µ-opioid receptor mRNA expressions in snakes and turtles did not differ. CONCLUSIONS AND CLINICAL RELEVANCE In ball pythons, TFP-12 application resulted in high PFCs, but there was no change in thermal antinociception, indicating resistance to µ-opioid-dependent antinociception in this species.

In vitro and in vivo assessment of the abuse potential of PF614, a novel BIO-MD™ prodrug of oxycodone.

OBJECTIVE: The need for pain medication which will not lead to abuse is well recognized. Ensysce has designed prodrug analogs of the commonly used pain medications including hydromorphone, oxycodone (OC), hydrocodone, and morphine that limit their use to oral delivery, two of which are in clinical development. This study was undertaken to demonstrate that PF614, an extended-release prodrug of OC, allows the release of OC as designed when delivered orally, yet it resists ex vivo extraction with household chemicals and is pharmacologically inactive when administered by nonoral routes (nasal and parenteral), thereby substantially reducing its intravenous (IV) and intranasal abuse potential. METHODS: In vitro and in vivo methods were used to determine release of OC from PF614 and to show potential µ-opioid receptor activity. Plasma and cerebral spinal fluid levels of OC were evaluated following in vivo IV administration of PF614 in rats. In vitro extraction of OC from PF614 was explored using enzymes, common solvents, and household chemicals at room temperature and elevated temperature over time to determine release of OC from the prodrug. RESULTS: PF614 was stable with in vitro exposure to human plasma, saliva, and liver microsomes or culinary enzyme preparations. PF614 was stable (≥90 percent remaining as intact prodrug) under all room temperature conditions evaluated for 24 hours. At 80°C for 1 hour, no OC was released. Incubation at 80°C for 24 hours in vinegar or vodka produced a conversion to OC of 6 percent. Incubation with trypsin at 37°C converted PF614 approximately stoichiometric to OC with half-life of 4 hours. PF614's penetration of the central nervous system was 83-fold lower than OC and it had a 6.5-fold reduced potency as a µ-opioid agonist. Finally, oral PF614 delivers OC into plasma with an extended-release profile in dogs (reduced Cmax; delayed Tmax). CONCLUSIONS: The Bio-Activated Molecular Delivery prodrug design limits the use of PF614 to the intended oral route of delivery with reduced potential for IV or nasal abuse, as it cannot be activated intravenously or nasally to provide an active opioid. Unlike existing opioid formulations, the extended-release profile of PF614 cannot be accelerated by chewing or ex vivo extraction to pharmacologically active substances.

Synthesis of tripeptides containing D-Trp substituted at the indole ring, assessment of opioid receptor binding and in vivo central antinociception.

The noncationizable tripeptide Ac-D-Trp-Phe-GlyNH2 was recently proposed as a novel minimal recognition motif for µ-opioid receptor. The introduction of different substituents (methyl, halogens, nitro, etc.) at the indole of D-Trp significantly influenced receptor affinities and resulted in serum stability and in a measurable effect on central antinociception in mice after ip administration.

Chronic methadone treatment shows a better cost/benefit ratio than chronic morphine in mice.

Chronic treatment of pain with opiate drugs can lead to analgesic tolerance and drug dependence. Although all opiate drugs can promote tolerance and dependence in practice, the severity of those unwanted side effects differs depending on the drug used. Although each opiate drug has its own unique set of pharmacological profiles, methadone is the only clinically used opioid drug that produces substantial receptor endocytosis at analgesic doses. Here, we examined whether moderate doses of methadone carry any benefits over chronic use of equianalgesic morphine, the prototypical opioid. Our data show that chronic administration of methadone produces significantly less analgesic tolerance than morphine. Furthermore, we found significantly reduced precipitated withdrawal symptoms after chronic methadone treatment than after chronic morphine treatment. Finally, using a novel animal model with a degrading µ-opioid receptor we showed that, although endocytosis seems to protect against tolerance development, endocytosis followed by receptor degradation produces a rapid onset of analgesic tolerance to methadone. Together, these data indicated that opioid drugs that promote receptor endocytosis and recycling, such as methadone, may be a better choice for chronic pain treatment than morphine and its derivatives that do not.

Uniform assessment and ranking of opioid µ receptor binding constants for selected opioid drugs.

The safe disposal of unused opioid drugs is an area of regulatory concern. While toilet flushing is recommended for some drugs to prevent accidental exposure, there is a need for data that can support a more consistent disposal policy based on an assessment of relative risk. For drugs acting at the Mu-opioid receptor (MOR), published measurements of binding affinity (K(i)) are incomplete and inconsistent due to differences in methodology and assay system, leading to a wide range of values for the same drug thus precluding a simple and meaningful relative ranking of drug potency. Experiments were conducted to obtain K(i)'s for 19 approved opioid drugs using a single binding assay in a cell membrane preparation expressing recombinant human MOR. The K(i) values obtained ranged from 0.1380 nM (sufentanil) to 12.486 µM (tramadol). The drugs were separated into three categories based upon their K(i) values: K(i) > 100 nM (tramadol, codeine, meperidine, propoxyphene and pentazocine), K(i)=1-100 nM (hydrocodone, oxycodone, diphenoxylate, alfentanil, methadone, nalbuphine, fentanyl and morphine) and K(i) < 1 nM (butorphanol, levorphanol, oxymorphone, hydromorphone, buprenorphine and sufentanil). These data add to the understanding of the pharmacology of opioid drugs and support the development of a more consistent labeling policies regarding safe disposal.

In vitro and in vivo assessment of mu opioid receptor constitutive activity.

Constitutive (basal) signaling has been described and characterized for numerous G protein coupled receptors (GPCRs). The relevance of this activity to disease, drug discovery and development, and to clinical pharmacotherapy is just beginning to emerge. Opioid receptors were the first GPCR systems for which there was definitive evidence presented for constitutive activity, with numerous studies now published on the regulation of this activity (e.g., structure/activity of the receptor as it relates to basal activity, pharmacology of ligands that act as agonists, inverse agonists and "neutral antagonists," etc.). This chapter summarizes some of the methods used to characterize constitutive activity at the mu opioid receptor (MOR) in preclinical in vitro and in vivo model systems. This includes cell-based systems that are useful for higher throughput screening of novel ligands and for studying variables that can impact basal tone in a system. In vivo assays are also described in which constitutive activity is increased in response to acute or chronic opioid agonist exposure and where withdrawal is precipitated with antagonists that may function as inverse agonists or "neutral" antagonists. The methods described have inherent advantages and disadvantages that need to be considered in any drug discovery/development program. A brief discussion of progress toward understanding the clinical implications of MOR constitutive activity in the management of opioid addiction and chronic pain is also included in this chapter.

Synthesis and characterizations of novel quinoline derivatives having mixed ligand activities at the kappa and mu receptors: Potential therapeutic efficacy against morphine dependence.

Based on an established 3D pharmacophore, a series of quinoline derivatives were synthesized. The opioidergic properties of these compounds were determined by a competitive binding assay using (125)I-Dynorphine, (3)H-DAMGO and (125)I-DADLE for kappa, mu, and delta receptors, respectively. Results showed varying degree of activities of the compounds to kappa and mu opioid receptors with negligible interactions at the delta receptor. The compound, S4 was the most successful in inhibiting the two most prominent quantitative features of naloxone precipitated withdrawal symptoms - stereotyped jumping and body weight loss. Determination of IC(50) of S4 revealed a greater affinity towards mu compared to kappa receptor. In conclusion, quinoline derivatives of S4 like structure offer potential tool for treatment of narcotic addictions.

Aerobic oxidation of indolomorphinan without the 4,5-epoxy bridge and subsequent rearrangement of the oxidation product to spiroindolinonyl-C-normorphinan derivative.

Aerobic oxidation of indolomorphinan 1 without a 4,5-epoxy bridge proceeded in the presence of platinum catalyst to give indoleninomorphinan 2 or quinolono-C-normorphinan 5. The 4-hydroxy group would play an important role in deciding the course of the reaction. Treatment of 2a with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave spiroindolinonyl-C-normorphinan 3a whose structure resembles that of delta opioid receptor agonist spiroindanyloxymorphone (SIOM). Boron trichloride was effective for the reverse reaction from 3a to 2a without side reaction. This practical interconversion method between hydroxyindolenine and spiroindolinone would be useful for the design and construction of drug-like compound libraries. Although the compound 3b was expected to show the selectivity for delta opioid receptor because of the structural resemblance to SIOM, it was rather selective for 1 opioid receptor (1: K(i)=0.75nM; delta: K(i)=2.90nM; kappa: K(i)=13.4nM). The result suggests that the slight difference of the spatial location of the benzene rings in these compounds may definitively affect the binding affinity for delta opioid receptor.