MOR Agonist Research Articles

A277 A NOVEL PH-SENSITIVE Μ-OPIOID RECEPTOR AGONIST THAT DOES NOT INDUCE TOLERANCE IN A COLITIS MOUSE MODEL

Abstract Background Adequate pain control in inflammatory bowel disease (IBD) can require opioids due to their high analgesic potency. The long-term use of opioids, however, is limited by the development of tolerance. This leads to reduced analgesic efficacy over time, resulting in escalating opioid dosing and thus increased risk of serious side effects. We previously demonstrated the safety and effectiveness of a novel pH-sensitive µ-opioid receptor (MOR) agonist, NFEPP, in a murine colitis pain model, but its tolerance potential with chronic administration is unknown. Purpose To assess the tolerance potential of NFEPP compared to its parent compound fentanyl during acute colitis in a preclinical mouse model. Method Acute colitis in C57BL/6 mice was induced using 2.5% dextran sulphate sodium for 5 days. NFEPP or fentanyl were then administered s.c. every 4 hours between 7am and 11pm over 5 days in daily increasing concentrations (0.4-1.5 mg/kg/d). Analgesic tolerance to opioids was assessed in conscious mice by measuring visceromotor responses (VMRs) to noxious colorectal distensions. Tolerance to the MOR agonist DAMGO in NFEPP or fentanyl treated mice was evaluated using patch-clamp recordings from dorsal root ganglion (DRG) neurons and extracellular recordings from lumbar splanchnic nerves that innervate the colon. Inflammation was assessed by macroscopic analyses, histological scoring and tissue-pH measurements of the inflamed colon. Group differences were analyzed using two-way ANOVA with Bonferroni′s or Tukey′s post-test (p-value <0.05). Result(s) NFEPP significantly reduced VMRs before and after chronic NFEPP treatment (39% reduction, p<0.05 vs. 41% reduction, p<0.05, compared to baseline at 80 µl). No differences of NFEPP induced antinociceptive actions were observed comparing VMR measurements before and after chronic administration (p=0.44). However, the analgesic activity of fentanyl decreased over time with less VMR inhibition observed after chronic treatment compared to fentanyl naïve mice (p<0.05). Cross tolerance testing between these two opioids revealed a loss of NFEPP- induced VMR inhibition in fentanyl treated mice, but fentanyl effects in the NFEPP treatment group remained unchanged. In patch-clamp recordings, DAMGO (100nM) evoked antinociceptive actions in DRG neurons from NFEPP treated mice (rheobase increase: 50%, p<0.05, compared to vehicle) whereas no change in rheobase with DAMGO was observed in DRG neurons from fentanyl treated mice. Afferent nerve activity from lumbar splanchnic nerves in response to von Frey filament probing decreased after DAMGO (100nM) application in NFEPP treated mice (p<0.05, compared to control probing), but showed no change in fentanyl treated mice. Colonic length, diameter, histological damage and tissue-pH did not differ between the two opioid treatment groups. Conclusion(s) Chronic administration of NFEPP during acute murine colitis does not lead to analgesic tolerance within the visceral nociceptive system, in contrast to its parent compound fentanyl. Please acknowledge all funding agencies by checking the applicable boxes below CCC Disclosure of Interest None Declared

Off-target activity of NBOMes and NBOMe analogs at the µ opioid receptor.

New psychoactive substances (NPS) are introduced on the illicit drug market at a rapid pace. Their molecular targets are often inadequately elucidated, which contributes to the delayed characterization of their pharmacological effects. Inspired by earlier findings, this study set out to investigate the µ opioid receptor (MOR) activation potential of a large set of psychedelics, substances which typically activate the serotonin (5-HT2A) receptor as their target receptor. We observed that some substances carrying the N-benzyl phenethylamine (NBOMe) structure activated MOR, as confirmed by both the NanoBiT® βarr2 recruitment assay and the G protein-based AequoScreen® Ca2+ release assay. The use of two orthogonal systems proved beneficial as some aspecific, receptor independent effects were found for various analogs when using the Ca2+ release assay. The specific 'off-target' effects at MOR could be blocked by the opioid antagonist naloxone, suggesting that these NBOMes occupy the same common opioid binding pocket as conventional opioids. This was corroborated by molecular docking, which revealed the plausibility of multiple interactions of 25I-NBOMe with MOR, similar to those observed for opioids. Additionally, structure-activity relationship findings seen in vitro were rationalized in silico for two 25I-NBOMe isomers. Overall, as MOR activity of these psychedelics was only noticed at high concentrations, we consider it unlikely that for the tested compounds there will be a relevant opioid toxicity in vivo at physiologically relevant concentrations. However, small modifications to the original NBOMe structure may result in a panel of more efficacious and potent MOR agonists, potentially exhibiting a dual MOR/5-HT2A activation potential.

Opioid-Induced Reductions in Amygdala Lateral Paracapsular GABA Neuron Circuit Activity.

Opioid use and withdrawal evokes behavioral adaptations such as drug seeking and anxiety, though the underlying neurocircuitry changes are unknown. The basolateral amygdala (BLA) regulates these behaviors through principal neuron activation. Excitatory BLA pyramidal neuron activity is controlled by feedforward inhibition provided, in part, by lateral paracapsular (LPC) GABAergic inhibitory neurons, residing along the BLA/external capsule border. LPC neurons express µ-opioid receptors (MORs) and are potential targets of opioids in the etiology of opioid-use disorders and anxiety-like behaviors. Here, we investigated the effects of opioid exposure on LPC neuron activity using immunohistochemical and electrophysiological approaches. We show that LPC neurons, and other nearby BLA GABA and non-GABA neurons, express MORs and δ-opioid receptors. Additionally, DAMGO, a selective MOR agonist, reduced GABA but not glutamate-mediated spontaneous postsynaptic currents in LPC neurons. Furthermore, in LPC neurons, abstinence from repeated morphine-exposure in vivo (10 mg/kg/day, 5 days, 2 days off) decrease the intrinsic membrane excitability, with a ~75% increase in afterhyperpolarization and ~40-50% enhanced adenylyl cyclase-dependent activity in LPC neurons. These data show that MORs in the BLA are a highly sensitive targets for opioid-induced inhibition and that repeated opioid exposure results in impaired LPC neuron excitability.

Bispecific sigma-1 receptor antagonism and mu-opioid receptor partial agonism: WLB-73502, an analgesic with improved efficacy and safety profile compared to strong opioids.

Opioids are the most effective painkillers, but their benefit-risk balance often hinder their therapeutic use. WLB-73502 is a dual, bispecific compound that binds sigma-1 (S1R) and mu-opioid (MOR) receptors. WLB-73502 is an antagonist at the S1R. It behaved as a partial MOR agonist at the G-protein pathway and produced no/unsignificant β-arrestin-2 recruitment, thus demonstrating low intrinsic efficacy on MOR at both signalling pathways. Despite its partial MOR agonism, WLB-73502 exerted full antinociceptive efficacy, with potency superior to morphine and similar to oxycodone against nociceptive, inflammatory and osteoarthritis pain, and superior to both morphine and oxycodone against neuropathic pain. WLB-73502 crosses the blood-brain barrier and binds brain S1R and MOR to an extent consistent with its antinociceptive effect. Contrary to morphine and oxycodone, tolerance to its antinociceptive effect did not develop after repeated 4-week administration. Also, contrary to opioid comparators, WLB-73502 did not inhibit gastrointestinal transit or respiratory function in rats at doses inducing full efficacy, and it was devoid of proemetic effect (retching and vomiting) in ferrets at potentially effective doses. WLB-73502 benefits from its bivalent S1R antagonist and partial MOR agonist nature to provide an improved antinociceptive and safety profile respect to strong opioid therapy.

Plasma pharmacokinetics and pharmacodynamic effects of the 2-benzylbenzimidazole synthetic opioid, isotonitazene, in male rats

Isotonitazene is an illicit synthetic opioid associated with many intoxications and fatalities. Recent studies show that isotonitazene is a potent µ-opioid receptor (MOR) agonist in vitro, but little information is available about its in vivo effects. The aims of the present study were to investigate the pharmacokinetics of isotonitazene in rats, and relate pharmacokinetic parameters to pharmacodynamic effects. Isotonitazene and its metabolites were identified and quantified by liquid chromatography tandem quadrupole mass spectrometry (LC-QQQ-MS). Male Sprague-Dawley rats with jugular catheters and subcutaneous (s.c.) temperature transponders received isotonitazene (3, 10, 30µg/kg, s.c.) or its vehicle. Blood samples were drawn at 15, 30, 60, 120, and 240min post-injection, and plasma was assayed using LC-QQQ-MS. At each blood draw, body temperature, catalepsy scores, and hot plate latencies were recorded. Maximum plasma concentrations of isotonitazene rose in parallel with increasing dose (range 0.2-9.8ng/mL) and half-life ranged from 23.4 to 63.3min. The metabolites 4'-hydroxy nitazene and N-desethyl isotonitazene were detected, and plasma concentrations were below the limit of quantitation (0.5ng/mL) but above the limit of detection (0.1ng/mL). Isotonitazene produced antinociception (ED50 = 4.22µg/kg), catalepsy-like symptoms (ED50 = 8.68µg/kg), and hypothermia (only at 30µg/kg) that were significantly correlated with concentrations of isotonitazene. Radioligand binding in rat brain tissue revealed that isotonitazene displays nM affinity for MOR (Ki = 15.8nM), while the N-desethyl metabolite shows even greater affinity (Ki = 2.2nM). In summary, isotonitazene is a potent MOR agonist whose pharmacodynamic effects are related to circulating concentrations of the parent drug. The high potency of isotonitazene portends substantial risk to users who are exposed to the drug.

Opioid modulation of T-type Ca2+ channel-dependent neuritogenesis/neurite outgrowth through the prostaglandin E2/EP4 receptor/protein kinase A pathway in mouse dorsal root ganglion neurons

Irregular regeneration or inappropriate remodeling of the axons of the primary afferent neurons after peripheral nerve trauma could be associated with the development of neuropathic pain. We analyzed the molecular mechanisms for the neuritogenesis and neurite outgrowth caused by prostaglandin E2 (PGE2) in mouse dorsal root ganglion (DRG) neurons, and evaluated their opioid modulation. PGE2 in combination with IBMX, a phosphodiesterase inhibitor, caused neuritogenesis/neurite outgrowth in DRG cells, an effect abolished by a prostanoid EP4, but not EP2, receptor antagonist, and inhibitors of adenylyl cyclase or protein kinase A (PKA). Blockers of T-type Ca2+ channels (T-channels), that are responsible for window currents involving the sustained low-level Ca2+ entry at voltages near the resting membrane potentials and can be functionally upregulated by PKA, inhibited the neuritogenesis/neurite outgrowth caused by PGE2/IBMX or dibutylyl cyclic AMP, a PKA activator, in DRG neurons, an inhibitory effect mimicked by ZnCl2 and ascorbic acid that block Cav3.2, but not Cav3.1 or Cav3.3, T-channels. Morphine and DAMGO, μ-opioid receptor (MOR) agonists, suppressed the neuritogenesis and/or neurite outgrowth induced by PGE2/IBMX in DRG neurons and also DRG neuron-like ND7/23 cells, an effect reversed by naloxone or β-funaltrexamine, a selective MOR antagonist. Our data suggest that the EP4 receptor/PKA/Cav3.2 pathway is involved in the PGE2-induced neuritogenesis/neurite outgrowth in DRG neurons, which can be suppressed by MOR stimulation. We propose that MOR agonists including morphine in the early phase after peripheral nerve trauma might delay the axonal regeneration of the primary afferent neurons but prevent the development of neuropathic pain.

Structurally diverse fentanyl analogs yield differential locomotor activities in mice

Synthetic narcotics have been implicated as the single greatest contributor to increases in opioid-related fatalities in recent years. This study evaluated the effects of nine fentanyl-related substances that have emerged in the recreational drug marketplace, and for which there are no existing or only limited in vivo data. Adult male Swiss Webster mice were administered fentanyl-related substances and their effects on locomotion as compared to MOR agonist standards were recorded. In locomotor activity tests, morphine (100, 180 mg/kg), buprenorphine (1, 10 mg/kg), fentanyl (1, 10 mg/kg), cyclopropylfentanyl (1, 10 mg/kg), cyclopentylfentanyl (10 mg/kg), (±)-cis-3-methylbutyrylfentanyl (0.1, 1, 10 mg/kg), ortho-methylacetylfentanyl (10 mg/kg), para-chloroisobutyrylfentanyl (100 mg/kg), ocfentanil (1, 10 mg/kg), and ortho-fluoroacrylfentanyl (0.1, 1, 10 mg/kg) elicited significant (p ≤ 0.05) dose-dependent increases in locomotion. However, 2,2,3,3-tetramethylcyclopropylfentanyl did not have any effects on locomotion, even when tested up to 100 mg/kg, and 4′-methylacetylfentanyl (10, 100 mg/kg) significantly decreased locomotion. The rank order of efficacy for stimulating locomotion (maximum effect as a % of fentanyl's maximum effect) for fentanyl-related substances relative to MOR agonist standards was cyclopropylfentanyl (108.84 ± 20.21) > fentanyl (100 ± 15.3) > ocfentanil (79.27 ± 16.92) > morphine (75.9 ± 14.5) > (±)-cis-3-methylbutyrylfentanyl (68.04 ± 10.08) > ortho-fluoroacrylfentanyl (63.56 ± 19.88) > cyclopentylfentanyl (56.46 ± 8.54) > para-chloroisobutyrylfentanyl (22.44 ± 8.51) > buprenorphine (11.26 ± 2.30) > ortho-methylacetylfentanyl (9.45 ± 2.92) > 2,2,3,3-tetramethylcyclopropylfentanyl (6.75 ± 1.43) > 4′-methylacetylfentanyl (3.47 ± 0.43). These findings extend in vivo results from previous reports documenting additional fentanyl related-related substances that stimulate locomotion similar to known abused opioids while also identifying some anomalies.

Discovery and Structural Explorations of G-Protein Biased μ-Opioid Receptor Agonists.

Compounds that activate only the G-protein signalling pathway represent an effective strategy for making safer opioids. In the present study, we report the design, synthesis and evaluation of two classes of novel PZM21 derivatives containing the benzothiophene ring and biphenyl ring group respectively as biased μ-opioid receptor (μOR) agonists. The new compound SWG-LX-33 showed potent μOR agonist activity and produced μOR-dependent analgesia. SWG-LX-33 does not activate the β-arrestin-2 signalling pathway in vitro even at high concentrations. Computational docking demonstrated the amino acid residue ASN150 to be critical for the weak efficacy and potency of μOR agonists in arrestin recruitment.

A Journey through Diastereomeric Space: The Design, Synthesis, In Vitro and In Vivo Pharmacological Activity, and Molecular Modeling of Novel Potent Diastereomeric MOR Agonists and Antagonists.

Four sets of diastereomeric C9-alkenyl 5-phenylmorphans, varying in the length of the C9-alkenyl chain, were designed to examine the effect of these spatially distinct ligands on opioid receptors. Functional activity was obtained by forskolin-induced cAMP accumulation assays and several compounds were examined in the [35S]GTPgS assay and in an assay for respiratory depression. In each of the four sets, similarities and differences were observed dependent on the length of their C9-alkenyl chain and, most importantly, their stereochemistry. Three MOR antagonists were found to be as or more potent than naltrexone and, unlike naltrexone, none had MOR, KOR, or DOR agonist activity. Several potent MOR full agonists were obtained, and, of particular interest partial agonists were found that exhibited less respiratory depression than that caused by morphine. The effect of stereochemistry and the length of the C9-alkenyl chain was also explored using molecular modeling. The MOR antagonists were found to interact with the inactive (4DKL) MOR crystal structures and agonists were found to interact with the active (6DDF) MOR crystal structures. The comparison of their binding modes at the mouse MOR was used to gain insight into the structural basis for their stereochemically induced pharmacological differences.

Interactive Effects of µ-Opioid and Adrenergic-α 2 Receptor Agonists in Rats: Pharmacological Investigation of the Primary Kratom Alkaloid Mitragynine and Its Metabolite 7-Hydroxymitragynine.

The primary kratom alkaloid mitragynine is proposed to act through multiple mechanisms, including actions at µ-opioid receptors (MORs) and adrenergic-α 2 receptors (Aα 2Rs), as well as conversion in vivo to a MOR agonist metabolite (i.e., 7-hydroxymitragynine). Aα 2R and MOR agonists can produce antinociceptive synergism. Here, contributions of both receptors to produce mitragynine-related effects were assessed by measuring receptor binding in cell membranes and, in rats, pharmacological behavioral effect antagonism studies. Mitragynine displayed binding affinity at both receptors, whereas 7-hydroxymitragynine only displayed MOR binding affinity. Compounds were tested for their capacity to decrease food-maintained responding and rectal temperature and to produce antinociception in a hotplate test. Prototypical MOR agonists and 7-hydroxymitragynine, but not mitragynine, produced antinociception. MOR agonist and 7-hydroxymitragynine rate-deceasing and antinociceptive effects were antagonized by the opioid antagonist naltrexone but not by the Aα 2R antagonist yohimbine. Hypothermia only resulted from reference Aα 2R agonists. The rate-deceasing and hypothermic effects of reference Aα 2R agonists were antagonized by yohimbine but not naltrexone. Neither naltrexone nor yohimbine antagonized the rate-decreasing effects of mitragynine. Mitragynine and 7-hydroxymitragynine increased the potency of the antinociceptive effects of Aα 2R but not MOR reference agonists. Only mitragynine produced hypothermic effects. Isobolographic analyses for the rate-decreasing effects of the reference Aα 2R and MOR agonists were also conducted. These results suggest mitragynine and 7-hydroxymitragynine may produce antinociceptive synergism with Aα 2R and MOR agonists. When combined with Aα 2R agonists, mitragynine could also produce hypothermic synergism. SIGNIFICANCE STATEMENT: Mitragynine is proposed to target the µ-opioid receptor (MOR) and adrenergic-α2 receptor (Aα2R) and to produce behavioral effects through conversion to its MOR agonist metabolite 7-hydroxymitragynine. Isobolographic analyses indicated supra-additivity in some dose ratio combinations. This study suggests mitragynine and 7-hydroxymitragynine may produce antinociceptive synergism with Aα2R and MOR agonists. When combined with Aα2R agonists, mitragynine could also produce hypothermic synergism.

Discovery of pyrazole-1-carboxamide derivatives as novel Gi-biased μ-opioid receptor agonists.

μ-Opioid receptor (MOR) Gi-biased agonists with no recruitment of β-arrestin were introduced as a new analgesic strategy to overcome the conventional undesirable side effects of opioid receptor-targeted drugs, such as tolerance, addiction, respiratory depression, and constipation. For the development of novel Gi-biased MOR agonists, the design, synthesis, and structure-activity relationship(SAR) analysis of the aminopyrazole core skeleton were conducted according to the current SAR data of PZM21 (2a) and its derivatives. New derivatives were biologically evaluated for their agonistic effects on cyclic adenosine monophosphate(cAMP) levels for the Gi pathway and β-arrestin recruitment in MOR/κ-opioid receptor/δ opioid receptor. An optimized selective Gi-biased agonist, Compound 17a, was discovered with potent cAMP inhibitory activities, with a 50% efficacy concentration valueof 87.1 nM and no activity in the MOR β-arrestin pathway and other subtypes. The in vivo pain relief efficacy of Compound 17a was confirmed in a dose-dependent manner with spinal nerve ligation and cisplatin-induced peripheral neuropathy rodent neuropathic pain models.

Coincident Regulation of PLCβ Signaling by Gq-Coupled and μ-Opioid Receptors Opposes Opioid-Mediated Antinociception.

Pain management is an important problem worldwide. The current frontline approach for pain management is the use of opioid analgesics. The primary analgesic target of opioids is the μ-opioid receptor (MOR). Deletion of phospholipase Cβ3 (PLCβ3) or selective inhibition of Gβγ regulation of PLCβ3 enhances the potency of the antinociceptive effects of morphine suggesting a novel strategy for achieving opioid-sparing effects. Here we investigated a potential mechanism for regulation of PLC signaling downstream of MOR in human embryonic kidney 293 cells and found that MOR alone could not stimulate PLC but rather required a coincident signal from a Gq-coupled receptor. Knockout of PLCβ3 or pharmacological inhibition of its upstream regulators, Gβγ or Gq, ex vivo in periaqueductal gray slices increased the potency of the selective MOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt in inhibiting presynaptic GABA release. Finally, inhibition of Gq- G protein-coupled receptor coupling in mice enhanced the antinociceptive effects of morphine. These data support a model where Gq and Gβγ-dependent signaling cooperatively regulate PLC activation to decrease MOR-dependent antinociceptive potency. Ultimately, this could lead to identification of new non-MOR targets that would allow for lower-dose utilization of opioid analgesics. SIGNIFICANCE STATEMENT: Previous work demonstrated that deletion of phospholipase Cβ3 (PLCβ3) in mice potentiates μ-opioid receptor (MOR)-dependent antinociception. How PLCβ3 is regulated downstream of MOR had not been clearly defined. We show that PLC-dependent diacylglycerol generation is cooperatively regulated by MOR-Gβγ and Gq-coupled receptor signaling through PLCβ3 and that blockade of either Gq-signaling or Gβγ signaling enhances the potency of opioids in ex vivo brain slices and in vivo. These results reveal potential novel strategies for improving opioid analgesic potency and safety.

Investigation of the μ and κ-opioid receptor activation by eight new synthetic opioids using the [35S]-GTPγS assay: U-47700, isopropyl U 47700, U-49900, U-47931E, N-methyl U-47931E, U-51754, U-48520 and U-48800

Since their first occurrence in 2008, a rapid increase of new psychoactive substances (NPS) on the recreational drug market has been observed. One class of NPS are synthetic opioids, which poses a health risk due to the high affinity and high potency of most of these compounds for the opioid receptors. Four opioid receptor subtypes are known, the mü-opioid (MOR), the delta-opioid (DOR), the kappa-opioid (KOR) and the nociceptin receptor, which are G protein-coupled receptors (GCPRs). MOR and DOR agonists often express mood-enhancing and euphoric properties. However, opioid overdoses can lead to respiratory depression and result in death. Nevertheless, for many NPS, including new synthetic opioids, data on toxicological and toxicokinetic properties are scarce. The aim of the present study was to investigate eight structurally closely related U-opioids (U-47700, isopropyl U-47700, U-49900, U-47931E, N-methyl U-47931E, U-48520, U-51754, and U-48800) for their potential of MOR and KOR activation, due to the relative lack of pharmacological data for the U-opioids. The [35S]-GTPγS assay, was used to study receptor activation and is based on the exchange of GDP with radiolabelled GTP. This exchange is one of the first events of the GPCR-signal cascade. For each receptor two respective and well-studied compounds were chosen as reference compounds: hydromorphone and fentanyl for MOR, and U-69593 and U-50488 for KOR. Owing to their close structural similarity of the investigated U-compounds, structure activity relationships have also been derived. The most prevalent compound of this set, U-47700, was also the most potent compound of the test set at MOR, with an EC50 value of 111 nM. However, the two MOR reference compounds hydromorphone and fentanyl had lower EC50 values of 6.75 nM and 24.9 nM, respectively, hence, being 16 and 4 times more potent than U-47700. At KOR, U-51754 was the most potent substance out of the test set, with an EC50 value of 120 nM. Nevertheless, the two U-opioids U-69593 and U-50488, which have been used as KOR reference compounds, were 4 and 5 times more potent than U-51754 at KOR, with EC50 of 29.3 nM and 24.8 nM, respectively. The studied compounds can be categorized into the U-47700 and U-50488 group. It has been previously reported that the compounds of the U-47700 group show higher receptor binding affinities for MOR and compounds of the U-50488 group for KOR, hence the presence or absence of the methylene group has an influence on the receptor binding affinity at MOR and KOR. In general, our results on the receptor activation of MOR and KOR are in good alignment with reported receptor binding affinities, confirming that the U-47700 group has a higher affinity and activity at MOR and the U-50488 group at KOR. The following structural features were advantageous for activating MOR: two chlorine substituents in 3,4-position at the aromatic ring, the absence of the methylene group between the amide group and the aromatic ring, a methyl group at the amide nitrogen, and/or a dimethylamine residue at the amine nitrogen of the cyclohexane ring. Further, the following structural features were beneficial for KOR activation: a methylene group between the amide group and the aromatic ring, a pyrrolidine residue at the amine nitrogen of the cyclohexane ring, a methyl group at the amide nitrogen, and/or chlorine substitution in 3,4-position at the aromatic ring. While synthetic opioids are often considered more potent than conventional opioids, there are some occurring on the NPS market, which are less potent. Hence, performing these studies is important to identify the effect of structural alterations on the receptor activity and in understanding the potential risk to the public health.

Off-target activity of NBOMes and NBOMe analogs at the μ opioid receptor

New psychoactive substances (NPS) are introduced on the illicit drug market at a rapid pace. They are usually poorly pharmacologically characterized, leaving users unaware of the potential harms they could be exposed to. Apart from the unknown effects and potencies of these new substances, their exact molecular targets are often inadequately elucidated. Guided by recent literature, we investigated the μ opioid receptor (MOR) activation potential of a large set of psychedelics, which typically activate their target receptor, the serotonin (5-HT2A) receptor. Screening for MOR activity of a panel of psychedelics was done using a cell-based NanoBiT® bioassay. The NanoBiT® assay monitors the recruitment of the intracellular signaling protein β-arrestin2 to the ligand activated MOR receptor. The principle exploits functional complementation of a split nanoluciferase enzyme of which the inactive subunits are either fused to the receptor and βarr2. Receptor activation causes the 2 subunits to come in close proximity, resulting in restoration of the nanoluciferase activity. Upon addition of the substrate, a luminescent signal can be detected using a luminometer. Based on the results of this initial screening, MOR activity of a selected panel of NBOMes and NBOMe analogs was evaluated using the both the NanoBiT® and AequoScreen® assay, the latter measuring the rapid intracellular calcium flux caused by receptor activation, a more downstream effect caused by G protein signaling. Using the NanoBiT® assay, 5 NBOMes showed MOR activation potential. Based on these findings, a panel of NBOMe analogs was selected and evaluated using 2 distinct assays. Comparing both assays, the same overall trends regarding MOR activity could be determined. The iodo analog 25I-NBOMe was more potent than the bromo substituted 25B-NBOMe, which was more potent than the chloro analog 25C-NBOMe, signifying that MOR activity increased with an increased size of the halogen atom. Similarly, MOR activity increased with increasing size of the alkyl side chain, with the ethylated 25E-NBOMe being more active than the methylated 25D-NBOMe. However, a propyl or isopropyl side chain might be too long or bulky, as the 25P-NBOMe and 25iP-NBOMe showed a substantial decrease in activity. NBOMe compounds were more active at MOR than their NBOH counterparts, whereas earlier data showed that they had a similar activity at their designated target receptor 5-HT2A. Moving the methoxy group from position 2 to position 3 or 4 resulted in an important decrease in MOR activity. Furthermore, the effects at MOR of the analyzed compounds could be blocked by the opioid antagonist naloxone, suggesting that these NBOMes occupy the same common opioid binding pocket as conventional opioids. Using the AequoScreen® assay, aspecific, receptor independent effects were found for the alkylated 25iP-NBOMe, 25E-NBOMe and 25D-NBOMe, indicating that these may directly interact with other targets in the downstream signaling pathway. A limited potential to activate the MOR was found for 10 NBOMes and NBOMe analogs, which was confirmed using 2 distinct cell-based assays. Structure-activity relationships could be determined, and additional experiments revealed that the effects of these psychedelics at MOR could be antagonized by naloxone, signifying that these substances occupy the same binding pocket as common opioids. Some compounds showed an aspecific effect in the AequoScreen® assay, which was not observed using the NanoBiT® assay. As MOR activity of these psychedelics was only noticed at high concentrations, it is unlikely that these will contribute to pronounced opioid toxicity at physiologically relevant concentrations. However, small modifications to the original NBOMe structure may result in a panel of more efficacious and potent MOR agonists.

Possible mechanism for improving the endogenous immune system through the blockade of peripheral μ-opioid receptors by treatment with naldemedine.

It has been considered that activation of peripheral μ-opioid receptors (MORs) induces side effects of opioids. In this study, we investigated the possible improvement of the immune system in tumour-bearing mice by systemic administration of the peripheral MOR antagonist naldemedine. The inhibitory effect of naldemedine on MOR-mediated signalling was tested by cAMP inhibition and β-arrestin recruitment assays using cultured cells. We assessed possible changes in tumour progression and the number of splenic lymphocytes in tumour-bearing mice under the repeated oral administration of naldemedine. Treatment with naldemedine produced a dose-dependent inhibition of both the decrease in the cAMP level and the increase in β-arrestin recruitment induced by the MOR agonists. Repeatedtreatment with naldemedine at a dose that reversed the morphine-induced inhibition of gastrointestinal transport, but not antinociception, significantly decreased tumour volume and prolonged survival in tumour-transplanted mice. Naldemedine administration significantly decreased the increased expression of immune checkpoint-related genes and recovered the decreased level of toll-like receptor 4 in splenic lymphocytes in tumour-bearing mice. The blockade of peripheral MOR may induce an anti-tumour effect through the recovery of T-cell exhaustion and promotion of the tumour-killing system.

Hydrogen Sulfide Increases the Analgesic Effects of µ- and δ-Opioid Receptors during Neuropathic Pain: Pathways Implicated.

Recent studies have revealed that hydrogen sulfide (H2S) increases the analgesic actions of the δ-opioid receptor (DOR) in inflammatory pain. However, the possible improvement of the analgesia of μ-opioid receptor (MOR) and DOR agonists during neuropathic pain, through pretreatment with two slow-releasing H2S donors—DADS (diallyl disulfide) and GYY4137 (morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate dichloromethane complex)—is still unknown. In male C57BL/6J mice with neuropathic pain incited by chronic constriction of the sciatic nerve (CCI), we evaluated: (1) the influence of DADS (3.5 mg/kg) and GYY4137 (0.7 mg/kg) on the inhibition of the allodynia and hyperalgesia produced by the systemic or local administration of morphine (3 mg/kg or 65 µg) and UFP-512 (1 mg/kg or 12.5 µg); (2) the reversion of the antinociceptive actions of high doses of DADS (30 mg/kg) and GYY4137 (24 mg/kg) with MOR and DOR antagonists; and (3) the effects of H2S donors on oxidative stress, apoptotic responses, and MOR and DOR expression in the medial septum (MS) and dorsal root ganglia (DRG). The results revealed that both DADS and GYY4137 improved the antiallodynic effects of morphine and UFP-512, possibly by up-regulating MOR and DOR expression in DRG. The administration of MOR and DOR antagonists blocked the analgesic properties of DADS and GYY4137, revealing the feasible participation of the endogenous opioid system in H2S analgesic effects. Moreover, both H2S donors inhibited oxidative stress and apoptosis generated by CCI in the MS and/or DRG. This study suggests the co-treatment of H2S donors with MOR or DOR agonists as a potential therapy for neuropathic pain.

Effects of Mitragynine and its Active Metabolites on the Reinforcing Effects of Remifentanil and Cocaine in Rats Self‐Administering Remifentanil

Kratom is being developed as a pharmacotherapy to mitigate withdrawal symptoms from opioids; however, the presence of active metabolites of the primary kratom alkaloid mitragynine (MG) at mu‐opioid receptors [(MORs), 7‐hydoxymitragynine (7‐OH‐MG) and mitragynine pseudoindoxyl (MG‐P)] may limit effectiveness due to their abuse potential. Yue et al (2018) indicated specificity of the antagonizing effects of MG for the reinforcing effects of the MOR agonist heroin relative to the non‐opioid methamphetamine. Using an intravenous drug self‐administration assay, the present study compared abuse potential of MG and its metabolites, and assessed specificity of MG to antagonize the reinforcing effects of the MOR agonist remifentanil and the non‐opioid cocaine in rats. In rats self‐administering remifentanil (0.1‐3.2 µg/kg/injection), reference MOR agonists (methadone and buprenorphine) and the dopamine uptake inhibitor cocaine maintained self‐administration responding above extinction (no injection) levels whereas antagonists at MOR (naltrexone and naloxone) did not. Self‐administration responding above extinction was maintained by 7‐OH‐MG (1.0‐10 µg/kg/injection) and MGP (0.32 µg/kg/injection), but not MG (up to 3.2 mg/kg/injection) when substituted for remifentanil. Self administration of remifentanil, cocaine, 7‐OH‐MG, and MG‐P was decreased by naltrexone (3.2 mg/kg, i.p.). Self administration of remifentanil was also decreased by MG, 7‐OH‐MG, and MG‐P (ED50 values: 34.5, 4.35, and 2.66 mg/kg, respectively). Self administration of cocaine was also decreased by MG, 7‐OH‐MG, and MG‐P (ED50 values: 59.2, 5.12, and 5.17 mg/kg, respectively). Despite the abuse potential of active metabolites of MG, these results suggest less abuse potential of MG than that of its metabolites in rats. The lack of obvious reinforcing effects of MG also suggests if any low contribution of 7‐OH‐MG and MGP to the in vivo MOR activity of MG in rats. Though MG was least potent to decrease the reinforcing effects of remifentanil and cocaine, the comparable ED50 values per compound suggest a lack of specificity of the antagonizing effects of MG and its metabolites in rats with opioid history. These results also indicate that drug history could affect the antagonizing effects of MG on the reinforcing effects of stimulants.

Irreversible and noncompetitive mu opioid receptor agonists

The ability of a ligand to preferentially promote engagement of one signaling pathway over another downstream of GPCR activation has been referred to as signaling bias, functional selectivity or biased agonism. The presentation of ligand bias reflects selectivity between active states of the receptor which may result in the display of preferential engagement with one signaling pathway over another. In this study, we provide evidence that the G protein‐biased MOR agonists, SR‐17018 and SR‐14968 stabilize the mu opioid receptor in a wash‐resistant, yet antagonist‐reversible, G protein signaling state. Furthermore, we demonstrate that these structurally related biased agonists, are noncompetitive for radiolabeled MOR antagonist binding and while they stimulate G protein signaling in mouse brain, partial agonists of this class do not compete with full agonist activation. Importantly, opioid antagonists can readily reverse their effects in vivo. Given that chronic treatment with SR‐17018 does not lead to tolerance in several mouse pain models, this feature may be desirable for the development of long lasting opioid analgesics that remain sensitive to antagonist reversal of respiratory suppression.

Activation of Brainstem mu‐Opioid Receptors (mORs) Increases Respiratory‐Related Motor Burst Amplitude in Neonatal Rat Brainstem‐Spinal Cord Preparations

Mu‐opioid receptors (mORs) located on brainstem respiratory neurons are part of an intricate peptidergic neuromodulatory system that regulates breathing. The endogenous role of mORs in respiratory motor control, however, is poorly understood. To explore this question, very low (nanomolar) concentrations of mOR agonist drugs were bath‐applied to neonatal rat (P1‐P3) brainstem‐spinal cord preparations while measuring respiratory‐related motor output from spinal C4‐C5 rootlets containing phrenic motor neuron axons. When DAMGO (synthetic mOR agonist) or endomorphin‐2 (endogenous mOR ligand) were bath‐applied for 15 min at 20‐100 nM, respiratory motor burst amplitude increased dramatically with a modest decrease in burst frequency. Burst amplitude facilitated by 45 ± 38% and 32 ± 24% (mean ± stdev) above baseline for 50 nM DAMGO (n=8) and 100 nM endomorphin‐2 (n=10), respectively (p<0.001). To test whether respiratory motor output on thoracic ventral roots was altered by mOR activation, brainstem‐spinal cord preparations were isolated to include spinal segments C1‐T8. Bath‐applied DAMGO (50 nM) induced burst amplitude facilitation on thoracic roots T4‐T6 and cervical roots C4‐C5 by 62 ± 19% and 38 ± 36%, respectively (p<0.012). To test whether mOR‐induced burst facilitation is due to brainstem or spinal mechanisms, a split‐bath chamber with a barrier at C1‐C2 was used to separately bathe the brainstem and spinal cord compartments. DAMGO (50 nM) application to the spinal cord (C2‐C8) had little effect on respiratory motor output measured from C4‐C5 rootlets, whereas DAMGO application to the brainstem increased C4‐C5 motor output by 34 ± 19% (p<0.004). These data suggest that mOR activation in the brainstem can transform the respiratory network to produce a motor pattern with decreased frequency balanced by increased amplitude, with relatively little change in overall neural output. Given that mOR‐induced facilitation was observed in both cervical and thoracic motor neuron pools, we hypothesize that burst amplitude facilitation is due to mOR activation on rhythm‐generating neurons or premotor neurons in the brainstem. This study reveals that endogenous mOR activation modulates the neural control of breathing in a manner that is potentially different from respiratory depression, which is more commonly associated with opioids.

Structure-Based Evolution of G Protein-Biased μ-Opioid Receptor Agonists.

The μ‐opioid receptor (μOR) is the major target for opioid analgesics. Activation of μOR initiates signaling through G protein pathways as well as through β‐arrestin recruitment. μOR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased μOR agonist. Here we report the cryoEM structure of PZM21 bound μOR in complex with Gi protein. Structure‐based evolution led to multiple PZM21 analogs with more pronounced Gi protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to μOR in complex with Gi protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce β‐arrestin recruitment by the μOR, and hold promise for developing next‐generation analgesics with fewer adverse effects.