RESUMEN

Oprm1, the gene encoding the µ-opioid receptor, has multiple reported transcripts, with a variable 3' region and many alternative sequences encoding the C-terminus of the protein. The functional implications of this variability remain mostly unexplored, though a recurring notion is that it could be exploited by developing selective ligands with improved clinical profiles. Here, we comprehensively examined Oprm1 transcriptional variants in the murine central nervous system, using long-read RNAseq as well as spatial and single-cell transcriptomics. The results were validated with RNAscope in situ hybridization. We found a mismatch between transcripts annotated in the mouse genome (GRCm38/mm10) and the RNA-seq results. Sequencing data indicated that the primary Oprm1 transcript has a 3' terminus located on chr10:6,860,027, which is âˆ¼ 9.5 kilobases downstream of the longest annotated exon 4 end. Long-read sequencing confirmed that the final Oprm1 exon included a 10.2 kilobase long 3' untranslated region, and the presence of the long variant was unambiguously confirmed using RNAscope in situ hybridization in the thalamus, striatum, cortex and spinal cord. Conversely, expression of the Oprm1 reference transcript or alternative transcripts of the Oprm1 gene was absent or close to the detection limit. Thus, the primary transcript of the Oprm1 mouse gene is a variant with a long 3' untranslated region, which is homologous to the human OPRM1 primary transcript and encodes the same conserved C-terminal amino acid sequence.

Asunto(s)

Prosencéfalo , Receptores Opioides mu , Médula Espinal , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Animales , Ratones , Médula Espinal/metabolismo , Prosencéfalo/metabolismo , Masculino , Ratones Endogámicos C57BL , Exones , Regiones no Traducidas 3'

RESUMEN

Knowledge of opioid tolerance in a deceased person is important for distinguishing between therapeutic and toxic opioid concentrations for that particular individual when interpreting postmortem toxicological results. However, no biomarkers for opioid tolerance are currently available. This review aimed to study the existing literature on mechanisms or changes in signaling pathways related to chronic opioid use, which could be relevant for further studies to identify biomarkers for opioid tolerance. We performed a systematic literature search using the PRISMA 2020 guidelines using the MeSH terms "opioid tolerance AND biomarkers" in PubMed, Embase, WebofScience, and the Cochrane library. A review of the search results yielded seven studies on animal models or humans, identifying and evaluating thirteen possible biomarkers in terms of specificity for changes induced by opioids and other aspects to be considered as potential biomarkers. We evaluated nine potential biomarkers as unlikely to be specific for opioid tolerance, and one had contradictory results in terms of upregulation or downregulation. However, methylation of the promoter region of the µ-opioid receptor gene, increased activity of soluble puromycin-sensitive aminopeptidase, altered miRNA profile, or other multiple component profiling may be interesting to study further as biomarkers for opioid tolerance in forensic postmortem cases.

Asunto(s)

Analgésicos Opioides , Biomarcadores , Tolerancia a Medicamentos , Humanos , Biomarcadores/metabolismo , Animales , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , MicroARNs/metabolismo , Toxicología Forense/métodos

RESUMEN

The emergence of new synthetic opioids (NSOs) has added complexity to recreational opioid markets worldwide. While NSOs with diverse chemical structures have emerged, brorphine currently remains the only NSO with a piperidine benzimidazolone scaffold. However, the emergence of new generations of NSOs, including brorphine analogues, can be anticipated. This study explored the pharmaco-toxicological, opioid-like effect profile of brorphine alongside its non-brominated analogue (orphine) and three other halogenated analogues (fluorphine, chlorphine, iodorphine). In vitro, radioligand binding assays in rat brain tissue indicated that all analogues bind to the µ-opioid receptor (MOR) with nM affinity. While analogues with smaller-sized substituents showed the highest MOR affinity, further in vitro characterization via two cell-based (HEK 293T) MOR activation (ß-arrestin 2 and mini-Gαi recruitment) assays indicated that chlorphine, brorphine, and iodorphine were generally the most active MOR agonists. None of the compounds showed significant in vitro biased agonism compared to hydromorphone. In vivo, we investigated the effects of intraperitoneal (IP) administration of the benzimidazolones (0.01-15 mg/kg) on mechanical and thermal antinociception in male CD-1 mice. Chlorphine and brorphine overall induced the highest levels of antinociception. Furthermore, the effects on respiratory changes induced by a fixed dose (15 mg/kg IP) of the compounds were investigated using non-invasive plethysmography. Fluorphine-, chlorphine-, and brorphine-induced respiratory depressant effects were the most pronounced. For some compounds, pretreatment with naloxone (6 mg/kg IP) could not reverse respiratory depression. Taken together, brorphine-like piperidine benzimidazolones are opioid agonists that have the potential to cause substantial harm to users should they emerge as NSOs. This article is part of the Special Issue on "Novel Synthetic Opioids (NSOs)".

Asunto(s)

Analgésicos Opioides , Animales , Humanos , Analgésicos Opioides/farmacología , Masculino , Células HEK293 , Ratones , Ratas , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Ratas Sprague-Dawley , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo

RESUMEN

Migraine is a debilitating neurological disorder impacting millions worldwide. Calcitonin Gene-Related Peptide (CGRP) has emerged as a key player in migraine pathophysiology, leading to the development of targeted therapies. This study reviews novel CGRP-targeted treatments, including monoclonal antibodies small molecule inhibitors/nutraceuticals and introduces Asarinin as a potential modulator of the pathway. Asarinin, a natural compound found in various plants, is examined for its pharmacological potential in migraine management. Pharmacokinetic assessments, toxicological modelling, molecular property analysis, and network pharmacology were conducted. Molecular docking and dynamics studies with CGRP reveal potential interactions, providing a foundation for understanding Asarinin's therapeutic effects. Asarinin's favourable pharmacokinetics, safety profile, and bioactivity, supporting its candidacy as a therapeutic agent. In-depth molecular docking studies with the CGRP receptor (PDB: 6ZHO) demonstrate strong binding affinity (- 10.3kcal/mol), while molecular dynamics simulations unveil the dynamic behavior of the Asarinin-CGRP complex, (- 10.53 kcal/mol) for Atogepant-CGRP complex. Network analysis highlights key proteins in migraine pathology, indicating Asarinin's potential efficacy. The groundwork for future investigations, suggests Asarinin as a promising candidate for migraine management by targeting OPRM1 pathway. The integration of diverse assessments provides a comprehensive understanding of Asarinin's potential and paves the way for further preclinical and clinical research.

Asunto(s)

Trastornos Migrañosos , Simulación del Acoplamiento Molecular , Receptores Opioides mu , Trastornos Migrañosos/tratamiento farmacológico , Trastornos Migrañosos/metabolismo , Humanos , Receptores Opioides mu/metabolismo , Transducción de Señal/efectos de los fármacos , Péptido Relacionado con Gen de Calcitonina/metabolismo , Simulación de Dinámica Molecular , Farmacología en Red , Animales

RESUMEN

BACKGROUND: Classical opioids are effective analgesics but carry various side effects, necessitating safer alternatives. Truncated six-transmembrane mu opioid receptors (6TM-µORs) mediate potent analgesia with fewer side effects and are a promising therapeutic target. However, few ligands known selectively target 6TM-µORs. Moreover, endogenous chaperones are believed essential for 6TM-µOR ligand binding and function. PURPOSE: To identify a 6TM-µOR selective agonist and elucidate requisite endogenous chaperones. METHODS: Virtual screening was used to identify promising selective 6TM-µOR agonists from traditional Chinese medicines. The role of 6TM-µOR in Exoticin analgesia was validated in loss- and gain-of-function models. APEX2 proteomics profiled proximal proteins under Exoticin or IBNtxA. Interactions were further characterized in vivo and in vitro. RESULTS: Exoticin was shortlisted for its selective binding to 6TM-µOR and ability to induce 6TM-µOR-dependent signal transduction. Exoticin analgesia was sensitive to ß-FNA and absent in E11 KO mice, but restored in mice infected with AAV-µOR1G. Slc3a2, Lrrc59, and Ppp1cb co-interacted with 6TM-µOR1G and were equally essential for Exoticin binding and 6TM-µOR1G activity. CONCLUSION: Exoticin is a promising selective agonist of 6TM µ opioid receptors with broad-spectrum analgesic efficacy but few side effects. Slc3a2, Lrrc59, Ppp1cb are endogenous chaperones essential for 6TM-µOR ligand binding and function.

Asunto(s)

Receptores Opioides mu , Animales , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Ratones , Humanos , Chaperonas Moleculares/metabolismo , Ratones Noqueados , Células HEK293 , Analgésicos Opioides/farmacología , Masculino , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología

RESUMEN

The OPRM1 gene codes for the mu opioid receptor (MOR) and polymorphisms are associated with complex patient clinical responses. The most studied single nucleotide polymorphism (SNP) in OPRM1 is adenine (A) substituted by guanine (G) at position 118 (118A>G, rs1799971) leading to a substitution of asparagine (Asn) for aspartic acid (Asp) at position 40 in the N terminus of the resulting protein. To date, no structural explanation for the associated clinical responses resulting from the 118A>G polymorphism has been proposed. We utilized computational modeling paired with functional cellular assays to predict unstructured N- and C-terminal regions of MOR-1. Using molecular docking and post-docking energy minimizations with morphine, we show that the extracellular substitution of Asn at position 40 alters the cytoplasmic C-terminal conformation, while leaving the G-protein binding interface unaffected. A real-time BRET assay measuring G-protein and ß-arrestin association with MOR r generated data that tested this prediction. Consistent with this in silico prediction, we show changes in morphine-mediated ß-arrestin association with receptor variants with little change in morphine-mediated G-protein association comparing MOR-1 wild type (WT) to MOR-1118A>G. We tested the system with different opioid agonists, the OPRM1 118A>G SNP, and different MOR splice variants (MOR-1 and MOR-1O). These results are consistent with the observation that patients with the 118A>G OPRM1 allele respond more readily to fentanyl than to morphine. In conclusion, the 118A>G substitution alters receptor responses to opioids through variable C-terminal domain movements that are agonist and splice variant dependent.

Asunto(s)

Simulación del Acoplamiento Molecular , Morfina , Polimorfismo de Nucleótido Simple , Receptores Opioides mu , beta-Arrestinas , Receptores Opioides mu/genética , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Humanos , beta-Arrestinas/metabolismo , Morfina/farmacología , Células HEK293 , Analgésicos Opioides/farmacología , Analgésicos Opioides/metabolismo , Unión Proteica

RESUMEN

The pathogenesis mechanism of acute gastric mucosal lesions (AGML) is still unclear; further exploration is urgently needed to find a new therapeutic target. This study aimed to investigate whether morphine might regulate the expression and function of transient receptor potential ankyrin 1 (TRPA1) through a cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-dependent pathway, thereby alleviating gastric mucosal lesions caused by water-immersion restraint stress (WIRS). Rats were administered with intrathecal morphine, TRPA1 antagonist (HC-030031), µ-opioid receptor antagonist, or protein kinase A inhibitor (H-89), respectively, before WIRS. After 6 hours of WIRS, microscopic lesions, hematoxylin and eosin staining, and transmission electron microscopy were applied to assess the damage of the gastric mucosa. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were conducted to detect the levels of TRPA1 and substance P (SP) in the dorsal root ganglia (DRG) and gastric tissues. In addition, immunofluorescence was used to explore the possible co-expression of TRPA1 and µ-opioid receptors in the DRG. The results indicated that WIRS upregulated TRPA1 and SP in gastric mucosa, and HC-030031 or H-89 could alleviate gastric mucosal lesions caused by WIRS (P < .0001). Morphine was found to suppress both WIRS-induced gastric mucosal lesions (P < .0001) and the upregulation of TRPA1 (P = .0086) and SP (P = .0013). Both TRPA1 and SP play important roles in the pathogenesis of WIRS-induced AGML. Exogenous gastroprotective strategies reduce elevated levels of TRPA1 via the cAMP/PKA-dependent pathway. Inhibition of TRPA1 upregulation in the DRG is critical for intrathecal morphine preconditioning-induced gastric protection.

Asunto(s)

Ganglios Espinales , Mucosa Gástrica , Isoquinolinas , Morfina , Ratas Sprague-Dawley , Restricción Física , Canal Catiónico TRPA1 , Regulación hacia Arriba , Animales , Morfina/farmacología , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Canal Catiónico TRPA1/metabolismo , Masculino , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Restricción Física/efectos adversos , Ratas , Isoquinolinas/farmacología , Acetanilidas/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Purinas/farmacología , Estrés Psicológico/complicaciones , Inmersión , Receptores Opioides mu/metabolismo , AMP Cíclico/metabolismo , Sulfonamidas

RESUMEN

How cellular adaptations give rise to opioid analgesic tolerance to opioids like morphine is not well understood. For one, pain is a complex phenomenon comprising both sensory and affective components, largely mediated through separate circuits. Glutamatergic projections from the medial thalamus (MThal) to the anterior cingulate cortex (ACC) are implicated in processing of affective pain, a relatively understudied component of the pain experience. The goal of this study was to determine the effects of chronic morphine exposure on mu-opioid receptor (MOR) signaling on MThal-ACC synaptic transmission within the excitatory and feedforward inhibitory pathways. Using whole cell patch-clamp electrophysiology and optogenetics to selectively target these projections, we measured morphine-mediated inhibition of optically evoked postsynaptic currents in ACC layer V pyramidal neurons in drug-naïve and chronically morphine-treated mice. We found that morphine perfusion inhibited the excitatory and feedforward inhibitory pathways similarly in females but caused greater inhibition of the inhibitory pathway in males. Chronic morphine treatment robustly attenuated morphine presynaptic inhibition within the inhibitory pathway in males, but not females, and mildly attenuated presynaptic inhibition within the excitatory pathway in both sexes. These effects were not observed in MOR phosphorylation-deficient mice. This study indicates that chronic morphine treatment induces cellular tolerance to morphine within a thalamo-cortical circuit relevant to pain and opioid analgesia. Furthermore, it suggests this tolerance may be driven by MOR phosphorylation. Overall, these findings improve our understanding of how chronic opioid exposure alters cellular signaling in ways that may contribute to opioid analgesic tolerance.NEW & NOTEWORTHY Opioid signaling within the anterior cingulate cortex (ACC) is important for opioid modulation of affective pain. Glutamatergic medial thalamus (MThal) neurons synapse in the ACC and opioids, acting through mu opioid receptors (MORs), acutely inhibit synaptic transmission from MThal synapses. However, the effect of chronic opioid exposure on MThal-ACC synaptic transmission is not known. Here, we demonstrate that chronic morphine treatment induces cellular tolerance at these synapses in a sex-specific and phosphorylation-dependent manner.

Asunto(s)

Analgésicos Opioides , Morfina , Receptores Opioides mu , Tálamo , Animales , Receptores Opioides mu/metabolismo , Morfina/farmacología , Morfina/administración & dosificación , Masculino , Femenino , Ratones , Analgésicos Opioides/farmacología , Analgésicos Opioides/administración & dosificación , Tálamo/efectos de los fármacos , Tálamo/fisiología , Tálamo/metabolismo , Giro del Cíngulo/efectos de los fármacos , Giro del Cíngulo/fisiología , Giro del Cíngulo/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/fisiología , Tolerancia a Medicamentos/fisiología , Ratones Endogámicos C57BL , Caracteres Sexuales , Transducción de Señal/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología

RESUMEN

The emergence of 2-benzylbenzimidazole "nitazene" opioids is stirring up the recreational synthetic opioid market. Many nitazene analogues act as potent agonists at the µ­opioid receptor (MOR), as demonstrated in various in vitro and in vivo studies. Severe intoxication and overdose deaths associated with nitazene analogues are increasingly being reported. Nitazene opioids are classified as a public health threat, stressing the need for close monitoring of new developments on the recreational drug market. This study reports on the detection of N-desethyl etonitazene in a sample handed in by a recreational drug user at a Swiss drug checking service in August 2023. The person bought the sample through an internet source where it was stated to contain isotonitazene. Chemical analyses were conducted to characterize the sample, i.e. nuclear magnetic resonance (NMR), capillary electrophoresis (CE), and high-resolution mass spectrometry (HRMS). The sample was additionally investigated using two different in vitro MOR activation assays. NMR and high-performance liquid chromatography (HPLC) coupled to HRMS confirmed the presence of N-desethyl etonitazene at a high purity and in the absence of isotonitazene and etonitazene. N-Desethyl nitazene analogues have been detected before as metabolites of isotonitazene and etonitazene. However, as first seen with N-desethyl isotonitazene, they are now emerging as standalone drugs. The applied bioassays demonstrated increased efficacy and approximately 6-9-fold higher potency of N-desethyl etonitazene at MOR compared to fentanyl. N-Desethyl etonitazene showed EC50 values of 3.35 nM and 0.500 nM in the ß-arrestin 2 recruitment and Aequoscreen® assays, respectively. The opioid activity present in the collected sample was additionally evaluated using the bioassays and showed good overlap with the reference standard, in line with the analytical purity assessment. This demonstrates the potential of these bioassays to provide a rapid opioid activity assessment of authentic samples. The emergence of other N-desethyl nitazene analogues must be considered during forensic and clinical toxicology casework, to avoid misclassification of intake of such analogues as metabolites. Finally, drug checking services enable the close monitoring of market developments and trends and are of great value for early warning and harm reduction purposes.

Asunto(s)

Analgésicos Opioides , Bencimidazoles , Drogas Ilícitas , Bencimidazoles/análisis , Bencimidazoles/química , Humanos , Analgésicos Opioides/análisis , Analgésicos Opioides/química , Drogas Ilícitas/análisis , Drogas Ilícitas/química , Cromatografía Líquida de Alta Presión/métodos , Espectroscopía de Resonancia Magnética/métodos , Receptores Opioides mu/metabolismo , Receptores Opioides mu/agonistas , Electroforesis Capilar/métodos , Nitrocompuestos/análisis , Espectrometría de Masas/métodos , Animales , Suiza

RESUMEN

The subiculum is a key region of the brain involved in the initiation of pathological activity in temporal lobe epilepsy, and local GABAergic inhibition is essential to prevent subicular-originated epileptiform discharges. Subicular pyramidal cells may be easily distinguished into two classes based on their different firing patterns. Here, we have compared the strength of the GABAa receptor-mediated inhibitory postsynaptic currents received by regular- vs. burst-firing subicular neurons and their dynamic modulation by the activation of µ opioid receptors. We have taken advantage of the sequential re-patching of the same cell to initially classify pyramidal neurons according to their firing patters, and then to measure GABAergic events triggered by the optogenetic stimulation of parvalbumin- and somatostatin-expressing interneurons. Activation of parvalbumin-expressing cells generated larger responses in postsynaptic burst-firing neurons whereas the opposite was observed for currents evoked by the stimulation of somatostatin-expressing interneurons. In all cases, events depended critically on ω-agatoxin IVA- but not on ω-conotoxin GVIA-sensitive calcium channels. Optogenetic GABAergic input originating from both parvalbumin- and somatostatin-expressing cells was reduced in amplitude following the exposure to a µ opioid receptor agonist. The kinetics of this pharmacological sensitivity was different in regular- vs. burst-firing neurons, but only when responses were evoked by the activation of parvalbumin-expressing neurons, whereas no differences were observed when somatostatin-expressing cells were stimulated. In conclusion, our results show that a high degree of complexity regulates the organizing principles of subicular GABAergic inhibition, with the interaction of pre- and postsynaptic diversity at multiple levels. KEY POINTS: Optogenetic stimulation of parvalbumin- and somatostatin-expressing interneurons (PVs and SOMs) triggers inhibitory postsynaptic currents (IPSCs) in both regular- and burst-firing (RFs and BFs) subicular pyramidal cells. The amplitude of optogenetically evoked IPSCs from PVs (PV-opto IPSCs) is larger in BFs whereas IPSCs generated by the light activation of SOMs (SOM-opto IPSCs) are larger in RFs. Both PV- and SOM-opto IPSCs critically depend on ω-agatoxin IVA-sensitive P/Q type voltage-gated calcium channels, whereas no major effects are observed following exposure to ω-conotoxin GVIA, suggesting no significant involvement of N-type channels. The amplitude of both PV- and SOM-opto IPSCs is reduced by the probable pharmacological activation of presynaptic µ opioid receptors, with a faster kinetics of the effect observed in PV-opto IPSCs from RFs vs. BFs, but not in SOM-opto IPSCs. These results help us understand the complex interactions between different layers of diversity regulating GABAergic input onto subicular microcircuits.

Asunto(s)

Parvalbúminas , Células Piramidales , Somatostatina , Animales , Células Piramidales/fisiología , Ratones , Somatostatina/metabolismo , Parvalbúminas/metabolismo , Interneuronas/fisiología , Potenciales Postsinápticos Inhibidores , Masculino , Neuronas GABAérgicas/fisiología , Neuronas GABAérgicas/metabolismo , Hipocampo/fisiología , Hipocampo/citología , Optogenética , Receptores Opioides mu/metabolismo , Receptores Opioides mu/fisiología , Ratones Endogámicos C57BL , Femenino , Receptores de GABA-A/metabolismo , Receptores de GABA-A/fisiología

RESUMEN

Desensitisation of the mu-opioid receptor (MOR) is proposed to underlie the initiation of opioid analgesic tolerance and previous work has shown that agonist-induced phosphorylation of the MOR C-tail contributes to this desensitisation. Moreover, phosphorylation is important for ß-arrestin recruitment to the receptor, and ligands of different efficacies induce distinct phosphorylation barcodes. The C-tail 370TREHPSTANT379 motif harbours Ser/Thr residues important for these regulatory functions. 375Ser is the primary phosphorylation site of a ligand-dependent, hierarchical, and sequential process, whereby flanking 370Thr, 376Thr and 379Thr get subsequently and rapidly phosphorylated. Here we used GRK KO cells, phosphosite specific antibodies and site-directed mutagenesis to evaluate the contribution of the different GRK subfamilies to ligand-induced phosphorylation barcodes and ß-arrestin2 recruitment. We show that both GRK2/3 and GRK5/6 subfamilies promote phosphorylation of 370Thr and 375Ser. Importantly, only GRK2/3 induce phosphorylation of 376Thr and 379Thr, and we identify these residues as key sites to promote robust ß-arrestin recruitment to the MOR. These data provide insight into the mechanisms of MOR regulation and suggest that the cellular complement of GRK subfamilies plays an important role in determining the tissue responses of opioid agonists.

Asunto(s)

Receptores Opioides mu , Arrestina beta 2 , Fosforilación , Arrestina beta 2/metabolismo , Arrestina beta 2/genética , Humanos , Receptores Opioides mu/metabolismo , Receptores Opioides mu/genética , Células HEK293 , Unión Proteica , Animales , Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/genética

RESUMEN

Neuromodulation in the retina is crucial for effective processing of retinal signal at different levels of illuminance. Intrinsically photosensitive retinal ganglion cells (ipRGCs), the neurons that drive nonimage-forming visual functions, express a variety of neuromodulatory receptors that tune intrinsic excitability as well as synaptic inputs. Past research has examined actions of neuromodulators on light responsiveness of ipRGCs, but less is known about how neuromodulation affects synaptic currents in ipRGCs. To better understand how neuromodulators affect synaptic processing in ipRGC, we examine actions of opioid and dopamine agonists have on inhibitory synaptic currents in ipRGCs. Although µ-opioid receptor (MOR) activation had no effect on γ-aminobutyric acid (GABA) currents, dopamine [via the D1-type dopamine receptor (D1R)]) amplified GABAergic currents in a subset of ipRGCs. Furthermore, this D1R-mediated facilitation of the GABA conductance in ipRGCs was mediated by a cAMP/PKA-dependent mechanism. Taken together, these findings reinforce the idea that dopamine's modulatory role in retinal adaptation affects both nonimage-forming and image-forming visual functions.NEW & NOTEWORTHY Neuromodulators such as dopamine are important regulators of retinal function. Here, we demonstrate that dopamine increases inhibitory inputs to intrinsically photosensitive retinal ganglion cells (ipRGCs), in addition to its previously established effect on intrinsic light responsiveness. This indicates that dopamine, in addition to its ability to intrinsically modulate ipRGC activity, can also affect synaptic inputs to ipRGCs, thereby tuning retina circuits involved in nonimage-forming visual functions.

Asunto(s)

Dopamina , Receptores de GABA-A , Células Ganglionares de la Retina , Animales , Células Ganglionares de la Retina/fisiología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/metabolismo , Dopamina/metabolismo , Dopamina/farmacología , Receptores de GABA-A/metabolismo , Ratones , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/antagonistas & inhibidores , Ratones Endogámicos C57BL , Receptores Opioides mu/metabolismo , Masculino , Potenciales Postsinápticos Inhibidores/fisiología , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Femenino , Agonistas de Dopamina/farmacología

RESUMEN

Psychopathy is characterized by antisocial behavior, poor behavioral control and lacking empathy, and structural alterations in the corresponding neural circuits. Molecular brain basis of psychopathy remains poorly characterized. Here we studied type 2 dopamine receptor (D2R) and mu-opioid receptor (MOR) availability in convicted violent offenders with high psychopathic traits (n = 11) and healthy matched controls (n = 17) using positron emission tomography (PET). D2R were measured with radioligand [11C]raclopride and MORs with radioligand [11C]carfentanil. Psychopathic subjects had lowered D2R availability in caudate and putamen, and striatal D2R availability was also associated with degree of psychopathic traits in this prisoner sample. No group differences were found in MOR availability, although in the prisoner sample, psychopathic traits were negatively correlated with MOR availability in the amygdala and nucleus accumbens. We conclude that D2R signaling could be the putative neuromolecular pathway for psychopathy, whereas evidence for alterations in the MOR system is more limited.

Asunto(s)

Trastorno de Personalidad Antisocial , Criminales , Tomografía de Emisión de Positrones , Receptores de Dopamina D2 , Violencia , Humanos , Receptores de Dopamina D2/metabolismo , Masculino , Trastorno de Personalidad Antisocial/diagnóstico por imagen , Trastorno de Personalidad Antisocial/metabolismo , Adulto , Tomografía de Emisión de Positrones/métodos , Receptores Opioides mu/metabolismo , Racloprida/farmacocinética , Adulto Joven , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagen , Fentanilo/análogos & derivados

RESUMEN

PURPOSE: Sex differences play a crucial role in understanding vulnerability to opioid addiction, yet there have been limited preclinical investigations of this effect during the transition from adolescence to adulthood. The present study compared the behaviors of male and female rodents in response to fentanyl treatment and targeted molecular correlates in the striatum and medial prefrontal cortex. MATERIALS AND METHODS: Thirty adolescent C57BL/6J mice underwent a 1-week fentanyl treatment with an escalating dose. In addition to evaluating locomotor activity and anxiety-related parameters, we also assessed naloxone-induced fentanyl acute withdrawal jumps. We employed real-time quantitative PCR (qPCR) to assess overall gene expression of dopaminergic receptors (Drd1, Drd2, Drd4 and Drd5) and the µ-opioid receptor Oprm1. The levels of epigenetic base modifications including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were assessed on CpG islands of relevant genes. RESULTS: Females had higher locomotor activity than males after chronic fentanyl treatment, and they exhibited higher fentanyl withdrawal jumping behavior induced by naloxone. Females also presented lower Drd4 gene expression and DNA methylation (5mC + 5hmC) in the striatum. We found that locomotor activity and fentanyl withdrawal jumps were negatively correlated with Drd4 methylation and gene expression in the striatum, respectively. CONCLUSIONS: The findings suggested that female mice displayed heightened sensitivity to the effects of fentanyl treatment during the transition from adolescence to adulthood. This effect may be associated with molecular alterations related to the Drd4 gene.

Asunto(s)

Fentanilo , Ratones Endogámicos C57BL , Receptores Opioides mu , Caracteres Sexuales , Animales , Fentanilo/farmacología , Masculino , Femenino , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Ratones , Metilación de ADN/efectos de los fármacos , Analgésicos Opioides/farmacología , Cuerpo Estriado/metabolismo , Cuerpo Estriado/efectos de los fármacos , Locomoción/efectos de los fármacos , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Naloxona/farmacología , Conducta Animal/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/genética , Síndrome de Abstinencia a Sustancias/metabolismo , Epigénesis Genética/efectos de los fármacos

RESUMEN

Opioids are small-molecule agonists of µ-opioid receptor (µOR), while reversal agents such as naloxone are antagonists of µOR. Here, we developed machine learning (ML) models to classify the intrinsic activities of ligands at the human µOR based on the SMILES strings and two-dimensional molecular descriptors. We first manually curated a database of 983 small molecules with measured Emax values at the human µOR. Analysis of the chemical space allowed identification of dominant scaffolds and structurally similar agonists and antagonists. Decision tree models and directed message passing neural networks (MPNNs) were then trained to classify agonistic and antagonistic ligands. The hold-out test AUCs (areas under the receiver operator curves) of the extra-tree (ET) and MPNN models are 91.5 ± 3.9% and 91.8 ± 4.4%, respectively. To overcome the challenge of a small data set, a student-teacher learning method called tritraining with disagreement was tested using an unlabeled data set comprised of 15,816 ligands of human, mouse, and rat µOR, κOR, and δOR. We found that the tritraining scheme was able to increase the hold-out AUC of MPNN models to as high as 95.7%. Our work demonstrates the feasibility of developing ML models to accurately predict the intrinsic activities of µOR ligands, even with limited data. We envisage potential applications of these models in evaluating uncharacterized substances for public safety risks and discovering new therapeutic agents to counteract opioid overdoses.

Asunto(s)

Aprendizaje Automático , Receptores Opioides mu , Humanos , Receptores Opioides mu/metabolismo , Receptores Opioides mu/agonistas , Ligandos , Animales , Analgésicos Opioides/farmacología , Ratones , Redes Neurales de la Computación , Ratas

RESUMEN

Mitragynine, an alkaloid present in the leaves of Mitragyna speciosa (kratom), has a complex pharmacology that includes low efficacy agonism at µ-opioid receptors (MORs). This study examined the activity of mitragynine at adrenergic α2 receptors (Aα2Rs) in vitro and in vivo. Mitragynine displaced a radiolabeled Aα2R antagonist ([3H]RX821002) from human Aα2ARs in vitro with lower affinity (Ki = 1260 nM) than the agonists (-)-epinephrine (Ki = 263 nM) or lofexidine (Ki = 7.42 nM). Mitragynine did not significantly stimulate [35S]GTPγS binding at Aα2ARs in vitro, but in rats trained to discriminate 32 mg/kg mitragynine from vehicle (intraperitoneally administered; i.p.), mitragynine exerted an Aα2R agonist-like effect. Both α2R antagonists (atipamezole and yohimbine) and MOR antagonists (naloxone and naltrexone) produced rightward shifts in mitragynine discrimination dose-effect function and Aα2R agonists lofexidine and clonidine produced leftward shifts. In the mitragynine trained rats, Aα2R agonists also produced leftward shifts in discrimination dose-effect functions for morphine and fentanyl. In a separate rat cohort trained to discriminate 3.2 mg/kg i.p. morphine from vehicle, naltrexone produced a rightward shift, but neither an Aα2R agonist or antagonist affected morphine discrimination. In a hypothermia assay, both lofexidine and clonidine produced marked effects antagonized by yohimbine. Mitragynine did not produce hypothermia. Together, these data demonstrate that mitragynine acts in vivo like an Aα2R agonist, although its failure to induce hypothermia or stimulate [35S]GTPγS binding in vitro, suggests that mitragynine maybe a low efficacy Aα2R agonist.

Asunto(s)

Mitragyna , Receptores Adrenérgicos alfa 2 , Alcaloides de Triptamina Secologanina , Animales , Alcaloides de Triptamina Secologanina/farmacología , Masculino , Humanos , Ratas , Receptores Adrenérgicos alfa 2/metabolismo , Mitragyna/química , Ratas Sprague-Dawley , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Antagonistas de Receptores Adrenérgicos alfa 2/farmacología , Células CHO , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Receptores Opioides mu/antagonistas & inhibidores

RESUMEN

Opioid-related overdoses account for almost half of all drug overdose deaths in the United States and cause more preventable deaths every year than car crashes. Fentanyl, a highly potent mu opioid receptor (MOR) agonist and its analogues (fentalogues) are increasingly found in illicit drug samples, both where the primary drug of abuse is an opioid and where it is not. The prevalence of fentalogues in the illicit drug market is thought to be the primary driver of the increased number of opioid-related overdose deaths since 2016. In fact, fentanyl and its analogues are involved in more than 70% of opioid-related overdoses. The standard opioid overdose rescue therapy naloxone is often insufficient to reverse opioid overdoses caused by fentalogue agonists under current treatment paradigms. However, the pharmacology of many fentalogues is unknown. Moreover, within the fentalogue series of compounds, it is possible that antagonists could be identified that might be superior to naloxone as opioid overdose reversal agents. In this report, we explore the pharmacology of 70 fentalogues and identify compounds that behave as MOR antagonists in vitro and demonstrate with one of these reversals of fentanyl-induced respiratory depression in the mouse. Such compounds could provide leads for the development of effective agents for the reversal of opioid overdose.

Asunto(s)

Analgésicos Opioides , Fentanilo , Naloxona , Antagonistas de Narcóticos , Sobredosis de Opiáceos , Fentanilo/farmacología , Fentanilo/análogos & derivados , Animales , Sobredosis de Opiáceos/tratamiento farmacológico , Ratones , Antagonistas de Narcóticos/farmacología , Analgésicos Opioides/farmacología , Relación Estructura-Actividad , Naloxona/farmacología , Receptores Opioides mu/metabolismo , Humanos , Masculino

RESUMEN

This study explored the potential of a series of PZM21 analogues for pain treatment. Specifically, the hydroxyphenyl ring of PZM21 was replaced with a naphthyl ring, the thienyl ring was substituted with either a phenyl ring or furan rings, and the essential dimethylamine and urea groups were retained. These compounds aimed to enhance safety and minimize the adverse effects associated with opioid drugs. The research findings suggest that compound 6a does not induce ß-arrestin recruitment at low-nanomolar concentrations but exhibits significant analgesic effects in established mouse models. Compared to morphine, 6a shows advantages in alleviating respiratory depression and minimizing physical dependence. Molecular docking studies underscore the pivotal role of the D147 amino acid residue in the analgesic mechanism of 6a. Consequently, 6a is a compelling candidate for the development of safer opioid analgesics and warrants further attention.

Asunto(s)

Analgésicos Opioides , Simulación del Acoplamiento Molecular , Receptores Opioides mu , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Animales , Ratones , Analgésicos Opioides/farmacología , Analgésicos Opioides/química , Analgésicos Opioides/síntesis química , Humanos , Relación Estructura-Actividad , Dolor/tratamiento farmacológico , Masculino , Estructura Molecular , Tiofenos , Urea/análogos & derivados

RESUMEN

Although opioid analgesics are indispensable in treating pain, these drugs are accompanied by life-threatening side effects. While clinically relevant opioid drugs target the µ opioid receptor (MOR), a heterodimer between the MOR and the δ opioid receptor (DOR) has emerged as another target to develop safer analgesics. Although some heterodimer-preferring agonists have been reported so far, it is still difficult to activate the MOR/DOR heterodimer selectively in the presence of MOR or DOR monomers/homodimers. To gain insights to develop selective agonists for MOR/DOR, herein we prepared analogs of CYM51010, one of the reported heterodimer-preferring agonists, and collected structure-activity relationship information. We found that the ethoxycarbonyl group was needed for the activity for the heterodimer, although this group could be substituted with functional groups with similar sizes, such as an ethoxycarbonyl group. As for the acetylaminophenyl group, not a type of substituent, but rather a substituent located at a specific position (para-position) was essential for the activity. Changing the linker length between the acetylaminophenyl group and the piperidine moiety also had deleterious effects on the activity. On the other hand, the substitution of the acetylamino group with a trifluoroacetylamino group and the substitution of the phenethyl group with a benzyl group diminished the activities for the monomers/homodimers while keeping the activity for MOR/DOR, which enhanced the selectivity. Our findings herein will play an important role in developing selective agonists for MOR/DOR and for elucidating the physiological roles of this heterodimer in analgesic processes and in the establishment of side effects.

Asunto(s)

Receptores Opioides delta , Receptores Opioides mu , Relación Estructura-Actividad , Receptores Opioides delta/agonistas , Receptores Opioides delta/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Humanos , Estructura Molecular , Animales , Analgésicos Opioides/química , Analgésicos Opioides/farmacología , Analgésicos Opioides/síntesis química , Relación Dosis-Respuesta a Droga , Cricetulus , Células CHO

RESUMEN

[11C]Carfentanil ([11C]CFN) is the only selective carbon-11 labeled radiotracer currently available for positron emission tomography (PET) imaging of mu opioid receptors (MORs). Though used extensively in clinical research, [11C]CFN has not been thoroughly characterized as a tool for preclinical PET imaging. As we were occasionally observing severe vital sign instability in rat [11C]CFN studies, we set out to investigate physiological effects of CFN mass and to explore its influence on MOR quantification. In anesthetized rats (n = 15), significant dose-dependent PCO2 increases and heart rate decreases were observed at a conventional tracer dose range (IV, > 100 ng/kg). Next, we conducted baseline and retest [11C]CFN PET scans over a wide range of molar activities. Baseline [11C]CFN PET studies (n = 27) found that nondisplaceable binding potential (BPND) in the thalamus was positively correlated to CFN injected mass, demonstrating increase of MOR availability at higher injected CFN mass. Consistently, when CFN injected mass was constrained < 40 ng/kg (~ 10% MOR occupancy in rats), baseline MOR availability was significantly decreased. For test-retest variability (TRTV), better reproducibility was achieved by controlling CFN injected mass to limit the difference between scans. Taken together, we report significant cardiorespiratory depression and a paradoxical influence on baseline MOR availability at conventional tracer doses in rats. Our findings might reflect changes in cerebral blood flow, changes in receptor affinity, or receptor internalization, and merits further mechanistic investigation. In conclusion, rat [11C]CFN PET requires stringent quality assurance of radiotracer synthesis and mass injected to avoid pharmacological effects and limit potential influences on MOR quantification and reproducibility.

Asunto(s)

Encéfalo , Radioisótopos de Carbono , Fentanilo , Tomografía de Emisión de Positrones , Receptores Opioides mu , Animales , Receptores Opioides mu/metabolismo , Fentanilo/análogos & derivados , Fentanilo/metabolismo , Fentanilo/farmacología , Ratas , Tomografía de Emisión de Positrones/métodos , Encéfalo/metabolismo , Encéfalo/diagnóstico por imagen , Masculino , Ratas Sprague-Dawley , Radiofármacos/farmacocinética