Transcriptional signatures of fentanyl use in the mouse ventral tegmental area.

Synthetic opioids such as fentanyl contribute to the vast majority of opioid-related overdose deaths, but fentanyl use remains broadly understudied. Like other substances with misuse potential, opioids cause lasting molecular adaptations to brain reward circuits, including neurons in the ventral tegmental area (VTA). The VTA contains numerous cell types that play diverse roles in opioid use and relapse; however, it is unknown how fentanyl experience alters the transcriptional landscape in specific subtypes. Here, we performed single nuclei RNA sequencing to study transcriptional programs in fentanyl-experienced mice. Male and female C57/BL6 mice self-administered intravenous fentanyl (1.5 µg/kg/infusion) or saline for 10 days. After 24 h abstinence, VTA nuclei were isolated and prepared for sequencing on the 10× platform. We identified different patterns of gene expression across cell types. In dopamine neurons, we found enrichment of genes involved in growth hormone signalling. In dopamine-glutamate-GABA combinatorial neurons, and some GABA neurons, we found enrichment of genes involved in Pi3k-Akt signalling. In glutamate neurons, we found enrichment of genes involved in cholinergic signalling. We identified transcriptional regulators for the differentially expressed genes in each neuron cluster, including downregulated transcriptional repressor Bcl6, and upregulated transcription factor Tcf4. We also compared the fentanyl-induced gene expression changes identified in mouse VTA with a published rat dataset in bulk VTA, and found overlap in genes related to GABAergic signalling and extracellular matrix interaction. Together, we provide a comprehensive picture of how fentanyl self-administration alters the transcriptional landscape of the mouse VTA that serves as the foundation for future mechanistic studies.

Perioperative analgesic effects of the erector spinae plane block with bupivacaine or bupivacaine-dexmedetomidine in dogs undergoing hemilaminectomy: A randomized controlled trial.

Objective: To compare the perioperative opioid requirements among dogs receiving an erector spinae plane (ESP) block with bupivacaine, with or without dexmedetomidine, and a control group. Animals and procedure: Thirty client-owned, healthy adult dogs undergoing hemilaminectomy were included in this randomized, prospective, blinded clinical study. Dogs were randomly assigned to 1 of 3 treatment groups: Group B, ESP block with bupivacaine; Group BD, ESP block with bupivacaine and dexmedetomidine; and Group C, control. Rescue intra- and postoperative analgesia consisted of fentanyl and methadone, respectively. Postoperative pain was evaluated using the short form of the Glasgow Composite Measure Pain Scale (CMPS-SF). Results: In Group BD, 0/10 dogs required intraoperative fentanyl, compared to 9/10 in Group C (P < 0.001), whereas 1/10 required postoperative methadone, compared to 9/10 in Group B (P = 0.003) and 10/10 in Group C (P < 0.001). The total amount of intraoperative fentanyl (µg/kg) was 0 (0 to 4) in Group B and 0 (0 to 0) in BD, compared to 6 (0 to 8) in C (P = 0.004 and P < 0.001, respectively). Postoperative methadone (mg/kg) required during the first 12 h was 0.5 (0 to 1.4) in Group B (P = 0.003) and 0 (0 to 0) in BD (P < 0.001), compared to C (P = 0.003 and P < 0.001, respectively). Conclusion: An ESP block with bupivacaine, with or without dexmedetomidine, was associated with a reduction in perioperative opioid consumption and provided effective acute pain control.

Effets analgésiques périopératoires du bloc des érecteurs du rachis avec de la bupivacaïne ou de la bupivacaïne-dexmédétomidine chez les chiens subissant une hémilaminectomie: un essai contrôlé randomisé. Objectif: Comparer les besoins périopératoires en opioïdes chez les chiens recevant un bloc des érecteurs de la colonne vertébrale (ESP) avec de la bupivacaïne, avec ou sans dexmédétomidine, et un groupe témoin. Animaux et procédure: Trente chiens adultes en bonne santé appartenant à des clients subissant une hémilaminectomie ont été inclus dans cette étude clinique randomisée, prospective et en aveugle. Les chiens ont été répartis au hasard dans 1 des 3 groupes de traitement: groupe B, bloc ESP avec bupivacaïne; groupe BD, bloc ESP avec bupivacaïne et dexmédétomidine; et groupe C, témoin. L'analgésie de secours peropératoire et postopératoire consistait respectivement en fentanyl et en méthadone. La douleur postopératoire a été évaluée à l'aide du formulaire abrégé de l'échelle de mesure de la douleur de Glasgow (CMPS-SF). Résultats: Dans le groupe BD, 0/10 chiens ont eu besoin de fentanyl peropératoire, contre 9/10 dans le groupe C (P < 0,001), tandis que 1/10 ont eu besoin de méthadone postopératoire, contre 9/10 dans le groupe B (P = 0,003) et 10/10 dans le groupe C (P < 0,001). La quantité totale de fentanyl peropératoire (µg/kg) était de 0 (0 à 4) dans le groupe B et de 0 (0 à 0) dans le groupe BD, contre 6 (0 à 8) dans le groupe C (P = 0,004 et P < 0,001, respectivement). La méthadone postopératoire (mg/kg) nécessaire au cours des 12 premières heures était de 0,5 (0 à 1,4) dans le groupe B (P = 0,003) et de 0 (0 à 0) dans le groupe BD (P < 0,001), par rapport au groupe C (P = 0,003). et P < 0,001, respectivement). Conclusion: Un bloc ESP avec de la bupivacaïne, avec ou sans dexmédétomidine, a été associé à une réduction de la consommation peropératoire d'opioïdes et a permis un contrôle efficace de la douleur aiguë.(Traduit par Dr Serge Messier).

Xylazine does not enhance fentanyl reinforcement in rats: A behavioral economic analysis.

The adulteration of illicit fentanyl with the alpha-2 agonist xylazine has been designated an emerging public health threat. The clinical rationale for combining fentanyl with xylazine is currently unclear, and the inability to study fentanyl/xylazine interactions in humans warrants the need for preclinical research. We studied fentanyl and xylazine pharmacodynamic and pharmacokinetic interactions in male and female rats using drug self-administration behavioral economic methods. Fentanyl, but not xylazine, functioned as a reinforcer under both fixed-ratio and progressive-ratio drug self-administration procedures. Xylazine combined with fentanyl at three fixed dose-proportion mixtures did not significantly alter fentanyl reinforcement as measured using behavioral economic analyses. Xylazine produced a proportion-dependent decrease in the behavioral economic Q0 endpoint compared to fentanyl alone. However, xylazine did not significantly alter fentanyl self-administration at FR1. Fentanyl and xylazine co-administration did not result in changes to pharmacokinetic endpoints. The present results demonstrate that xylazine does not enhance the addictive effects of fentanyl or alter fentanyl plasma concentrations. The premise for why illicitly manufacture fentanyl has been adulterated with xylazine remains to be determined.

Co-administration of naloxone and dexmedetomidine to simultaneously reverse acute effects of fentanyl and methamphetamine in rats.

BACKGROUND: The incidence of combination methamphetamine (METH)-opioid overdose has substantially increased in recent years. While agitation is uncommon after the naloxone (NLX) reversal of opioids, it is a major clinical concern in acute METH intoxication and can be physiologically antagonized by opioid-induced sedation. This study aimed to perform initial preclinical analysis of the safety and efficacy of dexmedetomidine (DEXMED) co-administered with NLX to attenuate METH-induced locomotor activity, as a rat model of agitation, after the reversal of fentanyl (FENT)-induced sedation. METHODS: Male Sprague Dawley rats were administered subcutaneous (SC) 0.1mg/kg FENT ± 1mg/kg METH. Fifteen min later, SC 0.1mg/kg NLX ± an increasing (0, 0.032, 0.056, and 0.1mg/kg) DEXMED dose was administered prior to the measurement of locomotor activity. After a washout period, the FENT ± METH and NLX ± DEXMED administration with the highest dose of DEXMED was administered for measurement of blood oxygen saturation and heart rate. RESULTS: After the NLX reversal of FENT-induced sedation, adjunct DEXMED substantially and significantly reduced METH-induced locomotor activity (p<0.05) at all doses tested. While the addition of DEXMED did not significantly reduce blood oxygenation in METH treated rats, it did so in the absence of METH. Also, DEXMED significantly reduced heart rate compared to non-DEXMED treated groups and resulted in further significant reductions in the animals not exposed to METH (p<0.05). CONCLUSIONS: These data provide preclinical evidence that DEXMED may be a safe and effective chemical restraint for METH-induced agitation after NLX opioid reversal.

L-NAC and L-NAC methyl ester prevent and overcome physical dependence to fentanyl in male rats.

N-acetyl-L-cysteine (L-NAC) is a proposed therapeutic for opioid use disorder. This study determined whether co-injections of L-NAC (500 µmol/kg, IV) or its highly cell-penetrant analogue, L-NAC methyl ester (L-NACme, 500 µmol/kg, IV), prevent acquisition of acute physical dependence induced by twice-daily injections of fentanyl (125 µg/kg, IV), and overcome acquired dependence to these injections in freely-moving male Sprague Dawley rats. The injection of the opioid receptor antagonist, naloxone HCl (NLX; 1.5 mg/kg, IV), elicited a series of withdrawal phenomena (i.e. behavioral and cardiorespiratory responses, hypothermia and body weight loss) in rats that received 5 or 10 injections of fentanyl and similar numbers of vehicle co-injections. With respect to the development of dependence, the NLX-precipitated withdrawal phenomena were reduced in rats that received had co-injections of L-NAC, and more greatly reduced in rats that received co-injections of L-NACme. In regard to overcoming established dependence, the NLX-precipitated withdrawal phenomena in rats that had received 10 injections of fentanyl (125 µg/kg, IV) were reduced in rats that had received co-injections of L-NAC, and more greatly reduced in rats that received co-injections of L-NACme beginning with injection 6 of fentanyl. This study provides compelling evidence that co-injections of L-NAC and L-NACme prevent the acquisition of physical dependence and overcome acquired dependence to fentanyl in male rats. The higher efficacy of L-NACme is likely due to its greater cell penetrability in brain regions mediating dependence to fentanyl and interaction with intracellular signaling cascades, including redox-dependent processes, responsible for the acquisition of physical dependence to fentanyl.

The role of morphine- and fentanyl-induced impairment of intestinal epithelial antibacterial activity in dysbiosis and its impact on the microbiota-gut-brain axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupts the intestinal epithelial layer and causes intestinal dysbiosis. Depleting gut bacteria can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. The mechanism underlying opioid-induced dysbiosis, however, remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine or fentanyl exposure reduces the antimicrobial activity in the ileum, resulting in changes in the composition of bacteria. Fecal samples from morphine-treated mice had increased levels of Akkermansia muciniphila with a shift in the abundance ratio of Firmicutes and Bacteroidetes. Fecal microbial transplant (FMT) from morphine-naïve mice or oral supplementation with butyrate restored (a) the antimicrobial activity, (b) the expression of the antimicrobial peptide, Reg3γ, (c) prevented the increase in intestinal permeability and (d) prevented the development of antinociceptive tolerance in morphine-dependent mice. Improved epithelial barrier function with FMT or butyrate prevented the enrichment of the mucin-degrading A. muciniphila in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which opioids disrupt the microbiota-gut-brain axis.

Fentanyl Analogs Exert Antinociceptive Effects via Sodium Channel Blockade in Mice.

The formalin test is one approach to studying acute pain in rodents. Similar to formalin, injection with glutamate and veratrine can also produce a nociceptive response. This study investigated whether opioid-related compounds could suppress glutamate- and veratrine-induced nociceptive responses in mice at the same dose. The administration of morphine (3 mg/kg), hydromorphone (0.4 mg/kg), or fentanyl (0.03 mg/kg) suppressed glutamate-induced nociceptive response, but not veratrine-induced nociceptive response at the same doses. However, high doses of morphine (10 mg/kg), hydromorphone (2 mg/kg), or fentanyl (0.1 mg/kg) produced a significant reduction in the veratrine-induced nociceptive response. These results indicate that high doses are required when using morphine, hydromorphone, or fentanyl for sodium channel-related neuropathic pain, such as ectopic activity. As a result, concerns have arisen about overdose and abuse if the dose of opioids is steadily increased to relieve pain. In contrast, trimebutine (100 mg/kg) and fentanyl analog isobutyrylfentanyl (iBF; 0.1 mg/kg) suppressed both glutamate- and veratrine-induced nociceptive response. Furthermore, nor-isobutyrylfentanyl (nor-iBF; 1 mg/kg), which is a metabolite of iBF, suppressed veratrine-induced nociceptive response. Besides, the optimal antinociceptive dose of iBF, unlike fentanyl, only slightly increased locomotor activity and did not slow gastrointestinal transit. Cancer pain is a complex condition driven by inflammatory, neuropathic, and cancer-specific mechanisms. Thus, iBF may have the potential to be a superior analgesic than fentanyl.

Melatonin attenuates fentanyl - induced behavioral sensitization and circadian rhythm disorders in mice.

Melatonin is a neurohormone synthesized by the pineal gland to regulate the circadian rhythms and has proven to be effective in treating drug addiction and dependence. However, the effects of melatonin to modulate the drug-seeking behavior of fentanyl and its underlying molecular mechanism is elusive. This study was designed to investigate the effects of melatonin on fentanyl - induced behavioral sensitization and circadian rhythm disorders in mice. The accompanying changes in the expression of Brain and Muscle Arnt-Like (BMAL1), tyrosine hydroxylase (TH), and monoamine oxidase A (MAO-A) in relevant brain regions including the suprachiasmatic nucleus (SCN), nucleus accumbens (NAc), prefrontal cortex (PFC), and hippocampus (Hip) were investigated by western blot assays to dissect the mechanism by which melatonin modulates fentanyl - induced behavioral sensitization and circadian rhythm disorders. The present study suggest that fentanyl (0.05, 0.1 and 0.2 mg/kg) could induce behavioral sensitization and melatonin (30.0 mg/kg) could attenuate the behavioral sensitization and circadian rhythm disorders in mice. Fentanyl treatment reduced the expression of BMAL1 and MAO-A and increased that of TH in relevant brain regions. Furthermore, melatonin treatment could reverse the expression levels of BMAL1, MAO-A, and TH. In conclusion, our study demonstrate for the first time that melatonin has therapeutic potential for fentanyl addiction.

Erector spinae plane block in dogs undergoing hemilaminectomy: A prospective randomized clinical trial.

OBJECTIVE: To compare the perioperative cumulative opioid consumption and the incidence of cardiovascular complications in dogs undergoing hemilaminectomy in which either an erector spinae plane (ESP) block or systemic opioids were administered. STUDY DESIGN: Prospective randomized clinical trial. ANIMALS: A total of 60 client-owned dogs. METHODS: Dogs were randomized to one of three groups: an ESP block (group ESP), a constant rate infusion of fentanyl (group FNT, positive control) or a single dose of methadone as premedication (group MTD, negative control). Intraoperative nociceptive response was treated with fentanyl [1 µg kg-1, intravenously (IV)] boli. Before closure of the surgical site, morphine (0.1 mg kg-1) was applied to the dura mater. The cumulative dose of opioids was recorded and compared between groups. The incidence of intraoperative bradycardia and/or hypotension and the time to extubation were compared between groups. The short form of the Glasgow Composite Pain Scale (SF-GCPS) was used to score nociception before anaesthetic induction and 1, 2, 6, 12,18 and 24 hours postoperatively. Methadone 0.2 mg kg-1 was administered IV if the SF-GCPS score was ≥ 5. RESULTS: Group MTD required more intraoperative rescue analgesia than groups ESP (p = 0.008) and FNT (p = 0.001). The total cumulative intraoperative dose of fentanyl was higher in groups FNT (p < 0.0001) and MTD (p = 0.002) than in group ESP. The incidence of cardiovascular complications was similar between groups. Extubation time was longer in group MTD (p = 0.03). Postoperatively, the time to first rescue analgesia was longer in group ESP than in group MTD (p = 0.03). The cumulative postoperative opioid consumption and pain scores were similar between groups. CONCLUSIONS AND CLINICAL RELEVANCE: The ESP block resulted in a reduced intraoperative opioid consumption compared with the control positive and negative groups.

Apneic response to fentanyl in adult rats: Role of laryngeal afferents.

Intravenous (systemic) bolus injection of fentanyl (FNT) reportedly induces an immediate vagal-mediated apnea; however, the precise origin of vagal afferents responsible for this apnea remains unknown. We tested whether intralaryngeal (local) application of FNT would also trigger an apnea and whether the apneic response to both local and systemic administration of FNT was laryngeal afferent-mediated. Cardiorespiratory responses to FNT were recorded in anesthetized male adult rats with and without bilateral sectioning of the superior laryngeal nerve (SLNx) or peri-SLN capsaicin treatment (SLNcap) to block local C-fiber signal conduction. Opioid mu-receptor (MOR)-immunoreactivity was detected in laryngeal C- and myelinated neurons. We found that local and systemic administration of FNT elicited an immediate apnea. SLNx, rather than SLNcap, abolished the apneic response to local FNT application though MORs were abundantly expressed in both laryngeal C- and myelinated neurons. Importantly, SLNx failed to affect the apneic response to systemic FNT administration. These results lead to the conclusion that laryngeal afferents' MORs are responsible for the apneic response to local, but not systemic, administration of FNT.

The impact of chronic fentanyl administration on the cerebral cortex in mice: Molecular and histological effects.

PURPOSE: Fentanyl, a fully synthetic opioid, is widely used for severe pain management and has a huge abuse potential for its psychostimulant effects. Unlike other opioids, the neurotoxic effects of chronic fentanyl administration are still unclear. In particular, little is known about its effect on the cerebral cortex. The current study aims to test the chronic toxicity of fentanyl in the mice model. METHODS: Adult male Balb/c mice were chronically treated with low (0.05 mg/kg, i.p) and high (0.1 mg/kg, i.p) doses of fentanyl for 5 consecutive weeks, and various neurotoxic parameters, including apoptosis, oxidative stress, and neuroinflammatory response were assessed in the cortex. Potential histological as well as neurochemical changes were also evaluated. RESULTS: The results of this study show that chronic fentanyl administration induced intense levels of apoptosis, oxidative stress, and neuroinflammation in the cerebral cortex. These findings were found to be correlated with histopathological characteristics of neural degeneration and white matter injury. Moreover, fentanyl administration was found to reduce the expression of both NMDA receptor subunits and dopamine receptors and elevate the level of epidermal growth factor (EGF). CONCLUSION: Fentanyl administration induced neurotoxic effects in the mouse cerebral cortex that could be primarily mediated by the evoked oxidative-inflammatory response. The altered expression of NMDA receptors, dopamine receptors, and EGF suggests the pernicious effects of fentanyl addiction that may end in the development of toxic psychosis.

Pharmacologic Characterization of Substituted Nitazenes at µ, κ, and Δ Opioid Receptors Suggests High Potential for Toxicity.

The benzimidazole opioids (substituted nitazenes) are highly potent µ opiod receptor (MOR) agonists with heroin- or fentanyl-like effects. These compounds have caused hospitalizations and fatal overdoses. We characterized the in vitro pharmacology and structure-activity relationships of 19 nitazenes with substitutions at three positions of the benzimidazole core. Affinities were assessed using agonist radioligand binding assays at human µ, κ, and Δ opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in CHO cells. Notably, for MOR binding, nine substituted nitazenes had significantly higher affinities than fentanyl including N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, and N-desethyl isotonitazene; 13 had subnanomolar affinities. Only metodesnitazene and flunitazene had significantly lower affinities than fentanyl. Affinities for the substituted nitazenes at KOR and DOR relative to MOR were 46- to 2580-fold and 180- to 1280-fold lower, respectively. Functional activities were assessed using [35S]GTPγS binding assays. Four nitazenes had subnanomolar potencies at MOR: N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, N-pyrrilidino protonitazene and N-desethyl isotonitazene. Ten substituted nitazenes had significantly higher potencies than fentanyl. All tested nitazenes were full MOR agonists. Potencies at KOR and DOR relative to MOR were 7.3- to 7920-fold and 24- to 9400-fold lower, respectively. Thus, many of these compounds are high affinity/high potency MOR agonists with elevated potential to elicit toxicity and overdose at low doses. SIGNIFICANCE STATEMENT: Substituted nitazenes are a growing public health threat. Although the 19 nitazenes tested vary in their opioid receptor pharmacology, a number are very high affinity, high potency, and high efficacy compounds- higher than fentanyl. Their pharmacology suggests high potential for harm.

Enantiomeric contributions to methamphetamine's bidirectional effects on basal and fentanyl-depressed respiration in mice.

RATIONALE: Fentanyl remains the primary cause of fatal overdoses, and its co-use with methamphetamine (METH) is a growing concern. We previously demonstrated that racemic METH can either enhance or mitigate opioid-induced respiratory depression (OIRD) dependent upon whether a low or high dose is administered. The optical isomers of METH, dextromethamphetamine (d-METH) and levomethamphetamine (l-METH), differ substantially in their selectivity and potency to activate various monoamine (MA) receptors, and these pharmacological differences may underlie the bidirectional effects of the racemate. Since it is unknown which of METH's MA receptor mechanisms mediate these respiratory effects, examination of METH's pharmacologically distinct enantiomers may provide insight into treatment targets for OIRD. METHODS: The two optical isomers of METH, d-METH and l-METH, were tested in adult male mice to determine their effects on basal and fentanyl-depressed respiratory frequency, tidal volume, and minute ventilation (MVb; i.e., respiratory frequency x tidal volume) using whole-body plethysmography. RESULTS: When tested at dose ranges of 1.0-10 mg/kg, d-METH elevated MVb and l-METH decreased basal MVb. A dose of 30 mg/kg l-METH increased basal MVb. Under fentanyl-depressed conditions, the bidirectional effects of racemic METH were observed with d-METH treatment while l-METH significantly exacerbated OIRD at 1.0 and 3.0 mg/kg. CONCLUSIONS: d-METH and l-METH differentially contribute to the bidirectional respiratory modulation observed by the racemate, with d-METH exhibiting predominantly stimulatory effects and l-METH exhibiting primarily depressant effects depending on dose.

Comparisons of fentanyl and sufentanil on recovery time after inguinal hernia repair in children: a randomized clinical trial.

BACKGROUND: Inguinal hernia repair is a common pediatric procedure. We studied postoperative recovery times in children undergoing laparoscopic inguinal hernia repair with anesthesia induced by fentanyl versus sufentanil. METHODS: We performed a pilot randomized clinical trial between February and December 2022. Eligible children were assigned into two age groups, 2-6 and 6-12 years old groups. Then, children in each age group were randomly assigned into either the fentanyl (2 µg/kg) or sufentanil (0.2 µg/kg) group for anesthesia induction. Baseline characteristics were collected. The primary outcome was the postoperative recovery time, which was recorded as the time period from extubation to a Steward recovery score reaching 6. Secondary outcomes included surgical duration, anesthetic duration, intubation duration, and intraoperative hemorrhage. RESULTS: There were 300 children, with 75 children in each group. In the 2-6 years old group, children who received fentanyl had statistically significantly shorter postoperative recovery times than children who received sufentanil (0.9 ± 0.4 versus 1.5 ± 0.3 h, P < 0.001). However, in the 6-12 years old group, children who received fentanyl had statistically significantly longer postoperative recovery times than children who received sufentanil (1.2 ± 0.4 versus 0.8 ± 0.4 h, P < 0.001). Baseline characteristics and secondary outcomes were comparable between two groups. CONCLUSIONS: Anesthesia induction with fentanyl or sufentanil resulted in different postoperative recovery times after laparoscopic inguinal hernia repair in children in different age groups. More studies are required to determine the appropriate induction anesthetic in children of different ages. TRIAL REGISTRATION: The study protocol was retrospectively registered online at the Chinese Clinical Trial Registry (registration number ChiCTR2300072177, retrospectively registered on 06/06/2023).

In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the µ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as µ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and ß-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and ß-arrestin2 recruitment pathways. Compared to the reference agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting ß-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and ß-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. In addition, the extremely high potency of many NSOs now infiltrating illicit drug markets further contributes to the danger posed to public health.

The blockade of neuropeptide FF receptor 1 and 2 differentially contributed to the modulating effects on fentanyl-induced analgesia and hyperalgesia in mice.

Neuropeptide FF (NPFF) plays a critical role in various physiological processes through the activation of neuropeptide FF receptor 1 and 2 (NPFFR1 and NPFFR2). Numerous evidence has indicated that NPFF exhibits opposite opioid-modulating effects on opioid-induced analgesia after supraspinal and spinal administrations, while the detailed role of NPFFR1 and NPFFR2 remains unclear. In this study, we employed pharmacological and genetic inhibition of NPFFR to investigate the modulating roles of central NPFFR1 and NPFFR2 in opioid-induced analgesia and hyperalgesia, using a male mouse model of acute fentanyl-induced analgesia and secondary hyperalgesia. Our findings revealed that intrathecal (i.t.) injection of the nonselective NPFFR antagonist RF9 significantly enhanced fentanyl-induced analgesia, whereas intracerebroventricular (i.c.v.) injection did not show the same effect. Moreover, NPFFR2 deficient (npffr2-/-) mice exhibited stronger analgesic responses to fentanyl compared to wild type (WT) or NPFFR1 knockout (npffr1-/-) mice. Intrathecal injection of RF9 in npffr1-/- mice also significantly enhanced fentanyl-induced analgesia. These results indicate a crucial role of spinal NPFFR2 in the enhancement of opioid analgesia. Contrastingly, hyperalgesia induced by fentanyl was markedly reversed in npffr1-/- mice but remained unaffected in npffr2-/- mice. Similarly, i.c.v. injection of the selective NPFFR1 antagonist RF3286 effectively prevented fentanyl-induced hyperalgesia in WT or npffr2-/- mice. Notably, co-administration of i.c.v. RF3286 and i.t. RF9 augmented fentanyl-induced analgesia while reducing hyperalgesia. Collectively, these findings highlight the modulating effects of blocking spinal NPFFR2 and supraspinal NPFFR1 on fentanyl-induced analgesia and hyperalgesia, respectively, which shed a light on understanding the pharmacological function of NPFF system in future studies.

A Comparison of Three Anesthetic Drug Combinations for Use in Inducing Surgical Anesthesia in Female Guinea Pigs (Cavia porcellus).

Guinea pigs are often used in translational research, but providing them with safe and effective anesthesia is a challenge. Common methods like inhalant anesthesia and injectable ketamine/xylazine induce surgical anesthesia but can negatively affect cardiovascular, respiratory, and thermoregulatory systems and complicate the interpretation of research outcomes. Several alternative anesthetic regimens have been investigated, but none have consistently achieved a surgical plane of anesthesia. Therefore, identifying an anesthetic regimen that achieves a stable state of the surgical plane of anesthesia while preserving cardiorespiratory function would be a valuable contribution. To address this issue, we compared the efficacy of 3 anesthetic combinations in female Dunkin-Hartley guinea pigs: 1) alfaxalone, dexmedetomidine, and fentanyl (ADF); 2) alfaxalone, midazolam, and fentanyl (AMF); and 3) alfaxalone, midazolam, fentanyl, and isoflurane (AMFIso). We monitored anesthetic depth, heart rate, oxygenation, respiratory rate, respiratory effort, blood pressure, and body temperature every 15 min from injection to recovery. We also recorded the time to loss of righting reflex, duration of anesthesia, and time to achieve a surgical plane. The results showed no statistically significant differences in induction and recovery times among the groups. In the AMFIso group, 100% of the animals achieved a surgical plane of anesthesia, whereas only 10% of the animals in the AMF group reached that level. None of the animals in ADF group reached a surgical plane of anesthesia. Respiratory rate was significantly lower in the AMFIso as compared with the ADF group (P < 0.001) but was not different between the AMF and ADF groups. Temperature was significantly lower in the AMFIso group as compared with both the ADF and AMF groups (P < 0.001). In conclusion, both combinations of solely injectable anesthetics assessed in this study can be used for short, nonpainful procedures without significant cardiorespiratory depression. However, for mildly to moderately painful surgical procedures, the addition of an inhalant anesthetic like isoflurane is necessary for female guinea pigs.

Effects of fentanyl and the adulterant levamisole on the rewarding and locomotor effects of methamphetamine in rats.

BACKGROUND: People who use psychostimulant substances can be exposed to unknown adulterants, such as the synthetic opioid fentanyl (FEN) and the anthelmintic cholinergic agent levamisole (LEV). This work explores the rewarding and locomotor effects of methamphetamine (METH) in combination with FEN or LEV. METHODS: We used adult male Wistar rats in the conditioned-place preference (CPP) paradigm (conditioning, extinction, and reinstatement phases) and in the open field test to study effective doses of METH, FEN, or LEV, or ineffective doses of METH+FEN or METH+LEV in combination. RESULTS: METH and LEV, at 1mg/kg METH each, and 30µg/kg FEN produced CPP. Extinction to METH- or LEV-induced CPP occurred after eight saline injections, but it took 8-26 sessions to extinguish FEN-induced CPP. A challenge dose of 0.5mg/kg METH reinstated CPP. The same occurred with 15µg/kg FEN but not with 0.5 or 1mg/kg LEV. Training animals with ineffective doses of METH (0.01mg/kg) combined with either FEN (0.3µg/kg) or LEV (0.01mg/kg) produced CPP. Sub-effective doses of METH or FEN alone did not induce reinstatement after extinction. However, animals challenged with LEV, METH+FEN, or METH+LEV mixtures did it. Combining FEN (3µg/kg) with 0.1mg/kg METH increased locomotor activity. CONCLUSION: Ineffective FEN and LEV doses mixed with METH produce effects larger than would be expected based on the effects of either drug alone. This outcome suggests a supra-additive interaction, which could increase the risk of developing a METH use disorder.

Fentanyl dysregulates neuroinflammation and disrupts blood-brain barrier integrity in HIV-1 Tat transgenic mice.

Opioid overdose deaths have dramatically increased by 781% from 1999 to 2021. In the setting of HIV, opioid drug abuse exacerbates neurotoxic effects of HIV in the brain, as opioids enhance viral replication, promote neuronal dysfunction and injury, and dysregulate an already compromised inflammatory response. Despite the rise in fentanyl abuse and the close association between opioid abuse and HIV infection, the interactive comorbidity between fentanyl abuse and HIV has yet to be examined in vivo. The HIV-1 Tat-transgenic mouse model was used to understand the interactive effects between fentanyl and HIV. Tat is an essential protein produced during HIV that drives the transcription of new virions and exerts neurotoxic effects within the brain. The Tat-transgenic mouse model uses a glial fibrillary acidic protein (GFAP)-driven tetracycline promoter which limits Tat production to the brain and this model is well used for examining mechanisms related to neuroHIV. After 7 days of fentanyl exposure, brains were harvested. Tight junction proteins, the vascular cell adhesion molecule, and platelet-derived growth factor receptor-ß were measured to examine the integrity of the blood brain barrier. The immune response was assessed using a mouse-specific multiplex chemokine assay. For the first time in vivo, we demonstrate that fentanyl by itself can severely disrupt the blood-brain barrier and dysregulate the immune response. In addition, we reveal associations between inflammatory markers and tight junction proteins at the blood-brain barrier.

SERUM FENTANYL CONCENTRATIONS AND BEHAVIOR ASSOCIATED WITH TRANSDERMAL FENTANYL APPLICATION ON HEALTHY CORN SNAKES (PANTHEROPHIS GUTTATUS).

Snakes are common household pets and frequently managed in zoos. Geriatric snakes commonly develop osteoarthritis, leading to a declining quality of life that often results in euthanasia. Anecdotally, the application of transdermal fentanyl patches (TFP) appears to contribute to clinical improvement, including increased activity level, in osteoarthritic snakes presumed to be in pain. This study evaluated serum fentanyl concentrations over time and the effects of TFP on the normal behavior of healthy, captive, adult corn snakes (Pantherophis guttatus) using constant video monitoring. Serum fentanyl concentrations were evaluated over 4 wk during 12.5 µg/h TFP application, and the results demonstrated long-lasting (>4 wk) serum concentrations that were consistent with analgesic efficacy in mammalian species during TFP application. At 4 wk of TFP application, mean serum fentanyl concentrations were 11.5 ± 5.5 ng/ml. Snakes were videotaped for 1 wk prior to and 2 wk after 12.5 µg/h TFP application, and behavior was evaluated by an ethogram. Behavioral changes associated with TFP application included decreased mean time spent active, decreased mean number of climbs, and decreased mean number of water visits; feeding behavior was unchanged. Overall, these results suggest that TFP application may provide safe, clinically effective analgesia in healthy corn snakes for at least 4 wk without inducing deleterious side effects, and may therefore be appropriate analgesia for management of osteoarthritic snakes.