Cardiorespiratory failure induced by inhalation of aerosolized fentanyl in anesthetized rats.

Intravenous rapid injection of fentanyl causes respiratory depression (severe apneas), leading to sudden death, which constitutes the deadliest drug reaction among overdoses of synthetic opioids. Here we asked whether acute inhalation of overdose fentanyl would also result in similar respiratory failure and death. The anesthetized and spontaneously breathing rats with tracheal cannulation were exposed to aerosolized fentanyl at 100 mg/m3 (FNTH) or 30 mg/m3 (FNTL) for 10 min. Minute ventilation (VE), electromyography (EMG) of the internal and external intercostal muscles and thyroarytenoid muscles (EMGII, EMGEI, and EMGTA), heart rate and arterial blood pressure were recorded. During the exposure, FNTH and FNTL immediately triggered bradypnea (40 % reduction, p < 0.05) with TE prolonged and then gradually decreased VE by 40 % (P < 0.05) after a brief VE recovery. The initial TE prolongation (apneas) were characterized by the cessation of EMGEI activity with enhanced tonic discharges of EMGTA and EMGII. After termination of the exposure, the cardiorespiratory responses to FNTL returned to the baseline values 30 min later, while those to FNTH were greatly exacerbated (P < 0.05), leading to ventilatory and cardiac arrest occurred 16.4 ± 4.7 min and 19.3 ± 4.5 min respectively after the onset of FNTH. The ventilatory arrest was featured by cessation of both EMGEI and EMGII and augmentation of tonic EMGTA. Our results suggest that acute exposure to an overdose of fentanyl aerosol leads to death through initially inducing a brief central and upper airway obstructive apnea as well as chest wall rigidity followed by gradual severe hypoventilation, bradycardia and hypotension, and eventual cardiorespiratory arrest in anesthetized rats.

Effect of fentanyl and remifentanil on neuron damage and oxidative stress during induction neurotoxicity.

Opioids can be used for medical and non-medical purposes. Chronic pain such as cancer, as well as the frequent use of such drugs in places such as operating rooms and intensive care units, and in non-medical areas like drug abuse the effects and side effects of these drugs need to be examined in more detail. For this purpose, the effects of fentanyl and remifentanil drugs on neuroinflammation, oxidative stress and cholinesterase metabolism were investigated. Neuron cells (CRL-10742) were used for the evaluation of the toxicity of fentanyl and remifentanil. MTT, PON1 activity and total thiol levels for its effect on oxidative stress, AChE and BChE activities for its effect on the cholinergic system, and TNF, IL-8 and IL-10 gene levels for its neuroinflammation effect were determined. The highest neurotoxic dose of fentanyl and remifentanil was determined as 10 µg/mL. It was observed that the rate of neuron cells in this dose has decreased by up to 61.80% and 56.89%, respectively. The IL-8 gene expression level in both opioids was down-regulated while IL 10 gene level was up-regulated in a dose-dependent manner compared to the control. In our results, the TNF gene expression level differs between the two opioids. In the fentanyl group, it was seen to be up-regulated in a dose-dependent manner compared to the control. Fentanyl and remifentanil showed an inhibitory effect against PON1, while remifentanil showed an increase in total thiol levels. PON1, BChE and total thiol activities showed similarity with MTT.

Fentanyl overdose: Temporal effects and prognostic factors in SKH1 mice.

Fentanyl exposure and overdose are growing concerns in public health and occupational safety. This study aimed to establish parameters of fentanyl lethality in SKH1 mice for future overdose research. Lethality was determined using the up-down procedure, with subjects monitored post-administration using pulse oximetry (5 min) and then whole-body plethysmography (40 min). Following the determination of subcutaneous dose-response, [18F]Fluorodeoxyglucose positron emission tomography (18 F-FDG PET) was performed after LD10 fentanyl at 40 min, 6 h, 24 h or 7 days post-dose. LD10 and LD50 were observed to be 110 and 135 mg/kg, respectively, and consistent with four-parameter logistic fit values of 111.2 and 134.6 mg/kg (r2  = 0.9996). Overdose (LD10 or greater) yielded three distinct cardiovascular groups: survival, non-survival with blood oxygen saturation (SpO2) minimum ≥37% and non-survival with SpO2 <37%. Breaths per minute, minute volume and inspiratory quotient were significantly different between surviving and non-surviving animals for up to 40 min post-injection. 18 F-FDG PET revealed decreased glucose uptake in the heart, lungs and brain for up to 24 h. These findings provide critical insights into fentanyl lethality in SKH1 mice, including non-invasive respiratory effects and organ-specific impacts that are invaluable for future translational studies investigating the temporal effects of fentanyl overdose.

Fentanyl-induced respiratory depression in rodents is inhibited by bioabsorbable, subcutaneous naltrexone implants at 3.5 months.

The aim of this study is to determine if extended-release, bioabsorbable, subcutaneous naltrexone (NTX) implants inhibit respiratory depression after an IV injection of fentanyl. Bioabsorbable implants fabricated from two different release-controlling polymers, poly-D-L-lactide (PDLLA) and polycaprolactone (PCL), alone (placebo) or containing NTX, were subcutaneously implanted in Sprague Dawley rats. After 3.5 months of implantation, the rodents were administered an IV bolus of fentanyl through the tail vein. The placebo implant rats received a dose of 4 micrograms (mcg) - (10 mcg/kg/dose), while the NTX implanted animals received a dose of 8 mcg (20 mcg/kg/dose). The minimum active dose of fentanyl that caused a > 50 ± 2% depression in the respiration rate in the placebo implanted rodents was 4 mcg. The respiration rate of the placebo implanted rats dropped from 208 ± 14 breaths/minute at predose, to 84 ± 12 breaths/minute (p = 0.0003) at 2 min. In contrast, all NTX implanted animals easily tolerated twice the dose of 8 mcg of fentanyl without any significant reduction in respiration rate. The mean respiration rate = increased from 164 ± 22 breaths/minute at predose to 178 ± 17 breaths/minute (p = 0.24) at 2 min. The mean plasma concentrations of NTX, 3.5 months after implantation, ranged from 7.4 (±1.1) ng/mL to 80.3 (±37.5) ng/mL. Bioabsorbable implants containing NTX effectively blocked fentanyl-induced respiratory depression in rodents as compared with placebo implants, 3.5 months after implantation.

Cytotoxic effects of psychoactive isobutyrylfentanyl and its halogenated derivatives on isolated rat hepatocytes.

The novel and numerous psychoactive compounds derived from the analgesic prescription drug N-phenyl-N-[1-(2-phenylethyl)piperidin-4-yl]propanamide (fentanyl) have been illegally abused as recreational drugs and caused numerous fatalities. Because some psychoactive/psychotropic drugs are known to be hepatotoxic in humans and experimental animals, the cytotoxic effects and mechanisms of 4-fluoroisobutyrylfentanyl (4F-iBF), 4-chloroisobutyrylfentanyl (4Cl-iBF), and the parent compound isobutyrylfentanyl (iBF) were studied in freshly isolated rat hepatocytes. 4F-iBF caused not only concentration (0-2.0 mM)- and time (0-3 h)-dependent cell death accompanied by the depletion of cellular ATP and reduced glutathione (GSH) and protein thiol levels but also the accumulation of oxidized glutathione. Of these fentanyls examined, 4Cl-iBF/4F-iBF-induced cytotoxicity with the loss of mitochondrial membrane potential at concentrations of 0.5 and 1.0 mM and the production of reactive oxygen species (ROS) at 0.5 mM were greater than those induced by iBF. The pretreatment of hepatocytes with N-acetyl-l-cysteine as a precursor of cellular GSH ameliorated, at least in part, cytotoxicity accompanied by insufficient ATP levels, the loss of mitochondrial membrane potential, and generation of ROS caused by 4Cl-iBF/4F-iBF, whereas pretreatment with diethyl maleate as a GSH depletor enhanced fentanyl-induced cytotoxicity accompanied by the rapid loss of cellular GSH. Taken collectively, these results indicate that the onset of cytotoxic effects caused by these fentanyls is partially attributable to cellular energy stress as well as oxidative stress.

Toxicity and behavioural effects of ocfentanil and 2-furanylfentanyl in zebrafish larvae and mice.

The introduction of the so-called New Psychoactive Substances represents a problem of global concern due to several factors, including multiplicity of structures, poorly known activity, short half-life in the market, lack of pure standards etc. Among these problems, of the highest relevance is also the lack of information about metabolism and adverse effects, which must be faced using simple and low-cost animal models. On these grounds, the present work has been carried out on 5 days post fertilization zebrafish (Danio rerio) larvae in comparison with adult mice (Mus musculus). Ocfentanil and 2-furanylfentanyl were administered at different concentrations to zebrafish larvae (1, 10 µM) and mice (0.1, 1, 6, 15 mg/kg). The behavioural assay showed a decrease in basal locomotor activity in zebrafish, whereas in mice this effect was evident only after the mechanical stimulus. Larva extracts and mice urine were analysed by using liquid chromatography coupled to high resolution mass spectrometry to identify the metabolic pathways of the fentanyl analogs. For 2-furanylfentanyl, the most common biotransformations observed were hydroxylation, hydration and oxidation in zebrafish larvae, whereas mice produced mainly the dihydrodiol metabolite. Hydroxylation was the major route of metabolism for ocfentanil in zebrafish larvae, while in mice the O-demethylated derivative was the main metabolite. In addition, a study was conducted to evaluate morphological effects of the two drugs on zebrafish larvae. Malformations were noticeable only at the highest concentration of 2-furanylfentanyl, whereas no significant damage was observed with ocfentanil. In conclusion, the two animal models show similarities in behavioral response and in metabolism, considering the different biological investigated.

Hepatic and renal toxicities and metabolism of fentanyl analogues in rats.

New synthetic opioids continue to emerge in the illicit market, and among them, fentanyl analogues pose a serious threat to the public health with their abuse and trafficking. We investigated the toxicity of fentanyl analogues on the liver and kidneys mediated by the µ-opioid receptor (MOR). Our study focused on 4-fluoro-isobutyrylfentanyl (4F-iBF), which is classified as a "narcotic" in Japan; structurally similar analogues 4-chloro-isobutyrylfentanyl (4Cl-iBF) and isobutyrylfentanyl (iBF) were also investigated. Rats that were intraperitoneally administered 4F-iBF (5 mg/kg (12.3 µmol/kg)) or iBF (12.3 µmol/kg) displayed hepatic and renal ischemic-like damage, but 4Cl-iBF (12.3 µmol/kg) did milder renal damage only. We found that the agonist activity of 4F-iBF, at MORs was approximately 7.2 times that of 4Cl-iBF, and that pretreatment with MOR antagonist naltrexone (0.8 mg/kg) alleviated liver and kidney injuries caused by 4F-iBF. These results suggested that 4F-iBF might cause ischemic damage to the liver and kidneys, induced by respiratory depression mediated by MORs. Furthermore, to elucidate the metabolism of fentanyl analogues, we investigated the change over time in the amount of 4F-iBF, 4Cl-iBF, iBF (6.15 µmol/kg, respectively), and their respective metabolites in serum after intraperitoneal administration to rats. The results showed that in 24-h post-dose serum, 4Cl-iBF and iBF were substantially eliminated while 4F-iBF remained at about 30% of the maximum level, and each of the N-dephenylethylated metabolites of 4F-iBF, 4Cl-iBF, and iBF was detected in 2-h post-dose serum. The results from this study revealed information on the hepatic and renal toxicities and metabolism related to fentanyl analogues.

The Genotoxicity of Acrylfentanyl, Ocfentanyl and Furanylfentanyl Raises the Concern of Long-Term Consequences.

Three fentanyl analogues Acrylfentanyl, Ocfentanyl and Furanylfentanyl are potent, rapid-acting synthetic analgesics that recently appeared on the illicit market of new psychoactive substances (NPS) under the class of new synthetic opioids (NSO). Pharmacotoxicological data on these three non-pharmaceutical fentanyl analogues are limited and studies on their genotoxicity are not yet available. Therefore, the aim of the present study was to investigate this property. The ability to induce structural and numerical chromosomal aberrations in human lymphoblastoid TK6 cells was evaluated by employing the flow cytometric protocol of the in vitro mammalian cell micronucleus test. Our study demonstrated the non-genotoxicity of Fentanyl, i.e., the pharmaceutical progenitor of the class, while its illicit non-pharmaceutical analogues were found to be genotoxic. In particular, Acrylfentanyl led to a statistically significant increase in the MNi frequency at the highest concentration tested (75 µM), while Ocfentanyl and Furanylfentnyl each did so at both concentrations tested (150, 200 µM and 25, 50 µM, respectively). The study ended by investigating reactive oxygen species (ROS) induction as a possible mechanism linked to the proved genotoxic effect. The results showed a non-statistically significant increase in ROS levels in the cultures treated with all molecules under study. Overall, the proved genotoxicity raises concern about the possibility of serious long-term consequences.

Fentanyl Induces Novel Conditioned Place Preference in Adult Zebrafish, Disrupts Neurotransmitter Homeostasis, and Triggers Behavioral Changes.

Abuse of new psychoactive substances increases risk of addiction, which can lead to serious brain disorders. Fentanyl is a synthetic opioid commonly used in clinical practice, and behavioral changes resulting from fentanyl addiction have rarely been studied with zebrafish models. In this study, we evaluated the rewarding effects of intraperitoneal injections of fentanyl at concentrations of 10, 100, and 1000 mg/L on the group shoaling behavior in adult zebrafish. Additional behavioral tests on individual zebrafish, including novel tank, novel object exploration, mirror attack, social preference, and T-maze memory, were utilized to evaluate fentanyl-induced neuro-behavioral toxicity. The high doses of 1000 mg/L fentanyl produced significant reward effects in zebrafish and altered the neuro-behavioral profiles: reduced cohesion in shoaling behavior, decreased anxiety levels, reduced exploratory behavior, increased aggression behavior, affected social preference, and suppressed memory in an appetitive associative learning task. Behavioral changes in zebrafish were shown to be associated with altered neurotransmitters, such as elevated glutamine (Gln), gamma-aminobutyric acid (GABA), dopamine hydrochloride (DA), and 5-hydroxytryptamine (5-HT). This study identified potential fentanyl-induced neurotoxicity through multiple neurobehavioral assessments, which provided a method for assessing risk of addiction to new psychoactive substances.

Advancing humanized monoclonal antibody for counteracting fentanyl toxicity towards clinical development.

Innovative therapies to complement current treatments are needed to curb the growing incidence of fatal overdoses related to synthetic opioids. Murine and chimeric monoclonal antibodies (mAb) specific for fentanyl and its analogs have demonstrated pre-clinical efficacy in preventing and reversing drug-induced toxicity in rodent models. However, mAb-based therapeutics require extensive engineering as well as in vitro and in vivo characterization to advance to first-in-human clinical trials. Here, novel murine anti-fentanyl mAbs were selected for development based on affinity for fentanyl, and efficacy in counteracting the pharmacological effects of fentanyl in mice. Humanization and evaluation of mutations designed to eliminate predicted post-translational modifications resulted in two humanized mAbs that were effective at preventing fentanyl-induced pharmacological effects in rats. These humanized mAbs showed favorable biophysical properties with respect to aggregation and hydrophobicity by chromatography-based assays, and thermostability by dynamic scanning fluorimetry. These results collectively support that the humanized anti-fentanyl mAbs developed herein warrant further clinical development for treatment of fentanyl toxicity.

Identification of a novel opioid, N-piperidinyl etonitazene (etonitazepipne), in patients with suspected opioid overdose.

BACKGROUND: Novel opioids in the illicit drug supply, such as the "nitazene" group of synthetic opioids, present an ongoing public health problem due to high potency and respiratory depressant effects. We describe three patients in whom N-piperidinyl etonitazene, a compound not previously reported in human exposure, was detected after suspected opioid overdose. Other substances that these patients tested for included fentanyl, cocaine, levamisole, phenacetin, benzoylecgonine, para-fluorofentanyl, presumptive heroin (tested as 6-monoacetylmorphine (6-MAM), morphine, and codeine), and tramadol. METHODS: This is a case series of patients with acute opioid overdose enrolled in an ongoing multicenter prospective cohort study. Data collected included reported substance use, clinical course, naloxone dose and response, outcome, and analytes detected in biological samples. RESULTS: Between October 6, 2020 and October 31, 2021, 1006 patients were screened and 412 met inclusion criteria. Of these, three patients (age 33-55) tested positive for N-piperidinyl etonitazene at one site in New Jersey over a period of three days in July 2021. Two patients reported the use of cocaine; one reported the use of heroin and alprazolam. All three patients received naloxone with improvement in their mental status (2 milligrams (mg) intranasally (IN); 8 mg IN; 0.08 mg intravenous (IV)). Two of three received subsequent doses for recurrence of opioid toxicity (0.4-0.6 mg IV). One patient was diagnosed with pneumonia and admitted to the intensive care unit, one was discharged from the Emergency Department (ED), and one used additional drug while in the ED and required admission for a naloxone infusion. None developed organ damage or sequelae. CONCLUSION: These cases represent a local outbreak of a novel "nitazene" opioid. Public health toxicosurveillance should incorporate routine testing of this emerging class of synthetic compounds in the illicit drug supply.

Toxicological and pharmacological characterization of novel cinnamylpiperazine synthetic opioids in humans and in vitro including 2-methyl AP-237 and AP-238.

The recent scheduling actions of fentanyl-related substances in both the United States and China have sparked the emergence and proliferation of other generations of "legal" opioids that are structurally distinct from fentanyl, including the recently emerged class of cinnamylpiperazines. In contrast to fentanyl, which contains a piperidine core and a phenethyl moiety, the primary structural components of cinnamylpiperazines are the piperazine core and a cinnamyl moiety. This manuscript reports on the toxicological profile for antemortem and postmortem cases where a cinnamylpiperazine was detected. Samples were quantitatively confirmed using liquid chromatography tandem mass spectrometry. The cases were received between February 2020 and April 2021. Concentrations of 2-methyl AP-237 from four postmortem cases ranged from 820 to 5800 ng/mL, and concentrations of AP-238 from two postmortem cases were 87 and 120 ng/mL. µ-Opioid receptor (MOR) activation potential for 2-methyl AP-237, AP-237, para-methyl AP-237, and AP-238 were studied using a ßarr2 recruitment assay. Efficacies (Emax, relative to hydromorphone) and potencies (EC50) were derived and of the compounds tested AP-238 was the most potent compound in the panel with an EC50 of 248 nM. 2-Methyl AP-237 was found to be the most efficacious drug (Emax = 125%) of the tested cinnamylpiperazines; however, it had substantially less efficacy than fentanyl. The in vitro MOR activation potential of the studied cinnamylpiperazines was lower than that of fentanyl and other novel synthetic opioids (NSOs), in line with the relatively higher concentrations observed in postmortem toxicology samples-an important observational link between in vitro pharmacology and in vivo toxicology.

Reddit discussions about buprenorphine associated precipitated withdrawal in the era of fentanyl.

BACKGROUND: Induction of buprenorphine, an evidence-based treatment for opioid use disorder (OUD), has been reported to be difficult for people with heavy use of fentanyl, the most prevalent opioid in many areas of the country. In this population, precipitated opioid withdrawal (POW) may occur even after individuals have completed a period of opioid abstinence prior to induction. Our objective was to study potential associations between fentanyl, buprenorphine induction, and POW, using social media data. METHODS: This is a mixed methods study of data from seven opioid-related forums (subreddits) on Reddit. We retrieved publicly available data from the subreddits via an application programming interface, and applied natural language processing to identify subsets of posts relevant to buprenorphine induction, POW, and fentanyl and analogs (F&A). We computed mention frequencies for keywords/phrases of interest specified by our medical toxicology experts. We further conducted manual, qualitative, and thematic analyses of automatically identified posts to characterize the information presented. Results: In 267,136 retrieved posts, substantial increases in mentions of F&A (3 in 2013 to 3870 in 2020) and POW (2 in 2012 to 332 in 2020) were observed. F&A mentions from 2013 to 2021 were strongly correlated with mentions of POW (Spearman's ρ: 0.882; p = .0016), and mentions of the Bernese method (BM), a microdosing induction strategy (Spearman's ρ: 0.917; p = .0005). Manual review of 384 POW- and 106 BM-mentioning posts revealed that common discussion themes included "specific triggers of POW" (55.1%), "buprenorphine dosing strategies" (38.2%) and "experiences of OUD" (36.1%). Many reported experiencing POW despite prolonged opioid abstinence periods, and recommended induction via microdosing, including specifically via the BM. CONCLUSIONS: Reddit subscribers often associate POW with F&A use and describe self-managed buprenorphine induction strategies involving microdosing to avoid POW. Further objective studies in patients with fentanyl use and OUD initiating buprenorphine are needed to corroborate these findings.HIGHLIGHTSIncrease in mentions of precipitated opioid withdrawal (POW) on Reddit from 2012 to 2021 was associated with the increase in fentanyl and analog mentions.Experiences of precipitated opioid withdrawal (POW) were described by individuals despite reporting prolonged periods of abstinence compared to standard buprenorphine induction protocols.People with Opioid Use Disorder (OUD) on Reddit are using and recommending microdosing strategies with buprenorphine to avoid POW.People who used fentanyl report experiencing POW following statistically longer periods of abstinence than people who use heroin.

Inhibition of Oligodendrocyte Apoptosis in the Prelimbic Medial Prefrontal Cortex Prevents Fentanyl-induced Hyperalgesia in Rats.

Opioid-induced hyperalgesia (OIH) is a problem associated with prolonged use of opioids in chronic pain management, and its effective treatment has been hampered by lack of mechanistic evidence. Oligodendrocytes have recently been linked with several pain-related diseases; however, little is known its role in OIH. The prelimbic medial prefrontal cortex (PL-mPFC) has emerged as a significant center of pain regulation, and is rich in oligodendrocytes. Herein we explored the effect of oligodendrocyte apoptosis of PL-mPFC on OIH. Using a fentanyl-induced rat model of OIH and proteomics analysis of the PL-mPFC, we observed a downregulation in 5 types of myelin-related proteins originating from oligodendrocytes; this was further verified by western blotting. Meanwhile, cleaved-caspase 3 (an apoptosis marker) was increased, whereas the oligodendrocyte precursor cell (OPC) marker NG2 remained unchanged. These results suggest that downregulated myelin-related proteins may be associated with oligodendrocyte apoptosis rather than a reduction in their generating source, and immunohistochemistry confirmed this hypothesis. Behaviorally, prophylactic blockade of oligodendrocyte apoptosis by microinjection of z-DEVD-fmk into the PL-mPFC prevented fentanyl-induced mechanical and thermal hyperalgesia, but downregulated myelin basic protein (mbp) gradually recovered in 12 h. We suggest that OIH may be primed in part via oligodendrocyte apoptosis in the PL-mPFC. PERSPECTIVE: In this study we showed that oligodendrocyte apoptosis in the PL-mPFC is a key trigger for fentanyl-induced hyperalgesia. Targeting oligodendrocyte apoptosis in the PL-mPFC may prevented hyperalgesia priming induced by fentanyl.

Pre-treatment with Tandospirone attenuates fentanyl-induced respiratory depression without affecting the analgesic effects of fentanyl in rodents.

Opioid analgesics are widely used to treat acute, postoperative, and chronic pain. However, opioid receptor activation can result in severe respiratory depression. In this study, we demonstrated that Tandospirone (TS), a selective serotonin-1A receptor partial agonist, is effective against opioid-induced respiratory depression. Fentanyl was used to establish a respiratory depression model in rodents. We observed the effects of TS on respiratory depression in rats by using plethysmographic recordings and arterial oxygen saturation. In addition, we evaluated the effects of TS on fentanyl-induced sedation and analgesia by using the loss of righting reflex (LORR) and hot-plate tests, respectively. Rats (n = 5) were treated with TS or saline 5 min prior to fentanyl administration. TS [2 mg/kg, intravenous (i.v.)] dose-dependently attenuated fentanyl-induced respiratory depression versus saline + fentanyl group. Furthermore, pre-treatment with TS (2 mg/kg, i.v.) increased arterial oxygen saturation to 76.5 ± 2.0% at 5 min after fentanyl injection, compared with 35.9 ± 2.5% in saline pre-treated rats (P < 0.001), whereas the time to induction of LORR (P > 0.99) and duration of LORR (P = 0.95) did not differ between the "TS + fentanyl" and "saline + fentanyl" group. The antinociceptive effect of fentanyl was not affected by the administration of TS (P = 0.99) in mice (n = 10). In conclusion, we found that TS, a novel non-benzodiazepine anxiolytic/antidepressant drug, could attenuate severe fentanyl-induced respiratory depression and did not affect the analgesic/sedative effect of fentanyl. The clinical application of TS could significantly improve pain management.

Respiratory depressant effects of fentanyl analogs are opioid receptor-mediated.

Opioid-related fatalities involving synthetic opioids have reached unprecedented levels. This study evaluated the respiratory depressant effects of seven fentanyl analogs that have either emerged in the illicit drug supply or been identified in toxicological analyses following fatal or non-fatal intoxications. Adult male Swiss Webster mice were administered fentanyl analogs (isobutyrylfentanyl, crotonylfentanyl, para-methoxyfentanyl, para-methoxybutyrylfentanyl, 3-furanylfentanyl, thiophenefentanyl, and benzodioxolefentanyl) and their effects on minute volume as compared to mu-opioid receptor (MOR) agonist standards (fentanyl, morphine, and buprenorphine) were measured using whole body plethysmography (WBP). All drugs elicited significant (p ≤ 0.05) hypoventilation relative to vehicle for at least one dose tested: morphine (1, 3.2, 10, 32 mg/kg), buprenorphine, (0.032, 0.1, 0.32, 1, 3.2 mg/kg), fentanyl (0.0032, 0.01, 0.032, 0.1, 1, 32 mg/kg), isobutyrylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), crotonylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxyfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxybutyrylfentanyl (0.32, 1, 3.2, 10 mg/kg), 3-furanylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), thiophenefentanyl (1, 3.2, 10, 32, 100 mg/kg), and benzodioxolefentanyl (3.2, 10, 32, 100 mg/kg). The ED50 values for hypoventilation showed a rank order of potency as follows: fentanyl (ED50 = 0.96 mg/kg) > 3-furanylfentanyl (ED50 = 2.60 mg/kg) > crotonylfentanyl (ED50 = 2.72 mg/kg) > para-methoxyfentanyl (ED50 = 3.31 mg/kg) > buprenorphine (ED50 = 10.8 mg/kg) > isobutyrylfentanyl (ED50 = 13.5 mg/kg) > para-methoxybutyrylfentanyl (ED50 = 16.1 mg/kg) > thiophenefentanyl (ED50 = 18.0 mg/kg) > morphine (ED50 = 55.3 mg/kg) > benzodioxolefentanyl (ED50 = 10,168 mg/kg). A naloxone pretreatment (10 mg/kg) attenuated the hypoventilatory effects of all drugs. These results establish that the respiratory depressant effects of these fentanyl analogs are at least in part mediated by the MOR.

Evaluation of fentanyl toxicity and metabolism using a zebrafish model.

The increased abuse of novel drugs has created a critical need for cheap and rapid in vivo models to understand whole organism drug-induced toxicity and metabolic impacts. One such model is zebrafish, which share many similarities to human. Assays have been developed for behavioral, toxicity, and metabolism elucidation following chemical exposure. The zebrafish model provides the advantage of assessing these parameters within a single study. Previous zebrafish studies have evaluated the behavioral effects of fentanyl, but not developmental toxicity and its relation to metabolism. In this study, we evaluate the effects of fentanyl on the development of wild-type (TL strain) zebrafish and its metabolism over 4 days. Fertilized eggs were exposed to six concentrations of fentanyl (0.01, 0.1, 1, 10, 50, and 100 µM) through embryo media incubated at 28-29°C. Observations included egg coagulation, somite formation, heartbeat, tail and yolk morphology, pericardial formation, and swim bladder inflation. The incubation media was analyzed for the presence of metabolites using a targeted metabolomics approach. Fentanyl concentration caused significant effects on survival and development, with notable defects to the tail, yolk, and pericardium at 50 and 100 µM. Despropionyl fentanyl (4-ANPP), ß-hydroxy fentanyl, and norfentanyl were detected in zebrafish larvae. We present a single in vivo model to assess toxicity and metabolism of fentanyl exposure in a vertebrate model system. Our findings provide a foundation for further investigations into fentanyl's mechanism of action and translation to human drug exposure.

Sodium chloride injection to treat opioid overdose; Does it work? A preclinical study.

Opioid overdoses (ODs) are increasing in Mexico's northern border. Because naloxone is usually not available, witnesses inject common salt (NaCl) into a vein of OD victims in an attempt to help them regain consciousness. Despite this widespread practice, no preclinical studies have addressed the efficacy of NaCl as an opioid antidote. Here we tested saline solutions at different concentrations. Because the highest (31.6 %) caused tail necrosis, we selected 17.7 % as a hypertonic saline solution (HSS) to determine if it could prevent the lethal effect of morphine (Mor), fentanyl (Fen), or Mor + Fen in adult Wistar male rats. We also evaluated if NaCl could modify the opioid antagonist effect of naloxone. Our results show that HSS: a) sensitizes animals to thermal but not mechanical stimuli; b) does not prevent mortality caused by high morphine or fentanyl doses; c) decreases the latency to recovery from the sedative effects caused by low doses of morphine or fentanyl; and d) increases naloxone's efficacy to prevent the lethality produced by Mor or Fen, but not by Mor + Fen. These results suggest that HSS is marginally effective in shortening the recovery time from nonfatal opioid ODs and increases naloxone's efficacy to counteract opioid-induced ODs.

Wooden Chest syndrome: The atypical pharmacology of fentanyl overdose.

WHAT IS KNOWN AND OBJECTIVE: A large percentage of opioid overdose fatalities involve fentanyl or one of its legal or illegal analogs (F/FAs). Is there something about the pharmacology of these drugs that make them unusually dangerous in an overdose? COMMENT: Some of the reasons for the dangers of overdose of F/FAs is their high potency and low cost (that leads to wide distribution). But it is rarely asked if the basic pharmacology of F/FAs differ in some fundamental way from conventional opioids such as morphine and heroin. In addition to centrally mediated respiratory depression via opioid receptors, F/FAs cause rigidity in the key respiratory muscles of the chest, upper airway and diaphragm ("wooden chest syndrome," WCS) by a non-opioid mechanism. WHAT IS NEW AND CONCLUSION: WCS is an atypical pharmacology of F/FAs. Because of its rapid onset and non-opioid mechanism, WCS makes F/FA overdose particularly dangerous.