Cardiorespiratory failure and sudden death induced by carfentanil exposure: Involvement of central mu1 opioid receptors.

The highly potent opioid carfentanil (CAR) represents a growing health risk. CAR acts via Gi/o-coupled µ opioid receptors (µOR) and exhibits ultra-high toxicity. So far, no clear association between pharmacodynamics and toxicity of CAR has been described. We created a HEK-293 cell line stably expressing the µOR and, determined ligand binding affinity (Ki) and potency (EC50) of CAR, fentanyl, remifentanil, morphine or the endogenous ligand endomorphin-1. We found that µOR bind CAR with ~ 10-times higher affinity than fentanyl or remifentanil and with ~ 70-times higher affinity than morphine. Potency of CAR to inhibit cAMP was ~ 85-times higher compared to the fentanyl’s and ~ 620 higher compared to morphine. Thus, CAR’s toxicity rather associates with receptor potency than affinity. When receptor occupancy at EC50-values was calculated, it appeared that CAR is ~ 8-times more efficient to inhibit cAMP in comparison to morphine, fentanyl or remifentanil. Hence, we postulate that CAR stabilizes µOR conformations that are ultra-efficient in inhibiting cAMP. The OR antagonists naloxone and nalmefene are used as antidotes against opioid intoxication. Both antagonists revealed 10 to 100-times higher IC50-values against CAR-mediated cAMP inhibition compared to the other opioids, indicating that µOR conformations stabilized by CAR are rather resistant towards clinically used antidotes. Of note, when the long acting OR antagonist naltrexone was tested, it exhibited a ~ 65-times higher potency to inhibit CAR but not fentanyl compared to naloxone. Our data highlight the unique nature of CAR’s interactions with µOR and provide first pharmacodynamic indication that naltrexone might be a superior antidote.

Previous studies have shown that inhalation of aerosolized CRF could result in rapid death in humans and respiratory depression in animals. The respiratory depression is mediated by activation of peripheral and particularly central opioid receptors (ORs), among which opioid mu 1 -recetpors (µ 1 -ORs) may be critical. To date, the mechanisms underlying CRF exposure-induced cardiorespiratory failure and sudden death remain unclear. In this study, we first compared the CRF exposure-induced cardiorespiratory failure and sudden death between sexes. Both male and female conscious rats were exposed to aerosolized CRF at a concentration of 12.86 mg/m 3 for 15 min in a double-chamber plethysmograph to record cardiorespiratory activities for 60 min. CRF exposure led to sudden death in 2/6 male rats, but not in any of the females (0/7). Compared to aerosolized vehicle, CRF exposure initially decreased minute ventilation (V E ) by 50% (primarily via reduction of respiratory frequency and prolongation of expiratory duration) and heart rate (HR) by 25% within 1 min and by 80% and 40% respectively 10 min after the onset of the exposure. These responses were generally maintained to the end of experiment and disappeared on the next day in the survived rats. The V E depression and bradycardia were much more aggravated after 15 min CRF exposure in the dead rats, leading to ventilatory and then cardiac arrest within ~60 min after the onset of the exposure. Secondly, the impacts of the following pretreatments on the cardiorespiratory responses and the mortality were tested in male rats: a) naloxone hydrochloride (NLX) and naloxone methiodide (NLM) to systemically and peripherally block ORs respectively; and b) β-funaltrexamine (β-FNA) and naloxonazine (NLZ) to systemically antagonize both µ 1 - and µ 2 -Rs and only µ 1 -Rs respectively. NLX blocked the CRF-induced VE depression, bradypnea and bradycardia, while NLM had limited effect. β-FNA and NLZ similarly and significantly alleviated VE depression, bradypnea and bradycardia. The pretreatments prevented CRF-caused death except that NLM still caused death in 3/7 rats. Therefore, our experiments not only establish a model of CRF exposure-induced cardiorespiratory failure and sudden death in conscious rats, but also reveal a key role of central ORs, especially µ 1 -Rs, in the CRF exposure-induced cardiorespiratory failure. This study is supported National Institutes of Health (NIH), Bethesda. Grant numbers: DA059063 and HL163512. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Our previous studies in rats have shown that exposure to CRF aerosol initially induces rapid ventilation (V E ) depression, bradypnea and bradycardia followed by subsequent persistent cardiorespiratory depression. Moreover, blockade of peripheral ORs only slightly attenuates the initial response, while systemic antagonism of ORs fully abolishes all of cardiorespiratory responses. It is known that peripheral ORs in vagal afferents play a significant role in triggering the immediate cardiorespiratory responses to fentanyl, while ORs in the central nervous system (CNS) are essential for generating the opioid-induced respiratory depression. Thus, this study aimed to further define contributions of ORs in vagal afferents and the CNS to cardiorespiratory responses to CRF exposure. The rats were placed in a double-chamber plethysmograph and exposed to CRF aerosol at a concentration of 12.86 mg/m 3 for 15 min. Prior to the exposure, four groups of rats were respectively pretreated with intra-vagal microinjection of saline (IVG SAL ) and naloxone (IVG NLX ), and intrathecal injection of saline (IT SAL ) and NLX (IT NLX ). We found that CRF exposure caused death within 20 min after the onset of the exposure in 2 of 6 rats pretreated with IVG SAL . In the remaining 4 survived rats, CRF exposure triggered an immediate cardiorespiratory depression during the first 2 min exposure and decreased V E by 80% (mainly via reducing respiratory frequency by 75%) associated with 35% decrease in heart rate at the end of the 15-min exposure. The cardiorespiratory depression was generally maintained 45 min after the onset of the exposure with limited recovery, and disappeared on the next day. IVG NLX only significantly reduced the initial bradypnea without remarkable impact on the subsequent cardiorespiratory depression with death occurring in 3/6 rats. In contrast, IT NLX blocked the bradypnea and bradycardia and markedly blunted V E depression without death. Our results suggest that the initial bradypnea and bradycardia are mediated by ORs in both vagal afferents and the CNS, while the subsequent cardiorespiratory depression and failure are predominantly mediated by central ORs. This study is supported National Institutes of Health (NIH), Bethesda.Grant numbers: DA059063 and HL163512. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Bilateral Inhalation of carfentanil (CRF) aerosol could rapidly induce death in humans and CRF aerosol exposure induces respiratory depression in animals; however, the dynamic progression of cardiorespiratory failure leading to sudden death induced by acute CRF aerosol exposure remains unclear. This study aimed to establish a rat model that allows dynamically characterizing the cardiorespiratory failure prior to death following acute exposure to a lethal concentration of CRF aerosol. Two groups of anesthetized and spontaneously breathing rats were exposed to aerosolized vehicle and CRF (4 mg/m3) for 10 minutes respectively. CRF exposure resulted in 100% mortality among the tested rats. The cardiorespiratory responses were characterized sequentially by: immediate ventilatory depression resulting from rapidly developed bradypnea, persistent ventilatory depression, and hypotension and significant irregularities in both breathing (ataxic breathing) and heart beat rhythms that ultimately caused ventilatory and cardiac arrest at 7.7 ± 0.6 and 9.0 ± 0.7 minutes, respectively, after the onset of CRF exposure. Our results establish a rat model of cardiorespiratory failure and sudden death resulted from acute exposure to CRF aerosol. This model may facilitate further investigation into the mechanisms underlying cardiorespiratory failure and the development of potential countermeasures.

Our previous study has shown that inhalation of fentanyl (100 mg/m3) for 10 min initially induces apnea, hypotension and bradycardia, leading to ventilatory and then cardiac arrest within 15 min in anesthetized rats (Zhuang J., et al, 2024). CRF acts on opioid mu-receptors (MORs) with a potency 100 times that of fentanyl. Inhalation of aerosolized CRF as a chemical weapon has been reported to cause sudden death in humans (Wax PM et al, 2003), but the cause of the death is not fully understood. Moreover, though the opioid-induced respiratory disorder is known to mainly result from the action of μ1Rs, the contribution of μ1Rs to the CRF-induced cardiorespiratory disorder remains unknown. Therefore, we asked whether acute inhalation of overdose CRF would induce cardiorespiratory disorder and sudden death via acting on μ1R in awake rats. The rats were exposed to aerosolized vehicle and CRF at 12.86 (low dose) or 20 mg/m3 (high dose) for 15 min with cardiorespiratory (VE, VT, fR, and HR) responses recorded for 60 min. The low and high doses of CRF induced sudden death in 1/6 and 2/2 rats respectively. Compared to aerosolized vehicle, the low dose of CRF initially decreased VE and HR by ~50% within 3 min exposure, which were maintained to the end of experiment, and all responses disappeared on the next day in the survived rats. Interestingly, the responses of breathing pattern to the CRF exposure were sequentially characterized by the rapid shallow breathing (within several seconds of the exposure), slow and deep breathing and then only slow breathing. In comparison, the dVE and bradycardia became much more aggravated after 15 min CRF aerosol-delivery in the dead rats than the survived ones, resulting in ventilatory and cardiac arrest within ~25 min post exposure. The cardiorespiratory responses to the low dose of CRF aerosol exposure were also performed after pretreatment with naloxone hydrochloride (NLX to systemically antagonize opioid receptors) or naloxonazine (NALZ to selectively antagonize μ1Rs). The CRF-induced dVE and bradycardia were fully blocked by NLX and largely alleviated by NALZ. In conclusion, our experiments not only establish a rat model of acute inhalation of CRF-induced cardiorespiratory disorder and sudden death, but also demonstrate a key role of opioid receptors, especially μ1Rs, in the CRF-induced cardiorespiratory disorder. Supported by NIH R01 grants HL163512 and DA059063. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pain-related opioidergic and dopaminergic neurotransmission: Dual meta-Analyses of PET radioligand studies

The detection of fentanyl (FEN) and FEN analogs has been widely communicated throughout the scientific community. While most of the reporting has been in relation to overdose deaths, these drugs are commonly detected in impaired driving cases. A retrospective study of impaired driving cases analyzed between 2017 and 2019 produced 270 cases positive for FEN, carfentanil (CFEN) and/or acetylfentanyl (AFEN). FEN was the predominant drug found in these 270 cases (65.5%) with concentrations ranging from less than 1.0 to 64 ng/mL. CFEN was found alone in 6.6% cases with three concentrations above 1.0 ng/mL. AFEN was always found when FEN was positive with concentrations ranging from <1.0 to 9.2 ng/mL. Detailed case histories are provided with corresponding toxicology results. Toxicology results show impaired drivers using multiple drugs with a wide range of observed behaviors. The inclusion of these drugs in routine impaired driver toxicology testing is extremely important when attempting to determine their overall prevalence.

Carfentanil (CRF) is a potent synthetic opioid primarily used in anaesthetizing large animals. Exposure to CRF can cause both respiratory and central nervous system depression. Respiratory depression can lead to hypoxia, which may lead to cardiac damage and ultimately death. Myocardial damage has been observed in ferrets exposed to aerosolized CRF. Troponin is a protein complex that plays an integral part in muscle contractions within the myocardium and is used as an indicator of myocardial damage. An increase in blood levels of troponin in exposed ferrets would suggest underlying cardiac damage. Select animals were treated with naloxone (NX), an opioid antagonist, to investigate whether reversing opioid effects altered cardiac function or troponin levels.Male telemetrized ferrets were exposed to aerosolized CRF in a whole‐body plethysmography chamber. Cardiac parameters and respiratory dynamics were collected before, during, and after CRF exposure. Ferrets received no treatment, an oral or intramuscular (i.m.) dose of NX (0.375, 0.75, 1.5, or 3 mg/mL), or water 30 minutes post‐exposure. Euthanasia occurred 24 hours post‐exposure, and blood was collected via descending aorta or heart stick. Serum was separated from blood and analyzed using a high sensitivity rat cardiac troponin‐I enzyme‐linked immunosorbent assay (ELISA).Average troponin levels for all animals ranged from 0.00 – 6.30 ng/mL. Troponin levels were not statistically different across treatment groups, though we observed an increase in mean arterial pressure (MAP) and increased incidents of premature junction complexes for animals exposed to CRF, indicating potential cardiac distress. The increase in MAP could be a compensatory mechanism to overcome hypoxia due to decreased respiration induced by CRF. Both control and exposed animals showed an increase in MAP following treatment; however, sham‐exposed animals had no rise in MAP post‐treatment. This rise in MAP could be due to a combination of NX treatment and a response to being handled for the injection. Overall, our results suggest that CRF does not directly cause myocardial damage, nor do surgically implanted cardiac telemetry devices cause an explicit, permanent increase in troponin levels. Further investigation is needed to determine if cardiac abnormalities observed in ferrets are caused via hypoxia or other unknown mechanisms.Support or Funding InformationOpinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army. This research complied with the Animal Welfare Act and implementing Animal Welfare Regulations and adhered to the principles noted in The Guide for the Care and Use of Laboratory Animals. This research was supported by DTRA RD‐CB. E.P., W.Y.T., M.R. S.P., A.D., and V.D.C. were supported in part by an appointment to the Postgraduate Research Participation Program at the US Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USAMRDC.

Changes in murine respiratory dynamics induced by aerosolized carfentanil inhalation: Efficacy of naloxone and naltrexone

Death of a psychonaut: First French carfentanil fatality

Carfentanil (CRF) is a powerful synthetic opioid that is approximately 100 times more potent than fentanyl and 10,000 times more potent than morphine. CRF originally was intended to be used as a sedative for big game animals in a veterinary setting, but is becoming increasingly common in the illicit drug trade. The increased use of CRF by people has put strain on first responders, who need to administer increased doses of naloxone (Naracan®, Evzio®), an FDA‐approved opioid antagonist, to counteract the effects. First responders may also worry about the potential of self‐exposure through inhalation or dermal contact with CRF. We set out to study the effectiveness of naloxone against a potentially lethal dose of inhaled CRF in male ferrets. Ferrets were implanted with telemetry devices to study cardiac parameters and exposed to aerosolized CRF in a whole‐body plethysmography chamber to record respiratory parameters. We observed profound depression of respiration which resulted in the animals becoming apneic 23% ± 9% of the time during exposure. We also observed cardiac abnormalities, in the form of premature junctional contractions (PJCs), in the ferrets during the apneic periods. At our acute exposure dose, which was lethal in 9% of our animals, ferrets were unresponsive and incapacitated for 105.6 ± 20.0 minutes following the end of exposure. Naloxone, when administered intramuscularly at human equivalent doses ranging from 4 to 40 mg, significantly reduced the time that ferrets were incapacitated following exposure. Although we saw no significant difference in the reduction of time that the animals were incapacitated between the treatment groups (average times between treatment and revival ranged from 6.5 to 9.7 minutes), higher doses of naloxone did revive a portion of the ferrets quicker than the lower doses (some as soon as 1.5–3.5 minutes post‐treatment). We also observed that naloxone was able to significantly reduce the amount of apneic episodes in CRF‐exposed ferrets and resolved the observed cardiac PJCs. Our results suggest that naloxone is a viable treatment against the effects of a potentially lethal dose of inhaled CRF in ferrets. These data have the potential to inform and aid treatment strategies for an acute inhalation exposure to synthetic opioids.Support or Funding InformationOpinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army. This research complied with the Animal Welfare Act and implementing Animal Welfare Regulations and adhered to the principles noted in The Guide for the Care and Use of Laboratory Animals. This research was supported by DTRA RD‐CB. M.R. and N.A.A. were supported in part by an appointment to the Postgraduate Research Participation Program at the US Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and USAMRMC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A case report of carfentanil-related fatality in France

The United States is in the middle of a historically unprecedented opioid epidemic. Today, more people die of drug overdoses than any other form of accidental death, and opioid overdose rates surpass historic peak death rates from human immunodeficiency virus (HIV), gun violence, and motor vehicle

Carfentanil (CFTN), a derivative of fentanyl, is highly effective as an analgesic, but its relatively poor solubility in water has limited its nasal application. The objective of this study was to develop the new CFTN-CD inclusion technology to increase the solubility of CFTN. The inclusion compound CFTN–DM-β-CD was prepared by the ultrasonic method and characterized using X-ray powder diffraction and morphological shapes analysis (the scanning electron microscopy). The in vitro dissolution profiles of CFTN–DM-β-CD were assessed in hydrochloric acid and phosphate buffer. Nasal ciliotoxicity studies were carried out using isolated toad palate. Rats were treated with CFTN–DM-β-CD (250 µg/kg) by intravenous, intramuscular injection, oral, or nasal drops. The results showed that CFTN was successfully enveloped by DM-β-CD. The in vitro cumulative dissolution of CFTN–DM-β-CD was obviously enhanced compared to free CFTN in two buffers. Nasal ciliotoxicity studies have shown that the CFTN–DM-β-CD does not exhibit higher nasal ciliotoxicity than that of free CFTN. Pharmacokinetic studies demonstrated that CFTN–DM-β-CD by nasal administration was absorbed more rapidly and has higher Cmax and bioavailability than that of either intramuscular injection or oral administration. In conclusion, the CFTN–DM-β-CD nasal spray was shown to be a relatively safe dosage form for the rapid and effective intranasal delivery of CFTN.

Molecular Imaging of Opioid System in Idiopathic Parkinson's Disease.

In July of 2016, carfentanil (CF) emerged in Northeast Ohio resulting in over 25 deaths within a 30-day period. A total of 125 deaths have occurred in Summit County and Cuyahoga County has reported 40 deaths, relating to the presence of CF either alone, or in combinations with heroin and fentanyl. Prior to this surge in CF cases, positive fentanyl enzyme-linked immunosorbent assay (ELISA) screening results were increasing in number. Many were negative for fentanyl confirmation by gas chromatography-mass spectrometry. Fentanyl analogs such as CF, acetyl fentanyl (AF), 2-furanyl fentanyl (2-Fu-F) and 3-methylfentanyl (3-MF) may be present in these cases. Some fentanyl analogs like CF and 3-MF do not cross-react with the Immunalysis ELISA fentanyl assay. With the emergence of potent synthetic fentanyl analogs, questions arose as to how to interpret their very low concentrations or absence in the blood in relation to cause of death. Driving under the influence of drugs (DUID) blood specimens had also tested positive for CF by reference laboratories. A liquid chromatography-tandem mass spectrometry method was developed to identify and quantify fentanyl, norfentanyl (NF) and four analogs: AF, 2-Fu-F, 3-MF and CF. The method has been utilized to quantify these fentanyl analogs in blood and vitreous humor in authentic antemortem and postmortem cases. Calibration curves were established between 0.10-4.0 ng/mL (NF, AF, 3-MF, 2-Fu-F and CF) and 1.0-40 ng/mL for fentanyl. In total, 98 postmortem cases analyzed produced the following blood concentration ranges: CF (0.11-0.88 ng/mL), 3-MF (0.15-1.7 ng/mL), 2-Fu-F (0.15-0.30 ng/mL), AF (0.14-0.16 ng/mL), fentanyl (1.1-15 ng/mL) and NF (0.10-3.7 ng/mL). Only CF, fentanyl and NF were detected in a statistically significant subset DUID population of 26 cases producing concentration ranges between 0.11 and 0.47 ng/mL, 1.0 and 9.8 ng/mL, and 0.11 and 3.5 ng/mL, respectively.

This study examined the real-time exposure–response effects of aerosolized carfentanil (CRF) on opioid-induced toxicity, respiratory dynamics and cardiac function in mice. Unrestrained, conscious male CD-1 mice (25–30 g) were exposed to 0.4 or 4.0 mg/m3 of aerosolized CRF for 15 min (Ct = 6 or 60 mg min/m3) in a whole-body plethysmograph chamber. Minute volume (MV), core body temperature (Tc), mean arterial blood pressure (MAP) and heart rate (HR) were evaluated in animals exposed to CRF or sterile H2O. Loss of consciousness and Straub tail were observed in before 1 min following initiation of exposure to 6 or 60 mg min/m3 of CRF. Clinical signs of opioid-induced toxicity were observed in a dose-dependent manner. Exposure to 6 or 60 mg min/m3 of CRF resulted in significant decrease in MV as compared to the controls. MAP, HR and Tc decreased 24 h in animals exposed to either 6 or 60 mg min/m3 of CRF as compared to the controls. Post-exposure administration of naloxone (NX, 0.05 mg/kg, i.m.) did not increase the MV of animals exposed to CRF to control levels within 24 h, but decreased clinical signs of opioid-induced toxicity and the duration of respiratory depression. This is the first study to evaluate real-time respiratory dynamics and cardiac function during exposure and up to 24 h post-exposure to CRF. The evaluation of toxicological signs and respiratory dynamics following exposure to CRF will be useful in the development of therapeutic strategies to counteract the ongoing threat of abuse and overuse of opioids and their synthetic variants.

Persons with a history of alcohol dependence are more likely to use tobacco and to meet criteria for nicotine dependence compared with social drinkers or non-drinkers. The high levels of comorbidity of nicotine and alcohol use and dependence are thought to be related to interactions between nicotinic, opioid and dopamine receptors in mesolimbic regions. The current study examined whether individual differences in regional μ-opioid receptor (MOR) availability were associated with tobacco use, nicotine dependence and level of nicotine craving in 25 alcohol-dependent (AD) subjects. AD subjects completed an inpatient protocol, which included medically supervised alcohol withdrawal, monitored alcohol abstinence, transdermal nicotine maintenance (21 mg/day) and Positron Emission Tomography (PET) imaging using the MOR agonist [(11) C]-carfentanil (CFN) before (basal scan) and during treatment with 50 mg/day naltrexone (naltrexone scan). Subjects who had higher scores on the Fagerström Nicotine Dependence Test had significantly lower basal scan binding potential (BPND ) across mesolimbic regions, including the amygdala, cingulate, globus pallidus, thalamus and insula. Likewise, the number of cigarettes per day was negatively associated with basal scan BPND in mesolimbic regions. Higher nicotine craving was significantly associated with lower BPND in amygdala, globus pallidus, putamen, thalamus and ventral striatum. Although blunted during naltrexone treatment, the negative association was maintained for nicotine dependence and cigarettes per day, but not for nicotine craving. These findings suggest that intensity of cigarette smoking and severity of nicotine dependence symptoms are systematically related to reduced BPND across multiple brain regions in AD subjects.

The relationship between naloxone-induced cortisol and mu opioid receptor availability in mesolimbic structures is disrupted in alcohol dependent subjects