Respiratory Rate Depression Research Articles

The Role of Somatostatin‐Expressing Cells in Opioid‐Induced Respiratory Depression

Introduction Opioids have the ability to cause respiratory depression, a severe decline in respiratory rate that can be lethal with overdose. While key brainstem respiratory regions have been shown to play a role in respiratory depression by opioids, the cells involved remain unknown. One region of interest is the preBötzinger Complex, a site that mediates respiratory depression and contains a subgroup of somatostatin (SST)-expressing cells that are required to maintain normal breathing. We aimed to determine the role of SST-expressing cells in respiratory depression by opioids. We hypothesized that deletion of mu-opioid receptors (MORs) in SST-expressing cells will prevent opioid-induced respiratory depression. Methods We developed transgenic knockout mice that lack MORs in SST-expressing cells (SST-MOR-/-) using Cre-lox recombination. SST-Cre+/+ mice (Ssttm2.1(cre)Zjh/J) were bred with Oprm1fl/fl mice (Oprm1tm1.1Cgrf/KffJ) to produce SST-MOR-/- mice. We used in situ hybridization to determine whether MOR mRNAs are co-expressed with SST mRNA in the brainstem of control (wild-type, SST-MOR+/+) and SST-MOR-/- mice. To determine the effect of removing MORs from SST-expressing cells, respiratory depression by intraperitoneal injection of fentanyl (0.3mg/kg) was quantified in control (wild-type, SST-MOR+/+) and SST-MOR-/- mice. Respiratory rate was recorded using whole-body plethysmography. Results MORs were expressed in several brainstem regions regulating breathing such as the preBötzinger Complex, the Bötzinger Complex, the nucleus ambiguus, the Kölliker-Fuse nucleus, the locus coeruleus and the nucleus tractus solitarius, while SST and MORs were co-expressed primarily in the preBötzinger Complex. Knockout of MORs in SST-expressing cells was confirmed by microscopy. Intraperitoneal injection of fentanyl (0.3mg/kg) induced a similar, significant respiratory rate depression in both wild-type (p<0.001, n=14) and SST-MOR-/- (p<0.001, n=12) mice. Conclusions SST and MORs are co-expressed in the preBötzinger Complex while MORs are expressed in other key brainstem respiratory sites. Preliminary data suggests that MORs in SST-expressing cells are not required for fentanyl-induced respiratory depression. Our previous work showed that arousal state can affect the severity of respiratory depression by opioids. For this reason, we are currently investigating the impact of arousal state (active versus inactive) on opioid-induced respiratory depression in control and SST-MOR-/- mice. We are also looking at the role of SST-expressing cells in respiratory depression by other opioid drugs such as morphine.

Investigating Opioid‐induced Respiratory Depression and Analgesia Using Larval Zebrafish

Introduction Opioid drugs produce both analgesia and severe respiratory depression which can be lethal. In 2019, over 50,000 North Americans died of an opioid overdose. Our understanding on how to differentiate between these two effects is limited due to a lack of simple animal models. Therefore, we aim to understand respiratory depression and analgesia by opioids using novel zebrafish models, allowing for drug screening. Using these models, we tested a variety of drugs including serotoninergic agonists, glutamatergic modulators, and calcium channel activators, for their ability to reverse opioid-induced respiratory depression. Methods We have established phenotype-based approaches using in-vivo zebrafish models of respiratory depression and analgesia by fentanyl. To quantify respiratory depression, we measured respiratory activity by quantifying mandible movements in 12-14 day post-fertilization larvae. To quantify analgesia, we measured the escape swimming response to nociceptive stimuli such as formalin or allyl isothiocyanate (AITC) combined with fentanyl to induce analgesia. We then introduced various candidate drugs to measure their effects on respiratory depression and analgesia. Results Respiratory rate was normalized to each fish's baseline (baseline=100%). Zebrafish strains were found to be differentially sensitive to opioids, where Tübingen (TU) fish did not respond to fentanyl (1µM, p=0.4240, n=9) and AB fish showed significant respiratory rate depression (p<0.001, n=11). AB and TU crosses did not show respiratory depression (n=7). In AB zebrafish, a dose-dependent decrease in respiratory rate was observed with 1μM and 3µM fentanyl (p=0.01, n=11 and p<0.001, n=27, respectively). The opioid antagonist naloxone (20µM) and selective antagonist CTAP (4μM) reversed respiratory depression by fentanyl (p<0.001, n=9 and p=0.049, n=11, respectively). Respiratory depression by 1μM fentanyl was reversed by the AMPA receptor positive allosteric modulator CX614 (5μM, p<0.001, n=9) but not the serotoninergic 5-HT4 agonist BIMU8 (10μM, n=21). Calcium channel activators nefiracetam (1µM) and FLP-64176 (1µM) reversed respiratory depression by fentanyl (1µM, p=0.005, n=11 and3µM, p<0.001, n=15, respectively). Formalin (0.075%) and AITC (100µM) significantly increased swimming velocity (p=0.005, n=21 and p<0.001, n=23, respectively), which was reduced by fentanyl (4μM, p<0.001, n=12and 6µM, p=0.004, n=14, respectively). FLP-64176 reversed fentanyl analgesia when administered with formalin (p<0.001, n=10). Discussion: Our models show that respiratory depression and analgesia by opioids can be measured in zebrafish larvae. The strain differences point to a potential genetic protection which we are investigating using quantitative real-time polymerase chain reaction (qPCR). Our novel zebrafish models can be used to investigate the effects of opioids, as well as for drug screening to find ways to treat respiratory depression while preserving analgesia.

Comparing Benzodiazepines-morphine-induced Respiratory Depression by Analyzing Respiratory Pattern in Rats

Background and Aim: Opioid and benzodiazepine family drugs are concurrently used in various patients. Considering the respiratory depressant effects of both classes, in this study, we investigated the effect of coadministration of morphine and several widely used benzodiazepines in the clinic on the rate of respiratory depression in rats. Methods &amp; Materials: Seventy adult male Wistar rats were randomly divided into 10 groups; morphine, midazolam, diazepam, lorazepam, alprazolam, morphine-midazolam, morphine-diazepam, morphine-lorazepam, and morphine-alprazolam. Respiration signal was recorded using whole-body plethysmography 15 minutes after the intraperitoneal injection of the drugs. The respiratory pattern was examined using several parameters; the mean value of inter-breath interval and the respiratory rate, as well as the coefficient of variation and sample entropy analysis of inter-breath interval. Ethical Considerations: This study was approved by the Ethics Committee of Arak University of Medical Sciences (Code: IR.ARAKMU.REC.1397.327). Results: Analyzing respiratory data revealed that injecting the anxiolytic dose of alprazolam, and the combination of morphine-alprazolam and morphine-midazolam, altered the respiratory pattern. Such changes were associated with a decrease in the number of breaths and an increase in the inter-breath interval in the explored test animals, compared with the controls. The obtained data also indicated that morphine-midazolam injection increased the variability of the breathing pattern; such an alternation was associated with increased irregularity and decreased coefficient of variation of the inter-breath interval. Conclusion: The present research results suggested that the short-term injection of morphine-midazolam changes the respiratory pattern more severely than morphine combined with other benzodiazepines.

The impact and mechanism of ampakine CX1739 on protection against respiratory depression in rats.

Background: Abuse of analgesic and sedative drugs often leads to severe respiratory depression and sometimes death. Approximately 69,000 people worldwide die annuallyfrom opioid overdoses. Purpose: This work aimed to investigate whether CX1739 can be used for emergency treatment of acute respiratory depression due to drug abuse. Results: First, theresults clarify that CX1739 is a low-impact ampakine that can safely activate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors without causing excito-neurotoxicity. Second, CX1739 rapidly crossed the blood-brain barrier (Tmax=2min), which meets the requirement of rapid onset of action in vivo. Our work providespreliminarily confirmation that high-dose intravenous administration of CX1739 can immediately reverse respiratory depression in animal models of respiratory depression caused by opioid agonist 030418, pentobarbital sodiumand ethanol.

CRITICAL CARE COMPLICATIONS OF LITHIUM TOXICITY

SESSION TITLE: Medical Student/Resident Critical Care Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Lithium is amongst the earliest agents with therapeutic benefit in the treatment of mania and remains employed in the management of bipolar disorder. However, its use is limited by a narrow therapeutic window, with numerous acute and acute-on-chronic toxicities with a spectrum of severity. Here, we discuss the critical care management of two cases of lithium toxicity-associated seizures. CASE PRESENTATION: Case 1:A 54-year-old female with schizophrenia on lithium and quetiapine recently initiated on a course of ibuprofen for sciatica presented with nausea, vomiting, diarrhea, and altered sensorium. Physical exam revealed disorientation to her surroundings with psychomotor slowing and speech latency with tremulousness and a depressed respiratory rate. Labs were notable for acute hypercapnic respiratory failure with concomitant non-anion gap metabolic acidosis and acute kidney injury. Serum lithium levels were elevated to 3.89 mmol/L. Initial electroencephalogram showed epileptiform waves concerning for seizure foci. Urgent dialysis was initiated with transition to continuous renal replacement therapy for lithium clearance. The patient was loaded on levetiracetam. Her metabolic encephalopathy slowly improved, with eventual transfer to the psychiatric ward for further medication titration.Case 2:A 67-year-old female with bipolar disorder on lithium and haloperidol recently discharged after titration of medications in the psychiatric ward presented with altered sensorium. Physical exam was notable for waxing and waning mental status with associated dysarthric speech. Urgent evaluation for stroke found no structural causes of her altered sensorium. Lithium levels returned elevated to 2.410 mmol/L. No renal replacement therapy was pursued after discussion with toxicology given lab value downtrend, but continuous electroencephalogram demonstrated non-convulsive status epilepticus suspicious for chronic lithium toxicity with blood brain barrier penetration. The trachea was intubated and anti-epileptics were initiated including a midazolam drip, levetiracetam load, lacosamide, and fosphenytoin. However, her seizures were refractory with prolonged seizure activity causing persistent global encephalopathy. Tracheostomy and percutaneous endoscopic gastrostomy tubes were placed. The patient remains hospitalized after a six-month course but is now stable off mechanical ventilation. DISCUSSION: Lithium toxicity is an acute medical condition with potentially profound neurologic consequences. The cornerstone of management of lithium toxicity is supportive care. Dialysis for lithium toxicity is recommended based upon current guidelines, with therapy directed by lithium levels, evidence of renal impairment, changes in mentation, and evidence of seizure activity or dysrhythmia [1]. CONCLUSIONS: Lithium is a high-risk medication necessitating regular monitoring, with potential severe neurotoxicity. Reference #1: Decker BS et al. Extracorporeal treatment for lithium poisoning: Systematic review and recommendations from the EXTRIP workgroup. 2015. Clin J Am Soc Nephrol 10: 875-887. DISCLOSURES: Speaker/Speaker's Bureau relationship with Boehringer-Ingelheim Pharmaceuticals Please note: $5001 - $20000 Added 04/07/2020 by Jaime Betancourt, source=Web Response, value=Honoraria Speaker/Speaker's Bureau relationship with Vapotherm, Inc Please note: $5001 - $20000 Added 04/07/2020 by Jaime Betancourt, source=Web Response, value=Honoraria No relevant relationships by Scott Oh, source=Web Response No relevant relationships by Alexander Yuen, source=Web Response

Genetic variation regulates opioid-induced respiratory depression in mice

In the U.S., opioid prescription for treatment of pain nearly quadrupled from 1999 to 2014. The diversion and misuse of prescription opioids along with increased use of drugs like heroin and fentanyl, has led to an epidemic in addiction and overdose deaths. The most common cause of opioid overdose and death is opioid-induced respiratory depression (OIRD), a life-threatening depression in respiratory rate thought to be caused by stimulation of opioid receptors in the inspiratory-generating regions of the brain. Studies in mice have revealed that variation in opiate lethality is associated with strain differences, suggesting that sensitivity to OIRD is genetically determined. We first tested the hypothesis that genetic variation in inbred strains of mice influences the innate variability in opioid-induced responses in respiratory depression, recovery time and survival time. Using the founders of the advanced, high-diversity mouse population, the Diversity Outbred (DO), we found substantial sex and genetic effects on respiratory sensitivity and opiate lethality. We used DO mice treated with morphine to map quantitative trait loci for respiratory depression, recovery time and survival time. Trait mapping and integrative functional genomic analysis in GeneWeaver has allowed us to implicate Galnt11, an N-acetylgalactosaminyltransferase, as a gene that regulates OIRD.

Effectivity Comparison of Ketamine and Morphine as Post-Operative Analgesic in Spinal Surgery

Introduction: Patients who undergo spinal procedure, experience the post-operative pain as the major problem. From the Visual Analog Scale (VAS), patients scale their pain around 8. An inadequate pain management could fasten the healing process and reduce patient life quality. Opioid group as the gold standard still inflicts several problems, such as respiratory depression. Moreover, the combination NSAID and opioid which used to suppress the side effect, still burden the healthcare cost. In a research of analgesic, through the discovery of N-Methyl D Aspartate receptor, researchers found an explanation of the ketamine effect in relieve chronic and intense pain which safer and cheaper. Method and Material: This research using single blind randomized control trial. Comparing 0.25mg/kg ketamine IV followed by ketamine 0,1mg/kg/h for the intervention group and 0.02mg/kg/h of morphine for the control group to manage the first 24 hours pain sensation. If patient VAS was more than 4, patient would get additional 0.5mg/kg ketamine (intervention group) and 25μg fentanyl (control group). Result and Discussion: From 17 patients each groups, the VAS values were better in control group rather than on intervention group. Low dose ketamine can’t be compared with morphine to manage post spinal procedure pain. There were no hemodynamic changes, respiratory rate depression, loss of consciousness and hallucination, nystagmus, vomiting and hyper salivation. Even though 11.8% of the subject were nausea. The morphine group tends to experience hemodynamic changes and loss of consciousness in the first 12 hours but still within normal range. In the control group, 47.1 % patients were having nausea in the first hour, but only 17.6% of them who actually vomited. Conclusion: The analgesic effect of morphine is higher than ketamine, but the amount effect of ketamine is lower than morphine so that ketamine is more effective and safer given in the room.

Effects of Different Systemic Opioid Doses on Subareas of the Ventral Respiratory Column

BackgroundThe Parabrachial Nucleus (PBN)/Kölliker‐Fuse (KF) complex are important subareas of the ventral respiratory column (VRC) that regulate inspiratory on‐ ( 1) and off‐switch ( 2). We have shown in vagus‐intact rabbits that respiratory rate depression caused by clinical opioid concentrations was in part mediated by the PBN ( 3), and that systemic opioid effects on respiratory phase timing could be partially reversed in the preBötzinger complex (preBC) albeit without reversing respiratory rate depression ( 4). The present study was to determine how much the combination of local microinjections of naloxone into the KF, PBN and preBC could reverse respiratory depression from intravenous remifentanil (REMI).MethodsThe study was approved by the local Animal Care Committee and conformed to NIH standards. Adult New Zealand White rabbits (3–4kg) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate inspiratory and expiratory duration and peak phrenic activity (PPA). Gridwise localized pressure microinjections of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA, 50 μM, 70 nl) utilizing a multibarrel glass pipette into the VRC caudal to the inferior collicle identified the PBN where AMPA injection caused significant tachypnea and the KF where AMPA caused transient bradypnea and a decrease in PPA. The tachypneic response to AMPA injection in the medulla was used to identify the preBC area. After functional identification of the PBN, KF and preBC areas, we injected a bolus of 10mcg REMI IV, which resulted in apnea. On return of the breathing pattern, a REMI infusion was started to achieve a steady‐state respiratory rate depression of 50% of control rate. After reaching steady‐state, we injected naloxone (1mM, 700nl) into the bilateral KF, PBN and preBC. We then repeated the REMI bolus injection with the apneic dose of 10mcg. In a subset of animals, the 10 mcg REMI bolus was repeated after naloxone injection into the KF/PBN and after injection into the preBC. Residual respiratory depression was reversed at the end of the protocol with IV naloxone (300μM). Drug effects were compared with one‐way RM ANOVA. Data are expressed as mean±SD.ResultsIn 11 animals, IV REMI depressed respiratory rate from 39±7 to 18±6 breaths per minute (BPM). Naloxone injection recovered respiratory rate to 20±9 in the KF (p=0.36), to 26±9 after additional injection into the PBN (p&lt;0.001), and to 28±7 bpm after injection into the preBC area (p=0.54). The IV REMI bolus caused apnea &gt;30s at control but only depressed respiratory rate to 12±6 bpm after naloxone injection into the KF/PBN (p&lt;0.001) and to 16±7 after additional naloxone reversal in the preBC area (p=0.21, n=5).ConclusionRespiratory rate depression from IV opioids is mostly mediated by the PBN, however, higher opioid doses may also affect other brainstem areas.Support or Funding InformationSupported by NIH R01GM112960701A1 (Dr. Stucke) and VA merit grant I01BX000721 (Dr. Zuperku)Figure 1

Methocinnamox (MCAM) Reverses and Provides Extended Protection from Fentanyl‐Induced Respiratory Depression in Awake Unrestrained Rats

Opioid use disorder affects over 2 million Americans with an increasing number of deaths due to overdose from the synthetic opioid fentanyl and its analogs. Opioid toxicity is due, in large part, to the suppression of respiratory function. The FDA‐approved opioid receptor antagonist naloxone (e.g. Narcan®) is used currently to reverse opioid‐induced respiratory depression; however, its short duration of action limits its clinical utility. Many abused opioids have a duration of action longer than naloxone and reemergence of respiratory depression is common after rescue with naloxone. Methocinnamox (MCAM) is a selective, pseudoirreversible mu opioid receptor antagonist with a long duration of action that may reverse and provide long‐term protection from the respiratory‐depressant effects of fentanyl. Previous research has shown that MCAM reverses and prevents the respiratory‐depressant effects of heroin in nonhuman primates. However, the effects of MCAM on opioid agonist‐induced respiratory depression has not been examined in other species. Moreover, its effectiveness in antagonizing the respiratory depressant effects of intravenous (i.v.) opioids, including fentanyl, has not been examined. This study compared the ability of naloxone and MCAM to reverse and protect from fentanyl‐induced respiratory depression in rats. On different occasions, male Sprague‐Dawley rats (N = 8) received 0.01, 0.032, 0.056, 0.1, or 0.178 mg/kg fentanyl i.v. and ventilatory parameters were recorded using a whole‐body plethysmograph. Fentanyl decreased respiration in a dose‐ and time‐dependent manner. Compared with respiration under control conditions, these doses of fentanyl decreased minute volume by 20–65% with 0.178 mg/kg of fentanyl producing the largest and longest decrease in minute volume. Within 1 minute after i.v. administration, naloxone (10 mg/kg) and MCAM (10 mg/kg) reversed fully the respiratory depressant effects (minute volume) of 0.178 mg/kg fentanyl. The day after antagonist administration, MCAM, but not naloxone, attenuated fully the respiratory depressant effects of 0.178 mg/kg fentanyl. This study extends to another species (rats), another route of administration (i.v.), and another mu opioid receptor agonist (fentanyl) the ability of MCAM to reverse and provide extended protection from the respiratory depressant effects of opioids. Prolonged antagonism by MCAM may provide a useful treatment option for the current opioid crisis, including toxicity from fentanyl and related highly potent analogs.Support or Funding InformationSupported by NIH (R01DA005018 and R01DA048417) and the Welch Foundation (AQ‐0039).

Investigating Opioid‐induced Respiratory Depression and Analgesia Using Larval Zebrafish

IntroductionOpioid drugs present the severe side‐effect of respiratory depression which can be lethal. In fact, every year, in North America, over 70,000 people die of opioid overdose. Our current understanding is limited due to a lack of simple and amenable animal models to study the effects of opioid drugs on the nervous system. We aim to understand respiratory depression and analgesia by opioid drugs using novel zebrafish models, allowing for high throughput drug screening and gene editing. Using these models, we will demonstrate whether respiratory depression can be reversed by serotonergic agonists, glutamatergic modulators and calcium channel activators.MethodsWe have established phenotype‐based approaches using in‐vivo zebrafish models of respiratory depression and analgesia by fentanyl, a clinically relevant opioid analgesic. To quantify respiratory depression, we measured respiratory activity by recording mandible movements in 12–14 day post‐fertilization larvae. To quantify analgesia, we measured the escape swimming response to nociceptive stimuli such as formalin and administered fentanyl to induce analgesia. To show that μ‐opioid receptors (MORs) are expressed in respiratory circuits in the brainstem, we used in‐situ hybridization for Oprm1 (mRNA of the MOR).ResultsIn comparing respiratory sensitivity to opioids between zebrafish strains, we showed that Tübingen (TU) fish did not respond to fentanyl (1μM, P=0.4240, n=9), whereas AB fish showed pronounced respiratory rate depression (54±7.7% decrease, P&lt;0.001, n=11). Zebrafish crossed between AB and TU did not show respiratory depression (P=0.104, n=7). In AB zebrafish, a dose‐dependent decrease in respiratory rate was observed, with 1μM fentanyl showing the most potent respiratory depression (P=0.01, n=11). The opioid receptor antagonist naloxone (5μM) or the selective MOR antagonist CTAP (4μM) reversed respiratory depression to baseline levels (P=0.002, n=8). Similarly, respiratory depression was reversed by the positive allosteric modulator of AMPA receptors CX614 (50μM, P=0.187, n=10) or the serotonergic 5‐HT4 agonist BIMU8 (10μM, P=0.024, n=6). To test the role of N‐type calcium channels, we showed that nefiracetam, a calcium channel activator, reversed respiratory depression by fentanyl (57±19%, P=0.002, n=9). Formalin (0.05%) significantly increased swimming velocity (320±313% of baseline, P=0.003, n=24–30), which was reduced by fentanyl (3μM, 117±151%, n=10). The effect of fentanyl was blocked by naloxone (5μM, P=0.001, n=16) and CTAP (4μM, P=0.029, n=16). MOR mRNA was expressed in the medulla where respiratory circuits are located.DiscussionOur models show that respiratory depression and analgesia by opioids can be mimicked in zebrafish larvae. The strain differences suggest a potential genetic protection against respiratory depression by opioids which can be uncovered using gene screening and knockout animals. Our novel zebrafish models are powerful models to investigate opioid‐induced respiratory depression and analgesia. Using them, we identified potential molecular targets to develop safe opioid pain therapies, such as calcium channels.Support or Funding InformationSt. Michael’s Hospital Foundation and the J.P. Bicknell Foundation Biomedical Grant

Endogenous glutamatergic inputs to the Parabrachial Nucleus/Kölliker-Fuse Complex determine respiratory rate

The Kölliker-Fuse Nucleus (KF) has been widely investigated for its contribution to “inspiratory off-switch” while more recent studies showed that activation of the Parabrachial Nucleus (PBN) shortened expiratory duration. This study used an adult, in vivo, decerebrate rabbit model to delineate the contribution of each site to inspiratory and expiratory duration through sequential block of glutamatergic excitation with the receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and d(–)-2-amino-5-phosphonopentanoic acid (AP5). Glutamatergic disfacilitation caused large increases in inspiratory and expiratory duration and minor decrease in peak phrenic activity (PPA). Hypoxia only partially reversed respiratory rate depression but PPA was increased to >200 % of control. The contribution of PBN activity to inspiratory and expiratory duration was equal while block of the KF affected inspiratory duration more than expiratory. We conclude that in the in vivo preparation respiratory rate greatly depends on PBN/KF activity, which contributes to the “inspiratory on- “and “off-switch”, but is of minor importance for the magnitude of phrenic motor output.

In vitro profiling of opioid ligands using the cAMP formation inhibition assay and the β-arrestin2 recruitment assay: No two ligands have the same profile

Previously, we showed that no two of seven opioids administered by the intracerebroventricular route had the same potency rank order for evoking antinociception, constipation and respiratory depression in rats. To gain insight at the cellular level, this study was designed to systematically investigate the activity profiles of six commonly used opioid ligands using the forskolin-stimulated cAMP assay and a β-arrestin2 recruitment assay in cultured HEK-293 cells transfected with MOP(μ), DOP(δ) or KOP(κ) receptors(-r).Morphine was a potent agonist at the MOP-r in the cAMP assay whereas it was a weak agonist at the KOP-r and DOP-r. Oxycodone had moderate efficacy and low potency at the MOP-r. Buprenorphine was a potent MOP-r and DOP-r agonist; its efficacy rank order was DOP > MOP > KOP. Fentanyl was a potent agonist at the MOP-r; its efficacy rank order was MOP > DOP > KOP. For DPDPE, its agonist efficacy was confined to the DOP-r, whereas for U69593, its efficacy rank order was KOP>> MOP.For the β-arrestin2 assay, fentanyl had full efficacy at the MOP-r whereas morphine and oxycodone were weak with insignificant efficacy at DOP and KOP receptors. Buprenorphine did not recruit β-arrestin2 at all three opioid-receptors. DPDPE and U69593 had full efficacy for β-arrestin2 recruitment to the DOP-r and KOP-r respectively.Despite the low efficacy and potency of morphine, oxycodone and buprenorphine in recruiting β-arrestin2 to the MOP-r herein, these opioids all evoked respiratory depression and constipation in rats. Together, our findings discount a key role for β-arrestin2 recruitment at the MOP-r in evoking opioid-related side-effects.

Intracerebroventricular administration of CYX-6, a potent μ-opioid receptor agonist, a δ- and κ-opioid receptor antagonist and a biased ligand at μ, δ & κ-opioid receptors, evokes antinociception with minimal constipation and respiratory depression in rats in contrast to morphine

Mu opioid receptor (MOPr) agonists are thought to produce analgesia via modulation of G-protein-coupled intracellular signalling pathways whereas the β-arrestin2 pathway is proposed to mediate opioid-related adverse effects. Here, we report the antinociception, constipation and respiratory depressant profile of CYX-6, a potent MOPr agonist that is also a delta and a kappa opioid receptor (DOPr/KOPr) antagonist and that lacks β-arrestin2 recruitment at each of the MOPr, DOPr and the KOPr. In anaesthetised male Sprague Dawley rats, an intracerebroventricular (i.c.v.) guide cannula was stereotaxically implanted. After 5–7 days post-surgical recovery, rats received a single i.c.v. bolus dose of CYX-6 (3–30 nmol), morphine (100 nmol) or vehicle. Antinociception was assessed using the warm water tail flick test (52.5 ± 0.5 °C). Constipation was assessed using the charcoal meal gut motility test and the castor oil-induced diarrhoea test. Respiratory depression was measured by whole-body plethysmography in awake, freely moving animals, upon exposure to a hypercapnic gas mixture (8% CO2, 21% O2 and 71% N2). The intrinsic pharmacology of CYX-6 given by the i.c.v. route in rats showed that it produced dose-dependent antinociception. It also produced respiratory stimulation rather than depression and it had a minimal effect on intestinal motility in contrast to the positive control, morphine. CYX-6 is an endomorphin-2 analogue that dissociates antinociception from constipation and respiratory depression in rats. Our findings provide useful insight to inform the discovery and development of novel opioid analgesics with a superior tolerability profile compared with morphine.

Kölliker-Fuse/Parabrachial complex mu opioid receptors contribute to fentanyl-induced apnea and respiratory rate depression

Overdoses caused by the opioid agonist fentanyl have increased exponentially in recent years. Identifying mechanisms to counter progression to fatal respiratory apnea during opioid overdose is desirable, but difficult to study in vivo. The pontine Kölliker-Fuse/Parabrachial complex (KF/PB) provides respiratory drive and contains opioid-sensitive neurons. The contribution of the KF/PB complex to fentanyl-induced apnea was investigated using the in situ arterially perfused preparation of rat. Systemic application of fentanyl resulted in concentration-dependent respiratory disturbances. At low concentrations, respiratory rate slowed and subsequently transitioned to an apneustic-like, 2-phase pattern. Higher concentrations caused prolonged apnea, interrupted by occasional apneustic-like bursts. Application of CTAP, a selective mu opioid receptor antagonist, directly into the KF/PB complex reversed and prevented fentanyl-induced apnea by increasing the frequency of apneustic-like bursting. These results demonstrate that countering opioid effects in the KF/PB complex is sufficient to restore phasic respiratory output at a rate similar to pre-fentanyl conditions, which could be beneficial in opioid overdose.

Antagonistic Effects of Tetramethylpyrazine on Hypoxic Respiratory Depression in Rats.

Objective Tetramethylpyrazine (TMP) is an alkaloid extracted from the root and stem of the traditional Chinese herbal medicine called Chuanxiong. The present study aims to study the effects of TMP on hypoxic respiratory depression in rats. Materials and methods. The effects of TMP on respiratory responses of rats induced by hypoxia were observed by diaphragm electromyogram (EMG) recording. The effects of TMP on the protein expression of FOS and acid sensing ion channel1a (ASIC1a) in the brainstem induced by hypoxia were investigated by immunohistochemistry. Results The respiration of rats was first excited and then depressed during hypoxia treatment, while TMP pretreatment could significantly antagonize the respiratory depression induced by hypoxia (P < 0.01). Hypoxia obviously induced the protein expression of FOS (P < 0.01) and ASIC1a(P < 0.05) in the brainstem, which can be also significantly inhibited by TMP pretreatment. Conclusions TMP has protective effects on hypoxic respiratory depression, and the mechanisms might be concerned with its downregulation of FOS and ASIC1a in the brainstem induced by hypoxia.

A high-throughput bioanalytical assay to support pharmacokinetic interaction study of oxycodone and diazepam in Sprague Dawley rats.

Benzodiazepines potentiate respiratory depression when combined with an opioid leading the U.S Food and Drug Administration (FDA) to recommend updating the labels of these products with a boxed warning for respiratory depression with co-use. Potential respiratory depression upon co-administration of opioids with some psychotropic drugs is not well understood. The FDA is currently investigating various psychotropic drug interactions with the commonly used opioid, oxycodone, in a rat model assessing respiratory depression. Pharmacokinetic and/or pharmacodynamic (PK/PD) interaction between oxycodone and diazepam was evaluated in a positive control arm of these experiments. Understanding the systemic exposure of these drugs alone and in combination exposures was used to identify PK/PD interactions. The authors developed a simple, high throughput liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the simultaneous determination of oxycodone and diazepam in rat plasma. Sample preparation was performed in 96-well protein precipitation plates using acetonitrile. Processed samples were analyzed using a C18 column with a gradient mobile phase composed of 2 mM aqueous ammonium formate with 0.1% formic acid and acetonitrile. A Thermo TSQ Quantum Ultra AM triple quadrupole mass spectrometer with multiple reaction monitoring (MRM) mode was used to acquire data. The method was validated for selectivity, specificity, linearity, precision and accuracy, dilution integrity and stability. The validated LC-MS/MS assay was utilized for quantifying oxycodone and diazepam in concomitantly treated Sprague Dawley (SD) rats.

Differential impact of two critical respiratory centres in opioid-induced respiratory depression in awake mice.

The main cause of death from opioid overdose is respiratory depression due to the activation of µ-opioid receptors (MORs). We conditionally deleted MORs from neurons in two key areas of the brainstem respiratory circuitry (the Kölliker-Fuse nucleus (KF) and pre-Bötzinger complex (preBötC)) to determine their role in opioid-induced respiratory disturbances in adult, awake mice. Deletion of MORs from KF neurons attenuated respiratory rate depression at all doses of morphine. Deletion of MORs from preBötC neurons attenuated rate depression at the low dose, but had no effect on rate following high doses of morphine. Instead, high doses of morphine increased the occurrence of apnoeas. The results indicate that opioids affect distributed key areas of the respiratory network in a dose-dependent manner and countering the respiratory effects of high dose opioids via the KF may be an effective approach to combat overdose. The primary cause of death from opioid overdose is respiratory failure. High doses of opioids cause severe rate depression and increased risk of fatal apnoea, which correlate with increasing irregularities in breathing pattern. µ-Opioid receptors (MORs) are widely distributed throughout the brainstem respiratory network, but the mechanisms underlying respiratory depression are poorly understood. The medullary pre-Bötzinger complex (preBötC) and the pontine Kölliker-Fuse nucleus (KF) are considered critical for inducing opioid-related respiratory disturbances. We used a conditional knockout approach to investigate the roles and relative contribution of MORs in KF and preBötC neurons in opioid-induced respiratory depression in awake adult mice. The results revealed dose-dependent and region-specific opioid effects on the control of both respiratory rate and pattern. Respiratory depression induced by an anti-nociceptive dose of morphine was significantly attenuated following deletion of MORs from either the KF or the preBötC, suggesting cumulative network effects on respiratory rate control at low opioid doses. Deletion of MORs from KF neurons also relieved rate depression at near-maximal respiratory depressant doses of morphine. Meanwhile, deletion of MORs from the preBötC had no effect on rate following administration of high doses of morphine. Instead, a severe ataxic breathing pattern emerged with many apnoeas. We conclude that opioids affect distributed areas of the respiratory network and opioid-induced respiratory depression cannot be attributed to only one area in isolation. However, countering the effects of near maximal respiratory depressant doses of opioids in the KF may be a powerful approach to combat opioid overdose.

Electrocortical changes associating sedation and respiratory depression by the opioid analgesic fentanyl

Opioid drugs are the mainstay of pain management but present the side-effect of respiratory depression that can be lethal with overdose. In addition to their respiratory effect, opioids also induce a profound sedative state and produce electrocortical features characteristic of a state of reduced brain arousal, similar to anaesthesia or sleep. In such states, respiratory activity depends more on the integrity of the brainstem respiratory network than it does during wakefulness. Accordingly, we propose that sedation by fentanyl induces specific electrocortical changes consistent with reduced brain arousal, and that the magnitude of respiratory depression is associated with distinct electrocortical changes. To these aims, we determined the effects of systemic injections of fentanyl (dosage 100 µg ·kg) versus control on electrocortical and respiratory activities of freely-behaving rats. We found that fentanyl induced electrocortical changes that differed from those observed in sleep or wakefulness. Fentanyl increased δ (1–3 Hz) frequency power (P < 0.001), but reduced α (7.5–13.5 Hz) and β2 (20–30 Hz) powers (P = 0.012 and P < 0.001, respectively), when compared to wakefulness. Interestingly, respiratory rate depression by fentanyl was significantly correlated with increased θ power (R = 0.61, P < 0.001), therefore showing a clear association between electrocortical activity and the magnitude of respiratory rate depression. Overall, we provide new evidence linking specific electrocortical changes to the severity of respiratory depression by opioids, which highlights the importance of considering the cortical and subcortical effects of opioids in addition to their impacts on breathing when evaluating opioid-induced respiratory depression.

Solving the Opioid Crisis: Respiratory Depression by Opioids as Critical End Point

Solving the Opioid Crisis: Respiratory Depression by Opioids as Critical End Point

Synergistic interaction between the agonism of cebranopadol at nociceptin/orphanin FQ and classical opioid receptors in the rat spinal nerve ligation model.

Cebranopadol (trans‐6′‐fluoro‐4′,9′‐dihydro‐N,N‐dimethyl‐4‐phenyl‐spiro[cyclohexane‐1,1′(3′H)‐pyrano[3,4‐b]indol]‐4‐amine) is a novel analgesic nociceptin/orphanin FQ opioid peptide (NOP) and classical opioid receptor (MOP, DOP, and KOP) agonist with highly efficacious and potent activity in a broad range of rodent models of nociceptive, inflammatory, and neuropathic pain as well as limited opioid‐type side effects such as respiratory depression. This study was designed to explore contribution and interaction of NOP and classical opioid receptor agonist components to cebranopadol analgesia in the rat spinal nerve ligation (SNL) model. Assessing antihypersensitive activity in SNL rats intraperitoneal (IP) administration of cebranopadol resulted in ED 50 values of 3.3 and 3.58 μg/kg in two independent experiments. Pretreatment (IP) with J‐113397 (4.64 mg/kg) a selective antagonist for the NOP receptor or naloxone (1 mg/kg), naltrindole (10 mg/kg), or nor‐BNI (10 mg/kg), selective antagonists for MOP, DOP, and KOP receptors, yielded ED 50 values of 14.1, 16.9, 17.3, and 15 μg/kg, respectively. This 4‐5 fold rightward shift of the dose‐response curves suggested agonistic contribution of all four receptors to the analgesic activity of cebranopadol. Combined pretreatment with a mixture of the antagonists for the three classical opioid receptors resulted in an 18‐fold potency shift with an ED 50 of 65.5 μg/kg. The concept of dose equivalence was used to calculate the expected additive effects of the parent compound for NOP and opioid receptor contribution and to compare them with the observed effects, respectively. This analysis revealed a statistically significant difference between the expected additive and the observed effects suggesting intrinsic synergistic analgesic interaction of the NOP and the classical opioid receptor components of cebranopadol. Together with the observation of limited respiratory depression in rats and humans the synergistic interaction of NOP and classical opioid receptor components in analgesia described in the current study may contribute to the favorable therapeutic index of cebranopadol observed in clinical trials.