Opioid Receptor Research Articles

Corticotropin-releasing hormone deficiency results in impaired analgesic response during CFA-induced inflammation

PurposeCorticotropin-releasing hormone (CRH) plays an important role in relief of pain by releasing analgesia-associated molecules in several inflammatory states. During inflammation, peripheral CRH acts on cells of the immune system to stimulate the local expression of proopiomelanocortin (POMC) and the production of β-endorphin, which in turn binds to opioid receptors on sensory neurons to produce antinociception. In the present study, we further investigated the role of endogenous CRH in inflammatory pain by determining the effects of Crh-deficiency on this process.MethodsFor this purpose, we used Crh-deficient (Crh-/-) mice and their wildtype (Crh + / +) littermates in the CFA (Complete Freund’s Adjuvant)-induced inflammatory pain model. Pain thresholds were evaluated with the Hargreaves apparatus.ResultsOur experiments showed that Crh deficiency led to increased pain response, which was associated with decreased POMC mRNA levels in locally inflamed paws of these mice. Furthermore, Crh-/- mice had higher paw edema than Crh + / + mice. Histological evaluation of inflamed paw tissues revealed increased inflammatory response in Crh-/- mice. Protein levels of proinflammatory cytokines, such as IL-6, TNF-α, and IL-1β, were higher in inflamed tissue of Crh-/- mice compared to wildtype mice. Corticosterone replacement increased the pain threshold of Crh-/- mice, restored their paw volume to the levels of wildtype mice, and significantly reduced their proinflammatory cytokine levels. Furthermore, glucocorticoid administration significantly increased POMC mRNA expression in the inflamed paw.ConclusionOur data suggest that genetic deficiency of CRH is associated with increased pain. This effect is likely attributable to the accompanying glucocorticoid insufficiency and is in part mediated by opioids expressed locally.

Pharmacological potential of 4-dimethylamino chalcone against acute and neuropathic pain in mice.

This work investigated the acute antinociceptive effect of a synthetic chalcone, 4-dimethylamino chalcone (DMAC), as well as its effects on vincristine-induced peripheral neuropathy (VIPN) in mice. The inhibitory activity of myeloperoxidase was assessed by measuring HOCl formation. Formalin and hot plate tests were used to study the acute antinociceptive effect of DMAC. VIPN was induced through the administration of vincristine sulphate (0.1 mg/kg, i.p., 14 days). Then, DMSO, DMAC (10 or 30 mg/kg; i.p.), or pregabalin (10 mg/kg, i.p.) were administered for 14 consecutive days. Thermal hyperalgesia and mechanical allodynia were evaluated before and after VIPN induction and on days 1, 3, 7, and 14 of treatment. Neurodegeneration and neuroinflammation were assessed through immunohistochemistry for NF200, iNOS, and arginase-1 within the sciatic nerve. DMAC inhibited myeloperoxidase activity in vitro and presented an acute antinociceptive effect in both formalin and hot plate tests, with the involvement of muscarinic and opioid receptors. Treatment with 30 mg/kg of DMAC significantly attenuated thermal hyperalgesia and mechanical allodynia and prevented macrophage proinflammatory polarisation in VIPN mice. Our results show that DMAC, acting through different mechanisms, effectively attenuates VIPN.

Synergistic Antinociceptive Effect of β-Caryophyllene Oxide in Combination with Paracetamol, and the Corresponding Gastroprotective Activity.

Pain is the most frequent symptom of disease. In treating pain, a lower incidence of adverse effects is found for paracetamol versus other non-steroidal anti-inflammatory drugs. Nevertheless, paracetamol can trigger side effects when taken regularly. Combined therapy is a common way of lowering the dose of a drug and thus of reducing adverse reactions. Since β-caryophyllene oxide (a natural bicyclic sesquiterpene) is known to produce an analgesic effect, this study aimed to determine the anti-nociceptive and gastroprotective activity of administering the combination of paracetamol plus β-caryophyllene oxide to CD1 mice. Anti-nociception was evaluated with the formalin model and gastroprotection with the model of ethanol-induced gastric lesions. According to the isobolographic analysis, the anti-nociceptive interaction of paracetamol and β-caryophyllene oxide was synergistic. Various pain-related pathways were explored for their possible participation in the mechanism of action of the anti-nociceptive effect of β-caryophyllene oxide, finding that NO, opioid receptors, serotonin receptors, and K+ATP channels are not involved. The combined treatment showed gastroprotective activity against ethanol-induced gastric damage. Hence, the synergistic anti-nociceptive effect of combining paracetamol with β-caryophyllene oxide could be advantageous for the management of inflammatory pain, and the gastroprotective activity should help to protect against the adverse effects of chronic use.

The use of deep eutectic solvent-liquid phase microextraction as green analytical sample treatment for the analysis of drugs in urine

In this paper, a natural deep eutectic solvent (NADES)-based liquid phase microextraction (LPME) of 20 pharmaceuticals from urine, employing liquid chromatography tandem mass spectrometry (LC-MS/MS) for precise quantification, has been developed. The chromatographic gradient was accurately evaluated to ensure the effective separation of tramadol, an opioid receptor agonist, and o-desmethylvenlafaxine, one active metabolite of the antidepressant venlafaxine, both compounds sharing identical molecular mass for the precursor ion (Q1: 264.2 m/z) and fragmentation patterns (Quantifier ion: 58.2 m/z; qualifier ion: 246.0 m/z). NADES consisted of menthol-octanoic acid in a molar ratio of 1:2. Using the appropriate extraction conditions, the developed methodology provided appropriate LOQ values, ranging from 15 to 208 ng L–1, with reasonable RSD values (below 11 %), recoveries ranging from 64 to 120 % and preconcentration factors up to 66.6 folds. Application of the developed method to the analysis of urine samples served to highlight its usefulness. An AGREEprep score of 0.64 was obtained despite the demand for substantial sample volumes and the use of LC-MS/MS for analyte determination.

Safe Management of Adverse Effects Associated with Prescription Opioids in the Palliative Care Population: A Narrative Review.

In the palliative care population, prescription opioids are often considered viable pain relief options. However, in this complex patient population, the adverse effects of opioid medications should be identified and managed without delay. Common adverse effects can include constipation, nausea, somnolence, dizziness, vomiting, and pruritus. Less common adverse effects can include potentially lethal respiratory depression and cardiovascular effects. Critical aspects of safe opioid prescribing are recognition of side effects and knowledge of effective management strategies; prompt management is necessary for uninterrupted pain relief. Most complications are managed with general approaches such as dose reduction, opioid rotation, alternate routes of administration, and symptomatic management. The only opioid-induced complication for which US Food and Drug Administration-approved treatments currently exist is constipation. Treating laxative-refractory opioid-induced constipation (OIC) with peripherally acting mu-opioid receptor antagonists (PAMORAs), which block gastrointestinal opioid receptors, can restore gastrointestinal motility and fluid secretion. This narrative review discusses key complications of prescription opioid treatment and their management in the palliative care setting.

Bioassay-guided isolation of anti-inflammatory and antinociceptive metabolites among three Moroccan Juniperus leaves extract supported with in vitro enzyme inhibitory assays

Ethnopharmacological relevanceHerbs of the genus Juniperus (family Cupressaceae) have been commonly used in ancestral folk medicine known as “Al'Araar” for treatment of rheumatism, diabetes, inflammation, pain, and fever. Bioassay-guided isolation of bioactives from medicinal plants is recognized as a potential approach for the discovery of novel drug candidates. In particular, non-addictive painkillers are of special interest among herbal phytochemicals. Aim of the studyThe current study aimed to assess the safety of J. thurifera, J. phoenicea, and J. oxycedrus aqueous extracts in oral treatments; validating the traditionally reported anti-inflammatory and analgesic effects. Further phytochemical investigations, especially for the most bioactive species, may lead to isolation of bioactive metabolites responsible for such bioactivities supported with in vitro enzyme inhibition assays. Materials and methodsFirstly, the acute toxicity study was investigated following the OECD Guidelines. Then, the antinociceptive, and anti-inflammatory bioactivities were evaluated based on chemical and mechanical trauma assays and investigated their underlying mechanisms. The most active J. thurifera n-butanol fraction was subjected to chromatographic studies for isolating the major anti-inflammatory metabolites. Moreover, several enzymatic inhibition assays (e.g., 5-lipoxygenase, protease, elastase, collagenase, and tyrosinase) were assessed for the crude extracts and isolated compounds. ResultsThe results showed that acute oral administration of the extracts (300–500 mg/kg, p. o.) inhibited both mechanically and chemically triggered inflammatory edema in mice (up to 70% in case of J. thurifera) with a dose-dependent antinociceptive (tail flick) and anti-inflammatory pain (formalin assay) activities. This effect was partially mediated by naloxone inhibition of the opioid receptor (2 mg/kg, i. p.). In addition, 3-methoxy gallic acid (1), quercetin (2), kaempferol (3), and ellagic acid (4) were successfully identified being involved most likely in J. thurifera extract bioactivities. Nevertheless, quercetin was found to be the most potent against 5-LOX, tyrosinase, and protease with IC50 of 1.52 ± 0.01, 192.90 ± 6.20, and 399 ± 9.05 μM, respectively. ConclusionJ. thurifera extract with its major metabolites are prospective drug candidates for inflammatory pain supported with inhibition of inflammatory enzymes. Interestingly, antagonism of opioid and non-opioid receptors is potentially involved.

Xylazine is an agonist at kappa opioid receptors and exhibits sex-specific responses to opioid antagonism

Xylazine is in the unregulated drug supply at increasing rates, usually combined with fentanyl, necessitating understanding of its pharmacology. Despite commentary from politicians, and public health officials, it is unknown how xylazine impacts naloxone efficacy, and. few studies have examined it alone. Here, we examine the impact of xylazine alone and in combination with fentanyl on several behaviors in mice. Surprisingly, naloxone precipitates withdrawal from xylazine and fentanyl/xylazine coadministration, with enhanced sensitivity in females. Further, xylazine is a full agonist at kappa opioid receptors, a potential mechanism for its naloxone sensitivity. Finally, we demonstrate surprising effects of xylazine to kappa opioid antagonism, which are relevant for public health considerations. These data address an ongoing health crisis and will help inform critical policy and healthcare decisions. One-sentence summaryWe present surprising new insights into xylazine and fentanyl pharmacology with immediate implications for clinical practice and frontline public health.

Delta opioid peptide [D-ala2, D-leu5]-Enkephalin’s ability to enhance mitophagy via TRPV4 to relieve ischemia/reperfusion injury in brain microvascular endothelial cells

BackgroundLocal brain tissue can suffer from ischaemia/reperfusion (I/R) injury, which lead to vascular endothelial damage. The peptide δ opioid receptor (δOR) agonist [D-ala2, D-leu5]-Enkephalin (DADLE) can reduce apoptosis caused by...

Topical naltrexone increases aquaporin 5 production in the lacrimal gland and restores tear production in diabetic rats.

Diabetes mellitus is a prevalent disease that is often accompanied by ocular surface abnormalities including delayed epithelial wound healing and decreased corneal sensitivity. The impact of diabetes on the lacrimal functional unit (LFU) and the structures responsible for maintaining tear homeostasis, is not completely known. It has been shown that the Opioid Growth Factor Receptor (OGFr), and its ligand, Opioid Growth Factor (OGF), is dysregulated in the ocular surface of diabetic rats leading to overproduction of the inhibitory growth peptide OGF. The opioid antagonist naltrexone hydrochloride (NTX) blocks the OGF-OGFr pathway, and complete blockade following systemic or topical treatment with NTX restores the rate of re-epithelialization of corneal epithelial wounds, normalizes corneal sensitivity, and reverses dry eye in diabetic animal models. These effects occur rapidly and within days of initiating treatment. The present study was designed to understand mechanisms related to the fast reversal (<5 days) of dry eye by NTX in type 1 diabetes (T1D) by investigating dysregulation of the LFU. The approach involved examination of the morphology of the LFU before and after NTX treatment. Male and female adult Sprague-Dawley rats were rendered hyperglycemic with streptozotocin, and after 6 weeks rats were considered to be a T1D model. Rats received topical NTX twice daily to one eye for 10 days. During the period of treatment, tear production and corneal sensitivity were recorded. On day 11, animals were euthanized and orbital tissues including conjunctiva, eyelids, and lacrimal glands, were removed and processed for histologic examination including immunohistochemistry. Male and female T1D rats had significantly decreased tear production and corneal insensitivity, significantly decreased number and size of lacrimal gland acini, decreased expression of aquaporin-5 (AQP5) protein and decreased goblet cell size. Thus, 10 days of NTX treatment restored tear production and corneal sensitivity to normal values, increased AQP5 expression, and restored the surface area of goblet cells to normal. NTX had no effect on the number of lacrimal gland acini or the number of conjunctival goblet cells. In summary, blockade of the OGF-OGFr pathway with NTX reversed corneal and lacrimal gland complications and restored some components of tear homeostasis confirming the efficacy of topical NTX as a treatment for ocular defects in diabetes.

Endomorphin-2 and oxycodone paradoxically potentiate the exercise pressor reflex via acid-sensing ion channel 3 (ASIC3) in decerebrated rat

The exercise pressor reflex is evoked by the stimulation of group III and IV muscle afferents in response to muscle contractions. When the contracting muscles are freely perfused, acid-sensing ion channel 3 (ASIC3) on the endings of group IV afferents appear to play a minor role in evoking the exercise pressor reflex. We recently showed in isolated dorsal root ganglion (DRG) neurons innervating the gastrocnemius muscles that two μ opioid receptor agonists, namely endomorphin-2, and oxycodone, potentiated the sustained inward ASIC3 current evoked by acidic saline (pH 6.0). This in vitro finding prompted us to investigate whether endomorphin-2 and oxycodone, when infused into the arterial supply of freely perfused contracting hindlimb muscles, could potentiate the exercise pressor reflex. We found that infusing endomorphin-2 and naloxone (a μ opioid receptor antagonist) into the superficial epigastric artery of decerebrated unanesthetized rats potentiated the peak and integrated pressor responses to static contraction of the triceps surae muscles. The opioid-induced potentiation of the pressor responses to contraction were prevented by infusion of APETx2, an ASIC3 antagonist. Specifically, the peak pressor response to contraction averaged 19.3 ± 5.6 mmHg for control (n=10), 27.2 ± 8.1 mmHg after naloxone and endomorphin-2 infusion (n=10), and 20 ± 8 mmHg after APETx2 and endomorphin-2 infusion (n=10). Infusion of endomorphin-2 and naloxone did not potentiate the pressor responses to contraction in ASIC3 knockout rats (n=6), averaging 21.5 ± 7.7 mmHg for control (n=6), 20 ± 6.2 mmHg after naloxone and endomorphin-2 infusion (n=6), and 19.8 ± 6.9 mmHg after APETx2 and endomorphin-2 infusion (n=4). Similar findings were observed when oxycodone was substituted for endomorphin-2. Specifically, the peak pressor response to contraction averaged 24.3 ± 8.9 mmHg for control (n=10), 33.9 ± 10.6 mmHg after naloxone and oxycodone were infused (n=10), and 26.8 ± 8.1 mmHg after APETx2 and oxycodone were infused (n=10). Altogether, our data strongly suggest that ASIC3 stimulation by mu opioid agonists can potentiate the exercise pressor reflex, provided that mu opioid receptors on group III and IV afferents are not operative. (Supported by HL156594 and HL 156513). HL156594 and HL 156513. 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.

Role of Opioid Mu1-Recptors (μ1Rs) in The Inhalation of Carfentanil (CRF) Aerosol-Induced Respiratory Disorder in Awake Rats

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.

The Role of OPRM1 in the Neonatal Autoresuscitation Reflex and Sudden Infant Death Syndrome

Opioid receptors are widely expressed throughout the brain and integral in nociception as well as homeostatic regulation of autonomic processes including respiration through modulation of respiratory rhythmogenesis and chemosensory responses. Perturbations to homeostatic mu opioid receptor (MOR) signaling via opiate overdose results in respiratory depression, aberrant breathing, and often death. With the rise of opioid use over the last two decades, many infants are being diagnosed with Neonatal Abstinence Syndrome (NAS), a congenital cardio-respiratory disorder resulting from prenatal exposure to opioids. Infants born with NAS have a fourfold increased risk for Sudden Infant Death Syndrome (SIDS). SIDS and related Sudden Unexpected Infant Death takes the lives of approximately 3,400 infants annually under the age of one and its etiology remains mostly unknown. Currently SIDS is explained via the triple risk model that proposes that SIDS occurs in a vulnerable infant during a critical developmental period when triggered by an external stressor, converging on the failure of the neonate autoresuscitation reflex in most cases. Recently a study identified an association between a mutation in the OPRM1 gene encoding MORs and SIDS cases. However, how this gene and perturbations in its native function modulates respiratory regulation are poorly understood. We hypothesize that native OPRM1 function is integral to protective neonate respiratory reflexes and that perturbation of native OPRM1 signaling negatively affects the neonatal autoresuscitation reflex. Male and female OPRM1 loss of function mice were assayed for their autoresuscitation reflex on our novel testbed, Looper. Mice were exposed to repeated bouts of anoxia and allowed to autoresuscitate until death occurred. Preliminary data suggest that loss of OPRM1 function results in a higher mortality rate on the autoresuscitation assay with OPRM1 KO mice surviving significantly less anoxic challenges compared to wild type controls. No sex differences were observed between male and female survival for all genotype groups. Results indicate that perturbation of the native function of OPRM1 produces a vulnerability to autoresuscitation dysfunction and mitigated survival. Our data demonstrate the first functional assessment of OPRM1 in neonatal cardiovascular function and autoresuscitation and reveal an important risk factor that may be implicated in perpetuating SIDS and NAS phenotypes. Additional analyses of distinct parameters from respiratory trace data for OPRM1 loss of function mice will aid in a deeper, mechanistic understanding of features implicated in autoresuscitation dysfunction. Future studies aim to characterize specific splice variants of OPRM1, known to produce neomorphic function and model the SIDS associated VUS, to further characterize the implication of OPRM1 and its variants in congenital respiratory diseases. Funding sources NIH/NHLBI R01HLN161142-01 and R01HL130249-07. 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.

HYPERTENSION AND DEXTROMETHORPHAN POISONING IN PATIENTS AGED BETWEEN 0 TO 18 YEARS- A 7 YEAR RETROSPECTIVE CASE SERIES

Objective: Coughing is a common symptom in the pediatric population. The wide variety of preparations available in pharmacies allows easy access to over-the-counter use. Dextromethorphan inhibits the cough reflex through an antagonistic mechanism at the NMDA-glutamate opioid receptor. Its effects are similar to those of drugs of abuse (phencyclidine and ketamine). In high doses, it produces tachycardia, hypertension, altered consciousness to coma, and neurological symptoms (agitation, hallucinations, seizures). According to the literature, patients between the ages of 12 and 20 years account for 50% of all dextromethorphan poisoning emergencies. The aim of our study is to determine the presence of arterial hypertension, its characteristics and the course of its development in dextromethorphan intoxication. Design and method: We present a series of 18 cases of acute dextromethorphan poisoning that were admitted to the Pediatric Antitoxic Center in Bucharest between January 2017 and November 2023. Results: The age range in the series of 18 patients is from 1 to 17 years. Voluntary intoxication was recorded in 13 cases (72.22%) Hypertension was noted in 15 cases (83.33%). Hypertension was present at the onset of symptoms. The poisoning severity score(PSS) showed that most of the cases were moderate (77.77%) Conclusions: Hypertension is a common symptom of dextromethorphan intoxication in patients between 0 and 18 years of age and may be accompanied by palpitations and precordialgia. It is mild to moderate in severity and does not require any specific treatment, as general management of the intoxication is sufficient. Severe cases or death are a rare event, while moderate cases are the most common.

Exploring Serotonergic mechanisms in Opioid Induced Respiratory Depression

Opioids profoundly depress breathing which can be fatal in the case of overdose. Despite growing knowledge of the pontine and medullary respiratory control circuits that are inhibited by opioids, the circuit and cellular mechanisms of opioid induced respiratory depression are not fully understood. Serotonin neurons located in the caudal medullary raphe modulate breathing and possess mu opioid receptors. Therefore, we hypothesized that serotonin-producing raphe neurons contribute to opioid induced respiratory depression. To test this, we employed to experimental approaches: 1) arterially perfused in situ working heart brainstem preparations and 2) ex vivo acute brain slice electrophysiology to identify and characterize serotonin neuron inputs onto brainstem respiratory neurons. To specifically activate serotonin neurons, we used ePet-Cre::Ai32 mice which Cre-dependently express channelrhodopsin-2, a light-activated cation channel, in serotonin neurons. At the circuit level, using the in situ preparation, we found that activation of caudal medullary raphe serotonin neurons increased respiratory rate by decreasing inspiratory and expiratory duration. Fentanyl application blocked the respiratory effects induced by serotonin neuron activation which were subsequently restored using naloxone. These results suggest serotonin neurons in the caudal medullary raphe are inhibited by opioids. To characterize the serotonin-mediated mechanisms at the cellular level, we made acute brain slices containing dorsal raphe neurons and lateral parabrachial neurons from ePet-Cre::Ai32 mice. We verified that optical stimulation resulted in action potentials and serotonin release from dorsal raphe serotonin neurons. Optically evoked serotonin release caused 5HT1A receptor-mediated outward currents in dorsal raphe serotonin neurons. To locate and characterize serotonin neuron inputs onto lateral parabrachial neurons, we performed whole-cell voltage clamp recordings from lateral parabrachial neurons and delivered paired light pulses (10 ms duration, 50 ms interval). We observed optically evoked inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA-A receptor antagonist gabazine (1 μM). Optically evoked IPSCs had long latency (&gt; 5 ms) and high jitter (&gt; 1 ms), indicating stimulation of serotonin terminals resulted in polysynaptic GABA release onto lateral parabrachial neurons. Future work will examine the influence of opioids on this raphe-parabrachial circuit. Uncovering the role of serotonin neurons in opioid induced respiratory depression may reveal targets for restoring breathing in the presence of opioids or other conditions where life-threatening apneas occur. This work was supported by the National Institutes of Health (NIDA) R01DA047978-05 grant to ESL. 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.

Navacaprant, a Novel Kappa Opioid Receptor Antagonist for Major Depressive Disorder: A Randomized Placebo-Controlled Phase 2 Trial

Navacaprant, a Novel Kappa Opioid Receptor Antagonist for Major Depressive Disorder: A Randomized Placebo-Controlled Phase 2 Trial

Naltrindole attenuates irreversible Hypercontracture during Myocardial Ischemia by a Novel Mechanism and elicits robust cardioprotective effects in Myocardial Ischemia/Reperfusion Injury

Introduction: Previous studies have shown that naltrindole (NTI) produced robust cardioprotective effects (e.g.~80% reduction of infarct size) in both ex-vivo (2.5-5μM) and in-vivo (3.75-7.5 mg/kg) rat myocardial ischemia-reperfusion (MIR) injury models. We recently showed that NTI 100-200μM attenuated phorbol 12-myristate 13-acetate (PMA) induced polymorphonuclear leukocytes (PMNs) superoxide (SO) release by a novel mechanism of action (MOA) devoid of opioid receptors. We hypothesize NTI’s novel MOA is a reduction of intracellular calcium (Ca2+). To test this hypothesis, we conducted PMN SO release and isolated perfused MIR assays using KB-R7943(KB). KB is known to reduce intracellular Ca2+ via inhibition of reverse mode Na+/Ca2+ exchanger and therefore should attenuate PMN SO release, myocardial ischemic hypercontracture, infarct size, and left ventricular end diastolic pressure (LVEDP). Methods: PMNs and hearts isolated from anesthetized male Sprague Dawley (SD) rats (~400g SO release assay, ~300g for perfused hearts). PMN SO release was stimulated using PMA (100nM) ± NTI 10-200 μM, KB 5-20 μM, or vehicle (0.5% DMSO; controls) and measured spectrophotometrically as a change in absorbance @ 550nm over 390 secs and cell viability was determined using 0.2% Trypan blue exclusion. We then infused NTI 1.25-5μM, KB 10-20μM, or naloxone (NX; negative control) 10μM for 5 min into perfused hearts, just prior to global I (30min)/R(45min). Cardiac LV function was measured via a pressure transducer and infarct size using 1% triphenyltetrazolium chloride staining in MIR assay. Data were analyzed using ANOVA and Fischers post-hoc analysis, with p&lt;0.05 considered statistically significant. Results: PMN SO release assay: KB 10 μM, KB 20 μM, NTI 100 μM (~3.75 mg/kg), and NTI 200 μM(~7.5 mg/kg) significantly attenuated PMA induced PMN SO release by ~40% (KB 10μM, n=7, p&lt;0.05), ~50% ((KB 20μM, n=7, p&lt;0.05), ~30% (NTI 100 μM, n=7, p&lt;0.05), 75% (NTI 200μM, n=7, p&lt;0.05) respectively when compared to untreated control (n=10). MIR assay: NTI 5μM, 2.5μM, 1.25μM and KB 20 μM reduced infarct size (4.8±3%, n=5, p&lt;0.05), (6.8±0.8%, n=6, p&lt;0.05), (12.2±3%, n=5), and (2.9±2%, n=5, p&lt;0.05) respectively compared to vehicle (14.1±2%, n=9) and NX 10μM controls (18±3%, n=3). NTI 5 μM and KB 20 μM had significantly improved LVEDP to (32.4±11%, n=5, p&lt;0.05) and (22.8±4%, n=5, p&lt;0.05) respectively compared to vehicle control (72.4±6%, n=9) and NX 10μM controls(54.2±8%, n=3) Conclusion: NTI and KB showed concentration-dependent effects on reducing PMN SO release. Both NTI (2.5μM, 5μM) and KB (20μM) reduced infarct size and improved LVEDP. The novel MOA of NTI is most likely mediated via a reduction in intracellular Ca2+ and subsequent hypercontracture during myocardial ischemia. The infarct reducing effect of NTI can benefit patients who will undergo elective PCI, CABG, and transplants. Funding: This study was funded by the Philadelphia College of Osteopathic Medicine, Department of Biomedical Sciences, Division of Research, and the Center for Chronic Diseases of Aging and Young Therapeutics, LLC. 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.

Central Inhibition of Antidiuretic Hormone by the Kappa Opioid Receptor Agonist Nalfurafine Prevents and Reverses Loop Diuretic Resistance

PURPOSE: Diuretic resistance is a failure to achieve a therapeutically desired increase in the renal excretion of sodium and water and reduction in edema despite a full dose of a loop diuretic. High levels of antidiuretic hormone (ADH) have been associated with reduced urine output in heart failure patients. However, ADH’s contribution to the development of diuretic resistance is not known. Assuming that ADH may be released in response to the diuresis produced by loop diuretics, we tested the premise that co-treatment of rats with nalfurafine, a centrally acting kappa opioid (KOR) agonist that inhibits the release of ADH would prevent and/or reverse diuretic resistance to the loop diuretic furosemide. METHODS: Changes in 5-hr urine output (metabolic cages; no water access during collection), urinary Na/K excretion, and osmolality were measured in male Sprague-Dawley rats administered twice-daily i.p. injections (9:00am, 2:00pm; n=6/group) for 11 days with 1) furosemide only (F, 10mg/kg, i.p.; days 1-11), 2) furosemide + nalfurafine (F+N, 10ug/kg, i.p.; days 1-11) or 3) F (days 1-5) followed by F+N (days 6-11). 5-hr urine collection was measured in between the morning and afternoon drug injections and rats were returned to home cages. Control 5-hr urine samples (saline, i.p.) were collected 2 days prior to beginning drug treatments. SUMMARY: Initial treatment (day 1) of rats with F alone and F+N markedly increased urine output. However, over days 6-10, urine output was significantly reduced (diuretic resistance) in rats that continued to receive F alone. Daily co-treatment of F+N beginning day 1 maintained a marked 5-hrdiuresis for all 11 days. Further, delaying addition of nalfurafine co-administration with F until day 6 reversed established diuretic resistance as noted by a marked increase in diuresis over days 6-10 with F+N cotreatment. Initial treatment (day 1) of rats with furosemide also produced a marked natriuresis and kaliuresis. However, on subsequent days (2-11) there was a significant decrease in the renal excretion of sodium and potassium that remained lower in all 3 treatment groups. In contrast, nalfurafine co-treatment beginning either day 1 (Group 2) or day 6 (Group 3) increased free-water clearance as compared to furosemide alone (Group 1). CONCLUSIONS: These results demonstrate that co-administration of the KOR agonist nalfurafine prevented and reversed diuretic resistance to furosemide without worsening electrolyte excretion. Further, these findings support the premise that in response to fluid loss caused by repeated loop diuretic administration, a compensatory release and renal action of ADH plays an important role in mediating loop diuretic resistance. NIH P30GM106392 (DK) LSUHSC REP243 (DK). 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.

Repeated Vagus nerve electrical stimulation alleviates constipation in a porcine model of opioid-induced constipation

Background: Chronic constipation (CC) is highly prevalent and affects approximately 15% of persons in the United States. Opioid-induced constipation (OIC) in particular, is a significant complication affecting up to 80% of non-cancer patients receiving opioids. Current treatments for CC suffer from adverse effects, poor compliance, and low effcacy. Progress in treatment of CC is hampered in part by the lack of studies in models of higher translational value and due to limited means to assess gut motility non-invasively. To palliate this lack of, we recently developed and validated a model of opioid-induced constipation in pigs using chronic administration of the mu-opioid agonist, loperamide[1]. Neuromodulation, of the autonomic nervous system, is now regarded as the future therapy for refractory diseases such as refractory OIC. Our previous work in naïve-anesthetized pigs indicate that acute electrical stimulation of the celiac branch of the Vagus nerve (CBVN) can increase colonic motility. It is, however, unknown whether repeated CBVN stimulation (R-CBVNS) in conscious pigs can relieve chronic constipation induced by repeated opioid exposure. Aims: To evaluate the effect of R-CBVNS in a porcine model of opioid-induced constipation induced by chronic administration of loperamide. Methods: Female minipig Yucatan (35-50 kg, 5-7 months) were surgically equipped with a cuff electrode around the CBVN and a cecal cannula. Fourteen days later, pigs received daily oral administration of loperamide at 0.2, 0.4 or 3 mg/kg/day in regular diet mixed with palatant (bananas, marshmallows or yogurt mixed with honey) for 14-30 days. After 2 days of loperamide, the effect of concurrent R-CBVNS (30 min/day, 2 Hz, 1 mA, 300 us, 30s on/90s off) was tested on chronic OIC. The intracolonic pressure signals were collected on day 2, 7 and 14, by manometric probes placed in the proximal (pc) and distal colon (dc) with a sampling frequency of 100Hz in awake pigs fitted with chronic cecal cannulas. The long-term effects of R-CBVNS was also tested on the highest dose of loperamide by monitoring motility on day 21 and day 28, 1 and 2 weeks respectively after discontinuing R-CBVNS. In the distal colon, four probes were inserted through the anus and aligned at 10, 13, 16, and 19 cm proximal to the anal verge, denoted as D10, D13, D16, D19. In the proximal colon, four probes were inserted about 10, 13, 16, and 19 cm below the ceco-colic junction, denoted as P10, P13, P16, and P19. Analysis of the intracolonic pressure signals was performed by custom scripts developed in the MATLAB version 2020b. The colon luminal pressure spectral analysis, signal power spectrum density as well as peak frequency and contraction bursts were characterized. Defecation response and stool water content changes (SWC, Bristol scale) were monitored. Results: Loperamide altered the power of dominant manometric contraction frequency band (2-7cpm) increasing it in the pc but reducing it in the dc suggesting colon-region specific responses (0.4 and 3mg/kg/day). R-CBVNS in pigs treated with 0.2, 0.4 or 3 mg/kg/day (for 14 days) increased primarily the proximal colon contraction frequency power during and post stimulation period. The increase in the proximal colon contraction power was mainly seen in the pig colon dominant frequency band (2-6 cpm). The effect of R-CBVNS was less apparent on distal colon motility. R-CBVNS improved stool shape and tended to increase stool water content, when compared to loperamide without concurrent stimulation. Moreover, after 2 weeks of R-CBVNS, the effect lasted one week after the stimulation protocol ended. Conclusions: OIC in pigs is characterized by delayed GI transit and decreased stool water content, alterations of colonic motility patterns including selective suppression of 5-6 cpm peak frequency while inducing 3 cpm peak akin to that reported in some CC patients. R-CBVNS demonstrates its effectiveness in alleviating chronic constipation in a swine model of OIC via restoring primary colonic functions, with a quasi-return of basal motility, but also probably through a pro-secretory effect as evidenced by an increase in stool water content. Reference: 1. Atmani, K., et al., A porcine model of opioid-induced constipation: colon motility characterization and evidence for intestinal opioid receptor modulation. Physiology, 2023. 38(S1): p. 5734528. Supported by NIH SPARC OT2-OD024899. 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.

Stocking and supplying naloxone: Findings from a representative sample of community pharmacies in Victoria, Australia.

Naloxone is an opioid receptor antagonist, which can rapidly reverse the effects of an opioid overdose. Community pharmacists may experience several barriers to stocking and supplying naloxone including a lack of confidence or knowledge and time constraints. The current study aimed to examine the extent to which Victorian community pharmacies stock and supply naloxone and determine specific characteristics associated with stocking naloxone. A representative sample of community pharmacists (n = 558) in Victoria, Australia, were contacted between October and November 2020 and invited to participate in an online survey. Data related to pharmacy- and pharmacist-related characteristics, including stocking and frequency of supplying naloxone in the past year. Multivariate logistic regression analysis was performed to examine the effect of various covariates on stocking naloxone. The sample comprised 265 pharmacists (response rate 47%). Most pharmacies were located in Melbourne (the capital city of Victoria, 59.6%) and were part of a pharmacy chain (61.5%). In total, 100 (38%) pharmacies stocked naloxone, a third of whom did not supply it in the past year. Pharmacies that provided opioid agonist treatment had 2.4 times higher odds of stocking naloxone (95% confidence interval 1.425-4.136; p = 0.001). Less than half of Victorian community pharmacies stock naloxone, with even fewer actually supplying it in the past year. Future efforts are needed to increase the number of pharmacies that stock naloxone and the frequency in which it is supplied, while also addressing possible barriers to stocking and supplying naloxone among community pharmacists.

Designer Receptor Exclusively Activated by Designer Drug (DREADD)-Mediated Activation of the Periaqueductal Gray Restores Nociceptive Descending Inhibition After Traumatic Brain Injury in Rats.

Traumatic brain injury (TBI) patients frequently experience chronic pain that can enhance their suffering and significantly impair rehabilitative efforts. Clinical studies suggest that damage to the periaqueductal gray matter (PAG) following TBI, a principal center involved in endogenous pain control, may underlie the development of chronic pain. We hypothesized that TBI would diminish the usual pain control functions of the PAG, but that directly stimulating this center using a chemogenetic approach would restore descending pain modulation. We used a well-characterized lateral fluid percussion model (1.3 ± 0.1 atm) of TBI in male rats (n = 271) and measured hindpaw mechanical nociceptive withdrawal thresholds using von Frey filaments. To investigate the role of the PAG in pain both before and after TBI, we activated the neurons of the PAG using a Designer Receptor Exclusively Activated by Designer Drug (DREADD) viral construct. Immunohistochemical analysis of brain tissue was used to assess the location and confirm the appropriate expression of the viral constructs in the PAG. Activation of the PAG DREADD using clozapine N-oxide (CNO) caused hindpaw analgesia that could be blocked using opioid receptor antagonist, naloxone, in uninjured but not TBI rats. Due to the importance of descending serotonergic signaling in modulating nociception, we ablated spinal serotonin signaling using 5,7-DHT. This treatment strongly reduced CNO-mediated anti-nociceptive effects in TBI but not uninjured rats. To define the serotonergic receptor(s) required for the CNO-stimulated effects in TBI rats, we administered 5-HT7 (SB-269970) and 5-HT1A (WAY-100635) receptor antagonists but observed no effects. The selective 5-HT2A receptor antagonist ketanserin, however, blocked CNO's effects in the DREADD expressing TBI but not DREADD expressing sham TBI animals. Blockade of alpha-1 adrenergic receptors with prazosin also had no effect after TBI. Descending pain control originating in the PAG is mediated through opioid receptors in uninjured rats. TBI, however, fundamentally alters the descending nociceptive control circuitry such that serotonergic influences predominate, and those are mediated by the 5-HT2A receptor. These results provide further evidence that the PAG is a key target for anti-nociception after TBI.