Potency Of Morphine Research Articles

Biasing Gβγ Downstream Signaling with Gallein Inhibits Development of Morphine Tolerance and Potentiates Morphine-Induced Nociception in a Tolerant State.

Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gβγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gβγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gβγ and inhibit interactions of Gβγ with phospholipase-Cβ3 (PLCβ3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCβ3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gβγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gβγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cβ is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.

Antinociceptive and adverse effects of morphine:ketamine mixtures in rats.

Prescription opioids are the gold standard for treating moderate to severe pain despite their well-documented adverse effects. Of all prescription medications, opioids are abused most widely, and fatal overdoses have reached epidemic levels. One strategy for improving the margin of safety of opioids is combining them with non-opioid drugs to decrease the opioid dose needed for pain relief, thereby reducing adverse effects that occur with larger doses. The N-methyl-D-aspartate receptor antagonist ketamine has been used safely as an analgesic but only under a very limited range of conditions. The current studies characterized the antinociceptive, behavioral suppressant, and gastrointestinal effects of morphine and ketamine alone and in mixtures to determine their interaction in 24 adult male Sprague-Dawley rats (n = 8 per assay). Given alone, both morphine and ketamine produced antinociception, decreased responding for food, and reduced gastrointestinal transit (i.e. produced constipation). The effects of morphine:ketamine mixtures generally were additive, except for the antinociceptive effects of 1:1 mixtures for which the difference in slope (i.e. non-parallel shift) between the observed and predicted effects suggested synergy at smaller doses and additivity at larger doses. The potency of morphine to produce constipation was not enhanced by administration of morphine:ketamine mixtures with antinociceptive effects. The nature of the interaction between morphine and ketamine for adverse effects such as dependence, withdrawal, abuse, or respiratory depression remains unknown but also might be related to the ratio of each drug in mixtures. It will be important to identify conditions that produce the largest potential therapeutic window in humans.

Interactions between lorcaserin and opioids: Ventilation and food-versus-drug choice

Lorcaserin, a selective serotonin 2C (5-HT2C) receptor agonist, was approved for treating obesity and has been investigated for treating substance use disorders including those involving opioids. Although lorcaserin was withdrawn from the market, interest in the therapeutic potential of drugs acting at 5-HT2C receptors continues, supporting the need to further characterize potential adverse effects especially when combined with drugs of abuse. This study examined acute effects of lorcaserin on opioid-induced ventilatory depression, which is the primary cause of overdose, and opioid self-administration, which models factors contributing to opioid abuse, in male and female rhesus monkeys. In one group (n = 4), effects of morphine (0.178 to 5.6 mg/kg, s.c.), fentanyl (0.0032 to 0.1 mg/kg, s.c.), and lorcaserin (0.1 to 1.78 mg/kg, s.c.) alone as well as effects of lorcaserin with each opioid on ventilation were determined using head plethysmography. Another group (n = 5) responded under a food versus fentanyl (0.1 to 3.2 μg/kg/infusion, i.v.) choice procedure, and lorcaserin (0.32 to 1.78 mg/kg, i.v.) was given as a pretreatment. Lorcaserin dose-dependently decreased minute volume to below 70 % of baseline when administered alone and increased the potency of morphine and fentanyl. Consistent with previous studies, lorcaserin failed to alter choice of fentanyl over food. This study demonstrates the novel finding that lorcaserin alone decreases ventilation and enhances the ventilatory-depressant effects of opioids. Taken together with previous studies, these results suggest that combining a 5-HT2C receptor agonist such as lorcaserin with an opioid could increase the risk of ventilatory depression without the benefit of decreasing abuse.

Telmisartan Is a Promising Agent for Managing Neuropathic Pain and Delaying Opioid Analgesic Tolerance in Rats.

Despite the large arsenal of analgesic medications, neuropathic pain (NP) management is not solved yet. Angiotensin II receptor type 1 (AT1) has been identified as a potential target in NP therapy. Here, we investigate the antiallodynic effect of AT1 blockers telmisartan and losartan, and particularly their combination with morphine on rat mononeuropathic pain following acute or chronic oral administration. The impact of telmisartan on morphine analgesic tolerance was also assessed using the rat tail-flick assay. Morphine potency and efficacy in spinal cord samples of treated neuropathic animals were assessed by [35S]GTPγS-binding assay. Finally, the glutamate content of the cerebrospinal fluid (CSF) was measured by capillary electrophoresis. Oral telmisartan or losartan in higher doses showed an acute antiallodynic effect. In the chronic treatment study, the combination of subanalgesic doses of telmisartan and morphine ameliorated allodynia and resulted in a leftward shift in the dose-response curve of morphine in the [35S]GTPγS binding assay and increased CSF glutamate content. Telmisartan delayed morphine analgesic-tolerance development. Our study has identified a promising combination therapy composed of telmisartan and morphine for NP and opioid tolerance. Since telmisartan is an inhibitor of AT1 and activator of PPAR-γ, future studies are needed to analyze the effect of each component.

Morphine and dopamine: Low dose apomorphine can prevent both the induction and expression of morphine locomotor sensitization and conditioning

The disinhibition of dopamine neurons in the VTA by morphine is considered an important contributor to the reward potency of morphine. In this report, three experiments were conducted in which a low dose of apomorphine (0.05 mg/kg) was used as a pretreatment to reduce dopamine activity. Locomotor hyperactivity was used as the behavioral response to morphine (10.0 mg/kg). In the first experiment, five treatments with morphine induced the development of locomotor and conditioned hyperactivity that were prevented by apomorphine given 10 min prior to morphine. Apomorphine before either vehicle or morphine induced equivalent reductions in locomotion. In the second experiment, the apomorphine pretreatment was initiated after induction of a conditioned hyperactivity and apomorphine prevented the expression of the conditioning. To assess the effects of apomorphine on VTA and the nucleus accumbens, ERK measurements were carried out after the induction of locomotor and conditioned hyperactivity. Increased ERK activation was found and these effects were prevented by the apomorphine in both experiments. A third experiment was conducted to assess the effects of acute morphine on ERK before locomotor stimulation was induced by morphine. Acute morphine did not increase locomotion, but a robust ERK response was produced indicating that the morphine-induced ERK activation was not secondary to locomotor stimulation. ERK activation was again prevented by the apomorphine pretreatment. We suggest that contiguity between the ongoing behavioral activity and the morphine activation of the dopamine reward system incentivizes and potentiates the ongoing behavior generating equivalent behavioral sensitization and conditioned effects.

Pharmacokinetic and Pharmacodynamic Consequences of Cytochrome P450 3A Inhibition on Mitragynine Metabolism in Rats.

Mitragynine, an opioidergic alkaloid present in Mitragyna speciosa (kratom), is metabolized by cytochrome P450 3A (CYP3A) to 7-hydroxymitragynine, a more potent opioid receptor agonist. The extent to which conversion to 7-hydroxymitragynine mediates the in vivo effects of mitragynine is unclear. The current study examined how CYP3A inhibition (ketoconazole) modifies the pharmacokinetics of mitragynine in rat liver microsomes in vitro. The study further examined how ketoconazole modifies the discriminative stimulus and antinociceptive effects of mitragynine in rats. Ketoconazole [30 mg/kg, oral gavage (o.g.)] increased systemic exposure to mitragynine (13.3 mg/kg, o.g.) by 120% and 7-hydroxymitragynine exposure by 130%. The unexpected increase in exposure to 7-hydroxymitragynine suggested that ketoconazole inhibits metabolism of both mitragynine and 7-hydroxymitragynine, a finding confirmed in rat liver microsomes. In rats discriminating 3.2 mg/kg morphine from vehicle under a fixed-ratio schedule of food delivery, ketoconazole pretreatment increased the potency of both mitragynine (4.7-fold) and 7-hydroxymitragynine (9.7-fold). Ketoconazole did not affect morphine's potency. Ketoconazole increased the antinociceptive potency of 7-hydroxymitragynine by 4.1-fold. Mitragynine (up to 56 mg/kg, i.p.) lacked antinociceptive effects both in the presence and absence of ketoconazole. These results suggest that both mitragynine and 7-hydroxymitragynine are cleared via CYP3A and that 7-hydroxymitragynine is formed as a metabolite of mitragynine by other routes. These results have implications for kratom use in combination with numerous medications and citrus juices that inhibit CYP3A. SIGNIFICANCE STATEMENT: Mitragynine is an abundant kratom alkaloid that exhibits low efficacy at the μ-opioid receptor (MOR). Its metabolite, 7-hydroxymitragynine, is also an MOR agonist but with higher affinity and efficacy than mitragynine. Our results in rats demonstrate that cytochrome P450 3A (CYP3A) inhibition can increase the systematic exposure of both mitragynine and 7-hydroxymitragynine and their potency to produce MOR-mediated behavioral effects. These data highlight potential interactions between kratom and CYP3A inhibitors, which include numerous medications and citrus juices.

Modulation of morphine physical dependence and discriminative stimulus effects by ovarian hormones: Role of estradiol

Ovarian hormones influence the activity of endogenous opioids, and exogenous administration of estradiol reduces opioid intake and opioid seeking in animal models of opioid reward and reinforcement. The purpose of this study was to examine the effects of ovarian hormones on the discriminative stimulus effects of morphine and naloxone-precipitated opioid withdrawal. To this end, separate groups of ovariectomized female rats were trained to discriminate the stimulus effects of either 3.0 or 10 mg/kg morphine, and substitution tests were conducted with estradiol or progesterone alone and in combination with morphine. At the conclusion of discrimination testing, rats were treated chronically with estradiol, progesterone, or their combination, and challenged with naloxone to measure opioid-like withdrawal symptoms. Finally, the effects of estradiol, progesterone, and their combination were examined on naloxone-precipitated withdrawal in morphine-dependent rats. Neither estradiol nor progesterone substituted for the morphine discriminative stimulus, but estradiol significantly increased the potency of morphine in rats trained to discriminate 10 mg/kg but not 3 mg/kg morphine. When administered chronically, neither hormone nor their combination produced an opioid-like withdrawal syndrome following a naloxone challenge. Acute administration of estradiol, but not progesterone or a combination of estradiol and progesterone, significantly reduced naloxone-precipitated weight loss in morphine-dependent rats. These data indicate that estradiol influences the behavioral effects of morphine, possibly by increasing endogenous tone at mu opioid receptors.

Antinociceptive Effects of Morphine:Ketamine Mixtures in Rats

Chronic pain is a serious public health concern with an economic burden of $600 million annually and personal burden for approximately 100 million Americans. Prescription opioids (mu opioid receptor agonists) are the “gold standard” for treating moderate to severe pain despite their well‐documented adverse effects (dependence, respiratory depression, constipation). Of all prescription medications, opioids are abused most widely, and fatal overdoses have reached epidemic levels. Thus, there is a dire need for safer, more effective treatments for pain. One strategy for improving the margin of safety of opioids is combining them with other analgesic drugs to decrease the opioid dose needed for pain relief, thereby avoiding adverse effects that occur with larger doses. The NMDA receptor antagonist ketamine has been used safely and effectively to treat pain, but only under a narrow range of conditions (in emergency departments, post‐operative recovery, and combat casualty). The current studies used a model of acute pain (warm water tail withdrawal) and a model of chronic inflammatory pain (Von Frey paw withdrawal) to determine the antinociceptive effects of morphine and ketamine alone and in mixtures (in 3:1, 1:1, and 1:3 ratios) in 16 male Sprague Dawley rats. Given alone, both morphine (1‐10 mg/kg) and ketamine (3.2‐32 mg/kg) dose‐dependently increased tail withdrawal latency, with morphine having greater potency and efficacy as compared to ketamine. Similarly, given alone, both morphine (0.56‐5.6 mg/kg) and ketamine (3.2‐32 mg/kg) dose‐dependency increased force required to elicit a paw withdrawal response, with morphine having greater potency as compared to ketamine. ED50 values were used to determine the doses for mixtures. In mixtures, the potency of morphine or ketamine to produce antinociception was enhanced by 2‐3 fold as compared to either drug given alone. Dose‐equivalence and dose‐additivity analyses showed that the effects of morphine:ketamine mixtures were additive. Furthermore, morphine:ketamine mixtures that relieved pain also were tested in an assay of constipation in a separate group of 8 rats. While both morphine (1‐10 mg/kg) alone and ketamine (3.2‐32 mg/kg) alone dose‐dependently decreased fecal output, morphine:ketamine mixtures did not enhance constipation. Therefore, morphine:ketamine mixtures appear to selectively enhance antinociception. It remains unknown whether morphine:ketamine mixtures have other adverse effects (abuse, physical dependence, respiratory depression) and whether interactions between morphine and ketamine on these outcomes might be related to the ratio of each drug in mixtures. Morphine:ketamine mixtures might have greater therapeutic potential than mu opioids alone for treating moderate to severe pain, but only if adverse effects of each drug are not enhanced.

Advanced Age and Sex Modulate Multiple Mu Opioid Receptor Signaling Mechanisms in the Rat Midbrain Periaqueductal Gray: Implications for Analgesia

Chronic pain is exceedingly prevalent in individuals over 65 years of age. Still, it is under-managed due in large part to a dearth of knowledge regarding the suitable dosing of opioids for chronic pain management. We have shown that advanced age and sex alter morphine modulation of persistent inflammatory pain (induced by intraplantar Complete Freund's adjuvant (CFA)). Specifically, morphine potency was highest in adult male rats (2mos), with a 2-fold rightward shift in the dose-response curve for aged males (18mos) and females regardless of age. In this study, we tested the hypothesis that advanced age attenuates μ-opioid receptor (MOR) signaling in the ventrolateral periaqueductal gray (vlPAG), a critical region for the modulation of pain. We find that aged males exhibit reduced vlPAG MOR expression (Bmax) compared to adult males, and both aged males and aged females exhibit reduced vlPAG MOR affinity (Kd) compared to adult males. Further, aged males and females, regardless of age, show reduced vlPAG MOR binding potential and reduced G-protein activation efficiency compared to adult males. Western blots of phosphorylated MOR at serine 375 revealed no age or sex differences in MOR phosphorylation. DAMGO-induced cAMP inhibition was 2-fold higher in adult males than aged males or females. The regulation of opioid-induced G-protein signaling was further assessed using RNAscope to analyze mRNA expression of Regulator of G-Protein Signaling (RGS) family members RGS4 and RGS9-2. We found increased expression of RGS4 and RGS9-2 in the vlPAG of aged animals compared to adults, indicating that MOR signaling is subjected to greater negative regulation in the aged vlPAG. The observed age-related reductions in vlPAG MOR expression, MOR binding potential, G-protein activation, and cAMP inhibition, along with the observed increase in vlPAG RGS4 and RGS9-2, have significant implications in pain management in the aged population. Grant support from NIH R01DA041529 (AZM), NIH R01DA052340 (JS) NIH P50MH100023 (LJY).

Characterizing the influence of Toxoplasma infection on opioid receptor expression using human neuroblastoma cells (SKNSH)

BackgroundToxoplasma gondii is an obligate intracellular parasite that has permanently infected billions of people. The parasite can convert to a latent form that is found preferentially in the brain and heart. Recent serological studies have linked Toxoplasma infection to many brain disorders including schizophrenia, depression, substance abuse and Alzheimer’s. However, the molecular mechanism by which Toxoplasma influences these brain disorders remains unclear. Previous studies have shown that Toxoplasmaincreases dopamine synthesis, and dopamine is an important neurotransmitter in the reward pathway, where the opioid system has been shown to play a major role in modulating this pathway. We aim to further explore these mechanisms by studying the influence of Toxoplasmainfection on opioid receptor expression.ObjectivesThe goal of our project is to elucidate the influence of Toxoplasma gondiion brain physiology through studying the impact of Toxoplasma infection on opioid receptor expression. The aim of this study is to determine the effect of Toxoplasma infection on opioid receptor expression in neuroblastoma cells (SKNSH) in the presence or absence of morphine.MethodsTo evaluate the potency of morphine against Toxoplasma, a doubling assay was performed. Briefly, Human foreskin fibroblasts (HFF) were infected with Toxoplasma. Six‐hours post‐infection, the uninvaded parasites were removed and the media was replaced with media containing 10 µM morphine, 10 µM pyrimethamine, or DMSO. The flasks were fixed with ice‐cold methanol and the number of parasites per vacuole were enumerated at 24‐ and 48‐hours post‐treatment. To evaluate the expression of opioid receptors, the neuroblastoma cell SKNSH, which has been shown to express functional mu and delta opioid receptors, was infected with Toxoplasma type 1. Four hours post‐infection, uninvaded parasites were removed and the medium was replaced with media containing DMSO or 1 µM morphine or 10 µM morphine. Thirty‐two hours post‐treatment, the cells were harvested and protein was isolated. The expression of mu and delta opioid receptors was evaluated using western blot analysis.ResultsWe observed changes in the morphology of SKNSH upon infection with Toxoplasma type 1. The results from our doubling assay demonstrate the lack of potency for morphine against Toxoplasma type 1 at 24 hours and 48 hours post treatment with the highest morphine concentration we have been using for these experiments. Preliminary results suggest that the expression of the delta opioid receptor is downregulated in the presence of Toxoplasma, independent of morphine administration. In addition, the expression of delta opioid receptor is increased in the presence of morphine alone. When pyrimethamine was added to cells containing both Toxoplasma and morphine, delta receptor expression was comparable to its expression in the presence of morphine alone.ConclusionWe have confirmed the expression of muand delta opioid receptors in SKNSH, as reported previously. In addition, we were able to successfully infect this cell line with Toxoplasma. Additionally, we have found that morphine lacks potency against Toxoplasma, unlike previously reported. Preliminary data suggest that the collective the presence of Toxoplasma can negatively influence the expression of delta opioid receptors. Future experiments are being designed to elucidate the molecular mechanism of these findings, as well as observing similar findings in the animal model of Toxoplasmosis.

Pharmacological evaluation and forensic case series of N-pyrrolidino etonitazene (etonitazepyne), a newly emerging 2-benzylbenzimidazole ‘nitazene’ synthetic opioid

Novel synthetic opioids continue to emerge on recreational drug markets worldwide. In response to legislative bans on fentanyl analogues, non-fentanyl structural templates, such as 2-benzylbenzimidazoles ('nitazenes'), are being exploited to create new μ-opioid receptor (MOR) agonists. Here, we pharmacologically characterize an emerging cyclic analogue of etonitazene, called N-pyrrolidino etonitazene (etonitazepyne), using in vitro and in vivo methods. A series of analytically confirmed fatalities is described to complement preclinical findings. Radioligandbinding assays in rat brain tissue revealed that N-pyrrolidino etonitazene has high affinity for MOR (Ki = 4.09nM) over δ-opioid (Ki = 959nM) and κ-opioid (Ki = 980nM) receptors. In a MOR-β-arrestin2 activation assay, N-pyrrolidino etonitazene displayed high potency (EC50 = 0.348nM), similar to etonitazene (EC50 = 0.360nM), and largely exceeding the potencies of fentanyl (EC50 = 14.9nM) and morphine (EC50 = 290nM). When administered s.c. to male Sprague Dawley rats, N-pyrrolidino etonitazene induced opioid-like antinociceptive, cataleptic, and thermic effects. Its potency in the hot plate test (ED50 = 0.0017mg/kg) was tenfold and 2,000-fold greater than fentanyl (ED50 = 0.0209mg/kg) and morphine (ED50 = 3.940mg/kg), respectively. Twenty-one overdose fatalities associated with N-pyrrolidino etonitazene were found to contain low blood concentrations of the drug (median = 2.2ng/mL), commonly in the context of polysubstance use. N-Pyrrolidino etonitazene was reported as a cause of death in at least two cases, demonstrating toxicity in humans. We demonstrate that N-pyrrolidino etonitazene is an extremely potent MOR agonist that is likely to present high risk to users. Continued vigilance is required to identify and characterize emergent 2-benzylbenzimidazoles, and other non-fentanyl opioids, as they appear in the marketplace.

Modulation of Morphine Analgesia, Antinociceptive Tolerance, and Mu-Opioid Receptor Binding by the Cannabinoid CB2 Receptor Agonist O-1966.

Acutely, non-selective cannabinoid (CB) agonists have been shown to increase morphine antinociceptive effects, and we and others have also demonstrated that non-selective CB agonists attenuate morphine antinociceptive tolerance. Activation of cannabinoid CB2 receptors reverses allodynia and hyperalgesia in models of chronic pain, and co-administration of morphine with CB2 receptor selective agonists has been shown to be synergistic. CB2 receptor activation has also been shown to reduce morphine-induced hyperalgesia in rodents, an effect attributed to CB2 receptor modulation of inflammation. In the present set of experiments, we tested both the acute and chronic interactions between morphine and the CB2 receptor selective agonist O-1966 treatments on antinociception and antinociceptive tolerance in C57Bl6 mice. Co-administration of morphine and O-1966 was tested under three dosing regimens: simultaneous administration, morphine pre-treated with O-1966, and O-1966 pre-treated with morphine. The effects of O-1966 on mu-opioid receptor binding were determined using [3H]DAMGO and [35S]GTPγS binding assays, and these interactions were further examined by FRET analysis linked to flow cytometry. Results yielded surprising evidence of interactions between the CB2 receptor selective agonist O-1966 and morphine that were dependent upon the order of administration. When O-1966 was administered prior to or simultaneous with morphine, morphine antinociception was attenuated and antinociceptive tolerance was exacerbated. When O-1966 was administered following morphine, morphine antinociception was not affected and antinociceptive tolerance was attenuated. The [35S]GTPγS results suggest that O-1966 interrupts functional activity of morphine at the mu-opioid receptor, leading to decreased potency of morphine to produce acute thermal antinociceptive effects and potentiation of morphine antinociceptive tolerance. However, O-1966 administered after morphine blocked morphine hyperalgesia and led to an attenuation of morphine tolerance, perhaps due to well-documented anti-inflammatory effects of CB2 receptor agonism.

Sulforaphane alleviates hyperalgesia and enhances analgesic potency of morphine in rats with cancer-induced bone pain

Due to the efficacy and tolerability of the available drugs, the current treatment for cancer-induced bone pain (CIBP) is not considered ideal, and new drugs are required for better treatment results. This study investigated whether intrathecal injection of sulforaphane (SFN) can modulates the noxious behavior associated with CIBP and enhances the analgesic effects of morphine and the possible mechanisms related to these effects were investigated. Walker256 breast cancer cells were injected into the bone marrow cavity of rats to establish the CIBP model. When CIBP rats began to exhibit painful behavior (CIBP 6 days), SFN was injected intrathecally for 7 days. The results showed that SFN alleviated the painful behavioral hypersensitivity caused by cancer, accompanied by nuclear factor, erythroid 2 like 2 (Nrf2), Haem oxygenase 1 (HO-1) activation, nuclear factor kappa B (NF-κB) inhibition and inflammation-related factors (tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-β), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) reduction. In addition, SFN treatment inhibited the proliferation of Walker 256 cells in a dose-dependent manner, promoted mu-opioid receptor (MOR) expression in SH-SY5Y cells and enhanced the antihyperalgesic effects of morphine on CIBP rats by restoring the downregulation of MOR expression in the spinal cord. Interestingly, the antihyperalgesic effects of SFN were partially blocked by opioid receptor antagonists. This study showed that SFN combined with morphine might be a new way to treat CIBP.

RETRACTED ARTICLE: Oxycodone versus morphine for analgesia after laparoscopic endometriosis resection

BackgroundThe objective of this study was to compare the analgesic potency of oxycodone versus morphine after laparoscopic deep infiltrating endometriosis resection.MethodsFifty patients undergoing laparoscopic deep infiltrating endometriosis resection were randomized to receive oxycodone or morphine intravenous-PCA after surgery. The primary outcome was opioid consumption during the 24 h after surgery. Secondary outcomes included time to first request for analgesia, the number of bolus, pain, sedation, nausea, vomiting, respiratory depression, and bradycardia. The prominent pain that caused patients to press the analgesic device was also recorded.ResultsOxycodone consumption (14.42 ± 2.83) was less than morphine consumption (20.14 ± 3.83). Compared with the morphine group, the total number of bolus (78 vs 123) was less and the average time to first request for analgesia (97.27 ± 59.79 vs 142.17 ± 51) was longer in the oxycodone group. The incidence of nausea was higher in the morphine group than in the oxycodone group at 0–2 h (45.45% vs 17.19%), 2–4 h (50% vs 17.19%),12–24 h (40.91% vs 13.04%) and 0–24 h (39.17% vs 19.13%). The overall incidence of vomiting was higher in the morphine group (27.27% vs 13.92%). There was no difference in visual analogue scale score, the incidence of respiratory depression, and bradycardia between groups. Of the three types of pain that prompted patients to request analgesia, the incidence of visceral pain was highest (59.9%, P < 0.01).ConclusionOxycodone was more potent than morphine for analgesia after laparoscopic endometriosis resection, and oxycodone has fewer side effects than morphine.Name of the registry: Chinese Clinical Trial RegistryTrial registration number: ChiCTR1900021870URL of trial registry record:http://www.chictr.org.cn/edit.aspx?pid=35799&htm=4Date of registration: 2019/3/13 0:00:00

The impact of advanced age and sex on Mu Opioid Receptor signaling in the midbrain periaqueductal gray: implications on analgesia

Chronic pain is under-managed in individuals over 65 years of age due to a dearth of knowledge regarding the impact of age on opioid efficacy in the elderly. We have previously shown that advanced age and sex alter morphine modulation of persistent inflammatory pain (induced by intraplantar administration of Complete Freund's adjuvant (CFA)), such that morphine potency is highest in adult male rats (2mos), with EC50 values 2-fold higher in aged males (18mos) and females regardless of age. Age-induced reductions in morphine potency were accompanied by reduced mu opioid receptor (MOR) expression in the ventrolateral periaqueductal gray (vlPAG), a CNS region critical in pain modulation. The present studies further explore the impact of age on opioid signaling within the PAG. MOR affinity, availability, and G-protein activation were assessed using radioligand binding assays and GTPγS assays in vlPAG tissue from adult and aged, male and female rats collected 72h following CFA administration. Regulation of opioid induced G-protein signaling was assessed using RNAscope to analyze mRNA expression of Regulator of G-Protein Signaling (RGS) proteins RGS4 and RGS9-2. We find that aged males and females (adult and aged) exhibit reduced vlPAG MOR binding potential and reduced G-protein activation efficiency compared to adult males, suggesting age- and sex- differences in MOR machinery drive reduced opioid potency. RNAscope revealed increased expression of RGS4 and RGS9-2 in the vlPAG of aged animals compared to adults, indicating that MOR signaling is subject to greater negative regulation in the aged vlPAG. The observed age-related reductions in vlPAG MOR agonist binding and opioid induced G-protein activation, along with the observed increase in vlPAG RGS expression have significant implications in pain management in the aged population. Our novel findings elucidate several mechanisms mediating reduced morphine potency in aged animals, and identify potential targets to improve pain management in the elderly. R01DA041529-04.

Mitragynine Attenuates the Development of Tolerance to and Withdrawal from Morphine in Rats

Mitragyna speciosa (kratom) has been used for centuries in Southeast Asia as a self-treatment for opioid dependence. However, scientific evidence to support the medical use of kratom has not been established. Kratom products contain numerous alkaloids, the most abundant of which is mitragynine (MG). This study assessed the potential for MG to attenuate the development and expression of opioid tolerance and dependence. In Sprague-Dawley rats, morphine (MOR) antinociception was measured prior to and following four days of escalating doses of morphine (10-40 mg/kg, every 12 hours). Chronic dosing tests were conducted with both morphine alone and co-treatment of morphine with mitragynine or other test compounds. Naltrexone-induced diarrhea was measured as a sign of opioid withdrawal. Chronic MG treatment alone (10, 32, and 56 mg/kg, every 12 hours) was assessed for the capacity to alter sensitivity to MOR and to result in naltrexone-induced withdrawal. Repeated MOR treatment conferred tolerance, as evidenced by a 7.9-fold rightward shift in the MOR antinociception dose-response function. Naltrexone produced diarrhea in the chronic MOR group and not the chronic saline group. Chronic administration of MG (10, 32, and 56 mg/kg) with MOR dose-dependently attenuated the development of MOR tolerance. Chronic MG alone produced up to a 2.4-fold rightward shift in the MOR antinociception dose-response function; chronic treatment with 32 mg/kg of mitragynine resulted in naltrexone-induced diarrhea. The effects of acutely administered MG on the expression of tolerance to and withdrawal from MOR were further compared to those of two FDA-approved medications for opioid use disorder: the μ-opioid receptor partial agonist buprenorphine and the α2-adrenergic receptor agonist lofexidine. In MOR-tolerant rats, lofexidine (1.0 mg/kg) significantly increased the potency of MOR to produce antinociception, buprenorphine (1.0 mg/kg) significantly antagonized morphine, and MG (32 and 56 mg/kg) did not modify the potency of MOR. Naltrexone-induced diarrhea was fully attenuated by lofexidine (1.0 mg/kg) and partially attenuated by MG (32 and 56 mg/kg) and buprenorphine (1.0 mg/kg). Collectively, these results support the potential of MG as a medication to attenuate the development of opioid tolerance and dependence. More studies are needed to assess the safety profile of mitragynine when administered in combination with opioids or other compounds.

The Hsp90 Isoform‐Selective Inhibitor KUNG65 Enhances Opioid Anti‐Nociception while Reducing Tolerance

Opioids are the gold standard for pain management, however, adverse side effects like respiratory depression and addiction limit their viability. One approach to improve opioid therapy focuses on a dose-reduction strategy by amplifying opioid analgesia without boosting side effects, so that less opioid can be given. To this end, we've focused on Heat shock protein 90 (Hsp90), a central protein regulator in the cell; ongoing studies from our lab have shown that inhibition of Hsp90 in the spinal cord improves morphine anti-nociceptive potency by 2-3 fold in acute and chronic pain while reducing tolerance, rescuing established tolerance, and not changing reward and constipation, enabling a dose-reduction strategy. However, our results also showed that non-selective Hsp90 inhibitors given systemically (intravenous, IV) have the opposite effect, blocking opioid pain relief. Seeking a means to surmount this roadblock, we determined which Hsp90 isoforms regulate opioid pain relief in brain and spinal cord in mice using selective small molecule inhibitors and CRISPR/Cas9 gene editing. We found that Hsp90α alone regulated opioid signaling and pain relief in the brain. However, in the spinal cord, we found that the isoforms Hsp90α, Hsp90β, and Grp94 all regulated opioid signaling and pain relief. This led to our hypothesis that targeting spinal cord Hsp90 with isoform-selective inhibitors given IV could boost opioid pain relief without altering side effects, enabling a dose-reduction strategy. We tested this hypothesis with the novel Grp94-selective inhibitor KUNG65, given at a 1 mg/kg dose IV in male and female CD-1 mice, followed by a 24 hr treatment time, then analysis of opioid anti-nociception and side effects. We found that systemic (IV) KUNG65 treatment resulted in a 1.9 fold increase in morphine potency to relieve tail flick pain, consistent with our earlier studies injecting inhibitor directly into spinal cord. We also found that KUNG65 boosted morphine potency in paw incision pain, with a 2.2 fold increase. Additionally, we found that KUNG65 injection could rescue established morphine tolerance in the tail flick assay, again as we found for direct spinal cord injection. These results support our hypothesis that isoform-selective Hsp90 inhibitors can selectively engage Hsp90 in the spinal cord when given systemically, resulting in improved opioid anti-nociception and side effects. These results strongly suggest that Hsp90 isoform-selective inhibitors could be a powerful new tool to improve opioid therapy through a dose-reduction strategy, and further show that this effect can be achieved through a translationally relevant dosing route.

Pharmacological Characterization of Mitragynine: Antinociception, Respiratory Depression, Self‐Administration, Drug Discrimination, Tolerance, and withdrawal in Rats

The primary kratom alkaloid mitragynine (MG) has low intrinsic activity at µ-opioid receptor (MOR). Here we characterized the MOR activity of MG using various in vivo assays in rats. Using a hotplate assay (52°C), MOR agonists fentanyl and morphine produced robust antinociception (i.v. cumulative ED50s: 0.015 and 8.1 mg/kg, respectively); MG (i.v.) was inactive up to 17.8 mg/kg and 32 mg/kg was lethal. The 32 mg/kg dose of MG (i.p.) did not significantly modify the dose-effect function (DEF) of morphine to produce antinociceptive effects. Using whole-body plethysmography under hypercapnic conditions (8% CO2), the maximum decreases of fentanyl, morphine, and MG in % control minute volume (mL/min./kg, normalized to ambient air) were 63% (i.v. cumulative ED25: 0.011 mg/kg), 46% (34 mg/kg), and 23%, respectively. In rats trained to self-administer remifentanil (RMF; 0.1-3.2 µg/kg/inj., fixed-ratio 5 schedule of reinforcement), the DEF of RMF self-administration was an inverted-U shaped curve and 1.0 µg/kg/inj. maintained maximum rates of responding (0.11 responses/sec. relative to 0.0071 responses/sec. without drug). When substituted for RMF, no dose of MG (0.1-3.2 mg/kg/inj.) maintained self-administration responding above control. A dose of 32 mg/kg MG (i.p.) decreased by 77% the maximum rates of responding maintained by RMF; 17.8 mg/kg MG was inactive. In contrast, the opioid antagonist naltrexone (3.2 mg/kg, i.p.) shifted the DEF of RMF to the right (i.e., appeared to produce surmountable antagonism). In rats discriminating morphine (3.2 mg/kg, i.p.; ED50=1.9 mg/kg) from vehicle, fentanyl and mitragynine produced 100% and 72% drug-lever responding (i.p. ED50s=0.022 and 30 mg/kg, respectively). The DEF of morphine was shifted 8.2-fold to the right (i.e., antagonized) by naltrexone (0.032 mg/kg, i.p.), and 3.9-fold to the left (i.e., potentiation) by MG (5.6 m/kg, i.p.). Tolerance to morphine antinociception (control ED50=14 mg/kg, i.p.) was assessed one day prior to (Day 1) and one day after (Day 6) escalating dosing of morphine (b.i.d. 10, 20, 30, and 40 mg/kg over 4 consecutive days, i.p.) or fixed dosing of MG (b.i.d. 32 mg/kg over 4 consecutive days, i.p.). The escalating dosing of morphine produced a 7.9-fold rightward shift of the morphine DEF (tolerance); in contrast, chronic MG treatment did not alter the potency of morphine (i.e., there was no cross-tolerance). One day after the tolerance test (Day 7), naltrexone (10 mg/kg, i.p.) produced diarrhea in 100% and 75% in morphine and MG groups (N=8 each), respectively. These data suggest that MG is a low efficacy MOR agonist in vivo that includes less respiratory and reinforcing effects as compared with other MOR agonists, as well as the capacity to reduce self-administration of those MOR agonists. Chronic MG does not appear to decrease the antinociceptive effectiveness of MOR agonists. Collectively, these data underscore the therapeutic potential of MG and novel chemical analogs of MG for opioid use disorder.

TRV130 partial agonism and capacity to induce anti-nociceptive tolerance revealed through reducing available μ-opioid receptor number.

β-Arrestin2 recruitment to μ-receptors may contribute to the development of opioid side effects. This possibility led to the development of TRV130 and PZM21, opioids reportedly biased against β-arrestin2 recruitment in favour of G-protein signalling. However, low efficacy β-arrestin2 recruitment by TRV130 and PZM21 may simply reflect partial agonism overlooked due to overexpression of μ-receptors. Efficacies and apparent potencies of DAMGO, morphine, PZM21 and TRV130 as stimulators of β-arrestin2 recruitment and inhibitors of cAMP accumulation were assessed in CHO cells stably expressing μ-receptors. Receptor availability was depleted through prior exposure of cells to the irreversible antagonist, β-FNA. We also examined whether μ-receptor availability influences TRV130 anti-nociception and/or tolerance using the tail withdrawal assay in wild-type C57BL/6 and μ+/- mice. Morphine, PZM21 and TRV130 were partial agonists in the β-arrestin2 recruitment assay. Only TRV130 exhibited partial agonism in the cAMP assay. Exposure to β-FNA to reduce μ-receptor availability further limited the efficacy of TRV130 and revealed morphine and PZM21 to be partial agonists. Despite having partial efficacy in vitro, TRV130 caused potent anti-nociception (ED50 : 0.33 mg·kg-1 ) in wild-type mice, without tolerance after daily administration for 10 days. TRV130 caused similar anti-nociception in μ+/- mice, with marked tolerance on day 4 of injections. Our findings emphasise the importance of receptor reserve when characterising μ-receptor agonists. Reduced receptor availability reveals that TRV130 is a partial agonist capable of tolerance, despite having limited efficacy for β-arrestin2 recruitment to the μ-receptor.

Advanced age attenuates the antihyperalgesic effect of morphine and decreases μ-opioid receptor expression and binding in the rat midbrain periaqueductal gray in male and female rats

The present study investigated the impact of advanced age on morphine modulation of persistent inflammatory pain in male and female rats. The impact of age, sex, and pain on μ-opioid receptor (MOR) expression and binding in the ventrolateral periaqueductal gray (vlPAG) was also examined using immunohistochemistry and receptor autoradiography. Intraplantar administration of complete Freund's adjuvant induced comparable levels of edema and hyperalgesia in adult (2–3 mos) and aged (16–18 mos) male and female rats. Morphine potency was highest in adult males, with a greater than two-fold increase in morphine EC50 observed in adult versus aged males (3.83 mg/kg vs. 10.16 mg/kg). Adult and aged female rats also exhibited significantly higher EC50 values (7.76 mg/kg and 8.74 mg/kg, respectively) than adult males. The upward shift in EC50 from adult to aged males was paralleled by a reduction in vlPAG MOR expression and binding. The observed age-related reductions in morphine potency and vlPAG MOR expression and binding have significant implications in pain management in the aged population.