ABSTRACT
The OPRM1 gene codes for the mu opioid receptor (MOR) and polymorphisms are associated with complex patient clinical responses. The most studied single nucleotide polymorphism (SNP) in OPRM1 is adenine (A) substituted by guanine (G) at position 118 (118A>G, rs1799971) leading to a substitution of asparagine (Asn) for aspartic acid (Asp) at position 40 in the N terminus of the resulting protein. To date, no structural explanation for the associated clinical responses resulting from the 118A>G polymorphism has been proposed. We utilized computational modeling paired with functional cellular assays to predict unstructured N- and C-terminal regions of MOR-1. Using molecular docking and post-docking energy minimizations with morphine, we show that the extracellular substitution of Asn at position 40 alters the cytoplasmic C-terminal conformation, while leaving the G-protein binding interface unaffected. A real-time BRET assay measuring G-protein and ß-arrestin association with MOR r generated data that tested this prediction. Consistent with this in silico prediction, we show changes in morphine-mediated ß-arrestin association with receptor variants with little change in morphine-mediated G-protein association comparing MOR-1 wild type (WT) to MOR-1118A>G. We tested the system with different opioid agonists, the OPRM1 118A>G SNP, and different MOR splice variants (MOR-1 and MOR-1O). These results are consistent with the observation that patients with the 118A>G OPRM1 allele respond more readily to fentanyl than to morphine. In conclusion, the 118A>G substitution alters receptor responses to opioids through variable C-terminal domain movements that are agonist and splice variant dependent.
Subject(s)
Molecular Docking Simulation , Morphine , Polymorphism, Single Nucleotide , Receptors, Opioid, mu , beta-Arrestins , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/metabolism , Humans , beta-Arrestins/metabolism , Morphine/pharmacology , HEK293 Cells , Analgesics, Opioid/pharmacology , Analgesics, Opioid/metabolism , Protein BindingABSTRACT
The gut-brain axis mediates the interaction pathway between microbiota and opioid addiction. In recent years, many studies have shown that molecular hydrogen has therapeutic and preventive effects on various diseases. This study aimed to investigate whether molecular hydrogen could serve as pharmacological intervention agent to reduce risks of reinstatement of opioid seeking and explore the mechanism of gut microbiota base on animal experiments and human studies. Morphine-induced conditioned place preference (CPP) was constructed to establish acquisition, extinction, and reinstatement stage, and the potential impact of H2 on the behaviors related to morphine-induced drug extinction was determined using both free accessible and confined CPP extinction paradigms. The effects of morphine on microbial diversity and composition of microbiota, as well as the subsequent changes after H2 intervention, were assessed using 16â¯S rRNA gene sequencing. Short-Chain Fatty Acids (SCFAs) in mice serum were detected by gas chromatography-mass spectrometry (GC-MS). Meanwhile, we also conducted molecular hydrogen intervention and gut microbiota testing in opioid-addicted individuals. Our results revealed that molecular hydrogen could enhance the extinction of morphine-related behavior, reducing morphine reinstatement. Gut microbes may be a potential mechanism behind the therapeutic effects of molecular hydrogen on morphine addiction. Additionally, molecular hydrogen improved symptoms of depression and anxiety, as well as gut microbial features, in individuals with opioid addiction. This study supports molecular hydrogen as a novel and effective intervention for morphine-induced addiction and reveals the mechanism of gut microbiota.
Subject(s)
Gastrointestinal Microbiome , Hydrogen , Morphine , Opioid-Related Disorders , Gastrointestinal Microbiome/drug effects , Animals , Hydrogen/pharmacology , Male , Mice , Humans , Opioid-Related Disorders/drug therapy , Morphine/pharmacology , Mice, Inbred C57BL , Brain-Gut Axis/drug effects , Extinction, Psychological/drug effects , Analgesics, Opioid/pharmacology , Adult , Fatty Acids, Volatile/metabolismABSTRACT
BACKGROUND: Classical opioids are effective analgesics but carry various side effects, necessitating safer alternatives. Truncated six-transmembrane mu opioid receptors (6TM-µORs) mediate potent analgesia with fewer side effects and are a promising therapeutic target. However, few ligands known selectively target 6TM-µORs. Moreover, endogenous chaperones are believed essential for 6TM-µOR ligand binding and function. PURPOSE: To identify a 6TM-µOR selective agonist and elucidate requisite endogenous chaperones. METHODS: Virtual screening was used to identify promising selective 6TM-µOR agonists from traditional Chinese medicines. The role of 6TM-µOR in Exoticin analgesia was validated in loss- and gain-of-function models. APEX2 proteomics profiled proximal proteins under Exoticin or IBNtxA. Interactions were further characterized in vivo and in vitro. RESULTS: Exoticin was shortlisted for its selective binding to 6TM-µOR and ability to induce 6TM-µOR-dependent signal transduction. Exoticin analgesia was sensitive to ß-FNA and absent in E11 KO mice, but restored in mice infected with AAV-µOR1G. Slc3a2, Lrrc59, and Ppp1cb co-interacted with 6TM-µOR1G and were equally essential for Exoticin binding and 6TM-µOR1G activity. CONCLUSION: Exoticin is a promising selective agonist of 6TM µ opioid receptors with broad-spectrum analgesic efficacy but few side effects. Slc3a2, Lrrc59, Ppp1cb are endogenous chaperones essential for 6TM-µOR ligand binding and function.
Subject(s)
Receptors, Opioid, mu , Animals , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/metabolism , Mice , Humans , Molecular Chaperones/metabolism , Mice, Knockout , HEK293 Cells , Analgesics, Opioid/pharmacology , Male , Mice, Inbred C57BL , Signal Transduction/drug effects , Drugs, Chinese Herbal/pharmacologyABSTRACT
Opioid addiction presents a relevant health challenge, with chronic heroin use linked to detrimental effects on various aspects of physical, mental, and sociological health. Opioid maintenance therapy (OMT), particularly using methadone, is the primary treatment option for heroin addiction. Previous studies using blood samples from current heroin addicts and OMT patients have shown immunomodulatory effects of heroin and methadone on T cell function. However, various additional factors beyond heroin and methadone affect these results, including the consumption of other substances, a stressful lifestyle, comorbid psychological and somatic disorders, as well as additional medications. Therefore, we here investigated the direct effects of heroin and methadone on purified human T cells in vitro. Our results reveal that both, heroin and methadone directly suppress Tcell activation and proliferation. Strikingly, this inhibitory effect was markedly stronger in the presence of methadone, correlating with a decrease in secretion of pro-inflammatory cytokines. While heroin did not interfere with the in vitro differentiation and expansion of regulatory Tcells (Tregs), methadone significantly impaired the proliferation of Tregs. Overall, our findings suggest a direct inhibitory impact of both opioids on effector T cell function in vitro, with methadone additionally interfering with Treg induction and expansion in contrast to heroin.
Subject(s)
Cell Proliferation , Cytokines , Heroin Dependence , Heroin , Lymphocyte Activation , Methadone , T-Lymphocytes, Regulatory , Methadone/pharmacology , Humans , Cell Proliferation/drug effects , Lymphocyte Activation/drug effects , Cells, Cultured , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/drug effects , Heroin Dependence/immunology , Heroin Dependence/drug therapy , Cytokines/metabolism , Opiate Substitution Treatment , Analgesics, Opioid/pharmacology , Male , Adult , Female , Cell Differentiation/drug effectsABSTRACT
Morphine is an important pain reliever employed in pain management, its extended utilize is hindered by the onset of analgesic tolerance and oxidative stress. Long-term morphine administration causes elevated production of reactive oxygen species (ROS), disrupting mitochondrial function and inducing oxidation. Sirtuin 3 (SIRT3), a mitochondrial protein, is essential in modulating ROS levels by regulating mitochondrial antioxidant enzymes as manganese superoxide dismutase (MnSOD). Our investigation focused on the impact of SIRT3 on hyperalgesia and morphine tolerance in mice, as evaluating the antioxidant effect of the polyphenolic fraction of bergamot (BPF). Mice were administered morphine twice daily for four consecutive days (20 mg/kg). On the fifth day, mice received an acute dose of morphine (3 mg/kg), either alone or in conjunction with BPF or Mn (III)tetrakis (4-benzoic acid) porphyrin (MnTBAP). We evaluated levels of malondialdehyde (MDA), nitration, and the activity of SIRT3, MnSOD, glutamine synthetase (GS), and glutamate 1 transporter (GLT1) in the spinal cord. Our findings demonstrate that administering repeated doses of morphine led to the development of antinociceptive tolerance in mice, accompanied by increased superoxide production, nitration, and inactivation of mitochondrial SIRT3, MnSOD, GS, and GLT1. The combined administration of morphine with either BPF or MnTBAP prevented these effects.
Subject(s)
Drug Tolerance , Hyperalgesia , Mitochondria , Morphine , Oxidative Stress , Polyphenols , Sirtuin 3 , Animals , Morphine/pharmacology , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Male , Hyperalgesia/drug therapy , Hyperalgesia/chemically induced , Polyphenols/pharmacology , Sirtuin 3/metabolism , Oxidative Stress/drug effects , Superoxide Dismutase/metabolism , Reactive Oxygen Species/metabolism , Spinal Cord/drug effects , Spinal Cord/metabolism , Antioxidants/pharmacology , Analgesics, Opioid/pharmacology , Malondialdehyde/metabolism , Glutamate-Ammonia Ligase/metabolism , Metalloporphyrins/pharmacologyABSTRACT
Opioids are widely used, effective analgesics to manage severe acute and chronic pain, although they have recently come under scrutiny because of epidemic levels of abuse. While these compounds act on numerous central and peripheral pain pathways, the neuroanatomical substrate for opioid analgesia is not fully understood. By means of single-cell transcriptomics and manipulation of morphine-responsive neurons, we have identified an ensemble of neurons in the rostral ventromedial medulla (RVM) that regulates mechanical nociception in mice. Among these, forced activation or silencing of excitatory RVMBDNF projection neurons mimicked or completely reversed morphine-induced mechanical antinociception, respectively, via a brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)-dependent mechanism and activation of inhibitory spinal galanin-positive neurons. Our results reveal a specific RVM-spinal circuit that scales mechanical nociception whose function confers the antinociceptive properties of morphine.
Subject(s)
Analgesics, Opioid , Brain-Derived Neurotrophic Factor , Medulla Oblongata , Morphine , Neurons , Nociception , Animals , Male , Mice , Analgesics, Opioid/pharmacology , Brain-Derived Neurotrophic Factor/metabolism , Brain-Derived Neurotrophic Factor/genetics , Medulla Oblongata/drug effects , Medulla Oblongata/metabolism , Mice, Inbred C57BL , Morphine/pharmacology , Neurons/drug effects , Neurons/metabolism , Neurons/physiology , Nociception/drug effects , Receptor, trkB/metabolism , Single-Cell Analysis , Spinal Cord/drug effects , Transcriptome , FemaleABSTRACT
Manipulation of neural circuits targeted by morphine enables pain relief without opioids.
Subject(s)
Analgesics, Opioid , Morphine , Neural Pathways , Nociception , Pain Management , Sensory Receptor Cells , Animals , Humans , Mice , Analgesics, Opioid/pharmacology , Morphine/pharmacology , Neural Pathways/drug effects , Neural Pathways/physiology , Nociception/drug effects , Nociception/physiology , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/physiologyABSTRACT
Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.
Subject(s)
Analgesics, Opioid , Cyclic AMP , Gabapentin , Neuralgia , Signal Transduction , Spinal Cord Injuries , Animals , Neuralgia/drug therapy , Neuralgia/metabolism , Cyclic AMP/metabolism , Rats , Spinal Cord Injuries/drug therapy , Spinal Cord Injuries/metabolism , Analgesics, Opioid/pharmacology , Gabapentin/pharmacology , Signal Transduction/drug effects , Rats, Sprague-Dawley , Male , Calcium Channels, L-Type/metabolism , Calcium/metabolism , Pregabalin/pharmacology , Pregabalin/therapeutic use , Drug Synergism , Sensory Receptor Cells/metabolism , Sensory Receptor Cells/drug effectsABSTRACT
The endocannabinoid system interacts with the reward system to modulate responsiveness to natural reinforcers, as well as drugs of abuse. Previous preclinical studies suggested that direct blockade of CB1 cannabinoid receptors (CB1R) could be leveraged as a potential pharmacological approach to treat substance use disorder, but this strategy failed during clinical trials due to severe psychiatric side effects. Alternative strategies have emerged to circumvent the side effects of direct CB1 binding through the development of allosteric modulators. We hypothesized that negative allosteric modulation of CB1R signalling would reduce the reinforcing properties of morphine and decrease behaviours associated with opioid misuse. By employing intravenous self-administration in mice, we studied the effects of GAT358, a functionally-biased CB1R negative allosteric modulator (NAM), on morphine intake, relapse-like behaviour and motivation to work for morphine infusions. GAT358 reduced morphine infusion intake during the maintenance phase of morphine self-administration under a fixed ratio 1 schedule of reinforcement. GAT358 also decreased morphine-seeking behaviour after forced abstinence. Moreover, GAT358 dose dependently decreased the motivation to obtain morphine infusions under a progressive ratio schedule of reinforcement. Strikingly, GAT358 did not affect the motivation to work for food rewards in an identical progressive ratio task, suggesting that the effect of GAT358 in decreasing opioid self-administration was reward specific. Furthermore, GAT58 did not produce motor ataxia in the rotarod test. Our results suggest that CB1R NAMs reduced the reinforcing properties of morphine and could represent a viable therapeutic route to safely decrease misuse of opioids.
Subject(s)
Morphine , Receptor, Cannabinoid, CB1 , Self Administration , Animals , Morphine/pharmacology , Morphine/administration & dosage , Receptor, Cannabinoid, CB1/drug effects , Mice , Allosteric Regulation/drug effects , Male , Drug-Seeking Behavior/drug effects , Recurrence , Reinforcement, Psychology , Motivation/drug effects , Analgesics, Opioid/pharmacology , Analgesics, Opioid/administration & dosage , Administration, Intravenous , Conditioning, Operant/drug effects , Signal Transduction/drug effectsABSTRACT
Tramadol (TR), a commonly prescribed pain reliever for moderate to severe pain, has been associated with kidney damage. This study investigates TR-induced nephrotoxicity mechanisms, focusing on its effects on renal proximal tubular cells (PTCs). The study findings demonstrate that TR disrupts PTC bioenergetic processes, leading to oxidative stress and inflammation. Significant toxicity to PTCs was observed with estimated effective concentration 50 values of 9.8 and 11.5 µM based on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, respectively. TR also interferes with the function of PTC transporters, including organic cation uptake transporter 1, organic cation transporter 2, P-glycoprotein, and multidrug resistance protein 2. Furthermore, bioenergetics assays showed that TR reduced the activities of mitochondrial complexes I and III, adenosine triphosphate production, mitochondrial membrane potential, and oxygen consumption rate while increasing lactate release. TR also increased the production of reactive oxygen species, lipid peroxidation thiobarbituric acid reactive substances end products, and the expression of the NRf2 gene while decreasing reduced glutathione (GSH-R) stores and catalase and superoxide dismutase antioxidant activities. Additionally, TR increased the production of inflammatory cytokines (TNF-α and IL-6) and their coding genes expression. Interestingly, the study found that antioxidants like GSH-R, inhibitors of IL-6 and TNF-α, and mitochondrial activating Co-Q10 could protect cells against TR-induced cytotoxicity. The study suggests that TR causes nephrotoxicity by disrupting bioenergetic processes, causing oxidative stress and inflammation, but antioxidants and agents targeting mitochondria may have protective and curative potential.
Subject(s)
Energy Metabolism , Inflammation , Oxidative Stress , Tramadol , Oxidative Stress/drug effects , Inflammation/chemically induced , Inflammation/metabolism , Tramadol/adverse effects , Tramadol/pharmacology , Energy Metabolism/drug effects , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/pathology , Humans , Cell Line , Animals , Analgesics, Opioid/adverse effects , Analgesics, Opioid/pharmacologyABSTRACT
Approximately 50 million Americans suffer from chronic pain, and nearly a quarter of chronic pain patients have reported misusing opioid prescriptions. Repeated drug seeking is associated with reactivation of an ensemble of neurons sparsely scattered throughout the dorsomedial prefrontal cortex (dmPFC). Prior research has demonstrated that chronic pain increases intrinsic excitability of dmPFC neurons, which may increase the likelihood of reactivation during drug seeking. We tested the hypothesis that chronic pain would increase oxycodone-seeking behaviour and that the pain state would differentially increase intrinsic excitability in dmPFC drug-seeking ensemble neurons. TetTag mice self-administered intravenous oxycodone. After 7 days of forced abstinence, a drug-seeking session was performed, and the ensemble was tagged. Mice received spared nerve injury (SNI) to induce chronic pain during the period between the first and second seeking session. Following the second seeking session, we performed electrophysiology on individual neurons within the dmPFC to assess intrinsic excitability of the drug-seeking ensemble and non-ensemble neurons. SNI had no impact on sucrose seeking or intrinsic excitability of dmPFC neurons from these mice. In females, SNI increased oxycodone seeking and intrinsic excitability of non-ensemble neurons. In males, SNI had no impact on oxycodone seeking or neuron excitability. Data from females are consistent with clinical reports that chronic pain can promote drug craving and relapse and support the hypothesis that chronic pain itself may lead to neuroadaptations which promote opioid seeking.
Subject(s)
Analgesics, Opioid , Drug-Seeking Behavior , Neuralgia , Neurons , Oxycodone , Prefrontal Cortex , Animals , Oxycodone/pharmacology , Prefrontal Cortex/drug effects , Prefrontal Cortex/physiopathology , Drug-Seeking Behavior/drug effects , Mice , Neuralgia/physiopathology , Neurons/drug effects , Male , Female , Analgesics, Opioid/pharmacology , Self Administration , Chronic Pain/physiopathology , Sex FactorsABSTRACT
BACKGROUND: The pleiotropic effects of the melanocortin system show promise in overcoming limitations associated with large variations in opioid analgesic effectiveness observed in equine practice. Of particular interest is variation in the melanocortin-1-receptor (MC1R) gene, which dictates pigment type expression through its epistatic interaction with the agouti signalling protein (ASIP) gene. MC1R has previously been implicated in opioid efficacy in other species; however, this relationship is yet to be explored in horses. In this study, analgesic effectiveness was scored (1-3) based on noted response to dura penetration during the performance of cerebrospinal fluid centisis after sedation and tested for association with known genetic regions responsible for pigmentation variation in horses. RESULTS: The chestnut phenotype was statistically significant (P < 0.05) in lowering analgesic effectiveness when compared to the bay base coat colour. The 11bp indel in ASIP known to cause the black base coat colour was not significant (P>0.05); however, six single nucleotide polymorphisms (SNPs) within the genomic region encoding the ASIP gene and one within MC1R were identified as being nominally significant (P<0.05) in association with opioid analgesic effectiveness. This included the location of the known e MC1R variant resulting in the chestnut coat colour. CONCLUSIONS: The current study provides promising evidence for important links between pigmentation genes and opioid effectiveness in horses. The application of an easily identifiable phenotype indicating variable sensitivity presents a promising opportunity for accessible precision medicine in the use of analgesics and warrants further investigation.
Subject(s)
Analgesics, Opioid , Polymorphism, Single Nucleotide , Receptor, Melanocortin, Type 1 , Animals , Horses , Analgesics, Opioid/pharmacology , Analgesics, Opioid/therapeutic use , Receptor, Melanocortin, Type 1/genetics , Pigmentation/genetics , Agouti Signaling Protein/genetics , Male , Female , Phenotype , Cerebrospinal Fluid/metabolismABSTRACT
This study explored the potential of a series of PZM21 analogues for pain treatment. Specifically, the hydroxyphenyl ring of PZM21 was replaced with a naphthyl ring, the thienyl ring was substituted with either a phenyl ring or furan rings, and the essential dimethylamine and urea groups were retained. These compounds aimed to enhance safety and minimize the adverse effects associated with opioid drugs. The research findings suggest that compound 6a does not induce ß-arrestin recruitment at low-nanomolar concentrations but exhibits significant analgesic effects in established mouse models. Compared to morphine, 6a shows advantages in alleviating respiratory depression and minimizing physical dependence. Molecular docking studies underscore the pivotal role of the D147 amino acid residue in the analgesic mechanism of 6a. Consequently, 6a is a compelling candidate for the development of safer opioid analgesics and warrants further attention.
Subject(s)
Analgesics, Opioid , Molecular Docking Simulation , Receptors, Opioid, mu , Receptors, Opioid, mu/agonists , Receptors, Opioid, mu/metabolism , Animals , Mice , Analgesics, Opioid/pharmacology , Analgesics, Opioid/chemistry , Analgesics, Opioid/chemical synthesis , Humans , Structure-Activity Relationship , Pain/drug therapy , Male , Molecular Structure , Thiophenes , Urea/analogs & derivativesABSTRACT
Opioid withdrawal is a liability of chronic opioid use and misuse, impacting people who use prescription or illicit opioids. Hyperactive autonomic output underlies many of the aversive withdrawal symptoms that make it difficult to discontinue chronic opioid use. The locus coeruleus (LC) is an important autonomic centre within the brain with a poorly defined role in opioid withdrawal. We show here that pannexin-1 (Panx1) channels expressed on microglia critically modulate LC activity during opioid withdrawal. Within the LC, we found that spinally projecting tyrosine hydroxylase (TH)-positive neurons (LCspinal) are hyperexcitable during morphine withdrawal, elevating cerebrospinal fluid (CSF) levels of norepinephrine. Pharmacological and chemogenetic silencing of LCspinal neurons or genetic ablation of Panx1 in microglia blunted CSF NE release, reduced LC neuron hyperexcitability, and concomitantly decreased opioid withdrawal behaviours in mice. Using probenecid as an initial lead compound, we designed a compound (EG-2184) with greater potency in blocking Panx1. Treatment with EG-2184 significantly reduced both the physical signs and conditioned place aversion caused by opioid withdrawal in mice, as well as suppressed cue-induced reinstatement of opioid seeking in rats. Together, these findings demonstrate that microglial Panx1 channels modulate LC noradrenergic circuitry during opioid withdrawal and reinstatement. Blocking Panx1 to dampen LC hyperexcitability may therefore provide a therapeutic strategy for alleviating the physical and aversive components of opioid withdrawal.
Subject(s)
Connexins , Locus Coeruleus , Nerve Tissue Proteins , Probenecid , Spinal Cord , Substance Withdrawal Syndrome , Animals , Locus Coeruleus/metabolism , Locus Coeruleus/drug effects , Connexins/metabolism , Connexins/genetics , Connexins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Substance Withdrawal Syndrome/metabolism , Substance Withdrawal Syndrome/drug therapy , Mice , Male , Rats , Spinal Cord/metabolism , Spinal Cord/drug effects , Probenecid/pharmacology , Morphine/pharmacology , Microglia/drug effects , Microglia/metabolism , Analgesics, Opioid/pharmacology , Norepinephrine/metabolism , Neurons/metabolism , Neurons/drug effects , Mice, Inbred C57BL , Rats, Sprague-Dawley , Tyrosine 3-Monooxygenase/metabolism , Mice, KnockoutABSTRACT
We aimed to examine the efficacy of combination therapies of Neurotropin® with tramadol and Neurotropin with mirogabalin for neuropathic pain management. A neuropathic pain model (L5 spinal nerve ligation model: L5-SNL) using male Wistar rats was generated through tight ligation of the left fifth lumbar nerve using silk sutures. Mechanical allodynia was assessed using the 50% paw withdrawal threshold. The combined antiallodynic effects were evaluated using isobolographic analyses. Small intestinal transit was evaluated using the charcoal meal test, and motor coordination using the rota-rod test. Neurotropin (50-200 NU/kg, p.o.), tramadol (7.5-60 mg/kg, p.o.), and mirogabalin (3-30 mg/kg, p.o.) showed a dose-dependent antiallodynic effect in L5-SNL rats. The combined antiallodynic effects of Neurotropin and tramadol were additive or synergistic, whereas those of Neurotropin and mirogabalin were additive. Neurotropin (100-400 NU/kg, p.o.) did not affect the small intestinal transit, whereas tramadol (30-100 mg/kg, p.o.) significantly inhibited it. Neurotropin (100-400 NU/kg, p.o.) did not affect the walking time, whereas mirogabalin (10-100 mg/kg, p.o.) significantly decreased it. Neurotropin dose-dependently ameliorated mechanical allodynia in rats, and combination therapy with Neurotropin-tramadol or Neurotropin-mirogabalin may alleviate neuropathic pain without aggravating the adverse effects of tramadol and mirogabalin.
Subject(s)
Disease Models, Animal , Hyperalgesia , Neuralgia , Rats, Wistar , Spinal Nerves , Tramadol , Animals , Tramadol/administration & dosage , Tramadol/pharmacology , Male , Neuralgia/drug therapy , Hyperalgesia/drug therapy , Spinal Nerves/drug effects , Ligation/adverse effects , Drug Therapy, Combination , Dose-Response Relationship, Drug , Rats , Gastrointestinal Transit/drug effects , Analgesics, Opioid/administration & dosage , Analgesics, Opioid/pharmacology , Bridged Bicyclo Compounds , PolysaccharidesABSTRACT
We recently developed a series of nalfurafine analogs (TK10, TK33, and TK35) that may serve as non-addictive candidate analgesics. These compounds are mixed-action agonists at the kappa and delta opioid receptors (KOR and DOR, respectively) and produce antinociception in a mouse warm-water tail-immersion test while failing to produce typical mu opioid receptor (MOR)-mediated side effects. The warm-water tail-immersion test is an assay of pain-stimulated behavior vulnerable to false-positive analgesic-like effects by drugs that produce motor impairment. Accordingly, this study evaluated TK10, TK33, and TK35 in a recently validated assay of pain-related behavioral depression in mice that are less vulnerable to false-positive effects. For comparison, we also evaluated the effects of the MOR agonist/analgesic hydrocodone (positive control), the neurokinin 1 receptor (NK1R) antagonist aprepitant (negative control), nalfurafine as a selective KOR agonist, SNC80 as a selective DOR agonist, and a nalfurafine/SNC80 mixture. Intraperitoneal injection of dilute lactic acid (IP lactic acid) served as a noxious stimulus to depress vertical and horizontal locomotor activity in male and female ICR mice. IP lactic acid-induced locomotor depression was alleviated by hydrocodone but not by aprepitant, nalfurafine, SNC80, the nalfurafine/SNC80 mixture, or the KOR/DOR agonists. These results suggest that caution is warranted in advancing mixed-action KOR/DOR agonists as candidate analgesics.
Subject(s)
Pain , Receptors, Opioid, delta , Receptors, Opioid, kappa , Animals , Receptors, Opioid, delta/agonists , Receptors, Opioid, delta/metabolism , Mice , Receptors, Opioid, kappa/agonists , Receptors, Opioid, kappa/metabolism , Pain/drug therapy , Pain/metabolism , Male , Depression/drug therapy , Depression/etiology , Morphinans/pharmacology , Behavior, Animal/drug effects , Analgesics, Opioid/pharmacology , Spiro Compounds/pharmacology , Spiro Compounds/chemistryABSTRACT
Animal studies modeling recreational opioid use show more severe withdrawal symptoms in male compared to female rats, whereas our study modeling opioid use for pain showed a greater withdrawal-induced decrease in wheel running in female rats. The objective of this experiment was to determine whether sex differences in spontaneous morphine withdrawal are caused by differences in assessment method (i.e., wheel running vs. somatic symptoms). Twice daily injections of morphine (5 - 20â¯mg/kg, s.c.) for 5 days produced a dose and time dependent decrease in wheel running that was greater in male compared to female rats. Termination of morphine administration resulted in an overall decrease in running and a decrease in the amount of running during the dark phase of the light cycle from 95â¯% to approximately 75â¯%. In male rats, this decrease in the percent of dark running was caused by a large decrease in dark phase running, whereas female rats had a slightly higher increase in light phase running. Withdrawal also reduced maximal running speed and caused a decrease in body weight that was larger in male than female rats. Withdrawal symptoms were greatest on the day following the last morphine injection, but persisted for all 3 days of assessment. Morphine withdrawal produced a greater decrease in dark phase wheel running and body weight in male rats and a greater increase in light phase running in female rats. Voluntary home cage wheel running provides a continuous measure of opioid withdrawal that is consistent with other measures of opioid withdrawal.
Subject(s)
Circadian Rhythm , Morphine , Motor Activity , Sex Characteristics , Substance Withdrawal Syndrome , Animals , Male , Female , Substance Withdrawal Syndrome/physiopathology , Morphine/pharmacology , Morphine/administration & dosage , Rats , Motor Activity/drug effects , Motor Activity/physiology , Circadian Rhythm/physiology , Circadian Rhythm/drug effects , Rats, Sprague-Dawley , Narcotics/administration & dosage , Narcotics/pharmacology , Body Weight/drug effects , Body Weight/physiology , Running/physiology , Dose-Response Relationship, Drug , Analgesics, Opioid/pharmacology , Analgesics, Opioid/administration & dosageABSTRACT
Agonists at µ opioid receptors relieve acute pain, however, their long-term use is limited by side effects, which may involve ß-arrestin2. Agonists biased against ß-arrestin2 recruitment may be advantageous. However, the classification of bias may be compromised by assays utilising overexpressed µ receptors which overestimate efficacy for G-protein activation. There is a need for re-evaluation with restricted receptor availability to determine accurate agonist efficacies. We depleted µ receptor availability in PathHunter CHO cells using the irreversible antagonist, ß-funaltrexamine (ß-FNA), and compared efficacies and apparent potencies of twelve agonists, including several previously reported as biased, in ß-arrestin2 recruitment and cAMP assays. With full receptor availability all agonists had partial efficacy for stimulating ß-arrestin2 recruitment relative to DAMGO, while only TRV130 and buprenorphine were partial agonists as inhibitors of cAMP accumulation. Limiting receptor availability by prior exposure to ß-FNA (100 nM) revealed morphine, oxycodone, PZM21, herkinorin, U47700, tianeptine and U47931e are also partial agonists in the cAMP assay. The efficacies of all agonists, except SR-17018, correlated between ß-arrestin2 recruitment and cAMP assays, with depleted receptor availability in the latter. Furthermore, naloxone and cyprodime exhibited non-competitive antagonism of SR-17018 in the ß-arrestin2 recruitment assay. Limited antagonism by naloxone was also non-competitive in the cAMP assay, while cyprodime was competitive. Furthermore, SR-17018 only negligibly diminished ß-arrestin2 recruitment stimulated by DAMGO (1 µM), whereas fentanyl, morphine and TRV130 all exhibited the anticipated competitive inhibition. The data suggest that SR-17018 achieves bias against ß-arrestin2 recruitment through interactions with µ receptors outside the orthosteric agonist site. This article is part of the Special Issue on "Ligand Bias".
Subject(s)
Analgesics, Opioid , Cricetulus , Cyclic AMP , Receptors, Opioid, mu , Animals , CHO Cells , Receptors, Opioid, mu/metabolism , Receptors, Opioid, mu/agonists , Analgesics, Opioid/pharmacology , Cyclic AMP/metabolism , Narcotic Antagonists/pharmacology , Naltrexone/pharmacology , Naltrexone/analogs & derivatives , Cricetinae , Humans , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , beta-Arrestins/metabolism , Dose-Response Relationship, Drug , beta-Arrestin 2/metabolism , Spiro Compounds , ThiophenesABSTRACT
The concept of ligand bias is based on the premise that different agonists can elicit distinct responses by selectively activating the same receptor. These responses often determine whether an agonist has therapeutic or undesirable effects. Therefore, it would be highly advantageous to have agonists that specifically trigger the therapeutic response. The last two decades have seen a growing trend towards the consideration of ligand bias in the development of ligands to target the κ-opioid receptor (κOR). Most of these ligands selectively favor G-protein signaling over ß-arrestin signaling to potentially provide effective pain and itch relief without adverse side effects associated with κOR activation. Importantly, the specific role of ß-arrestin 2 in mediating κOR agonist-induced side effects remains unknown, and similarly the therapeutic and side-effect profiles of G-protein-biased κOR agonists have not been established. Furthermore, some drugs previously labeled as G-protein-biased may not exhibit true bias but may instead be either low-intrinsic-efficacy or partial agonists. In this review, we discuss the established methods to test ligand bias, their limitations in measuring bias factors for κOR agonists, as well as recommend the consideration of other systematic factors to correlate the degree of bias signaling and pharmacological effects. This article is part of the Special Issue on "Ligand Bias".
Subject(s)
Pruritus , Receptors, Opioid, kappa , Receptors, Opioid, kappa/agonists , Humans , Animals , Pruritus/drug therapy , Pain/drug therapy , Analgesics, Opioid/therapeutic use , Analgesics, Opioid/pharmacology , Ligands , Pain Management/methodsABSTRACT
Opioids are small-molecule agonists of µ-opioid receptor (µOR), while reversal agents such as naloxone are antagonists of µOR. Here, we developed machine learning (ML) models to classify the intrinsic activities of ligands at the human µOR based on the SMILES strings and two-dimensional molecular descriptors. We first manually curated a database of 983 small molecules with measured Emax values at the human µOR. Analysis of the chemical space allowed identification of dominant scaffolds and structurally similar agonists and antagonists. Decision tree models and directed message passing neural networks (MPNNs) were then trained to classify agonistic and antagonistic ligands. The hold-out test AUCs (areas under the receiver operator curves) of the extra-tree (ET) and MPNN models are 91.5 ± 3.9% and 91.8 ± 4.4%, respectively. To overcome the challenge of a small data set, a student-teacher learning method called tritraining with disagreement was tested using an unlabeled data set comprised of 15,816 ligands of human, mouse, and rat µOR, κOR, and δOR. We found that the tritraining scheme was able to increase the hold-out AUC of MPNN models to as high as 95.7%. Our work demonstrates the feasibility of developing ML models to accurately predict the intrinsic activities of µOR ligands, even with limited data. We envisage potential applications of these models in evaluating uncharacterized substances for public safety risks and discovering new therapeutic agents to counteract opioid overdoses.