Warm-water Tail-withdrawal Assay Research Articles

Rats chasing the dragon: A new heroin inhalation method

BackgroundDespite extensive human use of inhalation for ingesting opioids, models in rodents have mostly been limited to parenteral injection and oral dosing. Methods using electronic drug delivery systems (EDDS; “e-cigarettes”) have shown efficacy in rodent models but these do not faithfully mimic the most popular human inhalation method of heating heroin to the point of vaporization. New methodMiddle aged rats were exposed to vapor created by direct heating of heroin HCl powder in a ceramic e-cigarette type atomizer. Efficacy was determined with a warm water tail withdrawal nociception assay, rectal temperature and self-administration. ResultsTen minutes of inhalation of vaporized heroin slowed response latency in a warm water tail withdrawal assay and increased rectal temperature in male rats, in a dose-dependent manner. Similar antinociceptive effects in female rats were attenuated by the opioid antagonist naloxone (1.0 mg/kg, s.c.). Female rats made operant responses for heroin vapor in 15-minute sessions, increased their response rate when the reinforcement ratio increased from FR1 to FR5, and further increased their responding when vapor delivery was omitted. Anti-nociceptive effects of self-administered volatilized heroin were of a similar magnitude as those produced by the 10-minute non-contingent exposure. Comparison with existing methodsInhalation of directly volatilized heroin successfully produces heroin-typical effects, comparable to EDDS inhalation delivery. ConclusionsThis study shows that “chasing the dragon” methods of inhalation of heroin can be modeled successfully in the rat. Inhalation techniques may be particularly useful for longer term studies deep into the middle age of rats.

Identification and Pharmacological Characterization of a Low-Liability Antinociceptive Bifunctional MOR/DOR Cyclic Peptide.

Peptide-based opioid ligands are important candidates for the development of novel, safer, and more effective analgesics to treat pain. To develop peptide-based safer analgesics, we synthesized a mixture-based cyclic pentapeptide library containing a total of 24,624 pentapeptides and screened the mixture-based library samples using a 55 °C warm water tail-withdrawal assay. Using this phenotypic screening approach, we deconvoluted the mixture-based samples to identify a novel cyclic peptide Tyr-[D-Lys-Dap(Ant)-Thr-Gly] (CycloAnt), which produced dose- and time-dependent antinociception with an ED50 (and 95% confidence interval) of 0.70 (0.52-0.97) mg/kg i.p. mediated by the mu-opioid receptor (MOR). Additionally, higher doses (≥3 mg/kg, i.p.) of CycloAnt antagonized delta-opioid receptors (DOR) for at least 3 h. Pharmacological characterization of CycloAnt showed the cyclic peptide did not reduce breathing rate in mice at doses up to 15 times the analgesic ED50 value, and produced dramatically less hyperlocomotion than the MOR agonist, morphine. While chronic administration of CycloAnt resulted in antinociceptive tolerance, it was without opioid-induced hyperalgesia and with significantly reduced signs of naloxone-precipitated withdrawal, which suggested reduced physical dependence compared to morphine. Collectively, the results suggest this dual MOR/DOR multifunctional ligand is an excellent lead for the development of peptide-based safer analgesics.

Tryptophan Substitution in CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) Introduces δ-Opioid Receptor Antagonism, Preventing Antinociceptive Tolerance and Stress-Induced Reinstatement of Extinguished Cocaine-Conditioned Place Preference.

The macrocyclic tetrapeptide CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer exhibit kappa opioid receptor (KOR) antagonism which prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference. Here, we evaluated the effects of substitution of Trp and D-Trp on the peptides' opioid activity, antinociceptive tolerance, and the ability to prevent relapse to extinguished drug-CPP. Six analogs were synthesized using a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, efficacy in the [35S]GTPγS assay, metabolic stability in mouse liver microsomes, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance, and conditioned place preference (CPP) were also assessed in vivo, and the ameliorating effect of analogs on the reinstatement of extinguished cocaine-place preference was assessed. Substitutions of other D-amino acids for D-Trp did not affect (or in one case increased) KOR affinity, while two of the three substitutions of an L-amino acid for Trp decreased KOR affinity. In contrast, all but one substitution increased mu opioid receptor (MOR) affinity in vitro. The metabolic stabilities of the analogs were similar to those of their respective parent peptides, with analogs containing a D-amino acid being much more rapidly metabolized than those containing an L-amino acid in this position. In vivo, CJ-15,208 analogs demonstrated antinociception, although potencies varied over an 80-fold range and the mediating opioid receptors differed by substitution. KOR antagonism was lost for all but the D-benzothienylalanine analog, and the 2'-naphthylalanine analog instead demonstrated significant delta opioid receptor (DOR) antagonism. Introduction of DOR antagonism coincided with reduced acute opioid antinociceptive tolerance and prevented stress-induced reinstatement of extinguished cocaine-CPP.

Nitro-benzylideneoxymorphone, a bifunctional mu and delta opioid receptor ligand with high mu opioid receptor efficacy.

Introduction: There is a major societal need for analgesics with less tolerance, dependence, and abuse liability. Preclinical rodent studies suggest that bifunctional ligands with both mu (MOPr) and delta (DOPr) opioid peptide receptor activity may produce analgesia with reduced tolerance and other side effects. This study explores the structure-activity relationships (SAR) of our previously reported MOPr/DOPr lead, benzylideneoxymorphone (BOM) with C7-methylene-substituted analogs. Methods: Analogs were synthesized and tested in vitro for opioid receptor binding and efficacy. One compound, nitro-BOM (NBOM, 12) was evaluated for antinociceptive effects in the warm water tail withdrawal assay in C57BL/6 mice. Acute and chronic antinociception was determined, as was toxicologic effects on chronic administration. Molecular modeling experiments were performed using the Site Identification by Ligand Competitive Saturation (SILCS) method. Results: NBOM was found to be a potent MOPr agonist/DOPr partial agonist that produces high-efficacy antinociception. Antinociceptive tolerance was observed, as was weight loss; this toxicity was only observed with NBOM and not with BOM. Modeling supports the hypothesis that the increased MOPr efficacy of NBOM is due to the substituted benzylidene ring occupying a nonpolar region within the MOPr agonist state. Discussion: Though antinociceptive tolerance and non-specific toxicity was observed on repeated administration, NBOM provides an important new tool for understanding MOPr/DOPr pharmacology.

Deuterated buprenorphine retains pharmacodynamic properties of buprenorphine and resists metabolism to the active metabolite norbuprenorphine in rats.

Introduction: An active metabolite of buprenorphine (BUP), called norbuprenorphine (NorBUP), is implicated in neonatal opioid withdrawal syndrome when BUP is taken during pregnancy. Therefore, reducing or eliminating metabolism of BUP to NorBUP is a novel strategy that will likely lower total fetal exposure to opioids and thus improve offspring outcomes. Precision deuteration alters pharmacokinetics of drugs without altering pharmacodynamics. Here, we report the synthesis and testing of deuterated buprenorphine (BUP-D2). Methods: We determined opioid receptor affinities of BUP-D2 relative to BUP with radioligand competition receptor binding assays, and the potency and efficacy of BUP-D2 relative to BUP to activate G-proteins via opioid receptors with [35S]GTPγS binding assays in homogenates containing the human mu, delta, or kappa opioid receptors. The antinociceptive effects of BUP-D2 and BUP were compared using the warm-water tail withdrawal assay in rats. Blood concentration versus time profiles of BUP, BUP-D2, and NorBUP were measured in rats following intravenous BUP-D2 or BUP injection. Results: The synthesis provided a 48% yield and the product was ≥99% deuterated. Like BUP, BUP-D2 had sub-nanomolar affinity for opioid receptors. BUP-D2 also activated opioid receptors and induced antinociception with equal potency and efficacy as BUP. The maximum concentration and the area under the curve of NorBUP in the blood of rats that received BUP-D2 were over 19- and 10-fold lower, respectively, than in rats that received BUP. Discussion: These results indicate that BUP-D2 retains key pharmacodynamic properties of BUP and resists metabolism to NorBUP and therefore holds promise as an alternative to BUP.

Effects of selective dopamine D3 receptor partial agonist/antagonists on oxycodone self-administration and antinociception in monkeys.

Recent studies suggest that dopamine D3 receptors (D3R) may be a therapeutic target for opioid use disorders (OUD). This study examined the effects of the D3Rpartial agonist (±)VK4-40 and the D3R-selective antagonist (±)VK4-116, compared to the mu-opioid receptor antagonist naltrexone (NTX), in nonhuman primate models of OUD and antinociception. Adult male and female (N = 4/sex) cynomolgus monkeys were trained to self-administer oxycodone (0.003-0.1 mg/kg/injection) first under a fixed-ratio (FR) and then a progressive-ratio (PR) schedule of reinforcement during daily 1- and 4-hr sessions, respectively. Under the FR schedule, intravenous NTX (0.01-0.1 mg/kg), (±)VK4-116 (1.0-10 mg/kg), and (±)VK4-40 (1.0-10 mg/kg) were studied in combination with the peak oxycodone dose and a dose on the descending limb of the dose-effect curve; NTX and (±)VK4-40 were also studied at the peak of the PR dose-response curve (N = 4). Following saline extinction, each compound was examined on oxycodone-induced reinstatement. Finally, these compounds were assessed in adult male rhesus monkeys (N = 3) in a warm-water (38 °C, 50 °C, 54 °C) tail withdrawal assay. NTX decreased responding on the peak of the FR oxycodone dose-response curve, but increased responding on the descending limb. (±)VK4-40, but not (±)VK4-116, significantly decreased peak oxycodone self-administration; (±)VK4-40 did not increase responding on the descending limb. NTX and (±)VK4-40, but not (±)VK4-116, attenuated oxycodone-induced reinstatement. Under PR responding, NTX and (±)VK4-40 decreased breakpoints. Oxycodone-induced antinociception was attenuated by NTX, but not by (±)VK4-40 or (±)VK4-116. Together, these results suggest that further research evaluating the effects of (±)VK4-40 as a novel pharmacotherapy for OUD is warranted.

Behavioral Effects of Vaporized Delta-8 Tetrahydrocannabinol, Cannabidiol, and Mixtures in Male Rats.

Background: The popularity of delta-8 tetrahydrocannabinol (THC) and cannabidiol (CBD) products has seen a sharp increase in use during recent years. Despite the rise in use of these minor cannabinoids, there are little to no pre-clinical behavioral data on their effects, with most pre-clinical cannabis research focusing on the behavioral effects of delta-9 THC. The current experiments aimed to characterize the behavioral effects of delta-8 THC, CBD, and mixtures of these two drugs using a whole-body vapor exposure route of administration in male rats. Methods: Rats were exposed to vapor that contained different concentrations of delta-8 THC, CBD, or CBD/delta-8 THC mixtures during 10 min of exposure. Following 10 min of vapor exposure, locomotor behavior was monitored, or the warm-water tail withdrawal assay was conducted to measure the acute analgesic effects of the vapor exposure. Results: CBD and CBD/delta-8 THC mixtures resulted in a significant increase in locomotion across the entire session. Although delta-8 THC alone had no significant effect on locomotion across the session, the 10 mg concentration of delta-8 THC had a hyperlocomotion effect in the first 30 min of the session followed by a hypolocomotor effect later in the session. In the tail withdrawal assay, a 3/1 mixture of CBD/delta-8 THC resulted in an immediate analgesic effect compared to vehicle vapor. Finally, immediately following vapor exposure, all drugs had a hypothermic effect on body temperature compared to vehicle. Conclusion: This experiment is the first to characterize the behavioral effects of vaporized delta-8 THC, CBD, and CBD/delta-8 THC in male rats. While data were generally congruent with previous research investigating delta-9 THC, future studies should explore abuse liability and validate plasma blood concentrations of these drugs following administration through whole-body vapor exposure.

The long-term effects of repeated heroin vapor inhalation during adolescence on measures of nociception and anxiety-like behavior in adult Wistar rats

RationaleAdolescents represent a vulnerable group due to increased experimentation with illicit substances that is often associated with the adolescent period, and because adolescent drug use can result in long-term effects that differ from those caused by drug use initiated during adulthood.ObjectivesThe purpose of the present study was to determine the effects of repeated heroin vapor inhalation during adolescence on measures of nociception, and anxiety-like behavior during adulthood in female and male Wistar rats.MethodsRats were exposed twice daily to 30 min of heroin vapor from post-natal day (PND) 36 to PND 45. At 12 weeks of age, baseline thermal nociception was assessed across a range of temperatures with a warm-water tail-withdrawal assay. Anxiety-like behavior was assessed in an elevated plus-maze (EPM) and activity was measured in an open-field arena. Starting at 23 weeks of age, baseline thermal nociception was re-assessed, nociception was determined after acute heroin or naloxone injection, and anxiety-like behavior was redetermined in the EPM.ResultsAdolescent heroin inhalation altered baseline thermal nociception in female rats at 12 weeks of age and in both female and male rats at ~ 23 weeks. Heroin-treated animals exhibited anxiety-like behavior when tested in the elevated plus-maze, showed blunted heroin-induced analgesia, but exhibited no effect on naloxone-induced hyperalgesia.ConclusionsThe present study demonstrates that heroin vapor inhalation during adolescence produces behavioral and physiological consequences in rats that persist well into adulthood.

Bis-Cyclic Guanidine Heterocyclic Peptidomimetics as Opioid Ligands with Mixed μ-, κ- and δ-Opioid Receptor Interactions: A Potential Approach to Novel Analgesics

The design and development of analgesics with mixed-opioid receptor interactions has been reported to decrease side effects, minimizing respiratory depression and reinforcing properties to generate safer analgesic therapeutics. We synthesized bis-cyclic guanidine heterocyclic peptidomimetics from reduced tripeptides. In vitro screening with radioligand competition binding assays demonstrated variable affinity for the mu-opioid receptor (MOR), delta-opioid receptor (DOR), and kappa-opioid receptor (KOR) across the series, with compound 1968-22 displaying good affinity for all three receptors. Central intracerebroventricular (i.c.v.) administration of 1968-22 produced dose-dependent, opioid receptor-mediated antinociception in the mouse 55 °C warm-water tail-withdrawal assay, and 1968-22 also produced significant antinociception up to 80 min after oral administration (10 mg/kg, p.o.). Compound 1968-22 was detected in the brain 5 min after intravenous administration and was shown to be stable in the blood for at least 30 min. Central administration of 1968-22 did not produce significant respiratory depression, locomotor effects or conditioned place preference or aversion. The data suggest these bis-cyclic guanidine heterocyclic peptidomimetics with multifunctional opioid receptor activity may hold potential as new analgesics with fewer liabilities of use.

OREX-1038: a potential new treatment for pain with low abuse liability and limited adverse effects.

Drugs targeting mu opioid receptors are the mainstay of clinical practice for treating moderate-to-severe pain. While they can offer excellent analgesia, their use can be limited by adverse effects, including constipation, respiratory depression, tolerance, and abuse liability. Multifunctional ligands acting at mu opioid and nociceptin/orphanin FQ peptide receptors might provide antinociception with substantially improved adverse-effect profiles. This study explored one of these ligands, OREX-1038 (BU10038), in several assays in rodents and nonhuman primates. Binding and functional studies confirmed OREX-1038 to be a low-efficacy agonist at mu opioid and nociceptin/orphanin FQ peptide receptors and an antagonist at delta and kappa opioid receptors with selectivity for opioid receptors over other proteins. OREX-1038 had long-acting antinociceptive effects in postsurgical and complete Freund's adjuvant (CFA)-induced thermal hyperalgesia assays in rats and a warm water tail-withdrawal assay in monkeys. OREX-1038 was active for at least 24 h in each antinociception assay, and its effects in monkeys did not diminish over 22 days of daily administration. This activity was coupled with limited effects on physiological signs (arterial pressure, heart rate, and body temperature) and no evidence of withdrawal after administration of naltrexone or discontinuation of treatment in monkeys receiving OREX-1038 daily. Over a range of doses, OREX-1038 was only transiently self-administered, which diminished rapidly to nonsignificant levels; overall, both OREX-1038 and buprenorphine maintained less responding than remifentanil. These results support the concept of dual mu and nociceptin/orphanin FQ peptide receptor partial agonists having improved pharmacological profiles compared with opioids currently used to treat pain.

Characterization of CM-398, a Novel Selective Sigma-2 Receptor Ligand, as a Potential Therapeutic for Neuropathic Pain.

Sigma receptors modulate nociception, offering a potential therapeutic target to treat pain, but relatively little is known regarding the role of sigma-2 receptors (S2R) in nociception. The purpose of this study was to investigate the in vivo analgesic and anti-allodynic activity and liabilities of a novel S2R selective ligand, 1-[4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)butyl]-3-methyl-1,3-dihydro-1,3-benzimidazol-2-one (CM-398). The inhibition of thermal, induced chemical, or inflammatory pain as well as the allodynia resulting from chronic nerve constriction injury (CCI) model of neuropathic pain were assessed in male mice. CM-398 dose-dependently (10–45 mg/kg i.p.) reduced mechanical allodynia in the CCI neuropathic pain model, equivalent at the higher dose to the effect of the control analgesic gabapentin (50 mg/kg i.p.). Likewise, pretreatment (i.p.) with CM-398 dose-dependently produced antinociception in the acetic acid writhing test (ED50 (and 95% C.I.) = 14.7 (10.6–20) mg/kg, i.p.) and the formalin assay (ED50 (and 95% C.I.) = 0.86 (0.44–1.81) mg/kg, i.p.) but was without effect in the 55 °C warm-water tail-withdrawal assay. A high dose of CM-398 (45 mg/kg, i.p.) exhibited modest locomotor impairment in a rotarod assay and conditioned place aversion, potentially complicating the interpretation of nociceptive testing. However, in an operant pain model resistant to these confounds, mice experiencing CCI and treated with CM-398 demonstrated robust conditioned place preference. Overall, these results demonstrate the S2R selective antagonist CM-398 produces antinociception and anti-allodynia with fewer liabilities than established therapeutics, adding to emerging data suggesting possible mediation of nociception by S2R, and the development of S2R ligands as potential treatments for chronic pain.

Positive Allosteric Modulation of the Mu Opioid Receptor

Opioid therapeutics, such as morphine, that act at the mu‐opioid receptor (MOR) are the clinical standard for patients struggling to manage symptoms associated with pain. It is widely understood that although opioids are effective at treating pain, their use leads to the development of severe adverse effects, such as constipation, addiction, and respiratory depression. Thus, there is a clear need for a safer alternative to manage pain. One such alternative is to enhance the effects of the body’s endogenous opioid system by positive allosteric modulation (PAM) of MOR. A known PAM, BMS‐986122, enhances MOR agonist potency in cellular models and MOR agonist mediated antinociception in vivo. In addition, this PAM is active alone in a variety of mouse pain assays by promoting the activity of endogenous opioid peptides. Moreover, at an effective antinociceptive dose, BMS‐986122 alone produces less severe adverse effects than morphine as determined by measures of constipation, respiratory depression, and conditioned place preference. However, we do not yet know how the overall pharmacology of opioids is affected by PAMs or if all opioid drugs are equally sensitive to PAM modulation. Here we compare the ability of BMS‐986122 to enhance the action of three structurally diverse opioid drugs, morphine, methadone, and fentanyl, in an acute pain assay and in an assay examining respiratory depression using CD‐1 male and female mice. We show that BMS‐986122 increases the antinociceptive effects of the opioid therapeutics in the warm water tail withdrawal assay without promoting the ability of the drugs to lower blood oxygen levels or heart rate. Future work will assess the effects of BMS‐986122 in additional acute and chronic pain models and with a more diverse group of opioids. If additional experiments support the concept that BMS‐986122 enhances MOR‐mediated antinociception but not MOR‐mediated respiratory depression, this will validate the development of MOR‐PAMs as standalone pain medications and support the use of PAMs as opioid‐sparing drugs for the effective management of pain.

Effects of prenatal opioid exposure and early life adversity on opioid‐induced antinociception

Diagnoses of maternal opioid use disorder (MOUD) at delivery increased more than 500% between 1999 and 2017 in the United States. Today, cases of in utero opioid exposure due to MOUD exceed cases of the most common birth defects, including cleft lip, cleft palate, clubfoot, and Down syndrome, combined. Children exposed to opioids in utero are at increased risk of adverse childhood experiences (ACEs) due to parental care being compromised by SUDs. These ACEs include abuse, neglect, loss of a parent from death or incarceration, parental divorce, and household domestic violence. Altered sensitivity to opioid analgesia later in life is one potential outcome shared by in utero opioid exposure and ACEs that may contribute to poorly controlled pain and increased risk of opioid addiction. Studies using rat models of prenatal opioid exposure (POE) or early life adversity (ELA) have shown that each decreases opioid‐induced antinociception long after exposure has ceased. However, the contributions of POE and ELA, alone and in combination, to opioid analgesic response in humans remains unknown. In this study, we used a rodent model of combined POE and ELA to disambiguate their long‐term effects more quickly and with greater experimental control than human studies. We hypothesized that combined POE and ELA will decrease morphine‐induced antinociception in adolescence relative to POE or ELA alone. We used a two‐by‐two between‐subjects factorial design in which timed‐pregnant Long‐Evans rats were exposed to morphine (15 mg/kg/day; “POE” group) or saline (“vehicle” group) via subcutaneous osmotic minipump from gestation day 9 until delivery. Litters were fostered to untreated dams then randomly assigned to normal housing or ELA conditions using a limited bedding and nesting procedure from postnatal days 3‐11. Morphine‐induced antinociception was measured between postnatal days 30 and 54 using a warm‐water tail withdrawal (WWTW) assay. Cumulative dose‐response of morphine‐induced antinociception was determined on test day 0 (1.0‐30 mg/kg morphine, s.c.) and test day 13 (3.0‐100 mg/kg, s.c.). On test days 1‐12, rats were dosed with 18 mg/kg, s.c., every 12 hours, and WWTW was conducted on odd test days. Interesting, preliminary results of this ongoing study indicate that morphine‐induced antinociception was enhanced on test day 0 in POE + normal‐housed males (n = 2). These rats exhibited 100% maximum possible effect (MPE) and significantly higher potency (mean ED50=5.96 mg/kg; 95% CI = ‐12.12, 24.05), while mean MPE for the other groups (n=2‐3) was less than 70% and mean ED50>17 mg/kg. Males exhibited partial tolerance throughout the 12‐day chronic morphine treatment period. In the POE + normal‐housed males, MPE decreased from 100% on test day 1 to 58% on test day 11. For the remaining groups, MPE decreased from 44‐57% on test day 1 to 12‐23% on test day 11. Morphine was more potent in the POE + normal‐housed males on test day 13 (mean ED50=66.21 mg/kg; 95% CI = ‐281.6, 414) than in other males (mean ED50>104.4 mg/kg). No clear group effect was observed in females. These unexpected results suggest a complex sex‐dependent interaction between POE and ELA on opioid antinociception.

Mice with high FGF21 serum levels had a reduced preference for morphine and an attenuated development of acute antinociceptive tolerance and physical dependence

Because of increased opioid misuse, there is a need to identify new targets for minimizing opioid tolerance, and physical and psychological dependence. Previous studies showed that fibroblast growth factor 21 (FGF21) decreased alcohol and sweet preference in mice. In this study, FGF21-transgenic (FGF21-Tg) mice, expressing high FGF21 serum levels, and wildtype (WT) C57BL/6J littermates were treated with morphine and saline to determine if differences exist in their physiological and behavioral responses to opioids. FGF21-Tg mice displayed reduced preference for morphine in the conditioned place preference assay compared to WT littermates. Similarly, FGF21-Tg mice had an attenuation of the magnitude and rate of acute morphine antinociceptive tolerance development, and acute and chronic morphine physical dependence, but exhibited no change in chronic morphine antinociceptive tolerance. The ED50 values for morphine-induced antinociception in the 55 °C hot plate and the 55 °C warm-water tail withdrawal assays were similar in both strains of mice. Likewise, FGF21-Tg and WT littermates had comparable responses to morphine-induced respiratory depression. Overall, FGF21-Tg mice had a decrease in the development of acute analgesic tolerance, and the development of physical dependence, and morphine preference. FGF21 and its receptor have therapeutic potential for reducing opioid withdrawal symptoms and craving, and augmenting opioid therapeutics for acute pain patients to minimize tolerance development.

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G-protein-biased agonists with reduced β-arrestin-2 activation are being investigated as safer alternatives to clinically-used opioids. β-arrestin-2 has been implicated in the mechanism of opioid-induced antinociceptive tolerance. Opioid-induced analgesic tolerance is classically considered as centrally-mediated, but recent reports implicate nociceptive dorsal root ganglia neurons as critical mediators in this process. Here, we investigated the role of β-arrestin-2 in the mechanism of opioid tolerance in dorsal root ganglia nociceptive neurons using β-arrestin-2 knockout mice and the G-protein-biased μ-opioid receptor agonist, TRV130. Whole-cell current-clamp electrophysiology experiments revealed that 15-18-h overnight exposure to 10 μM morphine in vitro induced acute tolerance in β-arrestin-2 wild-type but not knockout neurons. Furthermore, in wild-type neurons circumventing β-arrestin-2 activation by overnight treatment with 200 nM TRV130 attenuated tolerance. Similarly, acute morphine tolerance in vivo in β-arrestin-2 knockout mice was prevented in the warm-water tail-withdrawal assay. Treatment with 30 mg/kg TRV130 s.c. also inhibited acute antinociceptive tolerance in vivo in wild-type mice. Alternately, in β-arrestin-2 knockout neurons tolerance induced by 7-day in vivo exposure to 50 mg morphine pellet was conserved. Likewise, β-arrestin-2 deletion did not mitigate in vivo antinociceptive tolerance induced by 7-day exposure to 25 mg or 50 mg morphine pellet in both female or male mice, respectively. Consequently, these results indicated that β-arrestin-2 mediates acute but not chronic opioid tolerance in dorsal root ganglia neurons and to antinociception in vivo. This suggests that opioid-induced antinociceptive tolerance may develop even in the absence of β-arrestin-2 activation, and thus significantly affect the clinical utility of biased agonists.

Role of β-arrestin-2 in short- and long-term opioid tolerance in the dorsal root ganglia

G-protein-biased agonists with reduced β-arrestin-2 activation are being investigated as safer alternatives to clinically-used opioids. β-arrestin-2 has been implicated in the mechanism of opioid-induced antinociceptive tolerance. Opioid-induced analgesic tolerance is classically considered as centrally-mediated, but recent reports implicate nociceptive dorsal root ganglia neurons as critical mediators in this process. Here, we investigated the role of β-arrestin-2 in the mechanism of opioid tolerance in dorsal root ganglia nociceptive neurons using β-arrestin-2 knockout mice and the G-protein-biased μ-opioid receptor agonist, TRV130. Whole-cell current-clamp electrophysiology experiments revealed that 15-18-h overnight exposure to 10 μM morphine in vitro induced acute tolerance in β-arrestin-2 wild-type but not knockout neurons. Furthermore, in wild-type neurons circumventing β-arrestin-2 activation by overnight treatment with 200 nM TRV130 attenuated tolerance. Similarly, acute morphine tolerance in vivo in β-arrestin-2 knockout mice was prevented in the warm-water tail-withdrawal assay. Treatment with 30 mg/kg TRV130 s.c. also inhibited acute antinociceptive tolerance in vivo in wild-type mice. Alternately, in β-arrestin-2 knockout neurons tolerance induced by 7-day in vivo exposure to 50 mg morphine pellet was conserved. Likewise, β-arrestin-2 deletion did not mitigate in vivo antinociceptive tolerance induced by 7-day exposure to 25 mg or 50 mg morphine pellet in both female or male mice, respectively. Consequently, these results indicated that β-arrestin-2 mediates acute but not chronic opioid tolerance in dorsal root ganglia neurons and to antinociception in vivo. This suggests that opioid-induced antinociceptive tolerance may develop even in the absence of β-arrestin-2 activation, and thus significantly affect the clinical utility of biased agonists.

The mixed kappa and delta opioid receptor agonist, MP1104, attenuates chemotherapy-induced neuropathic pain

Effective treatments for chronic pain without abuse liability are urgently needed. One in 5 adults suffer chronic pain and half of these patients report inefficient treatment. Mu opioid receptor agonists (MOP), including oxycodone, tramadol and morphine, are often prescribed to treat chronic pain, however, use of drugs targeting MOP can lead to drug dependency, tolerance and overdose deaths. Kappa opioid receptor (KOP) agonists have antinociceptive effects without abuse potential; however, they have not been utilised clinically due to dysphoria and sedation. We hypothesise that mixed opioid receptor agonists targeting the KOP and delta opioid receptor (DOP) would have a wider therapeutic index, with the rewarding effects of DOP negating the negative effects of KOP. MP1104, an analogue of 3-Iodobenzoyl naltrexamine, is a novel mixed opioid receptor agonist with potent antinociceptive effects mediated via KOP and DOP in mice without rewarding or aversive effects. In this study, we show MP1104 has potent, long-acting antinociceptive effects in the warm-water tail-withdrawal assay in male and female mice and rats; and is longer acting than morphine. In the paclitaxel-induced neuropathic pain model in mice, MP1104 reduced both mechanical and cold allodynia and unlike morphine, did not produce tolerance when administered daily for 23 days. Moreover, MP1104 did not induce sedative effects in the open-field locomotor activity test, respiratory depression in mice using whole-body plethysmography, or have cross-tolerance with morphine. This data supports the therapeutic development of mixed opioid receptor agonists, particularly mixed KOP/DOP agonists, as non-addictive pain medications with reduced tolerance.

Lyophilized Kratom Tea as a Therapeutic Option for Opioid Dependence

BackgroundMade as a tea, the Thai traditional drug “kratom” reportedly possesses pharmacological actions that include both a coca-like stimulant effect and opium-like depressant effect. Kratom has been used as a substitute for opium in physically-dependent subjects. The objective of this study was to evaluate the antinociception, somatic and physical dependence produced by kratom tea, and then assess if the tea ameliorated withdrawal in opioid physically-dependent subjects. MethodsLyophilized kratom tea (LKT) was evaluated in C57BL/6J and opioid receptor knockout mice after oral administration. Antinociceptive activity was measured in the 55 °C warm-water tail-withdrawal assay. Potential locomotor impairment, respiratory depression and locomotor hyperlocomotion, and place preference induced by oral LKT were assessed in the rotarod, Comprehensive Lab Animal Monitoring System, and conditioned place preference assays, respectively. Naloxone-precipitated withdrawal was used to determine potential physical dependence in mice repeatedly treated with saline or escalating doses of morphine or LKT, and LKT amelioration of morphine withdrawal. Data were analyzed using one- and two-way ANOVA. ResultsOral administration of LKT resulted in dose-dependent antinociception (≥1 g/kg, p.o.) absent in mice lacking the mu-opioid receptor (MOR) and reduced in mice lacking the kappa-opioid receptor. These doses of LKT did not alter coordinated locomotion or induce conditioned place preference, and only briefly reduced respiration. Repeated administration of LKT did not produce physical dependence, but significantly decreased naloxone-precipitated withdrawal in morphine dependent mice. ConclusionsThe present study confirms the MOR agonist activity and therapeutic effect of LKT for the treatment of pain and opioid physical dependence.

Phenylalanine Stereoisomers of CJ-15,208 and [d-Trp]CJ-15,208 Exhibit Distinctly Different Opioid Activity Profiles.

The macrocyclic tetrapeptide cyclo[Phe-d-Pro-Phe-Trp] (CJ-15,208) and its stereoisomer cyclo[Phe-d-Pro-Phe-d-Trp] exhibit different opioid activity profiles in vivo. The present study evaluated the influence of the Phe residues’ stereochemistry on the peptides’ opioid activity. Five stereoisomers were synthesized by a combination of solid-phase peptide synthesis and cyclization in solution. The analogs were evaluated in vitro for opioid receptor affinity in radioligand competition binding assays, and for opioid activity and selectivity in vivo in the mouse 55 °C warm-water tail-withdrawal assay. Potential liabilities of locomotor impairment, respiratory depression, acute tolerance development, and place conditioning were also assessed in vivo. All of the stereoisomers exhibited antinociception following either intracerebroventricular or oral administration differentially mediated by multiple opioid receptors, with kappa opioid receptor (KOR) activity contributing for all of the peptides. However, unlike the parent peptides, KOR antagonism was exhibited by only one stereoisomer, while another isomer produced DOR antagonism. The stereoisomers of CJ-15,208 lacked significant respiratory effects, while the [d-Trp]CJ-15,208 stereoisomers did not elicit antinociceptive tolerance. Two isomers, cyclo[d-Phe-d-Pro-d-Phe-Trp] (3) and cyclo[Phe-d-Pro-d-Phe-d-Trp] (5), did not elicit either preference or aversion in a conditioned place preference assay. Collectively, these stereoisomers represent new lead compounds for further investigation in the development of safer opioid analgesics.

Evaluation of Biased and Balanced Salvinorin A Analogs in Preclinical Models of Pain.

In the search for safer, non-addictive analgesics, kappa opioid receptor (KOPr) agonists are a potential target, as unlike mu-opioid analgesics, they do not have abuse potential. Salvinorin A (SalA) is a potent and selective KOPr agonist, however, clinical utility is limited by the short duration of action and aversive side effects. Biasing KOPr signaling toward G-protein activation has been highlighted as a key cellular mechanism to reduce the side effects of KOPr agonists. The present study investigated KOPr signaling bias and the acute antinociceptive effects and side effects of two novel analogs of SalA, 16-Bromo SalA and 16-Ethynyl SalA. 16-Bromo SalA showed G-protein signaling bias, whereas 16-Ethynyl SalA displayed balanced signaling properties. In the dose-response tail-withdrawal assay, SalA, 16-Ethynyl SalA and 16-Bromo SalA were more potent than the traditional KOPr agonist U50,488, and 16-Ethynyl SalA was more efficacious. 16-Ethynyl SalA and 16-Bromo SalA both had a longer duration of action in the warm water tail-withdrawal assay, and 16-Ethynyl had greater antinociceptive effect in the hot-plate assay, compared to SalA. In the intraplantar 2% formaldehyde test, 16-Ethynyl SalA and 16-Bromo SalA significantly reduced both nociceptive and inflammatory pain-related behaviors. Moreover, 16-Ethynyl SalA and 16-Bromo SalA had no anxiogenic effects in the marble burying task, and 16-Bromo SalA did not alter behavior in the elevated zero maze. Overall, 16-Ethynyl SalA significantly attenuated acute pain-related behaviors in multiple preclinical models, while the biased KOPr agonist, 16-Bromo SalA, displayed modest antinociceptive effects, and lacked anxiogenic effects.