Development Of Antinociceptive Tolerance Research Articles

The role of morphine- and fentanyl-induced impairment of intestinal epithelial antibacterial activity in dysbiosis and its impact on the microbiota-gut-brain axis.

Recent evidence suggests that chronic exposure to opioid analgesics such as morphine disrupts the intestinal epithelial layer and causes intestinal dysbiosis. Depleting gut bacteria can preclude the development of tolerance to opioid-induced antinociception, suggesting an important role of the gut-brain axis in mediating opioid effects. The mechanism underlying opioid-induced dysbiosis, however, remains unclear. Host-produced antimicrobial peptides (AMPs) are critical for the integrity of the intestinal epithelial barrier as they prevent the pathogenesis of the enteric microbiota. Here, we report that chronic morphine or fentanyl exposure reduces the antimicrobial activity in the ileum, resulting in changes in the composition of bacteria. Fecal samples from morphine-treated mice had increased levels of Akkermansia muciniphila with a shift in the abundance ratio of Firmicutes and Bacteroidetes. Fecal microbial transplant (FMT) from morphine-naïve mice or oral supplementation with butyrate restored (a) the antimicrobial activity, (b) the expression of the antimicrobial peptide, Reg3γ, (c) prevented the increase in intestinal permeability and (d) prevented the development of antinociceptive tolerance in morphine-dependent mice. Improved epithelial barrier function with FMT or butyrate prevented the enrichment of the mucin-degrading A. muciniphila in morphine-dependent mice. These data implicate impairment of the antimicrobial activity of the intestinal epithelium as a mechanism by which opioids disrupt the microbiota-gut-brain axis.

Receptor Transporter Protein 4 (RTP4) in the Hypothalamus Is Involved in the Development of Antinociceptive Tolerance to Morphine.

Receptor transporter protein 4 (RTP4), one of the receptor chaperone proteins, contributes to the maturation and membrane trafficking of opioid receptor heteromers consisting of mu (MOPr) and delta (DOPr) opioid receptors (MOPr-DOPr). Although MOPr-DOPr is known to mediate the development of morphine tolerance, the extent to which RTP4 plays a role in this process has not been elucidated. Given that RTP4 can be upregulated by repeated administration of morphine, especially in the hypothalamus, here we investigated the effect of hypothalamus-selective ablation of RTP4 on the development of antinociceptive tolerance to morphine. In this study, we generated RTP4flox mice and selectively knocked-out RTP4 using local injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the hypothalamus. The AAV-Cre injection partially, but significantly, decreased the level of RTP4 expression, and suppressed the development of antinociceptive tolerance to morphine. Next, we examined the mechanism of regulation of RTP4 and found that, in neuronal cells, Rtp4 induction is via Gi and MAPK activation, while, in microglial cells, the induction is via Toll-like receptor 4. Together, these studies highlight the role of MOR activity in regulating RTP4, which, in turn, plays an important role in modulating morphine effects in vivo.

Allosteric modulators of the δ GABAA receptor subtype demonstrate a therapeutic effect in morphine-antinociceptive tolerance and withdrawal in mice

The present study evaluated the effects of compounds targeting extrasynaptic δ subunit-containing γ-aminobutyric acid type A receptors (δ*-GABAARs) to interrogate the role of tonic inhibition in the development of antinociceptive tolerance caused by repeated morphine administration. We investigated the effect of subchronic or acute treatment with non-steroidal positive allosteric modulators (PAMs) of δ*-GABAARs, such as 2–261, on the morphine-antinociceptive tolerance. Mice were treated twice daily with morphine for 9 days and antinociception was measured using the hot water tail immersion test. Co-treatment with 2–261 and morphine prevented morphine-antinociceptive tolerance and acute administration of 2–261 on day 9 was sufficient to reverse the tolerance. Other compounds with activity at δ*-GABAARs also reversed morphine tolerance, whereas an enaminone that lacked activity at δ*-GABAARs did not. Acute administration of 2–261 did not cause an additive or synergistic antinociceptive effect when combined with an acute submaximal dose of morphine. We then used Cre/LoxP recombination to generate GABAA δ-subunit knockout mice to corroborate the pharmacological results. Observations of male δ-knockout mice demonstrated that the δ*-GABAARs was necessary for 2–261 modulation of both analgesic tolerance and somatic withdrawal symptoms produced by subchronic morphine. While female mice still benefited from the positive effects of 2–261, the δ-subunit was not necessary for these effects, highlighting a distinction of the different pathways that could have implications for some of the sex-related differences seen in human opioid-induced outcomes. Consequently, subtype-specific allosteric modulators of GABAARs may warrant further investigation as pharmacological targets to manage tolerance and withdrawal from opioids.

Sex‐Differences in Acute Cannabinoid Response and Tolerance as a Function of Mouse Strain

Cannabinoids have been increasingly used to alleviate chronic pain; however, tolerance to the antinociceptive effects of cannabinoids, including delta‐9‐tetrahydrocannabinol (Δ9‐THC), may limit their therapeutic utility. Likewise, with more women than men now using medical cannabis for pain relief, it is imperative that we understand how sex may influence cannabinoid‐mediated antinociception and subsequent tolerance. While studies in rats have consistently found female rats to be more sensitive to the acute antinociceptive effects of cannabinoids compared to male rats, work in our lab consistently finds the opposite finding that male mice are more sensitive to the acute antinociceptive effects of both Δ9‐THC and CP55,940 compared to female littermates. Studies in our lab have consistently utilized mice on a C57BL6/J (B6) background. Therefore, the purpose of the present study is to examine whether our observed sex‐differences in Δ9‐THC‐induced antinociception and tolerance are consistent across multiple mouse strains or are strain‐dependent. Male and female B6 and DBA mice were first assessed for differences in acute Δ9‐THC‐induced antinociception using the tail‐flick assay across a range of doses of (0‐100 mg/kg). After a significant washout period, these mice were subsequently assessed for sex‐differences in antinociceptive tolerance development to 30 mg/kg Δ9‐THC following once‐daily treatment for seven consecutive days. Consistent with our previous findings, male B6 mice were more sensitive to the acute antinociceptive effects of Δ9‐THC than female B6 mice. Male and female DBA, however, mice did not differ in their antinociceptive response to Δ9‐THC, suggesting that sex‐differences in cannabinoid‐induced antinociception in mice is likely strain‐specific. These studies highlight the therapeutic potential of Δ9‐THC in pain management and underscore the importance of considering sex when evaluating their clinical utility.

Comparison of an Addictive Potential of μ-Opioid Receptor Agonists with G Protein Bias: Behavioral and Molecular Modeling Studies.

Among different approaches to the search for novel—safer and less addictive—opioid analgesics, biased agonism has received the most attention in recent years. Some μ-opioid receptor agonists with G protein bias, including SR compounds, were proposed to induce diminished side effects. However, in many aspects, behavioral effects of those compounds, as well as the mechanisms underlying differences in their action, remain unexplored. Here, we aimed to evaluate the effects of SR-14968 and SR-17018, highly G protein-biased opioid agonists, on antinociception, motor activity and addiction-like behaviors in C57BL/6J mice. The obtained results showed that the compounds induce strong and dose-dependent antinociception. SR-14968 causes high, and SR-17018 much lower, locomotor activity. Both agonists develop reward-associated behavior and physical dependence. The compounds also cause antinociceptive tolerance, however, developing more slowly when compared to morphine. Interestingly, SR compounds, in particular SR-17018, slow down the development of antinociceptive tolerance to morphine and inhibit some symptoms of morphine withdrawal. Therefore, our results indicate that SR agonists possess rewarding and addictive properties, but can positively modulate some symptoms of morphine dependence. Next, we have compared behavioral effects of SR-compounds and PZM21 and searched for a relationship to the substantial differences in molecular interactions that these compounds form with the µ-opioid receptor.

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.

Morphine‐induced kinase activation and localization in the periaqueductal gray of male and female mice

Morphine is a potent opioid analgesic with high propensity for the development of antinociceptive tolerance. Morphine antinociception and tolerance are partially regulated by the midbrain ventrolateral periaqueductal gray (vlPAG). However, the majority of research evaluating mu-opioid receptor signaling has focused on males. Here, we investigate kinase activation and localization patterns in the vlPAG following acute and chronic morphine treatment in both sexes. Male and female mice developed rapid antinociceptive tolerance to morphine (10mg/kg i.p.) on the hot plate assay, but tolerance did not develop in males on the tail flick assay. Quantitative fluorescence immunohistochemistry was used to map and evaluate the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2), protein kinase-C (PKC), and protein kinase-A (PKA). We observed significantly greater phosphorylated ERK 1/2 in the vlPAG of chronic morphine-treated animals which co-localized with the endosomal marker, Eea1. We note that pPKC is significantly elevated in the vlPAG of both sexes following chronic morphine treatment. We also observed that although PKA activity is elevated following chronic morphine treatment in both sexes, there is a significant reduction in the nuclear translocation of its phosphorylated substrate. Taken together, this study demonstrates increased activation of ERK 1/2, PKC, and PKA in response to repeated morphine treatment. The study opens avenues to explore the impact of chronic morphine treatment on G-protein signaling and kinase nuclear transport.

Protein Kinase‐C Inhibition Within the Periaqueductal Gray Counteracts the Development of Morphine Antinociceptive Tolerance

The ventrolateral periaqueductal gray (vlPAG) is an important midbrain region that regulates the descending pain modulatory system and is implicated in the development of antinociceptive tolerance to opioid analgesics. Protein Kinase-C (PKC) is a Ser/Thr kinase that phosphorylates the μ-opioid receptor (MOPr) following morphine activation. This phosphorylation is responsible for desensitization of the receptor, resulting in the termination of G-Protein signaling. It is speculated that PKC-mediated desensitization of the MOPr does not readily lead to the internalization of the receptor, thus contributing to tolerance. Previous studies have shown that intracerebroventricular administration of PKC inhibitors prevented the development of morphine tolerance in mice. Further, a non-specific PKC/GRK inhibitor was able to block the development of morphine tolerance in the vlPAG. However, the contribution of PKC to morphine tolerance in the vlPAG has not been directly explored. Using immunofluorescence staining, we observed increased phosphorylated PKC that is co-localized with the MOPr at the cell membrane of vlPAG neurons in morphine-tolerant animals. Further, we assessed the development of morphine antinociceptive tolerance following pre-treatment with the PKC inhibitor, Gö-7874, which targets conventional PKC isoforms. Repeated microinjections of the compound into the vlPAG of wild-type mice in combination with morphine led to a reduction in the development of morphine tolerance. Interestingly, repeated microinjections of Gö-7874 alone led to an enhancement of subsequent morphine antinociception. Lastly, morphine-induced changes in intracellular cAMP levels were assessed in-vitro, following pre-treatment with Gö-7874. This study contributes to the understanding of PKC-mediated MOPr desensitization processes leading to morphine tolerance.

Further investigation of the rapid-onset and short-duration action of the G protein-biased μ-ligand oliceridine

TRV130 (oliceridine), a G protein-biased ligand for μ-opioid receptor, has recently been synthesized. It is considered to have strong antinociceptive effects and only minor adverse effects. However, whether or not oliceridine actually exhibits an ideal pharmacological profile as an analgesic has not yet been fully clarified in animal studies. This study examined the pharmacological profile of oliceridine in cells and animals. Oliceridine (10 μM) did not produce any μ-opioid receptor internalization in cells even though it increased impedance, which reflects the activation of Gi protein using the CellKey™ system, and inhibited the formation of cAMP. In mice, oliceridine (0.3–10 mg/kg) produced a dose-dependent antinociceptive effect with a rapid-onset and short-duration action in the hot-plate test, as well as antihyperalgesia after sciatic nerve ligation without the development of antinociceptive tolerance using the thermal hyperalgesia test. On the other hand, oliceridine inhibited gastrointestinal transit. Furthermore, oliceridine produced rapid-onset hyperlocomotion at antinociceptive doses; sensitization developed in mice and an emetic effect was observed in ferrets. These results indicate that, although oliceridine may produce dopamine-related behaviors even through selective stimulation of the G-protein-biased μ-opioid receptor pathway, it still offers advantages for breakthrough pain without antinociceptive tolerance with adequate doses.

Neurophysiological response properties of medullary pain-control neurons following chronic treatment with morphine or oxycodone: modulation by acute ketamine.

Descending facilitatory circuitry that involves the rostroventromedial medulla (RVM) exerts a significant role in the development of antinociceptive tolerance and hyperalgesia following chronic morphine treatment. The role of the RVM in the development of antinociceptive tolerance to oxycodone, another clinically used strong opioid, is not yet known. Ketamine, an N-methyl-d-aspartate (NMDA) receptor antagonist, attenuates opioid antinociceptive tolerance, but its effect on RVM cell discharge in opioid-tolerant animals is not known. Here, we compared chronic effects of morphine and oxycodone on the discharge properties of RVM cells and attempted to attenuate chronic treatment-induced changes with ketamine. Parallel recordings of RVM cell discharge and limb withdrawal response were performed under light pentobarbital anesthesia in male rats following sustained systemic treatment with morphine or oxycodone at equianalgesic doses. Ongoing activity and the response to noxious heat and pinch were determined in pronociceptive RVM ON-cells and antinociceptive OFF-cells on the sixth treatment day. Proportions of RVM cell types were not changed. Chronic oxycodone induced antinociceptive tolerance both in limb withdrawal and RVM cell activity. Chronic morphine induced antinociceptive tolerance in limb withdrawal that was accompanied by pronociceptive heat response changes in RVM ON- and OFF-cells. A behaviorally subantinociceptive dose of acute ketamine reversed antinociceptive tolerance both to morphine and oxycodone in limb withdrawal and reversed the chronic morphine-induced pronociceptive discharge changes in RVM cells. The results indicate that an NMDA receptor-dependent descending pronociceptive circuitry involving the RVM has an important role in behavioral antinociceptive tolerance to morphine but not oxycodone.NEW & NOTEWORTHY Morphine and oxycodone are two clinically used strong opioids. Chronic treatment with oxycodone as well as morphine can lead to analgesic tolerance and paradoxical hyperalgesia. Here we show that an N-methyl-d-aspartate receptor-dependent pronociceptive change in discharge properties of rostroventromedial medullary neurons controlling spinal nociception has an important role in antinociceptive tolerance to morphine but not oxycodone. Interestingly, chronic oxycodone did not induce pronociceptive changes in the rostroventromedial medulla.

The Molecular Signatures of Acute-immobilization-induced Antinociception and Chronic-immobilization-induced Antinociceptive Tolerance.

In the present study, the productions of antinociception induced by acute and chronic immobilization stress were compared in several animal pain models. In the acute immobilization stress model (up to 1 hr immobilization), the antinociception was produced in writhing, tail-flick, and formalin-induced pain models. In chronic immobilization stress experiment, the mouse was enforced into immobilization for 1 hr/day for 3, 7, or 14 days, then analgesic tests were performed. The antinociceptive effect was gradually reduced after 3, 7 and 14 days of immobilization stress. To delineate the molecular mechanism involved in the antinociceptive tolerance development in the chronic stress model, the expressions of some signal molecules in dorsal root ganglia (DRG), spinal cord, hippocampus, and the hypothalamus were observed in acute and chronic immobilization models. The COX-2 in DRG, p-JNK, p-AMPKα1, and p-mTOR in the spinal cord, p-P38 in the hippocampus, and p-AMPKα1 in the hypothalamus were elevated in acute immobilization stress, but were reduced gradually after 3, 7 and 14 days of immobilization stress. Our results suggest that the chronic immobilization stress causes development of tolerance to the antinociception induced by acute immobilization stress. In addition, the COX-2 in DRG, p-JNK, p-AMPKα1, and p-mTOR in the spinal cord, p-P38 in the hippocampus, and p-AMPKα1 in the hypothalamus may play important roles in the regulation of antinociception induced by acute immobilization stress and the tolerance development induced by chronic immobilization stress.

The roles of several signal molecules in the production of antinociception by acute immobilization stress and the development of antinociceptive tolerance by chronic immobilization stress

The roles of several signal molecules in the production of antinociception by acute immobilization stress and the development of antinociceptive tolerance by chronic immobilization stress

Experimental Colitis Enhances the Rate of Antinociceptive Tolerance to Morphine via Peripheral Opioid Receptors.

Opioids are highly effective analgesics, however, their therapeutic use is limited by adverse effects that include respiratory depression, dependence, and tolerance. Inflammation has been implicated as a significant driver for the development of tolerance to opioids. Recent studies show that chronic morphine in mice results in gut microbial dysbiosis and inflammation in the colon. In the present study, we examined whether colonic inflammation results in tolerance to the antinociceptive effects of morphine. Colonic inflammation was induced in mice by intrarectal administration of 2,4,6-trinitro-benzene sulfonic acid. The development of antinociceptive tolerance was determined by warm-water tail-immersion assay in mice implanted with 25-, 50-, or 75-mg morphine pellet. Colonic inflammation significantly enhanced the rate at which tolerance developed in each cohort of chronic morphine-treated mice. At the lowest dose of morphine pellet (25 mg), antinociceptive tolerance only developed in the presence of colonic inflammation, whereas in 50- and 75-mg pelleted mice, tolerance developed faster in the inflamed animals than in the noninflamed mice. The enhanced antinociceptive tolerance was attenuated with daily administration of peripheral opioid receptor antagonist, 6β-N-heterocyclic-substituted naltrexamine derivative [17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'pyridyl)acetamido]morphinan (NAP)], irrespective of colonic inflammation. Collectively, these findings show that the rate of tolerance to morphine antinociception is exaggerated in the presence of colonic inflammation, and tolerance is prevented by a peripheral μ-opioid receptor antagonist. These studies suggest a peripheral component to the development of antinociceptive tolerance to opioids. Furthermore, peripherally selective opioid antagonists may be useful adjuncts in opioid-based pain management. SIGNIFICANCE STATEMENT: This study supports the notion that inflammation influences the development of antinociceptive tolerance to chronic morphine exposure. We found that, in the presence of colonic inflammation, the rate of development of tolerance to the antinociceptive effects of morphine increased. We also found that treatment with a peripheral opioid receptor antagonist prevented morphine antinociceptive tolerance. Increasing opioid intake during an inflammatory state would result in decreased analgesia and enhanced analgesic tolerance, which puts patients with inflammatory bowel diseases, inflammatory joint diseases, and sickle cell anemia at risk for heavy opioid use.

Mitochondria as a potential target for cadmium toxicity in the development of pancreatic cancer

Antinociceptive tolerance after repetitive administration of morphine severely limits its clinical use. Despite increased mechanistic understanding of morphine tolerance, little is known about the influence of dosing regimens in its development. We hypothesized that the starting dose of morphine, dosing frequency and dose increments, influence antinociception and the manifestation of antinociceptive tolerance in rats. Male rats were randomly divided into four groups with different intermittent starting-doses of daily morphine (b.i.d.) followed by different increments of single-dose morphine upon development of antinociceptive tolerance, for 2–3 weeks: 2.5 (b.i.d.)→5 → 10→15 mg/kg/day, 5 (b.i.d.)→10 mg/kg/day, 5 (b.i.d.)→15 mg/kg/day, 10 (b.i.d.)→20 mg/kg/day. Antinociception was assessed daily pre-treatment and at several time-points over 2 h post-administration, using tail-flick and hot-plate assays. Tolerance was defined as significant antinociceptive desensitization and was presented as significant reduction of the maximum and total antinociceptive efficacy upon morphine administration. Rats commenced on 2.5 mg/kg/day (b.i.d.) morphine developed tolerance faster than those started on 5 or 10 mg/kg/day (b.i.d.). Comparatively, higher starting and maintenance doses of morphine produced prolonged antinociception and delayed tolerance. Whereas, lower starting and maintenance doses of morphine produced less total antinociception during the course of treatment and did not delay the onset of tolerance, but require smaller dose-increments to reach antinociception after development of antinociceptive tolerance. These results suggest that morphine starting dose, dosing frequency, increments and timing determine the manifestation of antinociceptive tolerance and extent of antinociception. In addition, our results also highlight the need for generally standardized and validated assay protocols and procedures to compare different studies, as a prerequisite to translate pre-clinical results into the clinic.

Reversal of the Development of Antinociceptive Effects to Chronic Morphine in Mice by Fecal Microbiota Transplantation (FMT)

Morphine remains one of the most frequently prescribed drugs for the treatment of moderate to severe pain, including pain due to cancer or surgery. However, the long‐term use of this excellent pain reliever in man is limited by side‐effects that include analgesic tolerance, dependence and constipation. We have previously shown that depletion of gut bacteria by oral vancomycin results in the loss of antinociceptive tolerance, decreases epithelial permeability and reduces colonic inflammation by chronic morphine. The aim of this study was to investigate the effect of colonizing the gut with non‐opioid commensal microbiome on reversal of morphine‐induced antinociceptive tolerance. Gut bacteria were collected from 5 day placebo and morphine pelleted mice from fecal pellets designated as donor mice groups (PP‐FMT and MP‐FMT). The microbiome was then orally gavaged (1×109 cells/dose) daily for 5 days to placebo (PP) and morphine (MP) pelleted (75 mg pellet, 5 days) recipient mice groups, and the presence of antinociceptive tolerance was determined by morphine challenge (10 mg/kg, s.c.) in the tail immersion and hot plate tests. Daily gavage of the microbiome from morphine pelleted mice to the morphine‐pelleted recipient mice (MP+MP‐FMT) resulted in antinociceptive tolerance to morphine challenge. However, gavage of the microbiome from placebo pelleted donor mice to the morphine pelleted recipient mice (PP+MP‐FMT), prevented the development of antinociceptive tolerance. We further investigated whether an exogenous microbiome could reverse chronic morphine induced antinociceptive tolerance development. In these studies, oral gavage of gut microbiome was commenced after development of tolerance to morphine. Similar to the induction of tolerance, oral gavage of placebo microbiome to morphine tolerant mice (MP+PP‐FMT) reversed antinociceptive tolerance. In order to determine if transplantation of the gut microbiome also prevents the loss of epithelial integrity by chronic morphine treatment, intestinal permeability was measured by oral administration of FITC (Fluorescein isothiocyanate)‐dextran (44 mg/kg, p.o.) in placebo or morphine pelleted mice for 5 days. FITC levels were significantly higher in morphine pelleted mice (MP) treated with the microbiome from morphine donors (MP‐FMT) than placebo pelleted controls (PP, p<0.0001). However, the epithelial integrity was maintained in morphine pelleted mice (MP) treated with the commensal microbiome from placebo donors (PP‐FMT). These studies define a novel microbiome‐gut‐brain interaction in the development of opioid tolerance.Support or Funding InformationNIH R01 DA036975 and NIH T32 DA007027This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cannabinoid CB2 Agonist AM1710 Differentially Suppresses Distinct Pathological Pain States and Attenuates Morphine Tolerance and Withdrawal.

AM1710 (3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c) chromen-6-one), a cannabilactone cannabinoid receptor 2 (CB2) agonist, suppresses chemotherapy-induced neuropathic pain in rodents without producing tolerance or unwanted side effects associated with CB1 receptors; however, the signaling profile of AM1710 remains incompletely characterized. It is not known whether AM1710 behaves as a broad-spectrum analgesic and/or suppresses the development of opioid tolerance and physical dependence. In vitro, AM1710 inhibited forskolin-stimulated cAMP production and produced enduring activation of extracellular signal-regulated kinases 1/2 phosphorylation in human embryonic kidney (HEK) cells stably expressing mCB2. Only modest species differences in the signaling profile of AM1710 were observed between HEK cells stably expressing mCB2 and hCB2. In vivo, AM1710 produced a sustained inhibition of paclitaxel-induced allodynia in mice. In paclitaxel-treated mice, a history of AM1710 treatment (5 mg/kg per day × 12 day, i.p.) delayed the development of antinociceptive tolerance to morphine and attenuated morphine-induced physical dependence. AM1710 (10 mg/kg, i.p.) did not precipitate CB1 receptor-mediated withdrawal in mice rendered tolerant to Δ9-tetrahydrocannabinol, suggesting that AM1710 is not a functional CB1 antagonist in vivo. Furthermore, AM1710 (1, 3, 10 mg/kg, i.p.) did not suppress established mechanical allodynia induced by complete Freund's adjuvant (CFA) or by partial sciatic nerve ligation (PSNL). Similarly, prophylactic and chronic dosing with AM1710 (10 mg/kg, i.p.) did not produce antiallodynic efficacy in the CFA model. By contrast, gabapentin suppressed allodynia in both CFA and PSNL models. Our results indicate that AM1710 is not a broad-spectrum analgesic agent in mice and suggest the need to identify signaling pathways underlying CB2 therapeutic efficacy to identify appropriate indications for clinical translation.

Endomorphin-1 analogs with oligoarginine-conjugation at C-terminus produce potent antinociception with reduced opioid tolerance in paw withdrawal test

For clinical use, it is essential to develop potent endomorphin (EM) analogs with reduced antinociceptive tolerance. In the present study, the antinociceptive activities and tolerance development of four potent EM-1 analogs with C-terminal oligoarginine-conjugation was evaluated and compared in the radiant heat paw withdrawal test. Following intracerebroventricular (i.c.v.) administration, all analogs 1–4 produced potent and prolonged antinociceptive effects. Notably, analogs 2 and 4 with the introduction of D-Ala in position 2 exhibited relatively higher analgesic potencies than those of analogs 1 and 3 with β-Pro substitution, consistent with their μ-opioid binding characteristic. In addition, at a dose of 50 μmol/kg, endomorphin-1 (EM-1) failed to produce any significant antinociceptive activity after peripheral administration, whereas analogs 1–4 induced potent antinociceptive effects with an increased duration of action. Herein, our results indicated the development of antinociceptive tolerance to EM-1 and morphine at the supraspinal level on day 7. By contrast, analogs 1–4 decreased the antinociceptive tolerance. Furthermore, subcutaneous (s.c.) administration of morphine at 50 μmol/kg also developed the antinociceptive tolerance, whereas the extent of tolerance developed to analogs 1–4 was largely reduced. Especially, analog 4 exhibited non-tolerance-forming antinociception after peripheral administration. The present investigation gave the evidence that C-terminal conjugation of EM-1 with oligoarginine vector will facilitate the development of novel opioid analgesics with reduced opioid tolerance.

The Effect of Colonic Inflammation on Morphine Induced Antinociceptive Tolerance

Opioids are one of the most widely prescribed classes of drugs for the treatment of moderate to severe pain. Opioids are prescribed for abdominal pain, a major symptom associated with Crohn's Disease. Recent studies have reported that continued opioid use in patients with Crohn's Disease is associated with increased mortality rate, duration and severity of disease, as well as infection. However, it is not clear if this association is causal. We have previously demonstrated that chronic use of morphine, in addition to the development of antinociceptive tolerance, also causes the disruption of the intestinal barrier function resulting in bacterial translocation from the gut lumen into the peritoneal organs, thus inducing an inflammatory response in the colon. Therefore, we tested if inflammation in the colon, as in Crohn's disease, affects the rate of antinociceptive tolerance development to morphine. To test this hypothesis, we induced colonic inflammation in Swiss Webster male mice using 2,4,6‐trinitro‐benzene sulfonic acid (TNBS) or vehicle intrarectally and then surgically implanted 25 mg, 50 mg (2 × 25 mg), or 75 mg slow release morphine pellets or placebo pellets subcutaneously on the dorsum. Tail immersion assay was performed on day 3, 4, and 5 post treatment and colons were extracted from the animals to measure mRNA expression of Interleukin 1 beta (IL‐1β) and tumor necrosis factor alpha (TNF‐α) by real‐time polymerase chain reaction (RT‐PCR). A morphine (10 mg/kg) challenge was used to test for the development of antinociceptive tolerance at various times in morphine pelleted with (n= 7) or without TNBS (n= 5). In TNBS + morphine pelleted mice, tolerance developed by day 5 in 25 mg morphine pelleted mice, by day 4 in 50 mg (2 × 25 mg) morphine pelleted mice, and as early as day 2 in 75 mg morphine pelleted mice. In the absence of TNBS‐induced inflammation, tolerance to morphine did not develop to either 25 mg or 50 mg morphine pellets up to day 5, and at day 4 in 75 mg morphine pellets. Furthermore, IL‐1β production was significantly increased in the morphine and placebo pelleted treated animals with TNBS from day 1 compared to the vehicle treated groups. In summary, chronic morphine administration in the presence of colonic inflammation leads to an increased rate and extent of antinociceptive tolerance development.Support or Funding InformationAgency: NIDA Agency Number: 5R01DA036975‐04This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sex Differences in Antinociceptive Tolerance Development to Δ9‐THC and CP55,940 in Wild‐type and Desensitization‐Resistant S426A/S430A Mice

Tolerance to the antinociceptive effects of cannabinoid agonists limit their therapeutic potential. Male mice expressing a desensitization‐resistant form of the CB1 receptor (S426A/S430A) show delayed tolerance development to the hypothermic and antinociceptive effects of Δ9‐THC, a strongly desensitizing partial CB1 agonist. However, it remains unknown what effect this mutation might have on altering tolerance to the synthetic full CB1 agonist CP55,940 and whether tolerance development to Δ9‐THC and CP55,940 differ in a sex‐specific manner. The purpose of the current study was to investigate whether the S426A/S430A mutation alters antinociceptive and/or hypothermic tolerance to CP55,940 and Δ9‐THC and whether these effects are sex‐specific. Tolerance to Δ9‐THC (30 mg/kg) and CP55,940 (0.3–0.6 mg/kg) was assessed in two ways: following once‐daily injections across 7–10 consecutive days and via shifts in the cumulative dose response curves following 3–7 days of treatment with each agonist in both male and female wild‐type and S426A/S430A mice. Once‐daily drug administration resulted in slower tolerance development to the effects of CP55,940 versus Δ9‐THC in male and female wildtype mice. The S426A/S430A mutation delayed tolerance to the antinociceptive effect of CP55,940 in male mice and delayed tolerance to both the antinociceptive and hypothermic effects of CP55,940 and Δ9‐THC in female mice. In our cumulative dose responses, we found that the S426A/S430A mutation rendered both male and female mice more sensitive to the effects of Δ9‐THC compared to wild‐type mice. Overall, our results demonstrate that sex and agonist specific mechanisms may mediate cannabinoid tolerance. These studies showcase the antinociceptive therapeutic potential of cannabinoid agonists and offer potential avenues to better understand tolerance to their analgesic effects following prolonged exposure.Support or Funding InformationNIH grants DA036385 (DJM), DA037355 (DJM), Pennsylvania Department of Health using CURE Tobacco Settlement Funds (DJM), Penn State University College of Medicine Department of Anesthesiology and Perioperative Medicine Start Up FundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Morphine dosing strategy plays a key role in the generation and duration of the produced antinociceptive tolerance

Antinociceptive tolerance after repetitive administration of morphine severely limits its clinical use. Despite increased mechanistic understanding of morphine tolerance, little is known about the influence of dosing regimens in its development. We hypothesized that the starting dose of morphine, dosing frequency and dose increments, influence antinociception and the manifestation of antinociceptive tolerance in rats. Male rats were randomly divided into four groups with different intermittent starting-doses of daily morphine (b.i.d.) followed by different increments of single-dose morphine upon development of antinociceptive tolerance, for 2–3 weeks: 2.5 (b.i.d.)→5 → 10→15 mg/kg/day, 5 (b.i.d.)→10 mg/kg/day, 5 (b.i.d.)→15 mg/kg/day, 10 (b.i.d.)→20 mg/kg/day. Antinociception was assessed daily pre-treatment and at several time-points over 2 h post-administration, using tail-flick and hot-plate assays. Tolerance was defined as significant antinociceptive desensitization and was presented as significant reduction of the maximum and total antinociceptive efficacy upon morphine administration. Rats commenced on 2.5 mg/kg/day (b.i.d.) morphine developed tolerance faster than those started on 5 or 10 mg/kg/day (b.i.d.). Comparatively, higher starting and maintenance doses of morphine produced prolonged antinociception and delayed tolerance. Whereas, lower starting and maintenance doses of morphine produced less total antinociception during the course of treatment and did not delay the onset of tolerance, but require smaller dose-increments to reach antinociception after development of antinociceptive tolerance. These results suggest that morphine starting dose, dosing frequency, increments and timing determine the manifestation of antinociceptive tolerance and extent of antinociception. In addition, our results also highlight the need for generally standardized and validated assay protocols and procedures to compare different studies, as a prerequisite to translate pre-clinical results into the clinic.