Antinociceptive Synergy Research Articles

Dual Inhibition of FAAH and MAGL Counteracts Migraine-like Pain and Behavior in an Animal Model of Migraine.

The endocannabinoid system exerts an important role in pain processing and modulation. Modulation of the system with hydrolase inhibitors of anandamide (AEA) or 2-arachidonyl glycerol (2-AG) has proved effective in reducing migraine-like features in animal models of migraine. Here, we investigated the effect of dual inhibition of the AEA and 2-AG catabolic pathways in the nitroglycerin-based animal model of migraine. The dual inhibitor JZL195 was administered to rats 2 h after nitroglycerin or vehicle injection. Rats were then exposed to the open field test and the orofacial formalin test. At the end of the tests, they were sacrificed to evaluate calcitonin gene-related peptide (CGRP) serum levels and gene expression of CGRP and cytokines in the cervical spinal cord and the trigeminal ganglion. The dual inhibitor significantly reduced the nitroglycerin-induced trigeminal hyperalgesia and pain-associated behavior, possibly via cannabinoid 1 receptors-mediated action, but it did not change the hypomotility and the anxiety behaviors induced by nitroglycerin. The decreased hyperalgesia was associated with a reduction in CGRP and cytokine gene expression levels in central and peripheral structures and reduced CGRP serum levels. These data suggest an antinociceptive synergy of the endocannabinoid action in peripheral and central sites, confirming that this system participates in reduction of cephalic pain signals.

No antinociceptive synergy between morphine and delta-9-tetrahydrocannabinol in male and female rats with persistent inflammatory pain.

Studies have demonstrated antinociceptive synergy between morphine and delta-9-tetrahydrocannabinol (THC) in animals, but whether such synergy occurs against all types of pain and in humans is unclear. Because a majority of chronic pain patients are women, and sex differences in morphine and THC potencies have been observed in rodents, the present study examined sex-specific effects of morphine and THC given alone and in combination, in rats with persistent inflammatory pain. On day 1, baseline mechanical and thermal response thresholds, hindpaw weight-bearing, locomotor activity, and hindpaw thickness were determined. Inflammation was then induced via hindpaw injection of complete Freund's adjuvant (CFA). Three days later, morphine (s.c.), THC (i.p) or a morphine-THC combination (1:1, 3:1 and 1:3 dose ratios) was administered, and behavioral testing was conducted at 30-240 min postinjection. Morphine alone was antiallodynic and antihyperalgesic, with no sex differences, but at some doses increased weight-bearing on the CFA-treated paw more in males than females. THC alone reduced mechanical allodynia with similar potency in both sexes, but reduced thermal hyperalgesia and locomotor activity with greater potency in females than males. All morphine-THC combinations reduced allodynia and hyperalgesia, but isobolographic analysis of mechanical allodynia data showed no significant morphine-THC synergy in either sex. Additionally, whereas morphine alone was antinociceptive at doses that did not suppress locomotion, morphine-THC combinations suppressed locomotion and did not increase weight-bearing on the inflamed paw. These results suggest that THC is unlikely to be a beneficial adjuvant when given in combination with morphine for reducing established inflammatory pain.

Antinociceptive Synergy Between Metamizole and Hesperidin in a Model of Visceral Pain in Mice

Metamizole is used to relieve the visceral pain but its adverse effects limit its use. An alternative to improve its efficacy with lower doses is to combine it with a natural product as hesperidin. The aim of this study was to evaluate the antinociceptive interaction between metamizole and hesperidin in a visceral pain model using an isobolographic analysis. Antinociception was evaluated in the writhing model using acetic acid (1%) to induce writhes in mice. Metamizole (1-316mg/kg), hesperidin (3-300mg/kg), or combinations with a fixed-dose ratio of 1:1 were administered intraperitoneally 30min before the acetic acid and the number of writhes was counted for 30min. Isobolographic analysis was employed to define the nature of the compound interaction. Metamizole and hesperidin in individual administration induced dose-dependent antinociceptive effects, reached an efficacy of 84.2±5.9% and 66.3±7.4%, respectively. The ED50 values calculated from their dose-response curves were 84.5±22.7 and 108.9±17.9mg/kg, respectively. The analysis of DRC for the metamizole+hesperidin combination, in a ratio 1:1 showed a ED50 COMB value lower than the ED50 ADD estimated from the additivity line from the isobologram (46.7±6.3 vs. 96.7±11.9mg/kg, respectively). In addition, the pharmacological interaction calculated was of 0.48. These results suggest a synergistic interaction for the antinociceptive activity of metamizole+hesperidin combination. These data suggest that metamizole+hesperidin combination could be useful in treating visceral pain as it can interact synergistically using low dose of both drugs with the possibility of reducing the risk of adverse effects.

Positive allosteric modulation of the cannabinoid type-1 receptor (CB1R) in periaqueductal gray (PAG) antagonizes anti-nociceptive and cellular effects of a mu-opioid receptor agonist in morphine-withdrawn rats.

Opioid drugs are a first-line treatment for severe acute pain and other chronic pain conditions, but long-term opioid drug use produces opioid-induced hyperalgesia (OIH). Co-administration of cannabinoids with opioid receptor agonists produce anti-nociceptive synergy, but cannabinoid receptor agonists may also produce undesirable side effects. Therefore, positive allosteric modulators (PAM) of cannabinoid type-1 receptors (CB1R) may provide an option reducing pain and/or enhancing the anti-hyperalgesic effects of opioids without the side effects, tolerance, and dependence observed with the use of ligands that target the orthosteric binding sites. This study tested GAT211, a PAM of cannabinoid type-1 receptors (CB1R), for its ability to enhance the anti-hyperalgesic effects of the mu-opioid receptor (MOR) agonist DAMGO in rats treated chronically with morphine (or saline) and tested during withdrawal. We tested the effects of intra-periaqueductal gray (PAG) injections of (1) DAMGO, (2) GAT211, or (3) DAMGO + GAT211 on thermal nociception in chronic morphine-treated rats that were hyperalgesic and also in saline-treated control rats. We used slice electrophysiology to test the effects of DAMGO/GAT211 bath application on synaptic transmission in the vlPAG. Intra-PAG DAMGO infusions dose-dependently reversed chronic morphine-induced hyperalgesia, but intra-PAG GAT211 did not alter nociception at the doses we tested. When co-administered into the PAG, GAT211 antagonized the anti-nociceptive effects of DAMGO in morphine-withdrawn rats. DAMGO suppressed synaptic inhibition in the vlPAG of brain slices taken from saline- and morphine-treated rats, and GAT211 attenuated DAMGO-induced suppression of synaptic inhibition in vlPAG neurons via actions at CB1R. These findings show that positive allosteric modulation of CB1R antagonizes the behavioral and cellular effects of a MOR agonist in the PAG of rats.

Morphine and Delta‐9‐Tetrahydrocannabinol Antinociceptive Interactions in Male versus Female Rats with Inflammatory Pain

Previous studies have shown that morphine is more potent in male rats compared to females with chronic inflammatory pain. In contrast, delta‐9‐tetrahydrocannabinol (THC) may be more potent in females compared to males with chronic inflammatory pain. Some studies have demonstrated that morphine and THC produce synergistic antinociceptive effects in male animals. Therefore, the purpose of the present study was to determine sex‐specific potency of morphine and THC in male versus female rats with inflammatory pain, and then to test for antinociceptive synergy between morphine and THC.Male and female Sprague Dawley rats, 60–90 days old were used. On day 1 baseline measurements were taken on the von Frey test of mechanical allodynia, incapacitance test of biased weight bearing, a 10‐min locomotor activity test, and right hindpaw thickness. After baseline testing, inflammation was induced by an intraplantar injection of complete Freund's adjuvant (CFA) to the right hindpaw. Three days later, morphine (0, 0.1, 0.32, 1.0, or 3.2 mg/kg, s.c.) or THC (0, 0.5, 1.0, 2.0, or 4.0 mg/kg, i.p.) was administered and nociceptive testing was conducted at 30, 60, 120, and 240 min post‐injection. Paw thickness was also measured at 240 min post‐injection. A three‐way ANOVA was conducted on means of the 30–120 min time point data to determine sex differences in the antinociceptive effects of morphine alone and THC alone. The ED50 was calculated for morphine and for THC in each sex using data from the von Frey test. These data were used to determine 1:1, 3:1, and 1:3 dose‐addition ratios for each sex, which were administered in Experiment 2 under the same testing protocol as in Experiment 1.In both sexes, CFA increased right hindpaw thickness to approximately 200% of baseline, as well as causing mechanical allodynia, biased weight‐bearing, and decreased locomotor activity. In Experiment 1, morphine given alone dose‐dependently reduced mechanical allodynia similarly in both sexes. Morphine also dose‐dependently reduced biased weight‐bearing in both sexes, and was more potent in males than females. Morphine did not significantly alter paw thickness or locomotor activity in either sex. THC given alone dose‐dependently reduced allodynia and decreased locomotor activity similarly in males and females, but did not alter biased weight‐bearing or paw thickness in either sex. In Experiment 2, a 1:1 combination of morphine and THC produced large anti‐allodynic effects in both sexes. A dose‐addition analysis will be conducted when data collection is complete.Results thus far indicate that THC alone may be a beneficial analgesic to treat chronic inflammatory pain in both sexes, but may also produce sedation, a negative side effect. Similar to previous reports in rats, morphine alone also alleviates persistent inflammatory pain, but with greater potency in males than females. Given the sex differences observed with each drug alone, we predict that morphine‐THC combinations will produce greater antinociception in males compared to females.Support or Funding InformationThis research was supported by funds from the National Institute on Drug Abuse (DA016644).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Relative Contributions of Norepinephrine and Serotonin Transporters to Antinociceptive Synergy between Monoamine Reuptake Inhibitors and Morphine in the Rat Formalin Model

Multimodal analgesia is designed to optimize pain relief by coadministering drugs with distinct mechanisms of action or by combining multiple pharmacologies within a single molecule. In clinical settings, combinations of monoamine reuptake inhibitors and opioid receptor agonists have been explored and one currently available analgesic, tapentadol, functions as both a µ-opioid receptor agonist and a norepinephrine transporter inhibitor. However, it is unclear whether the combination of selective norepinephrine reuptake inhibition and µ-receptor agonism achieves an optimal antinociceptive synergy. In this study, we assessed the pharmacodynamic interactions between morphine and monoamine reuptake inhibitors that possess different affinities and selectivities for norepinephrine and serotonin transporters. Using the rat formalin model, in conjunction with measurements of ex vivo transporter occupancy, we show that neither the norepinephrine-selective inhibitor, esreboxetine, nor the serotonin-selective reuptake inhibitor, fluoxetine, produce antinociceptive synergy with morphine. Atomoxetine, a monoamine reuptake inhibitor that achieves higher levels of norepinephrine than serotonin transporter occupancy, exhibited robust antinociceptive synergy with morphine. Similarly, a fixed-dose combination of esreboxetine and fluoxetine which achieves comparable levels of transporter occupancy potentiated the antinociceptive response to morphine. By contrast, duloxetine, a monoamine reuptake inhibitor that achieves higher serotonin than norepinephrine transporter occupancy, failed to potentiate the antinociceptive response to morphine. However, when duloxetine was coadministered with the 5-HT3 receptor antagonist, ondansetron, potentiation of the antinociceptive response to morphine was revealed. These results support the notion that inhibition of both serotonin and norepinephrine transporters is required for monoamine reuptake inhibitor and opioid-mediated antinociceptive synergy; yet, excess serotonin, acting via 5-HT3 receptors, may reduce the potential for synergistic interactions. Thus, in the rat formalin model, the balance between norepinephrine and serotonin transporter inhibition influences the degree of antinociceptive synergy observed between monoamine reuptake inhibitors and morphine.

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PC-FACS

Protein Kinase Cϵ Is Required for Spinal Analgesic Synergy between Delta Opioid and Alpha-2A Adrenergic Receptor Agonist Pairs

We recently showed that spinal synergistic interactions between δ opioid receptors (δORs) and α2A adrenergic receptors (α2AARs) require protein kinase C (PKC). To identify which PKC isoforms contribute to analgesic synergy, we evaluated the effects of various PKC-isoform-specific peptide inhibitors on synergy between δORs and α2AARs using the tail flick assay of thermal nociception in mice. Only a PKCε inhibitor abolished synergy between a δOR agonist and an α2AAR agonist. We tested a panel of combinations of opioid and adrenergic agonists in PKCε knock-out mice and found that all four combinations of a δOR agonist and an α2AAR agonist required PKCε for antinociceptive synergy. None of the combinations of a μOR agonist with an α2AR agonist required PKCε. Immunohistochemistry confirmed that PKCε could be found in the population of peptidergic primary afferent nociceptors where δORs and α2AARs have been found to extensively colocalize. Immunoreactivity for PKCε was found in the majority of dorsal root ganglion neurons and intensely labeled laminae I and II of the spinal cord dorsal horn. PKCε is widespread in the spinal nociceptive system and in peptidergic primary afferents it appears to be specifically involved in mediating the synergistic interaction between δORs and α2AARs.

Antinociceptive synergy between diclofenac and morphine after local injection into the inflamed site

BackgroundCombinations of non-steroidal anti-inflammatory drugs with opioids are frequently used to reduce opioid doses required in the clinical management of acute pain. The present study was designed to evaluate the possible antinociceptive interaction between morphine and diclofenac at peripheral level in male rats. MethodsDrugs were chosen based of their efficacy in the treatment of this kind of pain and as representative drugs of their respective analgesic groups. For the formalin test, 50μl of 1% formalin solution was injected subcutaneously into the right hind paw. The interaction between morphine and diclofenac was evaluated by using isobolographic analysis and interaction index. Drug interaction was examined by administering fixed-ratio combinations of morphine-diclofenac (1 : 1 and 3 : 1) of their respective ED30 fractions. ResultsDiclofenac and morphine reduced flinching behavior in a dose-dependent manner during phase 2 but not phase 1 of the formalin test. Isobolographic analysis showed a synergistic interaction for the combination of morphine and diclofenac after local peripheral administration. ConclusionsData suggest that the combination of morphine with diclofenac at the site of injury is synergistic and could be useful in the treatment of wounds, bruises, rheumatisms and other painful peripheral conditions associated with an inflammatory process.

Morphine-induced antinociception in the rat: Supra-additive interactions with imidazoline I2 receptor ligands

Pain remains a significant clinical challenge and currently available analgesics are not adequate to meet clinical needs. Emerging evidence suggests the role of imidazoline I(2) receptors in pain modulation primarily from studies of the non-selective imidazoline receptor ligand, agmatine. However, little is known of the generality of the effect to selective I(2) receptor ligands. This study examined the antinociceptive effects of two selective I(2) receptor ligands 2-BFI and BU224 (>2000-fold selectivity for I(2) receptors over α(2) adrenoceptors) in a hypertonic (5%) saline-induced writhing test and analyzed their interaction with morphine using a dose-addition analysis. Morphine, 2-BFI and BU224 but not agmatine produced a dose-dependent antinociceptive effect. Both composite additive curve analyses and isobolographical plots revealed a supra-additive interaction between morphine and 2-BFI or BU224, whereas the interaction between 2-BFI and BU224 was additive. The antinociceptive effect of 2-BFI and BU224 was attenuated by the I(2) receptor antagonist/α(2) adrenoceptor antagonist idazoxan but not by the selective α(2) adrenoceptor antagonist yohimbine, suggesting an I(2) receptor-mediated mechanism. Agmatine enhanced the antinociceptive effect of morphine, 2-BFI and BU224 and the enhancement was prevented by yohimbine, suggesting that the effect was mediated by α(2) adrenoceptors. Taken together, these data represent the first report that selective I(2) receptor ligands have substantial antinociceptive activity and produce antinociceptive synergy with opioids in a rat model of acute pain. These data suggest that drugs acting on imidazoline I(2) receptors may be useful either alone or in combination with opioids for the treatment of pain.

Protein Kinase C Mediates the Synergistic Interaction Between Agonists Acting at α2-Adrenergic and Delta-Opioid Receptors in Spinal Cord

Coactivation of spinal alpha(2)-adrenergic receptors (ARs) and opioid receptors produces antinociceptive synergy. Antinociceptive synergy between intrathecally administered alpha(2)AR and opioid agonists is well documented, but the mechanism underlying this synergy remains unclear. The delta-opioid receptor (DOP) and the alpha(2A)ARs are coexpressed on the terminals of primary afferent fibers in the spinal cord where they may mediate this phenomenon. We evaluated the ability of the DOP-selective agonist deltorphin II (DELT), the alpha(2)AR agonist clonidine (CLON) or their combination to inhibit calcitonin gene-related peptide (CGRP) release from spinal cord slices. We then examined the possible underlying signaling mechanisms involved through coadministration of inhibitors of phospholipase C (PLC), protein kinase C (PKC) or protein kinase A (PKA). Potassium-evoked depolarization of spinal cord slices caused concentration-dependent release of CGRP. Coadministration of DELT and CLON inhibited the release of CGRP in a synergistic manner as confirmed statistically by isobolograpic analysis. Synergy was dependent on the activation of PLC and PKC, but not PKA, whereas the effect of agonist administration alone was only dependent on PLC. The importance of these findings was confirmed in vivo, using a thermal nociceptive test, demonstrating the PKC dependence of CLON-DELT antinociceptive synergy in mice. That inhibition of CGRP release by the combination was maintained in the presence of tetrodotoxin in spinal cord slices suggests that synergy does not rely on interneuronal signaling and may occur within single subcellular compartments. The present study reveals a novel signaling pathway underlying the synergistic analgesic interaction between DOP and alpha(2)AR agonists in the spinal cord.

Antinociception synergy between the peripheral and spinal sites of the heme oxygenase-carbon monoxide pathway

We have shown that the peripheral and spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase-cGMP pathways play an important role in antinociception in the rat experimental formalin model. Our objective was to determine if there is synergism between peripheral (paw) and spinal HO-CO pathways in nociception. Rats were handled and adapted to the experimental environment for a few days before the formalin test, in which 50 microL of a 1% formalin was injected subcutaneously into the dorsal surface of the right hind paw. The animals were then observed for 1 h and the frequency of flinching behavior was taken to represent the nociceptive response. Thirty minutes before the test, rats were pretreated with intrathecal injections of the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is a substrate of the HO pathway. The paw treatments took place 20 min before the test. Low doses of ZnDPBG did not increase nociception, while a low heme-lysinate dose did not change flinching behavior after paw or spinal injections. Combined subactive spinal (50 nmol) and peripheral (40 nmol) low doses of ZnDPBG induced hypernociception (increase of 80% in the first and 25% in the second phase flinching), whereas combined spinal-peripheral heme-lysinate (50 and 30 nmol) led to second phase antinociception (40% reduction in flinching). These findings suggest a synergy between the peripheral and spinal HO-CO pathways. Local activation of the HO system probably regulates the nociception initiation in peripheral tissue and participates in buffering the emerging nociceptive signals at the peripheral and spinal sites of action. In short, an antinociceptive synergy exists between peripheral and spinal HO pathways, which may reduce the doses required and side effects.

Interaction of the cannabinoid and opioid systems in the modulation of nociception

Cannabinoids and opioids produce antinociceptive synergy. Cannabinoids such as Δ-9-tetrahydrocannabinol (THC) release endogenous opioids and endocannabinoids such as anandamide (AEA) also alter endogenous opioid tone. Opioids and cannabinoids bind distinct receptors that co-localize in areas of the brain involved with the processing of pain signals. Therefore, it is logical to look at interactions of these two systems in the modulation of both acute and chronic pain. These drugs are often co-abused. In addition, the lack of continued effectiveness of opioids due to tolerance development limits the use of such drugs. The cost to society and patients in terms of dollars, loss of productivity, as well as quality of life, is staggering. This review summarizes the data indicating that with cannabinoid/opioid therapy one may be able to produce long-term antinociceptive effects at doses devoid of substantial side effects, while preventing the neuronal biochemical changes that accompany tolerance. The clinical utility of modulators of the endocannabinoid system as a potential mimic for THC-like drugs in analgesia and tolerance-sparing effects of opioids is a critical future direction also addressed in the review.

Role of delta opioid efficacy as a determinant of mu/delta opioid interactions in rhesus monkeys

Delta opioid agonists can selectively enhance the antinociceptive effects of mu opioid agonists without enhancing some other, potentially undesirable mu agonist effects. However, the degree of delta receptor efficacy required to produce this profile of interactions is unknown. To address this issue, the present study examined interactions produced by the mu agonist fentanyl and the intermediate-efficacy delta opioid MSF61 in rhesus monkeys. For comparison, interactions were also examined between fentanyl and the relatively high-efficacy delta agonist SNC243A and the delta antagonist naltrindole, which has negligible efficacy at delta receptors. Two different behavioral procedures were used: (a) a warm-water tail-withdrawal assay of thermal nociception, and (b) an assay of schedule-controlled responding for food reinforcement. Drug interactions within each procedure were evaluated using dose-addition analysis to compare experimental results with expected additivity. Drug interactions across procedures were evaluated using dose–ratio analysis to assess relative potencies to produce antinociception vs. response-rate suppression. As expected, dose-addition analysis found that fentanyl/SNC243A interactions were superadditive in the assay of antinociception but additive in the assay of schedule-controlled responding. Conversely, fentanyl/MSF61 interactions were generally additive in both procedures, and fentanyl/naltrindole interactions were additive or subadditive in both procedures. Dose–ratio analysis found that fentanyl alone produced antinociception and rate suppression with similar potencies. Some fentanyl/SNC243A mixtures produced antinociception with up to 4-fold greater potency than rate-suppression. However, fentanyl/MSF61 and fentanyl/naltrindole mixtures produced antinociception with lower potency than rate suppression. These results suggest that relatively high delta receptor efficacy is required for mu/delta antinociceptive synergy.

Nefopam and ketoprofen synergy in rodent models of antinociception

Combinations of analgesics with different mechanisms of action offer the possibility of efficient analgesia with a decrease in side effects as a result of reduced dosages of one or both compounds. Based on a clinical observation of synergism between nefopam, a centrally acting non-opioid that inhibits monoamines reuptake, and ketoprofen, a non-steroidal anti-inflammatory drug, the objective of this study was to further explore this antinociceptive synergy in four distinct animal models of pain (both drugs were administered subcutaneously). Strong antinociceptive properties were observed in the mouse writhing abdominal test with ED50 values of 2.56±0.38 and 1.41±0.41 mg/kg for nefopam and ketoprofen, respectively. In the inflammatory phase of the mouse formalin test, both compounds significantly inhibited the licking time of the injected hind-paw with ED50 of 4.32±0.17 mg/kg for nefopam and 49.56±15.81 mg/kg for ketoprofen. Isobolographic analysis revealed that this drug combination is synergistic in the formalin test and additive in the writhing test. In rat carrageenan-induced tactile allodynia, single administration of nefopam or ketoprofen only partially reduced allodynia. Combination of low analgesic doses of nefopam (10 or 30 mg/kg) with low analgesic doses of ketoprofen (30 or 100 mg/kg) significantly reduced or reversed allodynia, with a more pronounced anti-allodynic effect and a longer duration efficacy. In a rat model of postoperative thermal hyperalgesia induced by incision, co-administration of nefopam at a low analgesic dose (10 mg/kg) with ketoprofen at non-analgesic doses (30 or 100 mg/kg) showed the appearance of a strong anti-hyperalgesic effect, maintained during at least 3 h. In conclusion, co-administration of nefopam with ketoprofen is synergistic, and should allow either to increase their analgesic efficacy and/or to reduce their side effects.

Isobolographic analysis of multimodal analgesia in an animal model of visceral acute pain

Multiple analgesic–drug combinations are commonly used in the management of acute and chronic pain in humans during multimodal or balanced analgesia. At present, these combinations are used empirically in clinical practice and are considered to be beneficial for the patient. Interactions between two antinociceptive drugs have been thoroughly examined, but the nature of interactions between three analgesics has not been studied. The antinociceptive interaction of ketoprofen (K) with a mixture of morphine (M) and paracetamol (P) was evaluated using a model of visceral acute tonic pain, the acetic-acid writhing test in mice. The i.p. administration of the combination M/P + K resulted in a significant potentiation of the antinociception induced either by K or by the synergic two-drug mixtures M/K, P/K and M/P. The effect of opioid, cholinergic, adrenergic and serotonergic antagonists on the analgesic interaction was assessed. The pretreatment of mice with atropine (1 mg/kg) did not produce any change in the synergistic interaction of the triple combination. The pretreatment with naltrexone (1 mg/kg) or tropisetron (1 mg/kg) reduced the intensity of M/P + K synergic interaction, while prazosin (0.1 mg/kg) significantly potentiated the synergy. The findings of this work suggest that the two major pathways of descending inhibitory systems, noradrenergic and serotonergic are significantly involved in the mechanism of the antinociceptive synergy induced by the triple combination. On the other hand, opioid pathways also seem to participate, since pretreatment with naltrexone reduced the synergy. In conclusion, the triple combination M/P + K induced a strong synergistic antinociceptive effect, which could be of interest for optimal multimodal clinical analgesia.

ST91 [2-(2,6-Diethylphenylamino)-2-imidazoline Hydrochloride]-Mediated Spinal Antinociception and Synergy with Opioids Persists in the Absence of Functional α-2A- or α-2C-Adrenergic Receptors

Agonists acting at alpha2-adrenergic receptors (alpha2ARs) produce antinociception and synergize with opioids. The alpha2ARs are divided into three subtypes, alpha(2A)AR, alpha(2B)AR, and alpha(2C)AR. Most alpha2AR agonists require alpha(2A)AR activation to produce antinociception and opioid synergy. The same subtype also mediates the side effect of sedation, which limits the clinical utility of these compounds. Identification of a non-alpha(2A)AR-mediated antinociceptive agent would enhance the therapeutic utility of alpha2AR agonists in pain management. Previous studies have suggested that the alpha2AR agonist ST91 [2-(2,6-diethylphenylamino)-2-imidazoline hydrochloride] has a nonsedating, non-alpha(2A)AR mechanism of action. We examined the pharmacology of spinal ST91 and its interaction with the delta-opioid agonist deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly amide) in mice lacking either functional alpha(2A)ARs or alpha(2C)ARs. All drugs were administered by direct lumbar puncture, and drug interactions were evaluated using isobolographic analysis. In contrast to the majority of alpha2AR agonists, ST91 potency was only moderately reduced (3-fold) in the absence of the alpha(2A)AR. Studies with the alpha2AR subtype-preferring antagonists BRL-44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole maleate) and prazosin [[4-(4-amino-6,7-dimethoxy-quinazolin-2-yl) piperazin-1-yl]-(2-furyl)methanone] and the pan-alpha2AR antagonist SKF-86466 (6-chloro-2,3,4,5-tetrahydro-3-methyl-1-H-3-benzazepine) suggest a shift from alpha(2A)AR to the other alpha2AR subtype(s) in the absence of alpha(2A)AR. Antinociceptive synergy with deltorphin II was preserved in the absence of either alpha(2A)AR or alpha(2C)AR. In conclusion, ST91 activates both alpha(2A)AR and non-alpha(2A)AR subtypes to produce spinal antinociception and opioid synergy. This study confirms that the spinal pharmacology of ST91 differs from that of other alpha2AR agonists and extends those data to include spinal synergy with opioid agonists. The unique profile of ST91 may be advantageous in pain management.

Agmatine Induces Antihyperalgesic Effects in Diabetic Rats and a Superadditive Interaction with R(–)-3-(2-Carboxypiperazine-4-yl)-propyl-1-phosphonic Acid, a N-Methyl-d-aspartate-Receptor Antagonist

Agmatine, an endogenous cationic amine resulting from the decarboxylation of L-arginine, produces antihyperalgesic and antiallodynic effects in animal models of chronic neuropathic and inflammatory pain. We examined the effect of agmatine on tactile and thermal allodynia and on mechanical hyperalgesia in streptozocin-induced diabetic rats. To determine its mechanism of action and the potential interest of some of its combinations, the antihyperalgesic effect of agmatine was challenged with alpha(2)-adrenergic imidazoline and opioid-receptor antagonists, and its interaction with the opioid-receptor agonist morphine, the competitive N-methyl-D-aspartate receptor antagonist D-CPP [R(-)-3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid], and the nitric-oxide synthase inhibitor L-NAME (L-N(G)-nitro-L-arginine methyl ester) were examined. When intrathecally (i.t.) injected (4.4 to 438 nmol/rat), agmatine was ineffective in normal rats but suppressed tactile allodynia (von Frey hair test), thermal allodynia (tail immersion test), and mechanical hyperalgesia (paw-pressure test) in diabetic rats. This spinal antihyperalgesic effect was suppressed by idazoxan (40 micromol/rat i.t.) but not by yohimbine (40 micromol/rat i.t.) or naloxone (0.69 micromol/rat i.v.). In diabetic rats, an isobolographic analysis showed that combinations of i.t. agmatine with i.v. L-NAME or with i.t. morphine resulted in an additive antihyperalgesic effect, whereas the agmatine/D-CPP i.t. combination was superadditive. In summary, the present findings reveal that spinal agmatine produces antiallodynic and antihyperalgesic effects in diabetic neuropathic pain involving, at least for its antihyperalgesic effect, the imidazoline receptors. Moreover, agmatine combined with D-CPP produces an antinociceptive synergy in experimental neuropathy, opening opportunities in the development of new strategies for pain therapy.

Transcutaneous electrical nerve stimulation activates peripherally located alpha-2A adrenergic receptors

The alpha 2A and alpha 2C adrenergic receptor (AR) subtypes mediate antinociception when activated by the endogenous ligand norepinephrine. These receptors also produce antinociceptive synergy when activated concurrently with opioid receptor activation. The involvement of the opioid receptors in the mechanisms governing transcutaneous electrical nerve stimulation (TENS) has been well described. While spinal alpha-2 ARs do not appear to be involved in TENS antihyperalgesia in rats, the noradrenergic analgesic system also involves supraspinal and peripheral sites. Thus, a broader evaluation of the potential contribution of alpha-2 AR to TENS is warranted. The current study compared the antihyperalgesic efficacy of high (100 Hz) and low (4 Hz) frequency TENS in mutant mice lacking a functional alpha 2A AR against their respective wildtype counterparts. The degree of secondary heat hyperalgesia induced by intra-articular injection of carrageenan/kaolin (3%) mixture did not differ among the experimental groups. However, the antihyperalgesia induced by both low and high frequency TENS was significantly diminished in alpha 2A mutant mice compared to controls. The alpha 2 adrenergic receptor selective antagonist, SK&F 86466, reversed TENS-mediated antihyperalgesia when delivered intra-articularly, but not when delivered intrathecally or intracerebroventricularly. These data suggest that peripheral alpha 2 ARs contribute, in part, to TENS antihyperalgesia. This pharmacodynamic response is consistent with previous anatomical observations that alpha 2A ARs are expressed on primary afferent neurons and macrophages near injured tissue.

Co‐administration of oxycodone and morphine and analgesic synergy re‐examined

The paper by Grach et al. [1] in the Journal addresses the little explored potential benefit of combination opioids in analgesia. Compared with a published study documenting oxycodone-morphine synergy in rats [2], the antinociception study by Grach et al. in healthy subjects has significant design limitations to support their conclusions. In rats, antinociception dose–response curves were produced for each of oxycodone and morphine for the relief of noxious thermal pain [2]. Baseline responses increased 3-fold and antinociceptive ED50 doses of oxycodone and morphine were 2.8 and 8.5 mg kg−1, respectively. These ED50 values agreed with previously published data [3]. Dose–response curves employing oxycodone:morphine combination at ratios of 3 : 1, 1 : 1 and 1 : 3 relative to the morphine or oxycodone ED50 alone, were constructed. Marked antinociceptive synergy was demonstrated using isobolographic analysis and validated by using selective antagonists for each of the combination components. In their study, Grach et al. compared oral doses of 0.5 mg kg−1 morphine or 0.5 mg kg−1 oxycodone with 0.25 mg kg−1 morphine combined with 0.25 mg kg−1 oxycodone. The morphine dose selected was the lowest dose (0.43 mg kg−1) identified by Cleeland et al. [4] that produced significant antinociception in the cold pressor test. We have re-calculated the cold pressor tolerance (CPT) data as a percentage of the maximum possible response (see Figure 1). Figure 1 Cold pressure Test Tolerance response data expressed as percentage of the maximum possible response (▪) show 0.25 mg kg−1 of morphine corresponds to the ED10 According to the Cleeland study [4], only the largest dose of oral morphine tested (0.43 mg kg−1) produced significant antinociception compared with the active diphenhydramine placebo. Thus the assumptions were that the Median Baseline response was 30 s and the Minimum acceptable dynamic range was three times the baseline response, similar to the rodent antinociception criteria. The Percentage Maximum Possible Effect (%MPE) was calculated as the percentage difference between the measured response and the baseline response, divided by the difference between the maximum response and the baseline response. Figure 1 shows that 0.25 mg kg−1 morphine approximates the ED10. Using the same approach as Grach et al. (0.25 mg kg−1 morphine and oxycodone) represents, at best, the ED10 and ED25, respectively. Without additional dose–response data, it is not possible to confidently identify the effective doses of the individual opioids, the subantinociceptive dose range for cold pressor pain, or the doses needed to produce an adequate pharmacodynamic range in healthy subjects. Furthermore, their study highlights the poor sensitivity of this test for predicting clinically relevant doses of opioids as seen with morphine [4]. Thus, an alternative conclusion of this work is that the suitability of the cold pressor test to predict clinically relevant doses of opioids, administered alone or in combination, is questionable under the conditions of this study. Lauretttiet al. in 2003[5] reported opioid synergy in chronic cancer-related pain. Briefly, 22 patients received controlled-release morphine (CRM) and controlled-release oxycodone (CRO) in a crossover design involving two sequential 14-day treatment periods with immediate-release morphine (IRM) available for breakthrough pain. The requirement for breakthrough IRM was 38% higher in patients receiving CRM than in patients receiving CRO, suggesting that a synergistic analgesic interaction took place when morphine was administered to patients receiving CRO. In two double-blind, crossover studies involving 44 osteoarthritic patients with moderate to severe pain, the steady-state daily opioid requirements were approximately 40% less with morphine:oxycodone combinations compared with morphine alone (Smith et al. unpublished data). These findings suggest that morphine:oxycodone combinations produce greater than additive (and likely synergistic) pain relief in patients. In summary, extrapolation of findings from an antinociception study in healthy subjects using single doses of opioids either individually or in combination, without prior adequate identification of the pharmacodynamic range for the pain being tested, needs reassessment. In order to be of predictive value, studies of antinociceptive synergy in healthy subjects need to be carefully designed, taking into consideration the full pharmacological range of the drugs being studied.