Gabapentin Blocks and Reverses Antinociceptive Morphine Tolerance in the Rat Paw-pressure and Tail-flick Tests

From the Departments of Anesthesiology and Pharmacology & Toxicology, Kingston General Hospital, Queen's University, Kingston, Ontario, Canada.OPIOID tolerance is a diminution of analgesic effect or need for a higher dose to maintain the original effect following chronic opioid exposure. 1While its clinical importance is controversial, 2–5studies of opioid tolerance have advanced knowledge about analgesic mechanisms. In common with nerve or tissue injury, chronic opioid administration causes spinal changes involving translocation and activation of protein kinase C and production of nitric oxide (NO). 6Furthermore, mechanisms of opioid tolerance include N -methyl-d-aspartate (NMDA) receptor 6and 2-amino-3-hydroxy-5-methyl-4-isoxazole-proprionic acid (AMPA)/kainate receptor 7modulation, dynorphin activity, 8calcitonin gene–related peptide activity, 9and cyclooxygenase activity. 10In addition to suppressing opioid tolerance, drugs that modulate the previously mentioned mechanisms (such as NMDA receptor antagonists, 11AMPA/kainate receptor antagonists, 12and cyclooxygenase inhibitors 13) are also antihyperalgesic and/or antiallodynic. Gabapentin is a γ-aminobutyric acid (GABA) analog that reduces pain, hyperalgesia, and allodynia following tissue or nerve injury through several possible mechanisms. 14Previous data suggest that the effects of gabapentin are naloxone insensitive, chronic gabapentin administration does not lead to gabapentin tolerance, and morphine tolerance does not influence gabapentin analgesia in the rat formalin test. 15While previous preclinical investigations have evaluated gabapentin–opioid interactions, 16–18the effect of gabapentin on opioid tolerance has not been studied. Thus, the goal of this investigation is to test the hypothesis that gabapentin prevents and reverses chronic opioid tolerance.All experiments used adult, male Sprague-Dawley rats (250–300 g, Charles River, St. Constant, QC, Canada). Procedures were in accordance with the Animals for Research Act, the Guidelines of the Canadian Council of Animal Care, and the Queen's University Animal Care Committee. The paw-pressure 19,20and tail-flick 21,22tests were used to evaluate the response of the animals to nociceptive stimuli.Single intraperitoneal doses of a) 7.5 mg/kg morphine, b) 150 mg/kg gabapentin, c) 300 mg/kg gabapentin, and d) a combination of 7.5 mg/kg morphine and 150 mg/kg gabapentin were studied using the paw-pressure and tail-flick tests in naïve rats. Testing was performed every 10 min after drug administration for the first hour and every 30 min for the following 2 h.Rats received intraperitoneal injections of 15 mg/kg morphine once daily for 7 days. This dose has been shown previously to produce tolerance over 7 days following initial maximal antinociception. 23Testing was performed before and 30 min after drug administration. On day 8, cumulative dose-response curves were constructed, and the ED50values of morphine were determined as described previously. 23To obtain these curves, animals received increasing doses of morphine every 30 min, and testing followed 30 min after each drug injection. This protocol continued until maximal antinociception was obtained.To evaluate the effect of gabapentin on development of morphine tolerance, gabapentin (150 mg/kg, intraperitoneal) was coinjected with morphine (15 mg/kg, intraperitoneal) once daily for 7 days. Testing was performed once daily and cumulative dose–response curves were generated on day 8. To characterize the offset of the effect of gabapentin on morphine tolerance, another study evaluated gabapentin coinjected with morphine for days 1–3 followed by daily morphine alone on days 4–7.Morphine (15 mg/kg) was given once daily for 4 days to induce tolerance. On the following 3 days, gabapentin (150 mg/kg) was introduced in combination with morphine. Morphine dose–response curves were generated on day 8, and acute morphine ED50values were calculated.Morphine was obtained from BDH Pharmaceuticals (Toronto, ON, Canada) and gabapentin was obtained from Pfizer (Groton, CT). All drugs were dissolved in 0.9% saline.Tail-flick and paw-pressure values were converted to maximum percentage effect. All data are expressed as mean maximum percentage effect (± SEM). The ED50values were determined using nonlinear regression analysis. Statistical significance (P < 0.05) was determined using one-way ANOVA followed by a Dunnett post hoc test for multiple comparisons between groups.Submaximal doses of morphine (7.5 mg/kg) produced peak antinociception in both tail-flick and paw-pressure tests 30 min after administration. Gabapentin alone at doses of 150 mg/kg (figs. 1A, B) and 300 mg/kg (not shown) had no intrinsic effect in both tests. However, when given together, these doses of morphine and gabapentin resulted in maximal, and supra-additive, antinociception peaking 50 min after administration in the paw-pressure test and between 40 and 60 min after administration in the tail-flick test. The combination of gabapentin and morphine resulted in significantly larger responses than morphine alone, from 20 to 120 min for the paw-pressure test and from 40 to 150 min for the tail-flick test (figs. 1A, B). In both tests, responses returned to baseline by 150 to 180 min after injection. Visual inspection of treated animals revealed no signs of motor impairment.Administration of morphine (15 mg/kg) on day 1 produced maximal antinociception on day 1, which decreased to baseline levels by day 5. Coadministration of morphine with gabapentin (150 mg/kg) completely blocked the decrease in morphine effect throughout the entire 7-day period (figs. 2A, B). In a subsequent experiment, where gabapentin was coadministered with morphine only for days 1–3, maximal antinociception with morphine was still observed on day 4, but a subsequent decrease in effect was observed from days 5 to 7 (figs. 2A, B). Administration of morphine for 7 days significantly increased the ED50value three- to sixfold more than that observed in saline-treated animals (table 1). Coadministration of gabapentin with morphine for the entire 7-day period resulted in ED50values that were significantly lower than values for the morphine alone group (table 1).In this study, morphine plus gabapentin were administered on days 5–7. Chronic administration of morphine alone on days 1–4 resulted in a decrease in antinociception similar to that observed previously (figs. 2A, B). However, addition of gabapentin on days 5–7 resulted in a partial restoration of the morphine effect (figs. 2A, B) and significantly greater antinociception than for morphine alone on days 6 and 7 of the paw-pressure test. The ED50value on day 8 for this treatment group was significantly lower than for that of morphine alone with the paw-pressure test but not the tail-flick test (table 1).This study shows, for the first time, that gabapentin inhibits development of antinociceptive tolerance to morphine. This is evident in sustained responses to morphine in the presence of gabapentin for 7 days, a leftward shift of the acute morphine dose–response curve, and a decrease in the acute morphine ED50value compared to those of morphine tolerant animals. The tolerance to morphine, however, becomes apparent within 48 h of discontinuing gabapentin, indicating the need for continued gabapentin to maintain opioid potency. Finally, data from the paw-pressure test suggests that gabapentin can partially restore opioid potency in tolerant rats. Taken together, these results support a role for gabapentin–opioid combinations or for the addition of gabapentin to opioids in the setting of tolerance.Recent studies of gabapentin may explain its effects on opioid tolerance, which is mediated by l-glutamate action at spinal NMDA 6and AMPA/kainate 7receptors. Shimoyama et al. 24demonstrated that gabapentin presynaptically inhibits glutamate transmission and Chizh et al. 25showed that gabapentin antagonizes AMPA-evoked responses in vivo . Furthermore, a study in trigeminal nucleus slices showed that glutamate release activated by protein kinase C (also important in mediating opioid tolerance) is blocked by gabapentin. 26Also, chronic morphine has been shown to increase spinal dynorphin expression, which can be pronociceptive 27and, in this regard, Laughlin et al. 28have demonstrated that gaba-pentin reduces dynorphin-induced allodynia. Dynorphin expression following chronic morphine exposure involves activation of descending pain facilitory systems, 8suggesting the importance of supraspinal sites in the development of tolerance. In this regard, Andrews et al. 29showed that gabapentin blocked morphine-induced “conditioned place preference” (a test of psychological dependence) as well as morphine-induced dopamine release from nucleus accumbens. Finally, the effects of gabapentin on tolerance may be related to its unique binding to the (α)2(δ) calcium channel subunit. 30,31In this regard, a recent investigation by Luo et al. 32has demonstrated upregulation of this subunit following nerve injury, a condition which shares some similarities with opioid tolerance. 6In certain situations, tolerance may limit opioid efficacy and an understanding of the underlying mechanisms may improve pain management. This study suggests that gabapentin augments the antinociceptive action of both acute and chronic morphine therapy. Future studies are needed to further explain the sites and mechanisms of these actions. Also, clinical investigations are needed to identify specific settings and patient populations in which gabapentin–opioid combinations may be useful.

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