Administration of opioids may lead to an enhancement in pain sensitivity induced by central sensitization.1 This phenomenon is referred to as opioid-induced hyperalgesia (OIH). Preclinical studies have demonstrated that long-lasting hyperalgesia may occur even after acute administration of opioids in uninjured rats.2 Several clinical studies have shown that opioid-induced pain sensitivity can develop rapidly after a short-term exposure in humans and may, paradoxically, facilitate postoperative pain, leading to short-term tolerance.3–5 Consequently, therapies that can oppose early OIH and reduce subsequent exaggerated postoperative pain should be developed. However, OIH is one possible explanation of aggravation of postoperative pain; the alternative explanation is actual development of acute tolerance and, given the cross-sectional design of these clinical studies, it remains unclear whether OIH or tolerance was at play.
There are similarities in the mechanisms between neuropathic pain and OIH that reflect a sustained sensitization of the nervous system in which excitatory amino acid neurotransmitter systems have a critical role, especially via N-methyl-d-aspartate (NMDA) receptors.6 Therefore, drugs such as NMDA receptor antagonists or drugs acting indirectly on NMDA receptors by reducing the spinal release of excitatory amino acid neurotransmitters are likely to prevent OIH by inhibiting central sensitization.
Preclinical studies have shown that the NMDA receptor antagonist ketamine prevents the development of pain sensitivity induced by acute administration of fentanyl, an opioid widely used for surgery.2 We reported that gabapentin, a 3-alkylated analog of γ-amino butyric acid binding presynaptically to the α2δ1 subunit of the voltage-gated calcium channel (Cavα2δ1), prevents the delayed hyperalgesia induced by short-term use of systemic fentanyl in uninjured rats.7
The effects of the combination of drugs can be additive or synergistic (supraadditive). A synergistic response is possible when the drugs are acting through distinct mechanisms.8 This may improve the clinical effects of each drug, allowing a significant dose reduction thereby minimizing drug-specific adverse effects.9 Because ketamine and gabapentin are both effective in the prevention of OIH and seem to act through different mechanisms, we examined the effects of their combination on OIH. The goal of this study was to determine their type of pharmacologic interaction: synergism, additivity, or infraadditivity. In animals, the isobolographic analysis allows an evaluation of the interaction between 2 drugs, but no study using this analysis is available concerning ketamine and gabapentin. Therefore, we used a model of long-lasting hyperalgesia induced by acute systemic fentanyl in uninjured rats to define the median effective dose (ED50) of ketamine, gabapentin, and their combination, to perform an isobolographic analysis to determine their type of pharmacologic interaction.
Experiments were performed on male Sprague Dawley rats (250–275 g, CD1; Charles River, Iffa-Credo, L’Arbresle, France), housed in groups of 3 per cage, under a 12-hour light/12-hour dark cycle (lights on at 8:00 am), at a constant room temperature of 22°C ± 2°C, 1 week before experiments. Animals had access to food and water ad libitum. Experiments were approved by the Institution’s Animal Care and Use Committee and were performed in accordance with guidelines from the International Association for the Study of Pain, Committee for Research and Ethical Issues.10 This study, including care of the animals involved, was conducted according to the official edict presented by the French Ministry of Agriculture (Paris, France) and the recommendations of the Helsinki Declaration. At the end of the experiment, the rats were killed with isoflurane. Accordingly, these experiments were conducted in an authorized laboratory, and under the supervision of an authorized researcher (JXM).
The following drugs were used for the experiment: fentanyl citrate (Sigma-Aldrich Co., Saint-Quentin-Fallavier, France), gabapentin (Sigma-Aldrich Co., Steinheim, Germany), and ketamine hydrochloride (Sigma-Aldrich Co., Saint-Quentin-Fallavier, France). Gabapentin and ketamine were diluted with physiologic saline (0.9% NaCl). Gabapentin (50 mg/mL) was injected intraperitoneally (IP), whereas ketamine (2 mg/mL) was injected subcutaneously (SC). To induce acute OIH, fentanyl was injected SC (100 μL/100 g body weight). SC injections were performed on the back of nonanesthetized rats with a 25-gauge needle. For those animals given IP drugs, a single IP injection was accomplished by abdominal puncture, using a 25-gauge hypodermic needle.
Measurement of the Nociceptive Threshold
All animals were tested for quantitative changes in pain threshold, as measured by the use of von Frey filaments. The experiments were performed by an investigator blinded to the dose administered. Before the von Frey measurements were made, rats were confined in individual plastic boxes (20 × 20 × 15 cm) with elevated wire mesh floors. Animals were allowed 30 minutes to acclimatize. Thereafter, light mechanical stimuli using sufficient force to cause light buckling of the filament was applied from underneath to the midplantar area of the hindpaw using different strengths of von Frey filaments (Somedic, Hörby, Sweden). Care was taken to avoid stimulating the same spot repeatedly within this region and to avoid stimulating the tori/footpads. Mechanical sensitivity of the foot was determined by positive foot withdrawal in response to von Frey stimuli. In brief, von Frey filaments with incremental stiffness (buckling forces of 0.83, 1.42, 3.14, 10.8, 32.4, 50.0, and 81.4 mN) were applied serially to the hindpaw. The algorithm started with the lowest force in ascending fashion. As shown elsewhere,11 a single trial of stimuli consisted of 10 applications using the same von Frey filament to the plantar surface of the hindpaw for approximately 4 to 6 seconds each time. We allowed a period of 3 to 4 minutes before commencing with the next filament. Brisk foot withdrawals (at least 6 times of 10 applications) were considered positive for the von Frey filament used. If the value of this buckling force was equal to or higher than the basal buckling force value, the next administered dose of the drug was decreased 1 step, and von Frey filaments were applied in descending order of stiffness, beginning with the value of the buckling force found for the previous dose. Conversely, if the buckling force value found for the previous dose was lower than the basal buckling force value, the dose of the drug was increased 1 step, and von Frey filaments were applied in ascending order of stiffness beginning with the value of the buckling force found for the previous dose.
After arrival in the laboratory, animals were allowed 7 days to become accustomed to the colony room, gently handled daily for 5 minutes, and left in the test room for 2 hours (from 11:00 am to 1:00 pm). All experiments began at 11:00 am and were performed during the light part of the cycle. The basal nociceptive threshold was measured twice (with 30 minutes between the measurements) on the 2 days preceding the planned experimental day (i.e., D−2 and D−1), and on the experimental day (D0). Experiments with fentanyl, ketamine, and gabapentin were initiated only when no statistical changes in basal nociceptive thresholds were observed when estimated on D−2, D−1, and D0.3
Célèrier et al.,2 by performing the paw-pressure vocalization test with the Basile analgesimeter, have shown that high doses of fentanyl in rats induced delayed and long-lasting hyperalgesia for days, referred to as acute OIH. To evaluate the reliability of von Frey filaments for assessment of acute OIH, acute OIH was induced using the protocol of Célèrier et al. Fentanyl was injected 4 times (60 μg/kg per injection) at 15-minute intervals resulting in a total dose of 240 μg/kg administered over 1 hour.2 Supplemental O2 was administered via a facemask throughout the procedure. The nociceptive threshold was then measured using von Frey filaments twice daily (30 minutes between measurements) for 6 days (D+1–D+6), and compared with the basal nociceptive threshold measured on D0.
In a second set of experiments, we investigated the effects of ketamine, gabapentin, or their combination on OIH. Ketamine, gabapentin, or their combination was administered on D0 30 minutes before the first injection of fentanyl. The response was considered positive if the delayed decrease in nociceptive threshold was completely prevented on D+1. The dose of ketamine or of gabapentin received by a particular animal was determined by the response of the previous animal within the same group, using an up-and-down sequential allocation technique.12,13 Based on a previous study,2 the first animal in the ketamine group received 10 mg/kg SC ketamine. The dose adjustment interval was 1 mg/kg. Based on another previous study,7 the first animal in the gabapentin group received 300 mg/kg IP gabapentin. The dose adjustment interval was 30 mg/kg. Dose adjustment intervals were defined according to the expected standard deviation of the ED50.12,13 In the ketamine-gabapentin group, the first animal received ketamine and gabapentin at doses corresponding to slightly less than half the ED50 of ketamine alone and to half the ED50 of gabapentin alone (i.e., 5 mg/kg for ketamine, and 150 mg/kg for gabapentin). The doses were chosen to preserve a ratio of 1:30 between ketamine and gabapentin in the combination group.
To ensure that ketamine, gabapentin, or their combination actually prevented OIH without exerting independent analgesic effects, we performed saline placebo-controlled experiments, using the ED50 of gabapentin, or of ketamine, or of their combination. Experimenters were blinded to the treatment, and therefore to the dose administered.
Data and Statistical Analysis
The mean of the 2 measurements performed daily on D−2, D−1, and D0 were compared (1-way analysis of variance [ANOVA] for repeated measures). The basal reference value of the nociceptive threshold was chosen as the first measurement of the nociceptive threshold performed on D0.2 Normal distribution was verified with the Kolmogorov-Smirnov test. The nociceptive threshold was compared between days (D0, D+1–D+6) using ANOVA (2-way, for repeated measures) followed by a Tukey-Kramer test as appropriate. Data were expressed as the mean ± SEM, and P < 0.05 was considered statistically significant.
The ED50 of each drug leading to satisfactory antihyperalgesia in 50% of animals included in the study, and the 95% confidence interval (CI) of ED50 were calculated using Dixon’s up-and-down sequential allocation technique, according to the following formula:
where X1 is the last dose administered, k is the tabular value, and d is the interval between doses.12,13 The up-and-down method estimates the threshold for an all-or-none response, usually defined as a point above which 50% of the subjects respond to the stimulus and below which 50% of the subjects do not respond. Briefly, a certain dose is given to the first subject, and subsequent doses are given according to the following rule: if 1 subject responds positively, the dose is decreased 1 step for the next subject and, conversely, if the former subject does not respond, the latter dose is increased 1 step. Sampling is begun including the first pair of tests with opposite results. After the ED50 of each drug was determined, the drugs were administered in combination, and the up-and-down procedure was used again to determine the ED50 of the combination. The 95% CIs of ED50 were calculated for each drug separately and for their combination.13 ED50 and its 95% CI of the combination were calculated using 1 drug because the ratio was fixed. A classic isobolographic technique was used in a second stage to assess the possible interaction between the 2 drugs.14 The isobologram was constructed by connecting the ED50 of ketamine plotted on the abscissa with the ED50 of gabapentin on the ordinate to obtain the additive line. The 95% confidence contours of the joint action were drawn by joining the 95% CIs in each axis of the isobologram. The association of the 2 drugs was judged as additive if the contours overlapped, and supraadditive (synergistic) or infraadditive otherwise.
Long-Lasting Effect of Fentanyl on the Nociceptive Threshold (D+1–D+6)
The mean baseline nociceptive threshold value was 3.1 ± 0.3 mN (n =10). SC fentanyl caused a statistically significant long-lasting decrease in nociceptive thresholds for 4 days from D+1 to D+4 (1-way ANOVA followed by the Tukey-Kramer test, P < 0.001 from D+1 to D+3 and P < 0.05 on D+4; Fig. 1).
Antihyperalgesic Effect of Ketamine, Gabapentin, and Their Combination on OIH
The ED50 values of ketamine (n =18) and gabapentin (n =17) were 12.4 mg/kg (11.7–13.1 mg/kg) and 296.3 mg/kg (283.5–309.1 mg/kg), respectively. The ED50 values of the drug combination (n =18) were 4.1 mg/kg (3.7–4.6 mg/kg) for ketamine and 123.9 mg/kg (111.1–136.7 mg/kg) for gabapentin, thus demonstrating a supraadditive (synergistic) effect of the combination. The sequences of effective and ineffective antihyperalgesia are shown in Figure 2 and the isobolographic representation in Figure 3.
Independent Analgesic Effect of Ketamine, Gabapentin, and Their Combination on OIH
Neither ketamine (12 mg/kg) (n =8), gabapentin (300 mg/kg) (n =8), nor their combination (ketamine 4 mg/kg + gabapentin 125 mg/kg) (n =8) exerted independent analgesic effects at the time of OIH (i.e., D+1–D+4) (1-way ANOVA followed by the Tukey-Kramer test, P > 0.05; Fig. 4).
The major finding of this study was that the combination of ketamine and gabapentin was synergistic in preventing long-lasting hyperalgesia induced by acute systemic fentanyl in uninjured rats. This is the first study to offer an isobolographic analysis of the antihyperalgesic interaction between ketamine and gabapentin. These data suggest that ketamine and gabapentin given in combination might produce greater than additive effects in the treatment of some hyperalgesic states.
The paradoxical phenomenon of OIH has been described to develop rapidly after a single exposure in animals, volunteers, and surgical patients.15 There are similarities between neuropathic pain and OIH.6 Given that abnormal persistence of excitatory neuroplasticity is considered to be a major mechanism for the development of chronic pain,16 therapies that can oppose early postoperative hyperalgesia should be developed. In fact, the major benefit of attenuating hyperalgesia might be a reduction of the incidence of persistent pain after surgery. However, it is still unclear whether aggravated pain and increased opioid consumption in surgical patients receiving high intraoperative opioid doses are due to the onset of OIH or to the onset of tolerance. Further studies aimed to clarify this point would be valuable.
From a therapeutic viewpoint, this suggests that the early administration of antihyperalgesic drugs might be a fruitful strategy to prevent postoperative hyperalgesia, and may reduce postoperative opioid consumption. There is therefore a rationale to use NMDA receptor antagonists or drugs reducing the spinal release of excitatory amino acid neurotransmitters to prevent OIH. The NMDA receptor antagonist ketamine has been shown to prevent OIH in animals2 by NMDA receptor blockade2,17 and by inhibition of the morphine-induced enhancement of spinal long-term potentiation.18 We have demonstrated in the same model that the 3-alkylated analog of γ-amino butyric acid gabapentin prevents delayed hyperalgesia induced by short-term use of systemic fentanyl in uninjured rats.7 Induction of the α2δ1 subunit of the voltage-gated calcium channel (Cavα2δ1) in the spinal cord and dorsal root ganglia is likely regulated by factors that are specific for individual neuropathies and may contribute to gabapentin-sensitive allodynia.19 A supraadditive antihyperalgesic interaction between ketamine and gabapentin was detected in the current study in rats with OIH. This might be because these 2 drugs seem to act through different mechanisms. However, the mechanisms responsible for synergistic interactions are poorly understood, and a number of hypotheses have been developed to explain these effects.20 These include the interaction being pharmacokinetic, or pharmacodynamic. We did not examine the type of interaction between ketamine and gabapentin, and further work is required to elucidate the exact mechanism of synergy between these 2 drugs.
From a technical viewpoint, in the model of Célèrier et al.,2 the delayed and long-lasting hyperalgesia induced by fentanyl in rats was assessed using the paw-pressure test. In the current study, in the same model, we have shown that the delayed and long-lasting hyperalgesia induced by fentanyl in rats was similarly demonstrated using von Frey filaments.
From a methodological viewpoint, we used the Dixon up-and-down allocation technique that allows determination of ED50 with fewer subjects than conventional techniques. However, the Dixon method allows only determination of the median dose and its standard deviation, and does not allow drawing the entire dose-probability curve. Isobolographic analysis has been widely used to study the interaction of drugs, and its scientific use has been widely discussed.14,21 Therefore, in a second stage, based on previous studies,22–24 we searched for an interaction using a classic isobolographic analysis to compare the ED50 values of the drugs alone and in combination.
In conclusion, the essential observation in this study was that the combination of ketamine and gabapentin exhibited a supraadditive effect in preventing OIH. We postulate that the different sites of action of the 2 drugs may explain these results. As a whole, this suggests that the doses of the individual drugs can be reduced, thereby reducing drug-specific side effects. Therefore, the combination of ketamine and gabapentin may well have clinical virtue. Our results might provide a basis for clinical evaluation of a ketamine-gabapentin combination in patients with perioperative hyperalgesia. Further clinical studies in this field would be valuable.
Name: Alain C. Van Elstraete, MD.
Contribution: Study design, conduct of the study, data collection, data analysis, manuscript preparation.
Attestation: Dr. Van Elstraete is the archival author, has reviewed the original study data and data analysis, and attests to having approved the final manuscript.
Name: Philippe Sitbon, MD.
Contribution: Study design, conduct of the study, data collection, manuscript preparation.
Attestation: Dr. Sitbon has reviewed the original study data and data analysis, and attests to having approved the final manuscript.
Name: Dan Benhamou, MD.
Contribution: Study design, manuscript preparation.
Attestation: Dr. Benhamou has reviewed the original study data and data analysis, and attests to having approved the final manuscript.
Name: Jean-Xavier Mazoit, MD, PHD.
Contribution: Study design, data analysis, manuscript preparation.
Attestation: Dr. Mazoit has reviewed the original study data and data analysis, and attests to having approved the final manuscript.
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