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Abstract
BACKGROUND: The analgesic interaction between cannabinoids and local anesthetics has not been investigated. We sought to determine the nature of the interaction between the intrathecal cannabinoid receptor agonist (WIN 55,212-2) and bupivacaine using the formalin test.
METHODS: Lumbar intrathecal catheters were implanted in male Sprague-Dawley rats. After intrathecal administration of WIN 55,212-2, bupivacaine, or their combination, 50 μL of 5% formalin was injected subcutaneously into the hindpaw. Dose—response curves were established and the respective ED50 (50% effective dose) values were determined for each agent alone. Fixed-ratio combinations of WIN 55,212-2 and bupivacaine were tested for combined antinociceptive effects in the formalin test and an isobolographic analysis was performed to characterize the pharmacologic interaction of both drugs.
RESULTS: Intrathecally administered WIN 55,212-2, bupivacaine, or their combination produced a dose-dependent decrease in the number of flinches during Phase 1 and 2 of the formalin test. Isobolographic analysis revealed a synergistic interaction between intrathecal WIN 55,212-2 and bupivacaine in both phases of the formalin test. In combination, the ED50 value was significantly smaller than the theoretical additive value (P < 0.05).
CONCLUSIONS: These results demonstrate that intrathecally coadministered WIN 55,212-2 and bupivacaine provide synergistic antinociceptive interaction in both phases of the formalin test.
IMPLICATIONS: The pharmacologic nature of the interaction between intrathecal cannabinoid receptor agonist (WIN 55,212-2) and bupivacaine was determined using the formalin test. Intrathecally coadministered WIN 55,212-2 and bupivacaine produced synergistic antinociceptive interaction in both phases of the formalin test, with decreased side effects such as sedation and motor impairment.
Neuraxially administered local anesthetics produce antinociception and motor blockade by directly inhibiting neuronal voltage-gated sodium channels, thereby reducing axonal conduction (1). Although a motor blockade is sometimes helpful during surgical procedures, it is desirable to minimize unwanted motor blockade by local anesthetics in a variety of clinical areas related to pain management. Intrathecal coadministration of local anesthetics and other drugs, such as opioid analgesics or clonidine, produces supra-additive analgesic effects (2,3). These interactions allow a reduction in the dose of each drug, hence a reduction in potential side effects such as intense motor blockade. The aforementioned analgesic combination strategies also have been applied to clinical practice: neuraxially injected mixtures of an opioid or clonidine and a local anesthetic such as bupivacaine, at concentrations that do not produce motor blockade, are shown to offer advantages for the control of surgical, labor, and cancer pain (4—6). In addition, there have been numerous recent studies on other drug combinations producing analgesia in experimental animal models that may be useful in clinical populations.
Cannabinoids have been shown to produce antinociceptive and antihyperalgesic effects in various animal pain models (7,8). The antinociceptive effects of cannabinoids are believed to be exerted primarily via cannabinoid receptors (subtypes cannabinoid receptor Type 1 [CB1] and cannabinoid receptor Type 2 [CB2]) located on neurons within and outside the brain and spinal cord (9). However, unwanted central nervous system (CNS) effects, including hypothermia, hypoactivity, and catalepsy, can also be elicited by cannabinoids through stimulation of CB1 receptors within the brain (10). As these adverse CNS effects can limit the potential clinical application of cannabinoids, strategies to avoid or reduce such side effects have been sought. One of these strategies is to administer cannabinoid agonists by the intrathecal route (11,12), in view of the evidence that CB1 receptors are present in a nociceptive area of the spinal cord (13). Another strategy could be to exploit the synergistic interactions between cannabinoids and other agents for antinociception. In this experiment, we have used potent, synthetic aminoalkylindole cannabinoid WIN 55,212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone mesylate), with the molecular weight of 522.61. WIN 55,212-2 is the nonselective CB1/CB2 cannabinoid receptor agonist, and exhibits relatively high efficacy at both of these receptor types (14).
Despite the efforts to avoid unwanted side effects, no study, to our knowledge, has considered smaller doses through the interaction of cannabinoids with local anesthetics. The purpose of this study was to determine the characteristics of the drug interaction between intrathecal WIN 55,212-2 and a long-acting amino-amide local anesthetic (bupivacaine) using the formalin test, in an animal model of acute tissue injury-induced pain.
METHODS
All experiments were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of Ewha Womans University, Seoul, Korea. Male Sprague-Dawley rats (Orient, Seoul, Korea), weighing 250—300 g, were used in this study. The rats were housed in individual cages with free access to food and water, and maintained on a 12-h light—dark cycle (lights on 6:00 am) at an ambient temperature of 22 ± 1°C.
For drug administration, an intrathecal catheter was first implanted under ketamine—xylazine anesthesia (75 and 15 mg/kg, respectively, IM) by the direct lumbar catheterization method (15). Once anesthetized, a 2—3 cm midline longitudinal incision was made at the level of the iliac crests. This was followed by a catheter (polyethylene-10 tube, Becton Dickinson, Sparks, MD) with a metal wire that was advanced through the hole made by a 23-gauge needle in the space between the lumbar vertebrae L5 and L6. When the sign of intrathecal position (tail-flick or hindpaw retraction) was observed, the guidewire was withdrawn for about 2 cm and the catheter was pushed gently upward to reach L4 at the lumbar enlargement. After proper catheter placement, the guidewire was carefully removed, avoiding displacement of the catheter. The rostral end of the catheter was then heat-welded to another catheter (3 cm long, polyethylene-50 tube, Becton Dickinson, Sparks, MD) for drug administration. The external part of the catheter was tunneled subcutaneously to exit at the occipital region, flushed with saline and sealed by heat melting. The dead space of the catheter was 8.0 ± 1.6 μL. All incisions were sutured and the animals were allowed to recover from surgery for at least 7 days before the experiment. Rats exhibiting neurologic deficits or infection were excluded from the study.
Drugs used in this study included WIN 55,212-2 mesylate and bupivacaine hydrocholoride (Sigma Chemical CO, St. Louis, MO). WIN 55,212-2 was dissolved in 100% dimethylsulfoxide, and bupivacaine was dissolved in physiologic saline (0.9% sodium chloride). All drugs were administered intrathecally with a 10-μL microinjector syringe (Hamilton CO, Reno, NV) in a total volume of 10 μL over 20 s, followed by a 10 μL sterile saline to flush the catheter. Test drugs were given 10 min before the formalin test, as determined in pilot studies. Control rats received the drug solvent vehicle alone. The control study for the combination was done using 50% dimethylsulfoxide in saline.
Antinociception was assessed by the formalin test. Test sessions were conducted between 11:00 am and 5:00 pm. The animals were randomized by the sealed envelope method. In each group, 10 randomly selected rats were used and each animal was used only once. The rats were habituated to the open Plexiglas observation chamber (30 × 30 × 30 cm) for at least 30 min before testing. A mirror was placed under the chamber floor to provide an unobstructed view of the animal. Fifty μmicroliters of 5% formalin was then injected subcutaneously via a 29-gauge needle into the plantar surface of the right hindpaw while the rat was manually restrained. Immediately after receiving the injection, rats were placed back into the chamber and continuously observed by a blinded observer. Pain-related behavior was quantified by counting the spontaneous flinches for 60 min after formalin injection. Flinching was defined as rapid and brief withdrawal or flexing of the injected paw. The score was determined by the sum of incidences of flinching in a 5-min period. Formalin-induced flinching behavior is biphasic. The initial acute phase (Phase 1, during 0—5 min interval after the formalin injection) is followed by a quiescent period, which is then followed by a prolonged tonic phase (Phase 2, beginning approximately 10 min after the formalin injection). At the end of the experiments, rats were euthanized in a CO2 chamber, and the catheter position was verified by injection of indigo blue. Rats with incorrect placement were excluded from the data analysis.
The general behavior of each rat was carefully noted and recorded for 60 min after intrathecal drug administration by a blinded observer. Sedative effects were assessed at 10-min intervals using a 5-point scale proposed by Kawamata et al. (16): 0 = normal behavior, alert to the environment, standing, or grooming; 1 = sitting quietly, sometimes standing or grooming; 2 = sitting quietly, no spontaneous movement, but moved if touched; 3 = no spontaneous movement, did not move when touched; 4 = loss of righting reflex, unresponsive. Scores were totaled over the observation period. Motor function was evaluated at 5, 10, 15, 30, 45, and 60 min after treatment by the placing/stepping reflex and the righting reflex. The former was evoked by drawing the dorsum of either hindpaw across the edge of the table. The test result was considered normal if the animal placed the paw ahead into a position to walk. The righting reflex was assessed by placing the rat horizontally with its back on the table. The test result was considered normal if the animal returned to an upright position within 2 s. Based on the degree of impairment, motor blockade was scored as follows: 0, normal; 1, slight deficit; 2, moderate deficit; and 3, severe deficit. For relatively large doses of WIN 55,212-2 (3 and 10 μg), bupivacaine (30 and 100 μg) and the combination (1/32 ED50 and 1/128 ED50 doses), the behavioral effect of intrathecal drug was also evaluated without formalin injection.
To determine the time course and dose-dependency of the antinociceptive effects in the formalin test, several doses of WIN 55,212-2 (0.3, 1, 3, and 10 μg) or bupivacaine (1, 10, 30, and 100 μg) were tested. Experiments with each agent alone were performed first, so that doses of the combinations could be planned. From the dose—response curves of the two agents alone, ED50 (dose that produced 50% antinociception) values were determined. Subsequently, isobolographic analysis was conducted to determine the nature of the antinociceptive interaction between intrathecal WIN 55,212-2 and bupivacaine (17). This method is based on comparisons of dose ratios that were determined to be equally effective. WIN 55,212-2 and bupivacaine were coadministered in a constant dose ratio based on the ED50 values for each drug (combinations of each 1/2 ED50, 1/8 ED50, 1/32 ED50, or 1/128 ED50 doses). We decided to use these combination doses instead of the fraction usually used in many other isobolographic analyses (combinations of each 1/2 ED50, 1/4 ED50, 1/8 ED50, or 1/16 ED50 doses), because our preliminary study suggested very strong synergistic analgesic interaction (data not shown). The ED50 values of the combination were calculated from the dose—response curves of the combined drugs. The isobolograms were then constructed using the ED50 values obtained when the drugs were administered alone or combined. The ED50 values for each drug alone were plotted on the x and y axes, and the experimental ED50 value and 95% confidence interval (CI95) for the combination was plotted in the dose field. The diagonal line connecting axial points was presented as the theoretical line of additivity. The drug reactions were considered synergistic if the experimental ED50 point for the combination plotted in the dose field was below the theoretical line of additive. The theoretical additive total doses (Zadd), assuming simple additivity and CI95s, were calculated according to Tallarida and Murray (18). Zadd represents a total additive dose of the equipotent combination theoretically providing 50% antinociception. An experimental total dose (Zmix) significantly less than the Zadd indicated that the drugs produced a synergistic effect. To describe the degree of synergistic interaction, the interaction index (γ) was also calculated as follows:
If γ = 1, the interaction is additive; if γ < 1, it is synergistic interaction (the smaller the value, the greater the degree of synergy), and if γ > 1, it is antagonistic interaction.
Data are presented as means ± sd. The time— response data are presented as the mean number of flinches per min for each 5-min period. For the dose— response analysis, the cumulative instances of formalin-evoked flinches from Phase 1 and Phase 2 were calculated separately. To determine the ED50 values of each drug, the number of flinches was converted to a percentage of control according to the formula:
Statistical significance was determined for dose— responses by one-way analysis of variance (ANOVA) followed by the Tukey post hoc test. The log dose— response lines were fitted using least square linear regression and the ED50 values, and CI95 values were determined according to the methods described by Tallarida and Murray (19). The difference between the Zmix and the Zadd was compared using a t-test. A value of P < 0.05 was considered statistically significant.
RESULTS
A total of 234 rats were used. Fifteen rats had to be excluded before intrathecal drug treatment because of neurologic deficits or infection. Thirty-nine rats were excluded because of improper intrathecal catheter placement. Consequently, the results were calculated from a total of 180 rats.
Subcutaneous formalin injection into the plantar surface of the hindpaw produced a typical pattern of flinching behavior. Intrathecal administration of WIN 55,212-2, bupivacaine, or their combination produced dose-dependent decreases of the flinching response in both Phases 1 and 2 (Figs. 1 and 2). The ED50 (CI95) values for WIN 55,212-2 were calculated to be 2.15 μg (1.05—5.15 μg) in Phase 1 and 3.78 μg (1.96—12.55 μg) in Phase 2. The ED50 (CI95) values of bupivacaine in Phases 1 and 2 were 11.97 μg (5.34—25.76 μg) and 9.06 μg (5.75—13.64 μg), respectively.
Isobolographic analysis revealed a significant synergistic interaction between WIN 55,212-2 and bupivacaine in Phase 1 and Phase 2 of the formalin test. The isobolograms indicated that the experimental ED50 points lay below the theoretical line of additivity, and the CI95 values of the theoretical additive points and those of the experimental points did not overlap (Fig. 3). These graphical representations were confirmed statistically by comparison of the Zmix values to the Zadd values. As presented in Table 1, the Zmix values were significantly smaller than the Zadd values (P < 0.05) in both phases of the formalin test. The γ value of Phase 1 (0.126 ± 0.028) and Phase 2 (0.019 ± 0.004) also indicated synergistic antinociceptive interactions.
Mild sedation was observed in the rats that received large doses of WIN 55,212-2. After the intrathecal injection of 10 μg WIN 55,212-2, the range of sedation score at each time point was 1—3. The sum total sedation score for the 6-time points was 8.3 ± 6.4 in rats injected with formalin, and 5.5 ± 5.3 in rats not injected with formalin. Impairment of motor function (evaluated by the placing/stepping reflex and the righting reflex) occurred in rats that received large doses of bupivacaine. After intrathecal administration of 100 μg bupivacaine, the mean motor dysfunction score was 0.7 ± 1.2 in rats injected with formalin, and 0.8 ± 1.3 in rats without formalin injection. Motor disturbance lasted <10 min in all nine of these rats. No observable side effects were induced with the combination of WIN 55,212-2 and bupivacaine in the dose range used in the present study (Table 2).
DISCUSSION
The present study verified that intrathecally administered WIN 55,212-2 and bupivacaine produced dose-dependent decreases in the number of flinches for both Phase 1 and Phase 2 of the formalin test. Subcutaneous injection of diluted formalin into the rat hindpaw produces a typical biphasic nociceptive behavior. The initial acute phase is predominantly caused by the direct stimulation of peripheral nociceptors, while the prolonged tonic phase appears to be related to an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord (20). Electrophysiologic studies indicate that Phase 2 reflects sensitization and a wind-up of dorsal horn neurons (21). Therefore, the results of the current study confirm previous studies (22,23) showing that WIN 55,212-2 or bupivacaine can attenuate spinal sensitization, as well as acute nociceptive activity. The relative effectiveness of the drugs in Phase 1 and Phase 2 of the formalin test in our study was also consistent with previous data (22,23). Of note, the Phase 2 ED50 value was higher than the Phase 1 ED50 for WIN 55,212-2, whereas the ED50 value was higher in Phase 1 than in Phase 2 for bupivacaine.
Although many studies have reported on the analgesic interaction between different classes of analgesics, the antinociceptive interaction between cannabinoid receptor agonists and local anesthetics has not been previously performed. The results obtained in this study indicate that the intrathecal combination of WIN 55,212-2 and bupivacaine produced a synergistic antinociceptive interaction in both phases of the formalin test with decreased side effects. An interesting aspect of the results was the degree of synergism, measured as the interaction index (γ), between these two agents. The γ value was 0.126 ± 0.028 and 0.019 ± 0.004 in Phases 1 and 2, respectively. This finding shows a high degree of synergism between WIN 55,212-2 and bupivacaine, especially in Phase 2. As stated earlier, Phase 2 activity of the formalin test is thought to be the behavioral parallel of the wind-up phenomenon evoked by repetitive stimulation in dorsal horn neurons. Previous electrophysiological studies have shown that WIN 55,212-2 or local anesthetics produce a dose-dependent decrease in the wind-up of spinal wide dynamic range and nociceptive-specific neurons in response to repeated noxious stimulation (24,25). The Phase 2 activity is also mediated in part by the N-methyl-d-aspartate receptor (26), and the results from a previous study (27) suggest that local anesthetics, including bupivacaine, inhibit the N-methyl-d-aspartate receptor by various mechanisms. Although the reason for the greater degree of synergism in Phase 2 than in Phase 1 is unclear, the results of our study suggest that the combination therapy of cannabinoids and local anesthetics might have a role in the management of tonic inflammatory pain.
The mechanisms and sites of the synergistic antinociceptive interaction between WIN 55,212-2 and bupivacaine remain to be elucidated. However, there are several possible explanations for this synergy. First, the pharmacokinetics of each drug may be affected by coadministration. Bupivacaine might decrease spinal blood flow from a vasoconstrictive effect (28), thereby delaying absorption of WIN 55,212-2. Also, synergistic interactions can possibly occur when drugs affect different critical points along a common pathway (29). Local anesthetics provide analgesia by blockade of axonal conduction and synaptic transmission through their extensive effects on neuronal sodium channels, potassium channels, presynaptic calcium channels (30), and presynaptic muscarinic receptors (31). Although the mechanisms for spinal cannabinoid-induced antinociception are as yet unclear, several possible mechanisms have been proposed, including inhibition of neurotransmitter release via presynaptic CB1 receptors. Intrathecally administered cannabinoids appear to predominantly involve a spinal component in their antinociceptive action. Although WIN 55,212-2 is a mixed CB1 and CB2 cannabinoid receptor agonist, one study (22) has shown that spinal CB1 receptor, but not CB2 receptor, is involved in the antinociception of intrathecal WIN 55,212-2. Finally, WIN 55,212-2 produces membrane hyperpolarization through the activation of presynaptic CB1 receptors to inhibit N-type calcium channel activity. Therefore, WIN 55,212-2 might complement the action of bupivacaine on sodium channels, resulting in membrane hyperpolarization, which would inhibit neuronal excitability and integration of a synaptic response.
Unexpectedly, mild sedation was observed in rats that received large doses of WIN 55,212-2. CNS effects of intrathecally administered cannabinoids have not been reported. Although the reason for this sedation observed in our study is unclear, one possibility is that intrathecal WIN 55,212-2 might be absorbed into the blood or spread into the brain, thereby binding to the CB1 receptors within the brain. Therefore, some part of the antinociceptive effects of intrathecally administered WIN 55,212-2 might be mediated via a supraspinal mechanism.
Before considering a future clinical application, the neurotoxicity of the drugs should be determined. Several different laboratory models have proven that all local anesthetics can be neurotoxic when applied to neural tissues in clinically relevant concentrations (32). However, bupivacaine appears to have less potential for neurotoxicity than lidocaine and tetracaine, and is reported not to be neurotoxic when administered at a concentration of <0.75% (33). Although many studies have demonstrated either neuroprotective or neurotoxic effects of cannabinoids in human and animal research, no study has been performed to assess the potential spinal toxicity of intrathecal cannabinoids. This issue should be evaluated further in future studies.
One major limitation of the current study is that we only used the placing/stepping reflex and the righting reflex for the evaluation of motor impairment. Although these reflexes have been widely used in assessment of motor function, these measures represent fairly extreme impairment, and may be unaffected by cannabinoid agonist administration. Therefore, the measure of motor function in our investigation might not be sensitive enough to detect the full range of potential motor impairment. A thorough evaluation of motor function using more sophisticated tests is needed in future studies.
In the current study, we conducted isobolographic analysis by combining each drug at its equipotent ratio (i.e., 1:1 potency ratio), and showed synergistic interaction between WIN 55,212-2 and bupivacaine. However, because synergism is a function of the proportions in the combination, the nature of the pharmacologic interaction can differ if different fixed-dose ratios are tested. Thus, more experiments with different fixed-ratio mixtures are needed.
In conclusion, the current study demonstrated that intrathecally coadministered WIN 55,212-2 and bupivacaine produced synergistic antinociceptive interaction in both phases of the formalin test with decreased side effects. These findings suggest that intrathecal combination of WIN 55,212-2 and bupivacaine might be useful in the management of tissue injury-induced pain.
Source, Graphs and Figures: Antinociceptive Synergy Between the Cannabinoid Receptor Agonist WIN 55,212-2 and Bupivacaine in the Rat Formalin Test
BACKGROUND: The analgesic interaction between cannabinoids and local anesthetics has not been investigated. We sought to determine the nature of the interaction between the intrathecal cannabinoid receptor agonist (WIN 55,212-2) and bupivacaine using the formalin test.
METHODS: Lumbar intrathecal catheters were implanted in male Sprague-Dawley rats. After intrathecal administration of WIN 55,212-2, bupivacaine, or their combination, 50 μL of 5% formalin was injected subcutaneously into the hindpaw. Dose—response curves were established and the respective ED50 (50% effective dose) values were determined for each agent alone. Fixed-ratio combinations of WIN 55,212-2 and bupivacaine were tested for combined antinociceptive effects in the formalin test and an isobolographic analysis was performed to characterize the pharmacologic interaction of both drugs.
RESULTS: Intrathecally administered WIN 55,212-2, bupivacaine, or their combination produced a dose-dependent decrease in the number of flinches during Phase 1 and 2 of the formalin test. Isobolographic analysis revealed a synergistic interaction between intrathecal WIN 55,212-2 and bupivacaine in both phases of the formalin test. In combination, the ED50 value was significantly smaller than the theoretical additive value (P < 0.05).
CONCLUSIONS: These results demonstrate that intrathecally coadministered WIN 55,212-2 and bupivacaine provide synergistic antinociceptive interaction in both phases of the formalin test.
IMPLICATIONS: The pharmacologic nature of the interaction between intrathecal cannabinoid receptor agonist (WIN 55,212-2) and bupivacaine was determined using the formalin test. Intrathecally coadministered WIN 55,212-2 and bupivacaine produced synergistic antinociceptive interaction in both phases of the formalin test, with decreased side effects such as sedation and motor impairment.
Neuraxially administered local anesthetics produce antinociception and motor blockade by directly inhibiting neuronal voltage-gated sodium channels, thereby reducing axonal conduction (1). Although a motor blockade is sometimes helpful during surgical procedures, it is desirable to minimize unwanted motor blockade by local anesthetics in a variety of clinical areas related to pain management. Intrathecal coadministration of local anesthetics and other drugs, such as opioid analgesics or clonidine, produces supra-additive analgesic effects (2,3). These interactions allow a reduction in the dose of each drug, hence a reduction in potential side effects such as intense motor blockade. The aforementioned analgesic combination strategies also have been applied to clinical practice: neuraxially injected mixtures of an opioid or clonidine and a local anesthetic such as bupivacaine, at concentrations that do not produce motor blockade, are shown to offer advantages for the control of surgical, labor, and cancer pain (4—6). In addition, there have been numerous recent studies on other drug combinations producing analgesia in experimental animal models that may be useful in clinical populations.
Cannabinoids have been shown to produce antinociceptive and antihyperalgesic effects in various animal pain models (7,8). The antinociceptive effects of cannabinoids are believed to be exerted primarily via cannabinoid receptors (subtypes cannabinoid receptor Type 1 [CB1] and cannabinoid receptor Type 2 [CB2]) located on neurons within and outside the brain and spinal cord (9). However, unwanted central nervous system (CNS) effects, including hypothermia, hypoactivity, and catalepsy, can also be elicited by cannabinoids through stimulation of CB1 receptors within the brain (10). As these adverse CNS effects can limit the potential clinical application of cannabinoids, strategies to avoid or reduce such side effects have been sought. One of these strategies is to administer cannabinoid agonists by the intrathecal route (11,12), in view of the evidence that CB1 receptors are present in a nociceptive area of the spinal cord (13). Another strategy could be to exploit the synergistic interactions between cannabinoids and other agents for antinociception. In this experiment, we have used potent, synthetic aminoalkylindole cannabinoid WIN 55,212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone mesylate), with the molecular weight of 522.61. WIN 55,212-2 is the nonselective CB1/CB2 cannabinoid receptor agonist, and exhibits relatively high efficacy at both of these receptor types (14).
Despite the efforts to avoid unwanted side effects, no study, to our knowledge, has considered smaller doses through the interaction of cannabinoids with local anesthetics. The purpose of this study was to determine the characteristics of the drug interaction between intrathecal WIN 55,212-2 and a long-acting amino-amide local anesthetic (bupivacaine) using the formalin test, in an animal model of acute tissue injury-induced pain.
METHODS
All experiments were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of Ewha Womans University, Seoul, Korea. Male Sprague-Dawley rats (Orient, Seoul, Korea), weighing 250—300 g, were used in this study. The rats were housed in individual cages with free access to food and water, and maintained on a 12-h light—dark cycle (lights on 6:00 am) at an ambient temperature of 22 ± 1°C.
For drug administration, an intrathecal catheter was first implanted under ketamine—xylazine anesthesia (75 and 15 mg/kg, respectively, IM) by the direct lumbar catheterization method (15). Once anesthetized, a 2—3 cm midline longitudinal incision was made at the level of the iliac crests. This was followed by a catheter (polyethylene-10 tube, Becton Dickinson, Sparks, MD) with a metal wire that was advanced through the hole made by a 23-gauge needle in the space between the lumbar vertebrae L5 and L6. When the sign of intrathecal position (tail-flick or hindpaw retraction) was observed, the guidewire was withdrawn for about 2 cm and the catheter was pushed gently upward to reach L4 at the lumbar enlargement. After proper catheter placement, the guidewire was carefully removed, avoiding displacement of the catheter. The rostral end of the catheter was then heat-welded to another catheter (3 cm long, polyethylene-50 tube, Becton Dickinson, Sparks, MD) for drug administration. The external part of the catheter was tunneled subcutaneously to exit at the occipital region, flushed with saline and sealed by heat melting. The dead space of the catheter was 8.0 ± 1.6 μL. All incisions were sutured and the animals were allowed to recover from surgery for at least 7 days before the experiment. Rats exhibiting neurologic deficits or infection were excluded from the study.
Drugs used in this study included WIN 55,212-2 mesylate and bupivacaine hydrocholoride (Sigma Chemical CO, St. Louis, MO). WIN 55,212-2 was dissolved in 100% dimethylsulfoxide, and bupivacaine was dissolved in physiologic saline (0.9% sodium chloride). All drugs were administered intrathecally with a 10-μL microinjector syringe (Hamilton CO, Reno, NV) in a total volume of 10 μL over 20 s, followed by a 10 μL sterile saline to flush the catheter. Test drugs were given 10 min before the formalin test, as determined in pilot studies. Control rats received the drug solvent vehicle alone. The control study for the combination was done using 50% dimethylsulfoxide in saline.
Antinociception was assessed by the formalin test. Test sessions were conducted between 11:00 am and 5:00 pm. The animals were randomized by the sealed envelope method. In each group, 10 randomly selected rats were used and each animal was used only once. The rats were habituated to the open Plexiglas observation chamber (30 × 30 × 30 cm) for at least 30 min before testing. A mirror was placed under the chamber floor to provide an unobstructed view of the animal. Fifty μmicroliters of 5% formalin was then injected subcutaneously via a 29-gauge needle into the plantar surface of the right hindpaw while the rat was manually restrained. Immediately after receiving the injection, rats were placed back into the chamber and continuously observed by a blinded observer. Pain-related behavior was quantified by counting the spontaneous flinches for 60 min after formalin injection. Flinching was defined as rapid and brief withdrawal or flexing of the injected paw. The score was determined by the sum of incidences of flinching in a 5-min period. Formalin-induced flinching behavior is biphasic. The initial acute phase (Phase 1, during 0—5 min interval after the formalin injection) is followed by a quiescent period, which is then followed by a prolonged tonic phase (Phase 2, beginning approximately 10 min after the formalin injection). At the end of the experiments, rats were euthanized in a CO2 chamber, and the catheter position was verified by injection of indigo blue. Rats with incorrect placement were excluded from the data analysis.
The general behavior of each rat was carefully noted and recorded for 60 min after intrathecal drug administration by a blinded observer. Sedative effects were assessed at 10-min intervals using a 5-point scale proposed by Kawamata et al. (16): 0 = normal behavior, alert to the environment, standing, or grooming; 1 = sitting quietly, sometimes standing or grooming; 2 = sitting quietly, no spontaneous movement, but moved if touched; 3 = no spontaneous movement, did not move when touched; 4 = loss of righting reflex, unresponsive. Scores were totaled over the observation period. Motor function was evaluated at 5, 10, 15, 30, 45, and 60 min after treatment by the placing/stepping reflex and the righting reflex. The former was evoked by drawing the dorsum of either hindpaw across the edge of the table. The test result was considered normal if the animal placed the paw ahead into a position to walk. The righting reflex was assessed by placing the rat horizontally with its back on the table. The test result was considered normal if the animal returned to an upright position within 2 s. Based on the degree of impairment, motor blockade was scored as follows: 0, normal; 1, slight deficit; 2, moderate deficit; and 3, severe deficit. For relatively large doses of WIN 55,212-2 (3 and 10 μg), bupivacaine (30 and 100 μg) and the combination (1/32 ED50 and 1/128 ED50 doses), the behavioral effect of intrathecal drug was also evaluated without formalin injection.
To determine the time course and dose-dependency of the antinociceptive effects in the formalin test, several doses of WIN 55,212-2 (0.3, 1, 3, and 10 μg) or bupivacaine (1, 10, 30, and 100 μg) were tested. Experiments with each agent alone were performed first, so that doses of the combinations could be planned. From the dose—response curves of the two agents alone, ED50 (dose that produced 50% antinociception) values were determined. Subsequently, isobolographic analysis was conducted to determine the nature of the antinociceptive interaction between intrathecal WIN 55,212-2 and bupivacaine (17). This method is based on comparisons of dose ratios that were determined to be equally effective. WIN 55,212-2 and bupivacaine were coadministered in a constant dose ratio based on the ED50 values for each drug (combinations of each 1/2 ED50, 1/8 ED50, 1/32 ED50, or 1/128 ED50 doses). We decided to use these combination doses instead of the fraction usually used in many other isobolographic analyses (combinations of each 1/2 ED50, 1/4 ED50, 1/8 ED50, or 1/16 ED50 doses), because our preliminary study suggested very strong synergistic analgesic interaction (data not shown). The ED50 values of the combination were calculated from the dose—response curves of the combined drugs. The isobolograms were then constructed using the ED50 values obtained when the drugs were administered alone or combined. The ED50 values for each drug alone were plotted on the x and y axes, and the experimental ED50 value and 95% confidence interval (CI95) for the combination was plotted in the dose field. The diagonal line connecting axial points was presented as the theoretical line of additivity. The drug reactions were considered synergistic if the experimental ED50 point for the combination plotted in the dose field was below the theoretical line of additive. The theoretical additive total doses (Zadd), assuming simple additivity and CI95s, were calculated according to Tallarida and Murray (18). Zadd represents a total additive dose of the equipotent combination theoretically providing 50% antinociception. An experimental total dose (Zmix) significantly less than the Zadd indicated that the drugs produced a synergistic effect. To describe the degree of synergistic interaction, the interaction index (γ) was also calculated as follows:
If γ = 1, the interaction is additive; if γ < 1, it is synergistic interaction (the smaller the value, the greater the degree of synergy), and if γ > 1, it is antagonistic interaction.
Data are presented as means ± sd. The time— response data are presented as the mean number of flinches per min for each 5-min period. For the dose— response analysis, the cumulative instances of formalin-evoked flinches from Phase 1 and Phase 2 were calculated separately. To determine the ED50 values of each drug, the number of flinches was converted to a percentage of control according to the formula:
Statistical significance was determined for dose— responses by one-way analysis of variance (ANOVA) followed by the Tukey post hoc test. The log dose— response lines were fitted using least square linear regression and the ED50 values, and CI95 values were determined according to the methods described by Tallarida and Murray (19). The difference between the Zmix and the Zadd was compared using a t-test. A value of P < 0.05 was considered statistically significant.
RESULTS
A total of 234 rats were used. Fifteen rats had to be excluded before intrathecal drug treatment because of neurologic deficits or infection. Thirty-nine rats were excluded because of improper intrathecal catheter placement. Consequently, the results were calculated from a total of 180 rats.
Subcutaneous formalin injection into the plantar surface of the hindpaw produced a typical pattern of flinching behavior. Intrathecal administration of WIN 55,212-2, bupivacaine, or their combination produced dose-dependent decreases of the flinching response in both Phases 1 and 2 (Figs. 1 and 2). The ED50 (CI95) values for WIN 55,212-2 were calculated to be 2.15 μg (1.05—5.15 μg) in Phase 1 and 3.78 μg (1.96—12.55 μg) in Phase 2. The ED50 (CI95) values of bupivacaine in Phases 1 and 2 were 11.97 μg (5.34—25.76 μg) and 9.06 μg (5.75—13.64 μg), respectively.
Isobolographic analysis revealed a significant synergistic interaction between WIN 55,212-2 and bupivacaine in Phase 1 and Phase 2 of the formalin test. The isobolograms indicated that the experimental ED50 points lay below the theoretical line of additivity, and the CI95 values of the theoretical additive points and those of the experimental points did not overlap (Fig. 3). These graphical representations were confirmed statistically by comparison of the Zmix values to the Zadd values. As presented in Table 1, the Zmix values were significantly smaller than the Zadd values (P < 0.05) in both phases of the formalin test. The γ value of Phase 1 (0.126 ± 0.028) and Phase 2 (0.019 ± 0.004) also indicated synergistic antinociceptive interactions.
Mild sedation was observed in the rats that received large doses of WIN 55,212-2. After the intrathecal injection of 10 μg WIN 55,212-2, the range of sedation score at each time point was 1—3. The sum total sedation score for the 6-time points was 8.3 ± 6.4 in rats injected with formalin, and 5.5 ± 5.3 in rats not injected with formalin. Impairment of motor function (evaluated by the placing/stepping reflex and the righting reflex) occurred in rats that received large doses of bupivacaine. After intrathecal administration of 100 μg bupivacaine, the mean motor dysfunction score was 0.7 ± 1.2 in rats injected with formalin, and 0.8 ± 1.3 in rats without formalin injection. Motor disturbance lasted <10 min in all nine of these rats. No observable side effects were induced with the combination of WIN 55,212-2 and bupivacaine in the dose range used in the present study (Table 2).
DISCUSSION
The present study verified that intrathecally administered WIN 55,212-2 and bupivacaine produced dose-dependent decreases in the number of flinches for both Phase 1 and Phase 2 of the formalin test. Subcutaneous injection of diluted formalin into the rat hindpaw produces a typical biphasic nociceptive behavior. The initial acute phase is predominantly caused by the direct stimulation of peripheral nociceptors, while the prolonged tonic phase appears to be related to an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord (20). Electrophysiologic studies indicate that Phase 2 reflects sensitization and a wind-up of dorsal horn neurons (21). Therefore, the results of the current study confirm previous studies (22,23) showing that WIN 55,212-2 or bupivacaine can attenuate spinal sensitization, as well as acute nociceptive activity. The relative effectiveness of the drugs in Phase 1 and Phase 2 of the formalin test in our study was also consistent with previous data (22,23). Of note, the Phase 2 ED50 value was higher than the Phase 1 ED50 for WIN 55,212-2, whereas the ED50 value was higher in Phase 1 than in Phase 2 for bupivacaine.
Although many studies have reported on the analgesic interaction between different classes of analgesics, the antinociceptive interaction between cannabinoid receptor agonists and local anesthetics has not been previously performed. The results obtained in this study indicate that the intrathecal combination of WIN 55,212-2 and bupivacaine produced a synergistic antinociceptive interaction in both phases of the formalin test with decreased side effects. An interesting aspect of the results was the degree of synergism, measured as the interaction index (γ), between these two agents. The γ value was 0.126 ± 0.028 and 0.019 ± 0.004 in Phases 1 and 2, respectively. This finding shows a high degree of synergism between WIN 55,212-2 and bupivacaine, especially in Phase 2. As stated earlier, Phase 2 activity of the formalin test is thought to be the behavioral parallel of the wind-up phenomenon evoked by repetitive stimulation in dorsal horn neurons. Previous electrophysiological studies have shown that WIN 55,212-2 or local anesthetics produce a dose-dependent decrease in the wind-up of spinal wide dynamic range and nociceptive-specific neurons in response to repeated noxious stimulation (24,25). The Phase 2 activity is also mediated in part by the N-methyl-d-aspartate receptor (26), and the results from a previous study (27) suggest that local anesthetics, including bupivacaine, inhibit the N-methyl-d-aspartate receptor by various mechanisms. Although the reason for the greater degree of synergism in Phase 2 than in Phase 1 is unclear, the results of our study suggest that the combination therapy of cannabinoids and local anesthetics might have a role in the management of tonic inflammatory pain.
The mechanisms and sites of the synergistic antinociceptive interaction between WIN 55,212-2 and bupivacaine remain to be elucidated. However, there are several possible explanations for this synergy. First, the pharmacokinetics of each drug may be affected by coadministration. Bupivacaine might decrease spinal blood flow from a vasoconstrictive effect (28), thereby delaying absorption of WIN 55,212-2. Also, synergistic interactions can possibly occur when drugs affect different critical points along a common pathway (29). Local anesthetics provide analgesia by blockade of axonal conduction and synaptic transmission through their extensive effects on neuronal sodium channels, potassium channels, presynaptic calcium channels (30), and presynaptic muscarinic receptors (31). Although the mechanisms for spinal cannabinoid-induced antinociception are as yet unclear, several possible mechanisms have been proposed, including inhibition of neurotransmitter release via presynaptic CB1 receptors. Intrathecally administered cannabinoids appear to predominantly involve a spinal component in their antinociceptive action. Although WIN 55,212-2 is a mixed CB1 and CB2 cannabinoid receptor agonist, one study (22) has shown that spinal CB1 receptor, but not CB2 receptor, is involved in the antinociception of intrathecal WIN 55,212-2. Finally, WIN 55,212-2 produces membrane hyperpolarization through the activation of presynaptic CB1 receptors to inhibit N-type calcium channel activity. Therefore, WIN 55,212-2 might complement the action of bupivacaine on sodium channels, resulting in membrane hyperpolarization, which would inhibit neuronal excitability and integration of a synaptic response.
Unexpectedly, mild sedation was observed in rats that received large doses of WIN 55,212-2. CNS effects of intrathecally administered cannabinoids have not been reported. Although the reason for this sedation observed in our study is unclear, one possibility is that intrathecal WIN 55,212-2 might be absorbed into the blood or spread into the brain, thereby binding to the CB1 receptors within the brain. Therefore, some part of the antinociceptive effects of intrathecally administered WIN 55,212-2 might be mediated via a supraspinal mechanism.
Before considering a future clinical application, the neurotoxicity of the drugs should be determined. Several different laboratory models have proven that all local anesthetics can be neurotoxic when applied to neural tissues in clinically relevant concentrations (32). However, bupivacaine appears to have less potential for neurotoxicity than lidocaine and tetracaine, and is reported not to be neurotoxic when administered at a concentration of <0.75% (33). Although many studies have demonstrated either neuroprotective or neurotoxic effects of cannabinoids in human and animal research, no study has been performed to assess the potential spinal toxicity of intrathecal cannabinoids. This issue should be evaluated further in future studies.
One major limitation of the current study is that we only used the placing/stepping reflex and the righting reflex for the evaluation of motor impairment. Although these reflexes have been widely used in assessment of motor function, these measures represent fairly extreme impairment, and may be unaffected by cannabinoid agonist administration. Therefore, the measure of motor function in our investigation might not be sensitive enough to detect the full range of potential motor impairment. A thorough evaluation of motor function using more sophisticated tests is needed in future studies.
In the current study, we conducted isobolographic analysis by combining each drug at its equipotent ratio (i.e., 1:1 potency ratio), and showed synergistic interaction between WIN 55,212-2 and bupivacaine. However, because synergism is a function of the proportions in the combination, the nature of the pharmacologic interaction can differ if different fixed-dose ratios are tested. Thus, more experiments with different fixed-ratio mixtures are needed.
In conclusion, the current study demonstrated that intrathecally coadministered WIN 55,212-2 and bupivacaine produced synergistic antinociceptive interaction in both phases of the formalin test with decreased side effects. These findings suggest that intrathecal combination of WIN 55,212-2 and bupivacaine might be useful in the management of tissue injury-induced pain.
Source, Graphs and Figures: Antinociceptive Synergy Between the Cannabinoid Receptor Agonist WIN 55,212-2 and Bupivacaine in the Rat Formalin Test
