Pharmacologic Interaction between Cannabinoid and either Clonidine or Neostigmine in the Rat Formalin Test

The studies were reviewed and approved by the Institutional Animal Care Committee, Research Institute of Medical Science, Chonnam National University. Male Sprague-Dawley rats (250–300 g) were used for all experiments. The rats were maintained on a 12-h night/day cycle and allowed free access to food and water at all times. For drug administration, an intrathecal catheter was implanted during enflurane anesthesia, as previously described. 16The catheter was advanced caudally by 8.5 cm through an incision in the atlantooccipital membrane to the lumbar enlargement. The external end of the catheter was tunneled subcutaneously and exited at the top of head and plugged with a piece of steel wire. The skin was closed with 3-0 silk sutures. After surgery, rats were kept in individual cages. Only rats that displayed no postsurgical motor or sensory deficits were used. Animals showing neurologic dysfunction postoperatively were killed immediately. Studies were performed at least 4 or 5 days after intrathecal catheterization.

The following drugs were used in this study: WIN 55,212-2 mesylate (Tocris Cookson Ltd., Bristol, UK), clonidine hydrochloride (Sigma Chemical Co., St., Louis, MO), neostigmine bromide (Research Biochemical Internationals, Natick, MA), AM 251 (Tocris), AM 630 (Tocris), and JWH 133 (Tocris). WIN 55,212-2, AM 251, AM 630 and JWH 133 were dissolved in 100% dimethylsulfoxide, and clonidine and neostigmine were dissolved in normal saline. Intrathecal administration of these agents was performed using a hand-driven, gear-operated syringe pump. All drugs were delivered in a volume of 10 μl solution.

For the formalin test, 50 μl of 5% formalin solution was injected subcutaneously into the plantar surface of the hind paw using a 30-gauge needle. The formalin-injection produces characteristic pain behavior: biphasic flinching or shaking of the injected paw. Such pain behavior was therefore quantified by periodically counting the incident of spontaneous flinching or shaking of the injected paw. The number of flinches was counted for 1-min periods at 1 and 5 min and at 5-min intervals from 10 to 60 min. Two phases of spontaneous flinching were observed after the formalin injection. Phase 1 and 2 were defined as 0–9 and 10–60 min after formalin injection, respectively. After the observation period of 1 h, the animals were immediately killed.

Four to 5 days after surgery, rats were placed in a restraint cylinder for the experiment. After a 15- to 20-min adaptation, rats were then assigned to one of the drug treatment groups. The control study was done using intrathecal saline or dimethylsulfoxide depending on the solvent for experimental drug. Each animal was used in one experiment only. The total number of rats used was 298, and each group comprised 6–9 rats. The investigator was unaware of which drug was administered into each animal.

For evaluation of the dose-response of the antinociceptive action of cannabinoid receptor agonist (WIN 55,212-2 0.3, 1, 3, 10 μg), α2 receptor agonist (clonidine 1, 3, 10, 30 μg), and cholinesterase inhibitor (neostigmine 0.1, 0.3, 1, 3 μg), each of three agents was intrathecally administered. Intrathecal drugs were injected 10 min before formalin injection. Each ED⁵⁰value (effective dose producing a 50% reduction of control formalin response) of three agents was separately determined in two phases.

An isobolographic analysis 17was used to determine the nature of pharmacologic interaction between spinal WIN 55,212-2 and clonidine, and spinal WIN 55,212-2 and neostigmine. This method is based on comparisons of doses that are determined to be equieffective. First, each ED⁵⁰value was determined from the dose-response curves of three agents alone. Next, WIN 55,212-2 and either clonidine or neostigmine were intrathecally coadministered at a dose of the ED⁵⁰values and fractions (1/2, 1/4, 1/8) of ED⁵⁰of each drug. From the dose-response curves of the combined drugs, the ED⁵⁰values of the mixture were calculated and these dose combinations were used for plotting the isobologram. In this experiment, the isobolograms were undertaken to characterize the effect of WIN 55,212-2-clonidine and WIN 55,212-2-neostigmine combinations. The isobologram was constructed by plotting the ED⁵⁰values of the single agents on the x and y axes, respectively. The theoretical additive dose combination was calculated. From the variance of the total dose, individual variances for the agents in the combination were obtained. Furthermore, to describe the magnitude of the interaction, a total fraction value 16was calculated:

The fractional values indicate what portion of the single ED⁵⁰value was accounted for by the corresponding ED⁵⁰value for the combination. Values near 1 indicate additive interaction, values greater than 1 imply an antagonistic interaction, and values less than 1 indicate a synergistic interaction. The mixture was delivered intrathecally 10 min before the formalin test.

To determine whether the effect of intrathecal WIN 55,212-2 was mediated through certain subtypes of cannabinoid receptors, CB1 receptors antagonist (AM 251, 30 μg) and CB2 receptors antagonist (AM 630, 100 μg) were intrathecally administered 10 min before the delivery of WIN 55,212-2 (ED⁵⁰). The maximal doses of cannabinoid receptor antagonists without affecting the control formalin response were determined from the pilot experiments. The formalin test was done 10 min after administration of WIN 55,212-2. These experiments were conducted in phase 1 and 2, respectively. Furthermore, we examined the effect of the CB2 receptor agonist (JWH 133). Because rats showed motor dysfunction at more than 100 μg of JWH 133, we evaluated its effect at 100 μg.

For evaluation of behavioral change of WIN 55,212-2, clonidine, and neostigmine, additional rats were received the highest doses of three agents used in this study, and examined at 5, 10, 20, 30, 40, 50, and 60 min after intrathecal administration. Motor function was assessed by the righting reflex and placing-stepping reflex. The former was evaluated by placing the rat horizontally with its back on the table, which normally gives rise to an immediate coordinated twisting of the body to an upright position. Drawing the dorsum of either hind paw across the edge of the table evoked the latter. Normally, rats try to put the paw ahead into a position to walk. Changes in motor function were scored as follows: 0, normal; 1, slight deficit; 2, moderate deficit; 3, severe deficit. Pinna reflex and corneal reflex were also evaluated and judged as present or absent.

Data are expressed as means ± SD. The time-response data are presented as the number of flinches, whereas the dose-response data are presented as percentage of control in each phase. To calculate the ED⁵⁰values of each drug, the number of flinches was converted to percentage of control as follows:

Dose-response data were analyzed by one-way ANOVA with Scheffé for post hoc . The dose-response lines were fitted using least-squares linear regression and ED⁵⁰and its 95% confidence intervals were calculated according to the method described by Tallarida and Murray. 18The difference between theoretical and experimental ED⁵⁰, the antagonism for the effect of WIN 55,212-2, and the effect of CB2 receptor agonist were analyzed by t  test. P < 0.05 was considered statistically significant.

No change in pinna reflex, corneal reflex, and motor function was seen after intrathecal administration of WIN 55,212-2, clonidine, and neostigmine. The sum of the number of flinches in the saline or dimethylsulfoxide control group was not statistically different from each other in phase 1 (21 ± 4 vs.  19 ± 3) or phase 2 (166 ± 44 vs.  158 ± 38). Figure 1displays the time course of intrathecal WIN 55,212-2, clonidine, and neostigmine, administered 10 min before formalin injection.

Intrathecal WIN 55,212-2, clonidine, and neostigmine produced a dose-dependent suppression of the flinching response during phase 1 and 2 in the formalin test (fig. 2). The ED⁵⁰values (95% confidence intervals) of WIN 55,212-2, clonidine, and neostigmine in phase 1 were 1.4 (0.7–2.8), 10.3 (4.4–23.8), and 0.6 μg (0.4–0.8 μg), respectively. The phase 2 ED⁵⁰values (95% confidence intervals) of WIN 55,212-2, clonidine, and neostigmine were 2.8 (1.3–6.1), 4.7 (3.1–7.2), and 0.3 μg (0.2–0.4 μg), respectively.

Isobolographic analysis revealed a synergistic interaction between intrathecal WIN 55,212-2 and clonidine, as well as intrathecal WIN 55,212-2 and neostigmine during phase 1 and 2 in the formalin test (figs. 3 and 4).

The experimental ED⁵⁰values were significantly lower than the calculated ED⁵⁰values. Accordingly, the phase 1 ED⁵⁰values (95% confidence intervals) of the WIN 55,212-2 in the mixture of WIN 55,212-2-clonidine and WIN 55,212-2-neostigmine were 0.02 (0–52.2), 0.08 μg (0.01–1.0 μg), respectively. The phase 2 ED⁵⁰values (95% confidence intervals) of the WIN 55,212-2 in the mixture of WIN 55,212-2-clonidine and WIN 55,212-2-neostigmine were 0.3 (0.03–1.7), 0.6 μg (0.2–1.8 μg), respectively. The total fraction values of the mixture of WIN 55,212-2-clonidine were 0.03 in phase 1 and 0.19 in phase 2, and those of WIN 55,212-2-neostigmine were 0.12 in phase 1 and 0.41 in phase 2, indicating synergistic interactions in both phases.

Intrathecal AM 251 and AM 630 alone did not affect the control flinching response evoked by formalin injection (fig. 5, A). Intrathecal AM 251 reversed the antinociceptive effect of intrathecal WIN 55,212-2 during phase 1 and 2 in the formalin test (fig. 5, B and C). However, the antinociceptive effect of intrathecal WIN 55,212-2 was not antagonized by intrathecal AM 630 in both phases. Intrathecal JWH 133 did not alter the formalin-evoked flinching response in both phases (fig. 6).

In the current study, intrathecal WIN 55,212-2 attenuated the flinching response during phase 1 and 2 in the formalin test. In the formalin test, phase 1 response seems to result from the immediate and intense increase of primary afferent activity. However, phase 2 response mirrors the activation of wide dynamic range of dorsal horn neurons with very low level of ongoing activity of primary afferent. Therefore, phase 2 reflects a facilitated state that seems to be prominent, considering the decreased level of afferent input. These observations suggest that WIN 55,212-2 may alter the facilitated state and the acute nociception at the spinal level. An interesting aspect of the results was the relative effectiveness of WIN 55,212-2 on phase 1 and 2 of the formalin responses. A twofold-higher ED⁵⁰for phase 2 was observed compared to that for phase 1 with WIN 55,212-2. This effect was further evident in the combination studies when WIN 55,212-2 was combined with clonidine or neostigmine. The phase 2 to phase 1 ratio was 8–15 under the combined administrations. However, for both clonidine and neostigmine, the phase 2 to phase 1 ratio was 0.5. These data suggest that WIN 55,212-2 seems to be much more effective on the acute pain than on the facilitated state. However, previous studies 19,20showed different effects on formalin-induced response, in which injection of a cannabinoid receptor agonist attenuated phase 2 response, but not phase 1 or vice versa . Such discrepancy may be caused by the use of a different drug, the route of drugs given, the kind of animal, the concentration of formalin solution, and the type of measures made.

Cannabinoids are involved in the control of nociception at almost all relays of pain transmission, especially at the spinal level where they act through cannabinoid receptors. 21Cannabinoids activate two receptor subtypes, the CB1 and CB2 receptors. Expression of the CB1 receptor is essentially restricted to neurons and is coupled with G-proteins, 22whereas CB2 receptor expression is essentially restricted to immune cell lines. 23Moreover, CB1 receptor has been identified in the dorsal horn of the spinal cord intimately concerned with the processing of nociceptive information and the modulation. 1,12–15However, no reports have been made to date regarding the expression of the CB2 receptor at the spinal cord. The antinociceptive actions of peripheral CB1 and CB2 receptor agonists have been reported, 19,24which implicates that peripheral CB1 and CB2 receptors participate in the control of nociception. However, the role of each subtype of CB receptors was not examined at the spinal level. In the current experiments, the CB1 antagonist, not the CB2 antagonist, antagonized the antinociceptive effect of WIN 55,212-2, and further administration of intrathecal CB2 agonist did not produce antinociception. These findings suggest that the antinociceptive action of cannabinoids is mediated through just the CB1 receptor at the spinal level.

There are at least three possible mechanisms by which cannabinoid may act spinally to reduce the nociceptive transmission. 1First, it may prevent the afferent barrage by acting presynaptically to inhibit neurotransmitter release. Activation of CB1 receptor leads to reduction of calcium channel currents through N-type 25,26and P/Q-type 27channels. Activation of the cannabinoid receptor has been reported to inhibit adenylyl cyclase activity. 28In addition, CB1 receptor activation has been shown to enhance potassium channel “A” currents 29and activate potassium currents 30through G-protein-coupled inwardly rectifying K⁺channels. Initiating these currents or second messenger systems aids in decreasing the cellular excitability and reducing subsequent neurosecretion. Indeed, activation of the CB1 receptor inhibits the release of transmitter from primary afferent fibers in the dorsal horn of the spinal cord. 1,3Electrophysiologic studies have shown that cannabinoids suppress noxious stimulus-evoked activity of nociceptive neurons in the spinal cord, 31and that activity-dependent facilitation of nociceptive dorsal horn neurons is decreased after the application of cannabinoids to the spinal cord. 32Second, cannabinoids may act postsynaptically to stabilize membrane potentials at subthreshold levels via  the enhancement of K⁺currents and thus prevent the transduction of the nociceptive message. Third, cannabinoids may lead to disinhibition of an inhibitory circuit. Inhibition of terminal A removes the inhibition of terminal B resulting in the release of an antihyperalgesic substance.

Isobolographic analysis of this study revealed the synergistic interaction between intrathecal WIN 55,212-2 and clonidine, and between intrathecal WIN 55,212-2 and neostigmine during phase 1 and 2 in the formalin test. These results indicate that spinal combination of WIN 55,212-2 with clonidine or neostigmine can augment the antinociceptive effect of each drug alone, in both an acute nociceptive state and a tissue-injury state evoked by formalin stimulus. Although a pharmacologic interaction between two kinds of drugs is most likely complicated to characterize, several explanations could be possible for this synergy. First, drugs may interact by altering the kinetics of each other. One agent may alter the actions of the other agent at the receptor or channel. Second, such interaction may occur when both drugs affect different critical points along a common pathway. 33Cannabinoids, clonidine, and neostigmine act on receptors that are G-protein coupled. Hence, the action of these three agents may independently alter intracellular second messenger systems coupled with G-protein activation and mediate a synergistic interaction. 34Third, functional interaction may result from distinct drug effects at separate anatomic sites that may act independently and together to inhibit spinal nociceptive processing. 35As mentioned previously, cannabinoids, clonidine, and neostigmine possesses both presynaptic and postsynaptic actions. Therefore, simultaneous engagement of presynatptic and postsynaptic mechanisms may augment the antinociceptive action produced by either drug acting at one site independently. 36 

Clinically, spinal cannabinoids are not yet available. However, in the future they can be used with clonidine or neostigmine to manage pain because their combination with clonidine or neostigmine may provide a decreased dose of either drug or an increased maximum achievable. Taken together, intrathecal WIN 55,212-2, clonidine, and neostigmine reduce the pain behavior evoked by formalin stimulus, and WIN 55,212-2 interacts with clonidine or neostigmine in a synergistic fashion. Spinal CB1 receptor, but not CB2 receptor, is involved in the antinociception of intrathecal WIN 55,212-2.