Characteristic of Interactions Between Intrathecal Gabapentin and Either Clonidine or Neostigmine in the Formalin Test

Abstract

Intrathecal gabapentin is effective for phase 2 of the formalin response but not for acute pain. Unlike gabapentin, intrathecal clonidine and neostigmine attenuate both acute pain and phase 2 of the formalin response. We evaluated gabapentin’s interactions with either clonidine or neostigmine in the formalin test. Male Sprague-Dawley rats were used. For the formalin test, 50 μL of 5% formalin solution was injected into the hindpaw. The interaction of drugs was investigated by a fixed-dose analysis or an isobolographic analysis. Intrathecal gabapentin produced a suppression of the phase 2 flinching response, but not the phase 1 response, in the formalin test. Intrathecal clonidine and neostigmine resulted in a reduction of the pain behavior in both phases. A fixed-dose analysis in phase 1 showed that gabapentin potentiated the antinociceptive effect of clonidine and neostigmine. An isobolographic analysis in phase 2 revealed a synergistic interaction after intrathecal administration of gabapentin-clonidine or gabapentin-neostigmine mixture. We conclude that the combination of gabapentin with either clonidine or neostigmine at the level of the spinal cord could play a major role not only in acute pain, but also in phase 2 of the formalin response.

In Brief

IMPLICATIONS: We determined the pharmacological properties of gabapentin combined with either clonidine or neostigmine in the formalin test. Spinal gabapentin reinforced the effects of clonidine and neostigmine in the formalin test. The hitherto unreported action of gabapentin on acute nociceptive stimulus could be of considerable significance.

The formalin test is an experimental pain model that shows the two distinctive nociceptive states. The advantage of this model is that it may provide a tool for observing the effect of analgesics for two types of pain at a time.

Gabapentin is a structural analog to γ-aminobutyric acid (GABA) and originally developed as an anticonvulsant. Previous studies (^1–4) indicate that systemically and intrathecally administered gabapentin attenuates hyperalgesia in models of tissue injury pain. However, gabapentin is without substantive effect in models of acute pain (^1,2,4,5). The dose of intrathecal gabapentin required to reduce nociception is less than that of systemic gabapentin (³), which implies that the spinal cord may be a critical site of action. Although the mechanism of the antinociceptive action of gabapentin is not clear, involvement of GABA, N-methyl-d-aspartic (NMDA), adenosine receptors, and L-arginine nitric oxide pathways (^6–8) or calcium channels (⁹) have been suggested. Meanwhile, intrathecal clonidine and neostigmine reduce both acute pain and tissue injury hyperalgesia (^10,11). The antinociceptive effect of intrathecal clonidine and neostigmine is mediated through spinal α-2 adrenoceptors and muscarinic receptor, respectively (^12,13). The above-mentioned findings suggest that spinal gabapentin, clonidine, and neostigmine may exert comparable actions on phase 2 of the formalin test. However, the interaction between gabapentin and clonidine, or gabapentin and neostigmine, has not been evaluated in this formalin-induced phase 2 response. In addition, the influence of gabapentin on the effect of clonidine and neostigmine in acute pain has not been examined.

Thus, the purpose of the current study was to determine the manner of the drug interactions between intrathecal gabapentin and clonidine or neostigmine in phase 2 of the formalin test and to further clarify the consequence of gabapentin on the effect of clonidine and neostigmine in phase 1 of the formalin test.

Methods

The studies were conducted under a protocol approved by the Institutional Animal Care Committee, Research Institute of Medical Science, and Chonnam National University. Male Sprague-Dawley rats (250–300 g) were used. Rats were housed in group cages on a 12-h night/day cycle with access to food and water at all times. Catheter implantation into the subarachnoid space was performed, as previously described (¹⁴). The catheter (PE-10) was carefully advanced caudally by 8 cm through an incision in the atlantooccipital membrane to the lumbar enlargement. The exterior part of the catheter was tunneled under the skin and externalized on the top of the head and plugged with a stainless steel wire. The skin was closed with 3–0 silk sutures. Rats showing postoperative neurologic deficits were killed. After the catheter implantation, normal rats were kept individually.

The following drugs were used in this study: clonidine hydrochloride (Sigma, St Louis, MO), neostigmine bromide (Sigma), and gabapentin (1-[aminomethyl] cyclohexanacetic acid; Sigma). All drugs were prepared by dissolving them in normal saline. Intrathecal administration of these agents was performed using a hand-driven, gear-operated syringe pump. Drugs were intrathecally delivered in a volume of 10 μL, followed by an additional 10 μL of normal saline to flush the catheter.

The formalin test was used as a model of nociception. The nociceptive stimulus was induced by subcutaneous injection of formalin (5%; 50 μL) solution into the plantar surface of the hindpaw using a 30-gauge needle. Flinching or shaking response of the affected paw was regarded as an indicator of pain behavior. Therefore, the number of flinching responses was recorded for 1-min periods at 1 and 5 min and at 5-min intervals from 10 to 60 min. Because a biphasic flinching response was observed after formalin injection, each response was categorized as phase 1 (0–9 min) and phase 2 (10–60 min). After the observation period of 1 h, animals were immediately killed.

Four to five days after intrathecal catheterization, rats were placed in a restraint cylinder for the experiment. After a 15–20 min acclimation, rats were then assigned to one of the drug treatment groups. Control experiments were performed with saline. Formalin injection was never repeated in the same animal. For evaluation of the time course and dose-response of the antinociceptive action of gabapentin (10, 30, 100, and 300 μg), clonidine (1, 3, 10, and 30 μg), and neostigmine (0.1, 0.3, 1, and 3 μg), all three drugs were intrathecally administered. Intrathecal drugs were injected 10 min before formalin injection. Each ED₅₀ value (effective dose producing a 50% reduction of control formalin response) of the three drugs was separately determined for phase 1 and 2. For evaluation of the type of pharmacologic interactions between gabapentin and either clonidine or neostigmine, a fixed-dose analysis and an isobolographic analysis were used (¹⁵). After intrathecal administration of gabapentin, no antinociceptive effect was seen during phase 1 of the formalin response. Therefore, in phase 1, the fixed dose of gabapentin (300 μg) was coadministered with various doses of clonidine or neostigmine to assess the modulatory effect of gabapentin on the antinociception of clonidine and neostigmine alone. Additionally, to characterize the interaction during phase 2, an isobolographic analysis was used. The method is based on comparisons of doses that are determined to be equieffective. At first, each ED₅₀ value was obtained from the dose-response curves of the drugs alone. Next, gabapentin and either clonidine or neostigmine were simultaneously coadministered at doses of the ED₅₀ values and fractions (1/2, 1/4, and 1/8) of the ED₅₀ of each drug. From the dose-response curves of the combined drugs, the ED₅₀ values of the mixture were calculated, and these dose combinations were used for plotting the isobologram. The isobologram was constructed by plotting the ED₅₀ values of the single drugs 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 drugs in the combination were obtained. Furthermore, to describe the magnitude of the interaction, a total fraction value was calculated according to the formula:

The fractional values indicate what portion of the single ED₅₀ value was accounted for by the corresponding ED₅₀ value for the combination. Values near 1 indicate additive interaction, values more than 1 imply an antagonistic interaction, and values <1 indicate a synergistic interaction. The mixture was delivered intrathecally 10 min before the formalin test.

For examination of motor impairment by gabapentin, clonidine, and neostigmine, the largest dose of each drug was given intrathecally to the additional rats (n = 15). Motor function was assessed by the placing-stepping reflex and the righting reflex. The first was evoked by drawing the dorsum of either hindpaw across the edge of the table. Normal rats try to put the paw ahead into a position to walk. The other was evaluated by placing the rat horizontally with its back on the table. Normal rats immediately and coordinate twisting of the body to an upright position.

Data are expressed as the mean ± sem. The time response data are presented as the number of flinching responses. The dose-response data are presented as the sum of the number of flinches. To calculate the ED₅₀ values of each drug, the number of flinches was converted to a percentage of control according to the formula:

Dose-response data were analyzed by one-way analysis of variance with Scheffe post hoc analysis. The dose-response lines were fitted using least-squares linear regression and ED₅₀, and its 95% confidence intervals were calculated according to the method described by Tallarida and Murray (¹⁶).

The difference between theoretical ED₅₀ and experimental ED₅₀ was examined by t-test. P < 0.05 was considered statistically significant.

Results

Intrathecal gabapentin, clonidine, and neostigmine were not associated with changes of motor function. Subcutaneous injection of formalin into the hindpaw resulted in a biphasic flinching response of the injected paw. The time course effect of intrathecal gabapentin, clonidine, and neostigmine, administered 10 min before formalin injection, is shown in Figure 1. Intrathecal clonidine and neostigmine produced a suppression of the flinching during phase 1, but gabapentin did not alter the flinching response. During phase 2, all three drugs produced a dose-dependent suppression of flinching response (Fig. 2).

Figure 1.: Time course curves of intrathecal gabapentin (A), clonidine (B), and neostigmine (C) for flinching in the formalin test. Drugs were administered 10 min before formalin injection. Data are presented as the number of flinches. Each point on the graph represents the mean ± sem of six to seven rats.

Figure 2.: Dose-response curve of gabapentin, clonidine, and neostigmine for flinching during phase 1 (A) and phase 2 (B) in the formalin test. Dara are presented as the sum of the number of flinches. Gabapentin dose-dependently decreased flinching during phase 2, but not phase 1. Both clonidine and neostigmine produced a dose-dependent inhibition of flinching in both phases. Each point on the graph represents the mean ± sem of six to seven rats. Compared with control: *P < 0.01 and †P < 0.001.

The gabapentin-clonidine and gabapentin-neostigmine combinations did not show motor dysfunction. Intrathecal coadministration of gabapentin (300 μg) with clonidine or neostigmine in phase 1 augmented the antinociceptive effects of clonidine and neostigmine alone (Fig. 3).

Figure 3.: Effect of gabapentin (300 μg) given intrathecally with various doses of clonidine (A) or neostigmine (B) during phase 1 in the formalin test. Data are presented as the sum of the number of flinches. The addition of gabapentin increased the antinociceptive effect of clonidine and neostigmine alone. Each point on the graph represents the mean ± sem of six to seven rats. Compared with clonidine or neostigmine: *P < 0.05.

Isobolographic analysis revealed a synergistic interaction between intrathecal gabapentin and clonidine, as well as intrathecal gabapentin and neostigmine, during phase 2 in the formalin test. The experimental ED₅₀ values were significantly smaller than the calculated ED₅₀ values (Fig. 4) with a total fraction value of < 1, indicating a synergistic interaction (Table 1).

Table 1: ED₅₀ Values (μg) with 95% Confidence Intervals and TFV of Intrathecal Drugs

Figure 4.: Isobologram for the interaction between gabapentin and either clonidine (A) or neostigmine (B) during phase 2 in the formalin test. The ED₅₀ values for each drug are plotted on the x and y axes, respectively, and thick lines represent the sem of the ED₅₀. The straight line connecting each ED₅₀ value is the theoretical additive line, and the point on this line is the theoretical additive ED₅₀. The experimental ED₅₀ point was significantly different from the theoretical ED₅₀ point, indicating a synergism.

Discussion

In the current study, intrathecal gabapentin had no effect on phase 1 pain behavior. In contrast, during phase 2 intrathecal gabapentin attenuated the flinching response. These observations are consistent with previous data (^2,4).

Subcutaneous injection of formalin produces a biphasic behavioral reaction. This behavior consists of an initial phase and a second phase. Phase 1 results essentially from the direct stimulation of nociceptors, whereas phase 2 involves a period of sensitization during which inflammatory phenomena occur. Phase 2 has been attributed to central or peripheral mechanisms (^17,18).

The mechanisms for the antinociception of gabapentin have not been established. However, several hypotheses have been proposed. It has been reported that gabapentin increases the concentration, the rate of synthesis, and the release of GABA (⁶). But intrathecal injection of either GABA_A or GABA_B receptor antagonists did not reverse the antinociceptive effect of gabapentin (⁷). Thus, gabapentin may not act directly on the GABA receptor at the spinal cord. Although there is no direct evidence of gabapentin’s binding to the spinal NMDA receptor, intrathecal D-serine, an agonist at the nonstrychnine site of NMDA receptor, reversed the antinociceptive effect of gabapentin (⁷). These findings suggest that the action of gabapentin is mediated through the NMDA receptor. It has been suggested that gabapentin has subtle actions on calcium channels because gabapentin binds with high affinity to the α2δ subunit of voltage-sensitive calcium channels (⁹). In addition, a calcium channel blocker potentiates the antinociception of gabapentin (¹⁹). However, a calcium reuptake inhibitor had little influence on the effect of gabapentin (⁷). Other receptors, such as muscarinic, adenosine and opioid receptors, also seem to be involved in the antinociception of gabapentin (⁷). Moreover, the antinociceptive effect of gabapentin is more powerful after intrathecal rather than systemic administration (³), emphasizing that the spinal cord may be a major site of action.

In the current study, intrathecal clonidine and neostigmine attenuated both phases of the flinching response. Previous studies (^12,20) have demonstrated that there is a high density of α-2 adrenoceptor and cholinergic receptors in lamina I and II of the dorsal horn, areas important in nociceptive transmission. Furthermore, intrathecal clonidine and neostigmine increase the cerebrospinal fluid concentration of nor-epinephrine and acetylcholine, respectively (^21,22). The above observations suggest that intrathecal clonidine and neostigmine increase the level of spinal norepinephrine or acetylcholine, thereby producing an antinociceptive effect, which is mediated by the spinal α-2 adrenoceptor or cholinergic receptor. Additionally, clonidine is also a potent α-2 adrenoceptor agonist that undoubtedly contributes to an analgesic action (¹⁰)

Although intrathecal clonidine and neostigmine were effective for phase 1 of the formalin test in the present study, intrathecal gabapentin had no effect on the same state. However, gabapentin, clonidine, and neostigmine exhibited parallel profiles of spinal antinociception in phase 2 of the formalin test. Therefore, we sought to determine the nature of drug interaction using two different methods according to each phase of the formalin test. In phase 1, a fixed-dose analysis was used. Because the maximal dose (300 μg) of intrathecal gabapentin used in this experiment did not affect the phase 1 flinching response of the formalin test, the fixed dose of gabapentin (300 μg) was added to various doses of clonidine and neostigmine alone. In this manner, we examined whether intrathecal gabapentin could increase the effects of intrathecal clonidine and neostigmine. In phase 2, an isobolographic analysis was used. From our experiments, the addition of intrathecal gabapentin increased the antinociceptive effects of intrathecal clonidine and neostigmine alone for the phase 1 response, and concurrent delivery of intrathecal gabapentin-clonidine or gabapentin-neostigmine produced a synergism in the phase 2 response. These results indicate that spinal gabapentin potentiates the antinociceptive effects of clonidine and neostigmine in acute nociception. In addition, the spinal combination of gabapentin with either clonidine or neostigmine reinforces the effects of clonidine and neostigmine for the phase 2 response evoked by formalin stimulus. This synergy may result from a different drug interaction that acts independently to block nociceptive processing. Intrathecal or systemic gabapentin interacts synergistically with other analgesics, such as clonidine, naproxen, and morphine in various nociceptive models (^23–25). The advantage of this synergy is that we can obtain the same effect with a smaller dosage of either drug or an increased maximum achievable effect with a decreased incidence of side effects.

Gabapentin for spinal administration is not available in clinics. However, in the future, spinal administration of gabapentin alone or in combination with either clonidine or neostigmine may be useful in the treatment of tissue injury pain.

In conclusion, intrathecal gabapentin increases the effectiveness of clonidine and neostigmine in phase 1 of the formalin test and interacts in a synergistic fashion with both clonidine and neostigmine in phase 2 of the formalin test.