The interaction of intrathecal neostigmine and N-cyclohexyladenosine on anti-allodynic effects in rats with a nerve ligation injury

Abstract

Background: Nerve ligation injury in rats produces a pain syndrome that includes mechanical allodynia. Intrathecal administration of cholinesterase inhibitors or adenosine receptor agonists have anti-allodynic effects in this model. Therefore, we tested the interaction between intrathecal neostigmine and N(6)-cyclohexyladenosine (CHA) in a rat behavioral model of neuropathic pain.

Methods: Male Sprague-Dawley rats were prepared with tight ligation of the spinal nerves for producing allodynia and with a lumbar intrathecal catheter for drug administration. Allodynia thresholds for hindpaw withdrawal against mechanical stimuli were assessed and converted to percent maximal possible effect. Neostigmine (0.3-10 microg) and CHA (0.03-3 microg) were administered to obtain the dose-response curves and the 50% effective dose (ED(50)). Equal fractions (1/2, 1/4 and 1/8 ED(50)s) of the two drugs were administered to establish the ED(50) of neostigmine-CHA combination. Side effects were also assessed. The drug interaction was evaluated by isobolographic and fractional analyses.

Results: Neostigmine, CHA, and the neostigmine-CHA combination dose-dependently produced anti-allodynia effects. Side effects such as sedation and motor weakness were similar in the three groups. In the isobolographic analysis, the experimental ED(50) for the combination of neostigmine-CHA lay far below and to the left of the theoretical additive line. Fractional analysis indicated that the total combination fraction of the two drugs was 0.39.

Conclusions: Intrathecal co-administration of neostigmine and CHA showed a synergistic anti-allodynia effect.

Keywords: Allodynia; Drug interaction; Intrathecal injection; N6-cyclohexyladenosine; Neostigmine.

Fig. 1

Dose-response curves from the peak effects of percent maximal possible effect (%MPE) for anti-allodynia in the neostigmine, N⁶-cyclohexyladenosine, and their combination subgroups. These curves show a dose-dependent anti-allodynic effect. Data are expressed as mean ± SEM. Doses (µg) are represented logarithmically on the x axis and peak %MPE of each group is represented on the y axis. CHA: N⁶-cyclohexyladenosine, Neo: neostigmine, Neo-CHA: combination of neostigmine and N⁶-cyclohexyladenosine. ^*P < 0.05 compared with baseline value in each group.

Fig. 2

Isobologram for the interaction between intrathecal neostigmine and N⁶-cyclohexyladenosine. Horizontal and vertical bars indicate SEM. The diagonal line connecting two 50% effective dose (ED₅₀) points is the theoretical additive line. The ED₅₀ point A is calculated from the ED₅₀ values and 95% confidence intervals of each drug. The experimental ED₅₀ point B lies far below the line of additivity, indicating significant synergism. CHA: N⁶-cyclohexyladenosine, Neo: neostigmine. ^*P < 0.05 compared with theoretical ED₅₀ point A.

Fig. 3

Antagonistic study of the combination subgroup by pirenzepine. Pretreatment with the muscarinic M1 receptor antagonist, pirenzepine, decreases the anti-allodynic effect. Data are expressed as mean ± SEM. ED₅₀ = 50% effective dose, Neo-CHA: combination of neostigmine and N⁶-cyclohexyladenosine, Pir: pirenzepine. ^*P < 0.05 compared with baseline value in each group. ^†P < 0.05 compared with pirenzepine pretreatment group. ^‡P < 0.05 compared with control group.