It has been reported that cyclic guanosine monophosphate (cGMP) may play a critical role in the regulation of the nociceptive processing, and a local injection of dibutyryl-cGMP has been demonstrated to produce antinociception in a modification of the Randall-Selitto hyperalgesia . Additionally, it was shown intrathecal 8-bromo-cGMP reduced mechanical allodynia in the neuropathic rats . Guanylyl cyclase catalyzes the formation of cGMP from guanosine triphosphate (GTP), whereas cGMP-specific phosphodiesterase catalyzes the hydrolysis of cGMP to GMP . Therefore, intracellular cGMP concentrations are regulated by the action of guanylyl cyclase and by the rate of degradation by cGMP-specific phosphodiesterase [3,4]. These findings suggest that the activation of guanylyl cyclase or the inhibition of phosphodiesterase may result in an increase of cGMP content, thereby leading to an antinociceptive effect. A local injection of sildenafil, a phosphodiesterase inhibitor, produced antinociception in various animal pain models [5-9], and increased the antinociceptive effect of local morphine [5,8]. However, local zaprinast, another phosphodiesterase inhibitor, had no effect on prostaglandin E2 or carrageenan-induced hyperalgesia [10,11], and the effect of combination of zaprinast with morphine in the spinal cord has not been reported.
The aims of this study was to determine the nature of the drug interaction between zaprinast and morphine in the spinal cord against formalin-induced nociception, which is characterized by two different nociceptive states, acute nociception followed by a facilitated state .
This study was carried out using a protocol approved by the Institutional Animal Care Committee, Research Institute of Medical Science, Chonnam National University.
Adult male Sprague-Dawley rats weighing 250-300 g at the beginning of the experiment were used in this study. The animals were housed in groups of four in an animal facility with a temperature-controlled condition (20-22°C) and were maintained on a 12 h night/day cycle with food and water provided ad libitum.
Rats received an indwelling intrathecal catheter for spinal drug delivery during enflurane anaesthesia, as previously described . A polyethylene-10 catheter was advanced 8.5 cm caudal through an incision in the atlantooccipital membrane into the subarachnoid space extending to the level of the rostral lumbar enlargement. The external end of the catheter was tunnelled 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 and the rats were placed in individual cages for recovery. Only animals with no evidence of neurological deficits after catheter insertion were studied. Behavioural testing occurred 4-5 days after intrathecal catheter implantation.
The drugs used in this study were as follows: zaprinast (Tocris Cookson Ltd., Bristol, UK), morphine sulphate (Sigma, St Louis, USA). Zaprinast and morphine were dissolved in dimethylsulphoxide (DMSO) and normal saline, respectively. These agents were intrathecally administered using a hand-driven, gear-operated syringe pump. All drugs were administered in a volume of 10 μL solution, which was followed by the administration of an additional 10 μL of normal saline to flush the catheter.
Behavioural nociceptive test
The formalin test was used as a nociceptive test . The rats were given a subcutaneous injection of 50 μL of a 5% formalin solution into the plantar surface of the left hind paw with a 30-G needle. Observation started immediately after the formalin injection. After the injection, the rats exhibited a characteristic pain behaviour, which included a rapid, brief flexion of the injected paw. This pain behaviour was defined as flinching and appeared biphasically. Such pain behaviour was quantified by counting the number of flinches of the affected paw periodically during the observation period. 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 after the formalin injection. The formalin-induced pain were divided into phase 1 (0-9 min) and phase 2 (10-60 min). The rats were killed immediately after each experiment. In the formalin test, the phase 1 response seems to result from an immediate and intense increase in the primary afferent activity. In contrast, the phase 2 response reflects the activation of a wide dynamic range of dorsal horn neurons with very low levels of ongoing activity of the primary afferents. The afferent input generated by formalin is believed to release of glutamate, substance P and nitric oxide metabolites, which leads to a state of facilitation that appears to be greater than anticipated, considering the diminished level of afferent input [14,15]. The merit of this formalin pain model is that investigators may sequentially assess the effect of the drug on the two types of pain in the same animal.
On the behavioural test day, the rats were held for 15-20 min for adaptation and were randomly assigned to one of the drug treatment groups. The control study was done using intrathecal saline or DMSO depending on the solvent for experimental drug. In total, 107 rats (5-9 rats per group) were tested in this study. The formalin test was never repeated in the same rat. The researcher responsible for assessing behavioural testing was blind to experimental circumstances of each rat.
Effects of intrathecal zaprinast and morphine
The time course and dose-dependency of intrathecal zaprinast (37, 111, 369 nmol) and morphine (1, 4, 10, 40 nmol) were examined in the formalin test. Intrathecal drugs were given 10 min before the formalin injection. Doses of zaprinast and morphine were chosen based on our preliminary data or previous study . The ED50 values (effective dose producing a 50% decrease in the control formalin response) of zaprinast and morphine were determined in phase 1 and phase 2, respectively.
For determination of the characteristics of interaction between zaprinast and morphine, an isobolographic analysis was used . This method is based on a comparison of the doses that were determined to be equally effective. At first, each ED50 value was determined from the dose-response curves of two agents alone. Then, a dose-response curve is obtained by concurrent delivery of the two drugs in a constant dose ratio based on the ED50 values of the single agent. Thus, separate groups received: zaprinast ED50 + morphine ED50; (zaprinast ED50 + morphine ED50)/2; (zaprinast ED50 + morphine ED50)/4; and (zaprinast ED50 + morphine ED50)/8. 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 agents on the x- and y-axes, respectively. The theoretical additive dose combination was then calculated, and the individual variances for the agents in combination were obtained from the variance of the total dose . Moreover, to describe the magnitude of the interaction, a total fraction value was calculated.
Total fraction value = (ED50 dose of drug 1 in combination)/(ED50 value for drug 1 given alone) + (ED50 dose of drug 2 in combination)/(ED50 value for drug 2 given alone) . Values near 1 indicate an additive interaction, values greater than 1 signify an antagonistic interaction and values less than 1 indicate a synergistic interaction. The mixtures were intrathecally administered 10 min prior to the formalin test.
Motor function was evaluated by the performance of two specific behavioural reflexes, such as the placing-stepping reflex and the righting reflex, in additional rats . The rats were received the highest doses of zaprinast and morphine used here, and examined at 5, 10, 20, 30, 40, 50 and 60 min after intrathecal administration. The placing-stepping reflex was evoked by drawing the dorsum of either hind paw of the rat across the edge of the table. Normally rats attempt to place the paw ahead into a position to walk. The righting reflex was evoked by placing the rat horizontally with its back on the table. Normally rats immediately undergo coordinated twisting of the body until they reach the upright position. To quantify the changes in motor function, both reflexes were scored as follows: 0, normal; 1, slight deficit; 2, moderate deficit; 3, severe deficit.
Data are expressed as the mean ± SD. The time response data are presented as the number of flinching. The dose-response data are presented as percent of control in each phase. To calculate the ED50 values of each drug, the numbers of flinches were converted to percent inhibitory effect according to the following formula.
Percent inhibitory effect = (sum of phase 1(2) count with drug)/(sum of control phase 1(2) count) × 100. Dose-response data were analysed by one-way analysis of variance with Scheffe for post hoc. 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 . The difference between theoretical ED50 and experimental ED50 was examined by t-test. Differences were considered to be statistically significant when P < 0.05.
Motor tone, based on the placing-stepping and righting reflexes, were normal in zaprinast- and morphine-treated rats. Thus all animals scored 0. The vehicle (control) groups exhibited a typical biphasic flinching response of the injected paw after the formalin injection. And the sum of the number of flinches did not differ from each other in both phases (saline: DMSO; 18 ± 4: 18 ± 3 in phase 1, 152 ± 18: 147 ± 19 in phase 2). Figure 1 displays the time course of intrathecal zaprinast and morphine, administered 10 min before the formalin injection.
Intrathecal zaprinast and morphine resulted in a dose-dependent suppression of the flinching response during phase 1 and phase 2 in the formalin test (Fig. 2). The phase 1 ED50 values (95% confidence intervals) of zaprinast and morphine were 161.9 (87.9-298.3) and 11.6 nmol (4.8-27.9 nmol), respectively. The ED50 values (95% confidence intervals) of zaprinast and morphine for phase 2 were 229.9 (142.5-370.9) and 3.9 nmol (1.9-7.6 nmol), respectively.
Isobolographic analysis revealed a synergistic interaction between intrathecal zaprinast and morphine during phase 1 and phase 2 in the formalin test (Fig. 3). The experimental ED50 value was significantly lower than the theoretical ED50 value. Accordingly, the ED50 values (95% confidence intervals) of zaprinast in the mixture of zaprinast and morphine for phase 1 and phase 2 were 14.2 (4.9-40.6) and 10.4 nmol (3-35.9 nmol), respectively. Each total fraction value for the mixture of zaprinast and morphine in phase 1 and phase 2 were 0.16 and 0.09, which indicated a synergistic interaction.
Results of the current study presented here demonstrated that intrathecal zaprinast, decreased the flinching response during phase 1 and phase 2 in the formalin test. These findings suggest that spinal phosphodiesterase may play an important role in the modulation of acute pain and the facilitated state evoked by formalin injection.
Phosphodiesterase enzymes occur widely in biological systems and are present in several tissues . To date, at least nine distinct nucleotide phospodiesterase isoenzymes (1-9) have been identified on the basis of their functional characteristics, such as substrate specificity, cellular distribution and the susceptibility to selective inhibitors . It was reported that phosphodiesterases 5, 6 and 9 are specific for the degradation of cGMP , playing an important role in the modulation of nociception [1,2]. Furthermore, locally administered sildenafil, which is a phosphodiesterase 5 inhibitor, caused antinociception in carrageenan-induced hyperalgesia, the writhing test and the second phase of the formalin test [5-9]. These findings suggest that the inhibition of this enzyme, in turn, may increase the level of cGMP, thereby producing antinociception. However, the intraplantar injection of sildenafil did not affect the phase 1 response in the formalin test [5,7]. Additionally, local zaprinast had no effect on prostaglandin E2 or carrageenan-induced hyperalgesia [10,11]. Moreover, sildenafil did not alter the nociceptive threshold in the tail-flick and hot-plate assays . Such a discrepancy between this study and those reported in the literature may be caused by the different drugs, the route of the drugs given, the kinds of animal, the concentration of formalin solution and the different pain models used. Zaprinast inhibits cGMP-specific phosphodiesterase 5, 6 and 9. Phosphodiesterase 5 appears to be the most relevant enzyme in cGMP in cells [3,4]. Therefore, it can be assumed that the effect of zaprinast in this research was achieved by blocking the phosphodiesterase 5 isoenzyme.
In this study, intrathecal morphine reduced a flinching response in both phases of the formalin test which concurs with previous results . Therefore, opioid receptors are involved in the modulation of acute pain as well as the facilitated state. Previous report also indicated that morphine blocks wind up by acting at opioid receptors located postsynaptically to nociceptive primary afferents .
Isobolographic analysis conducted in this study revealed the synergistic interaction between intrathecal zaprinast and morphine during phase 1 and phase 2 in the formalin test. These results indicate that the combination of zaprinast with morphine is able to augment the antinociceptive effect for each drug alone, in both acute pain and the facilitated state evoked by the formalin injection. The present study demonstrate for the first time that an interaction between zaprinast and morphine at the spinal level. The mechanism of pharmacologic interaction between zaprinast and morphine is clearly of interest but cannot be established from the current results. Previous reports have shown that sildenafil, a phosphodiesterase inhibitor, enhanced the antinociception of morphine at a local level, and this enhancement of antinociception may develop through the inhibition of cGMP degradation [5,8]. It has been shown that the activation of opioid receptor by morphine produces an increase in nitric oxide, which in turn activates soluble guanylyl cyclase, leading an increase of cGMP formation [26,27]. In addition, narcotics increase cGMP accumulation . Hence, concurrent delivery of zaprinast and morphine would further increase the level of cGMP, thereby causing a synergism between zaprinast and morphine in the spinal cord.
Spinal phosphodiesterase inhibitors are not currently available in clinics. However, because a combination may enable to use of a reduced dose of either drug or an increased maximum achievable effect, they may be used in combination with morphine in the treatment of pain in the future. However, further studies for toxicity of intrathecal zaprinast will be needed.
In summary, the inhibition of phosphodiesterase by zaprinast increases cGMP level in the spinal cord, which can alleviate not only acute pain but also the facilitated state induced by injection of formalin. In addition, zaprinast interacts with morphine in a synergistic manner at the spinal level. Accordingly, a combination of zaprinast with morphine may be useful in the management of pain at the spinal level.
This work was supported in part by Research Institute of Medical Science, Chonnam National University.
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