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© 1999 International Anesthesia Research Society
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Abstract |
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A new competitive
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, (2,3-dioxo-7-[1H-imidazol-1-yl]-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl) acetic acid (YM872) has analgesic effects on acute thermal- and formalin-induced nociception by intrathecal administration. The purpose of this study was to determine the analgesic effects of systemically administered YM872 in both acute thermal- and irritant-induced pain. Sprague-Dawley rats were tested for tail withdrawal response by the tail flick test and for paw flinches by formalin injection after intraperitoneal administration of YM872. The tail flick latency increased dose-dependently with a 50% effective dose value of 156.3 µg. The number of flinches in both first and second phases of the formalin test decreased with increasing the dose of YM872. The 50% effective dose values were 1.0 µg in the first phase and 38.7 µg in the second phase. Transiently, intraperitoneal administration of 1 and 10 mg of YM872 induced motor disturbance and 10 mg induced loss of pinna reflex. We conclude that intraperitoneal administration of YM872 had analgesic effects on both acute thermal- and formalin-induced nociceptions in rats. Transient motor disturbance and loss of pinna reflex occurred only with large doses.
Implications: Intraperitoneally administered YM872, a new -amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, had analgesic effects on thermal- and formalin-induced pain in rats. Larger doses induced transient motor disturbance and loss of pinna reflex mediated in the brain.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are involved in nociceptive mechanisms in the spinal cord (1). Intrathecal injection of AMPA receptor antagonists have dose-dependent antinociceptive effects on thermally induced acute pain in the rat model (2,3). However, the effects of AMPA receptor antagonists are controversial on facilitated states of pain processing, characteristic of the "formalin test." It has been reported that the AMPA receptor antagonist inhibits the acute first phase (4), but not the tonic second phase (5). Conversely, Simmons et al. (6) reported that the AMPA receptor antagonist reduced the second phase but not the first phase. Our previous study has shown that both the first and the second phases were suppressed by the intrathecal administration of AMPA receptor antagonist, (2,3-dioxo-7-[1H-imidazol-1-yl]-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl) acetic acid (YM872) (3). These differences might be attributable to the differences of the morphology or affinity of the receptor subtypes. YM872 may be more useful than other antagonists considering the effects on the first and the second phases in the formalin test. Although the AMPA receptor antagonists have analgesic effects, they are not available clinically because of their side effects. 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline, which is often used experimentally, exhibits nephrotoxicity (7) and is not water soluble. Recently, a new AMPA/kainate receptor antagonist, LY293558 ([3s, 4aR, 6R, 8aR]-6-{2-[1(2)H-tetrazole-5-yl] ethyl} decahydroisoquinolone-3-carboxylic acid monohydrate), has been developed as a water soluble compound (8). Systemically administered LY293558 induces sedation and ataxia in rabbits (8) and blurred vision and sedation in humans (9). Intrathecally administered YM872 (2,3-dioxo-7-[1H-imidazol-1-yl]-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl) acetic acid; molecular weight, 349.26 (Yamanouchi Pharmaceutical Co., Ltd., Tsukuba, Japan) induced transient motor disturbance and flaccidity in our previous study (3).
The therapeutic ratio, the ratio between the 50% effective dose (ED50) and the minimum dose that induced side effects, by intrathecally administered YM872 was 10 in thermal nociception and approximately 50 in formalin-induced nociception (3). Currently no data exists concerning the effects of YM872 after systemic administration. Therefore, we studied the analgesic effects of systemically administered YM872 (intraperitoneally) on acute thermal and formalin-induced facilitated states of pain processing.
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Methods |
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The study protocol was approved by the Research and Education Institute of Harbor–University of California, Los Angeles Medical Center. Male Sprague-Dawley rats (300 to 350 g) were injected intraperitoneally with the agent in a total volume of 0.3 mL using a 1-mL syringe through a 30-gauge needle.
YM872 10 mg was dissolved in 0.29 mL of distilled water and 0.01 mL of 1N NaOH to adjust the pH to 7.3 to 7.5. Solutions of 0.01 (0.029), 0.1 (0.29), 1 (2.9), or 10 (29) mg (µmol) per 0.3 mL were made using normal saline, and injected intraperitoneally. In each dose group, eight randomly selected rats were used. Normal saline 0.3 mL was injected in the control group.
Tail Flick Test
The rats were placed in a clear plastic cylindrical tube with their tails extending through a slot provided in the rear of the device. Noxious stimulation was provided by a beam of high intensity light (Tail-Flick Analgesia Meter 0570–001L; Columbus Instruments International Co., Ltd., Columbus, OH) focused on the tail 2 to 3 cm proximal to the end. The response time was measured and defined as the interval between the onset of the thermal stimulation and the tail flick. The cut-off time in the absence of a response was set to 14 s, to prevent tissue injury. The response time was measured before, and 5, 10, 15, 30, 60, 90, 120, and 240 min after drug injection. YM872 0.01, 0.1, 1, and 10 mg were tested.
Formalin Test
Ten minutes after the intraperitoneal administration of YM872, the rats were anesthetized with 3% halothane until transient loss of spontaneous movement was observed, then quickly removed from the anesthesia box. Fifty microliters of 5% formalin was injected subcutaneously into the dorsal surface of the right hindpaw with a 30-gauge needle. Immediately after injection, the rats were placed in an open plexiglas chamber and observed for 60 min. Quantification of pain behavior was made by counting the incidence of spontaneous flinches/shaking of the injected paw at intervals of 1 to 2 min, 5 to 6 min, and 5 min during a period of 60 min after formalin injection. The animals were then killed with an overdose of halothane. As described previously (10), two distinct phases were observed: phase 1 (during a 0- to 6-min interval after injection) and phase 2 (beginning approximately 10 min after injection). YM872 0.01, 0.1, and 1 mg were tested.
Behavioral and Motor Function Test
The general behavior (including agitation and allodynia), motor function, pinna reflex, and corneal reflex were examined; their presence or absence was recorded. Agitation was judged to be present when the rat spontaneously vocalized or became restless. The presence of allodynia was examined by looking for agitation (escape or vocalization) evoked by lightly stroking the flank with a pencil. Motor function was evaluated by the "placing"/"stepping" reflex and by the "righting" reflex. The former was evoked by drawing the dorsum of either hindpaw across the edge of the table. The latter was applied by placing the rat horizontally with its back on the table, which normally gives rise to an immediate, coordinated turning of the body back to an upright position. YM872 0.01, 0.1, 1, and 10 mg were tested.
Data Analysis and Statistics
The response latency data from the tail flick measurements were converted to percent maximum possible effect (%MPE) according to the formula: %MPE = [(postdrug latency – baseline latency)/(cutoff time – baseline latency)] x 100. ED50 was calculated by a computer program as the dose that produced a value of 50% MPE.
Differences among doses were analyzed with two-way analysis of variance followed by Student-Newman-Keuls test. The number of rats with side effects was compared by using the 
2 test. A P value <0.05 was considered statistically significant.
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Results |
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Tail Flick Test
The baseline latency (before drug injection) in the tail flick test was 3.0 ± 0.2 s (mean ± SE). Intraperitoneal administration of YM872 resulted in dose-dependent increases in the tail flick response latency (Fig. 1). Peak effects were obtained in 10 to 30 min. ED50 value was 156.3 µg (95% confidence interval [CI]: 87.5–279.2 µg).
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= YM872, 0.01 mg/rat; 
= YM872, 0.1 mg/rat; 
= YM872, 1 mg/rat; 
= YM872, 10 mg/rat. Each point presents the mean ± SEM of eight rats. *P < 0.05 versus control (time 0); **P < 0.01 versus control (time 0); +P < 0.05 versus saline; ++P < 0.01 versus saline.Formalin Test
The number of flinches in both phase 1 (0 to 6 min) and phase 2 (10 to 60 min) in the formalin test decreased as the dose of intraperitoneal YM872 increased (Fig. 2). ED50 values were 1.0 µg (95% CI: 0.02–44.5 µg) in phase 1 and 38.7 µg (95% CI: 1.9–768 µg) in phase 2.
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Behavioral and Motor Function Test
Intraperitoneal administration of 0.01, 1, and 10 mg of YM872 induced agitation or allodynia in one of eight rats for each dose tested. A dose of 1 mg or more of YM872 induced a motor disturbance. Loss of pinna reflex was observed in one rat with a dose of 10 mg of YM872. All side effects resolved within 90 min (Fig. 3).
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] = YM872, 0.01 mg/rat; 
= YM872, 10 mg/rat. Each bar presents the number of rats that showed each side effect. The total number of rats tested in each dose was eight. |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In the present study, systemic (intraperitoneal) administration of YM872, a competitive AMPA receptor antagonist, showed dose-dependent analgesic effects on thermal and formalin-induced pain in rats. The ED50 values were 156 micrograms for thermal stimulation and one microgram in the first phase and 39 micrograms in the second phase of the formalin test. Transient motor disturbance also occurred with a dose of more than or equal to one milligram of YM872.
AMPA receptors mediate acute excitation of A and C fibers to dorsal horn neurons, whereas the windup of dorsal horn neurons after tissue damage is considered to be mediated by N-methyl-D-aspartate receptors (11,12). AMPA receptor antagonists are reported to have antinociceptive effects on acute thermal stimulation (2,13) and on the first phase of the formalin test (4). However, Simmons et al. (6) reported that AMPA receptor antagonist (LY293558) was effective only on the second phase of the formalin test. Sang et al. (9) applied the same AMPA antagonist (LY293558) as Simmons et al. (6) to humans. In that study, IV administration of LY293558 could reduce capsaicin-evoked sensitization but not acute pain (6). Our results showed analgesic effects of a new AMPA receptor antagonist (YM872) on both the first and the second phases of the formalin test by systemic administration. This result agrees with the previous finding that intrathecal administration of YM872 also had the effects on both phases of the formalin test (3). In a study by Hunter and Singh (4), an analgesic effect of an AMPA receptor antagonist was seen during the second phase of the formalin test but only with a large dose, which induced motor disturbance. The discrepancies among the studies may be attributed to different affinities of the compounds to kainate receptors, AMPA receptors, or their subtypes. This likely is attributed to their diverse chemical structures or to small therapeutic ratios of some compounds which prevent testing with larger doses. Because YM872 was effective by systemic administration, it might have a systemic, i.e., peripheral site of action. Further study is necessary to confirm the site of action of YM872.
In the present study, the ED50 values of the second phase of the formalin test were four to five times lower than those in the tail flick test. This is in agreement with intrathecal administration of YM872 (3). These results suggest that YM872 is more effective on the facilitated states of pain processing than on acute pain. Onset time of analgesia for thermally induced pain in our study was found to be the same as intrathecal administration. The analgesic effect lasted longer after intrathecal (3) than intraperitoneal administration.
Regarding side effects, one rat out of eight in each dose (0.01, 1, and 10 mg) of YM872 showed agitation or allodynia at 30 minutes after systemic administration. The number of rats with agitation or allodynia did not increase together with the increased dose of YM872. Therefore, we could not confirm that agitation and allodynia were the effects of YM872. The differences in side effects between the intrathecal and intraperitoneal routes were loss of pinna reflex and flaccidity. The former was observed only by the intraperitoneal route whereas the latter was seen only by the intrathecal route. Loss of pinna reflex is an effect of YM872 on the brain. Lack of loss of pinna reflex by intrathecal administration indicates that spinally administered YM872 did not cause brain concentrations in sufficient quantities to inhibit the pinna reflex. In contrast, flaccidity might be spinal cord mediated because only intrathecally administered YM872 induced it. Motor disturbances, judged by the disturbances of placing/stepping or righting reflex, which might be due to spinal action, recovered faster after systemic than after intrathecal administration, as did analgesic effects (3). Further pharmacokinetic studies are necessary to validate these suppositions on the mechanisms of action of YM872.
The therapeutic ratio determined by motor disturbance and the tail flick test (calculated as ED50 dose/minimum dose that induces side effects) was 0.1 by intrathecal administration (3) and 0.16 by systemic administration. The therapeutic ratio in the formalin test was 0.024 (first phase) and 0.021 (second phase) by intrathecal administration (3) and 0.001 (first phase) and 0.039 (second phase) by systemic administration. Except for the first phase of the formalin test, ED50 values are compatible between intrathecal and intraperitoneal administration. These results suggest that the systemic route is more effective than the intrathecal route in preventing acute pain induced by chemical stimulation. In contrast, both routes are equally effective on thermally induced acute pain and on facilitated pain processing.
In conclusion, the systemic (intraperitoneal) administration of a competitive AMPA receptor antagonist, YM872, was more effective on chemically induced acute nociception than the intrathecal administration. Thus, systemic administration of an AMPA receptor antagonist may be the route of choice for acute pain management.
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Footnotes |
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This work was conducted at the Department of Anesthesiology, Harbor–University of California, Los Angeles Medical Center.
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References |
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- Yoshimura M, Nishi S. Excitatory amino acid receptors involved in primary afferent-evoked polysynaptic EPSPs of substantia gelatinosa neurons in the adult rat spinal cord. Neurosci Lett 1992;143:131–4.[Web of Science][Medline]
- Nishiyama T, Yaksh TL, Weber E. Effects of intrathecal NMDA and non-NMDA antagonists on acute thermal nociception and their interaction with morphine. Anesthesiology 1998;89:715–22.[Web of Science][Medline]
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Nishiyama T, Gyermek L, Lee C, et al. The spinal antinociceptive effects of a novel competitive AMPA receptor antagonist, YM872, on thermal or formalin-induced pain in rats. Anesth Analg 1999;89:143–7.
[Abstract/Free Full Text] - Hunter JC, Singh L. Role of excitatory amino acid receptors in the mediation of the nociceptive response to formalin in the rat. Neurosci Lett 1994;174:217–21.[Web of Science][Medline]
- Coderre TJ, Melzack R. The contribution of excitatory amino acids to central sensitization and persistent nociception after formalin-induced tissue injury. Neurosci 1992;12:3665–70.[Abstract]
- Simmons RM, Li DL, Hoo KH, et al. Kainate GluR5 receptor subtype mediates the nociceptive response to formalin. Neuropharmacology 1998;37:25–36.[Web of Science][Medline]
- Xue D, Huang Z-G, Barnes K, et al. Delayed treatment with AMPA, but not NMDA, antagonists reduces neocortical infarction. Cereb Blood Flow Metab 1994;14:251–61.[Web of Science][Medline]
- Bowes MP, Swanson S, Zivin JA. The AMPA antagonist LY293558 improves functional neurological outcome following reversible spinal cord ischemia in rabbits. Metab 1996;16:967–72.
- Sang CN, Hostetter MP, Gracely RH, et al. AMPA/kainate antagonist LY293558 reduces capsaicin-evoked hyperalgesia but not pain in normal skin in humans. Anesthesiology 1998;89:1060–7.[Web of Science][Medline]
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- Dickenson AH, Sullivan AF. Differential effects of excitatory amino acid antagonists on dorsal horn nociceptive neurones in the rat. Brain Res 1990;506:31–9.[Web of Science][Medline]
- Advokat C, Rutherford D. Selective antinociceptive effect of excitatory amino acid antagonists in intact and acute spinal rats. Pharmacol Biochem Behav 1995;51:855–60.[Web of Science][Medline]
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