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Anesth Analg 1999;89:143
© 1999 International Anesthesia Research Society

REGIONAL ANESTHESIA AND PAIN MANAGEMENT

The Spinal Antinociceptive Effects of a Novel Competitive AMPA Receptor Antagonist, YM872, on Thermal or Formalin-Induced Pain in Rats

Tomoki Nishiyama, MD, PhD*,{dagger}, Laszlo Gyermek, MD, PhD*, Chingmuh Lee, MD*, Sachiko Kawasaki-Yatsugi, BS{ddagger}, and Tokio Yamaguchi, PhD{ddagger}

*Department of Anesthesiology, Harbor-University of California, Los Angeles Medical Center, Torrance, California; {dagger}Department of Anesthesiology, The University of Tokyo, Tokyo; and {ddagger}Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., Tsukuba, Japan

Address correspondence and reprint requests to Tomoki Nishiyama, MD, PhD, Department of Anesthesiology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan.


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
{alpha}-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonists have spinally mediated analgesic effects on acute nociception; however, their current formulations are not water-soluble and have toxic side effects. A new competitive AMPA antagonist, YM872 (2,3-dioxo-7-[1H-imidazol-1-yl]-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl acetic acid) is water-soluble and may have fewer side effects. The purpose of this study was to investigate the analgesic effects of YM872 on both acute thermal and irritant-induced pain. Sprague-Dawley rats were implanted with chronic lumbar intrathecal catheters and were tested for their tail withdrawal response by the tail flick test and for their paw flinches by formalin injection after the intrathecal administration of YM872. The tail flick latency increased dose-dependently with a 50% effective dose (ED50) value of 1.0 µg. The number of flinches in both Phase 1 and Phase 2 of the formalin test decreased with increasing dose of YM872. ED50 values were 0.24 µg in Phase 1 and 0.21 µg in Phase 2. YM872 10 and 30 µg induced motor disturbance and flaccidity. In rats, the intrathecal administration of YM872 had analgesic effects on both acute thermal and formalin-induced nociceptions. Transient motor disturbance and flaccidity occurred only with large doses. YM872 may have potential in the clinical management of both acute and chronic pain.

Implications: A novel {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, YM872, may have an analgesic effect on both acute and chronic pain when administered intrathecally.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Excessive glutamate receptor activation plays a major role in spinally mediated nociception. The glutamate receptors involved in nociception are mainly the N-methyl-D-aspartate (NMDA) receptors and the {alpha}-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. AMPA receptors mediate fast excitatory transmission involving both innocuous and acute nociceptive input, whereas NMDA receptors are implicated specifically in nociceptive reactions, particularly those induced by intense, prolonged stimulation sufficient to produce the hyperalgesic state underlying neuropathic pain (1–3). Thus, NMDA receptor antagonists are effective in neuropathic pain, whereas AMPA receptor antagonists have an analgesic effect on acute pain in animal models (4–6).

NMDA receptor antagonists may alleviate pain and provide therapy for some neurological disorders (7). Serious side effects of these compounds and their limited effectiveness have, however, directed interest toward drugs acting via non-NMDA receptors, especially AMPA receptors (8,9). Current formulations of AMPA receptor antagonists also have toxic side effects that prevent their clinical application. For example, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline (NBQX) is associated with nephrotoxicity (10) and is not water-soluble.

A new compound, 2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl acetic acid (YM872; molecular weight 349.26; Yamanouchi Pharmaceutical Co. Ltd., Tsukuba, Japan) is a potent and water-soluble competitive AMPA receptor antagonist (11) originally developed as a neuroprotective drug for brain ischemia (12).

For the above reasons, we explored the analgesic effects of YM872 on the two different types of nociception—acute thermal stimulation and formalin-induced facilitated states of pain processing—using rats with chronic intrathecal catheters.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The protocol was approved by the Research and Education Institute of Harbor-University of California, Los Angeles Medical Center. Sprague-Dawley rats were implanted with chronic lumbar intrathecal catheters under halothane (2%) anesthesia according to the method described by Yaksh and Rudy (13). Briefly, an 8.5-cm polyethylene (PE-10; Clay Adams, Parsippany, NJ) catheter was advanced caudally through an incision in the atlantooccipital membrane to the thoracolumbar level of the spinal cord. The external part of the catheter was tunneled subcutaneously to exit on the top of the skull and was plugged with a 28-gauge stainless steel wire. Only rats with normal motor function and behavior were used 5 days after surgery.

YM872 10 mg was dissolved in 0.97 mL of distilled water and 30 µL of 1N NaOH to adjust pH to 7.3–7.5. Solutions of 0.3 (0.86), 1 (2.86), 3 (8.59), 10 (28.63), or 30 (85.89) µg (nMol) per 10 µL were made using isotonic sodium chloride solution and were injected intrathecally. After drug injection, the catheter was flushed with 10 µL of isotonic sodium chloride solution to clear the dead space of the catheter (7 ± 0.4 µL, mean ± SE). Microinjector syringes were used for all injections. In each dose group, eight randomly selected rats were used. Isotonic sodium chloride solution 10 µL was injected in the control group.

The rats were placed in a clear plastic cylindrical cage with their tails extending through a slot provided in the rear of the tube. 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 2–3 cm proximal to the end. The response time was measured and defined as the interval between the onset of thermal stimulation and the abrupt flick of the tail. The cutoff time in the absence of a response was 14 s to prevent tissue injury. The response time was measured before and 5, 10, 15, 30, 60, 90, and 120 min after drug injection and at 1-h intervals until the response time returned to the baseline (maximum 360 min).

Ten minutes after the intrathecal administration of YM872, the rats were anesthetized with 3% halothane until transient loss of spontaneous movements was observed; they were then quickly removed from the 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 rat was placed in an open clear plastic chamber and observed for 60 min. Pain behavior was quantified by counting the incidences of spontaneous flinches/shaking of the injected paw 1–2 min, 5–6 min, and at intervals of 5 min 10–60 min after formalin injection. Animals were then killed with an overdose of halothane. As previously described (14), two distinct phases were observed: Phase 1 (0–6 min after injection) and Phase 2 (beginning approximately 10 min after injection).

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 assessed by placing the rat horizontally with its back on the table, which normally causes an immediate, coordinated turning of the body back to an upright position.

Response latency data from tail flick measurements were converted to maximal possible effect (%MPE) according to the formula: %MPE = (postdrug latency - baseline latency)/(cutoff time - baseline latency) x100. The 50% effective dose (ED50) was calculated using a computer program as the dose that produces a value of 50% MPE.

Differences between doses were analyzed by using two-way analysis of variance, followed by the Newman-Keuls test. The number of rats with side effects was compared by using the {chi}2 test. P < 0.05 was considered statistically significant.


   Results
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 Methods
 Results
Discussion
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The baseline latency (before drug injection) in the tail flick test was 3.1 ± 0.1 s (mean ± SE). The intrathecal administration of YM872 resulted in dose-dependent increases in the tail flick response latency (Fig. 1). The ED50 value (95% CI) was 1.0 µg (0.37–2.76 µg).



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  		Figure 1. Time course of the maximum possible effect (% MPE) of intrathecal YM872 on the tail flick latency. Each point presents the mean ± SEM of eight animals. *P < 0.05 versus control (time 0). **P < 0.01 versus control (time 0). +P < 0.05 versus saline. ++P < 0.01 versus saline. A dose of 30 µg induced a significantly longer duration of antinociception than smaller doses. The tail flick latency of 30 µg at 360 min was still greater than the control (time 0) (P = 0.031).

 
The number of flinches in both Phase 1 and Phase 2 in the formalin test decreased as the dose of intrathecal YM872 increased (Fig. 2). ED50 values were 0.24 µg (0.08–0.75 µg) in Phase 1 and 0.21 µg (0.06–0.73 µg) in Phase 2.



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  		Figure 2. Time course of the number of hindpaw flinches of the formalin test. Each point presents the mean ± SEM of eight animals. *P < 0.05 versus 1 min. **P < 0.01 versus 1 min. +P < 0.05 versus saline. ++P < 0.01 versus saline. The numbers of flinches in both Phase 1 and Phase 2 of the saline group was significantly lower than that in other groups with YM872. Doses of 10 and 30 µg produced fewer flinches than a 0.3-µg dose in both Phase 1 and Phase 2.

 
The intrathecal administration of YM872 10 µg produced a disturbance of placing and stepping for 30 min, a disturbance of the righting reflex for 60 min, and flaccidity for 60 min after injection (Fig. 3). More rats showed these side effects in the YM872 30 µg group. These side effects were reversible in 120 min. YM872 <=3 µg produced no observable side effects.



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  		Figure 3. Side effects of intrathecal YM872. Only doses of 10 and 30 µg induced a disturbance of placing and stepping, a disturbance of righting reflex, and flaccidity. The data for 0.3–3 µg and saline are not shown because they induced no side effects. Each bar presents the percentages of eight animals.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the present study, intrathecally administered YM872, a novel selective AMPA receptor antagonist, produced a dose-dependent increase in the tail flick latency with an ED50 value of 1.0 µg and dose-dependent decreases in the number of flinches in both Phase 1 (ED50 0.24 µg) and Phase 2 (ED50 0.21µg) of the formalin test. Only large doses (10 µg and 30 µg) induced transient motor disturbance and flaccidity.

AMPA receptors may be involved in mediating the acute excitation from primary afferent fibers to dorsal horn neurons evoked by high-intensity stimuli, whereas NMDA receptors seem to be involved centrally in the production of enhanced responsiveness of dorsal horn neurons after tissue damaging stimuli (1). Both A and C fiber-evoked responses of dorsal horn nociceptive neurons are mediated by AMPA receptors, whereas the C fiber-evoked wind-up of deep dorsal horn cells involves the NMDA receptor (15). NMDA is not more effective than AMPA or kainate at enhancing the synaptic responses of wide dynamic range neurons. There seems to be no specific involvement of NMDA receptors in the nociceptive responses of these neurons (16). Non-NMDA ionotropic receptor agonists are reported to be as effective as NMDA at enhancing responses of spinal neurons to both noxious and nonnoxious stimuli (16).

In general, the intrathecal injection of AMPA receptor antagonists produce dose-dependent antinociception on acute pain, as shown with the tail flick test (4) and with the hot plate test (5) in rats. AMPA receptor antagonists are also analgesic in the hyperalgesic thermal paw withdrawal test after peripheral mononeuropathy (17) and in affecting electrophysiological nociceptive indices (2). In the formalin test, antagonists of the NMDA receptor and of the nonstrychnine-sensitive glycine site will attenuate only the tonic second phase. Previous reports concerning the antinociceptive effects of AMPA receptor antagonists are conflicting. The AMPA receptor antagonists inhibit the acute first phase (18), but not the tonic second phase (19); whereas Simmons et al. (20)reported that a different type of AMPA receptor antagonist reduced the second phase but not the first phase.

In the present study, YM872 showed an apparent analgesic effect in both Phase 1 and Phase 2 of the formalin test. The reason for these discrepancies is unknown but could be drug-specific. The Phase 2 reaction in the formalin test is evoked by a polysynaptic response involving sensitization of deeper laminae dorsal horn neurons, which is induced by continuing activity of monosynaptic primary afferent output into the superficial laminae of the spinal cord during Phase 1 (21). AMPA/kainate receptor antagonists produced a marked decrease in pain behaviors in a rat model of postoperative pain (6), which, like the formalin test, is one of the models of continuous pain induced by inflammation. These discrepancies of antinociceptive actions of AMPA receptor antagonists between acute and chronic pain could be a result of a variation of their affinities to different subunits of the AMPA receptors. It is not known to which subunits of the AMPA receptors YM872 has a high affinity. The precise participation of the subunits of the AMPA receptors in acute and chronic nociception is a subject for future investigations.

YM872 can be considered as a more useful analgesic than NMDA receptor antagonists because it has analgesic effects on both acute and chronic pain, whereas NMDA receptor antagonists are effective only for facilitated pain.

The clinical usefulness of NMDA receptor antagonists is limited by their adverse effects, such as psychotomimetic effects (22), cognitive impairment (23), and neurotoxicity (24). In place of NMDA receptor antagonists, two classes of selective AMPA receptor antagonists have been developed. The first class comprises competitive quinoxalinedione antagonists such as NBQX (25). The second comprises a group of 2,3-benzodiazepines such as 1-(4'-aminophenyl)-3-methylcarbamoyl-4-methyl-3,4-dihydro-7,8-methylene-dioxy-5H-2,3-benzodiazepine HCl (GYKI 53655) (26) that act via an allosteric site. Although AMPA receptor antagonists do not share the side effects of NMDA receptor antagonists, their clinical application has also been limited because the first-generation compounds (such as NBQX) have poor water solubility and nephrotoxicity (10).

YM872, a novel competitive AMPA receptor antagonist, is approximately 1000-fold more water-soluble than NBQX and has a similar potency and selectivity for AMPA receptors (11). The effective doses induced no behavioral side effects. YM872 had no neurotoxicity in cat brain (12), although further toxicological studies should be performed to include other organs. In the present study, large doses of YM872 induced transient motor disturbance and flaccidity, which completely disappeared within 120 min. Furthermore, the doses that induced these side effects were >10 times the ED50.

In conclusion, the intrathecal administration of a novel competitive AMPA receptor antagonist, YM872, exhibited dose-dependent analgesic effects on acute thermal nociception and on formalin-induced nociception. Transient motor disturbance and flaccidity occurred with doses >10 times of ED50. These results suggest that, depending on whether a safety profile can be established, YM872 can be considered an important new drug that may be suitable for the clinical management of acute and chronic pain.


   References
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 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication March 8, 1999.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 1999 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press