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A novel AMPA receptor antagonist, YM872, reduces infarct size after middle cerebral artery occlusion in rats
Brain Research 793 Ž1998. 39–46
Research report
A novel AMPA receptor antagonist, YM872, reduces infarct size after middle cerebral artery occlusion in rats Sachiko Kawasaki-Yatsugi a, ) , Shin-ichi Yatsugi a , Masayasu Takahashi a , Takashi Toya a , Chikako Ichiki a , Masao Shimizu-Sasamata a , Tokio Yamaguchi a , Kazuo Minematsu b a
Neuroscience Research, Pharmacological Laboratory, Institute for Drug DiscoÕery Research, Yamanouchi Pharmaceutical, Tsukuba, Japan b CerebroÕascular DiÕision, Department of Medicine, National CardioÕascular Center, Osaka, Japan Accepted 27 January 1998
Abstract The neuroprotective effect of YM872 Žw2.3-dioxo-7-Ž1 H-imidazol-1-yl. 6-nitro-1,2,3,4-tetrahydro-1-quinoxalinylxacetic acid monohydrate., a novel a-amino-3-hydroxy-5-methyl-4-isoxazole propionate ŽAMPA. receptor antagonist with improved water solubility, was examined in a rat focal cerebral ischemia model. Rats were subjected to permanent middle cerebral artery ŽMCA. occlusion using the intraluminal suture occlusion method for 24 h. YM872 was intravenously infused for 4 h Ž20 and 40 mgrkgrh. or 24 h Ž10 and 20 mgrkgrh., starting 5 min after the MCA occlusion, to investigate the effect of prolonged duration of the treatment on infarct volume. In the 4 h infusion study, YM872 reduced the cortical infarct volume by 48% at a dose of 40 mgrkgrh. YM872 did not significantly reduce the infarct at 20 mgrkgrh for 4 h. In the 24 h infusion study, however, YM872 markedly reduced the cortical infarct volume by 62%, even at 20 mgrkgrh. The present study indicates that the neuroprotective effect of YM872 is enhanced by extending the duration of treatment, and demonstrates the benefit of the prolonged treatment with AMPA antagonists following focal cerebral ischemia. YM872, a highly water soluble compound, is applicable to investigate the role of AMPA receptors in ischemic models without concern about nephrotoxicity and could be useful in the treatment of human stroke. q 1998 Elsevier Science B.V. All rights reserved. Keywords: AMPA antagonist; Focal cerebral ischemia; Rat
1. Introduction Glutamate is thought to mediate the neuronal damage after cerebral ischemia by acting on both N-methyl-Daspartate ŽNMDA. and non-NMDA Ž a-amino-3-hydroxy5-methyl-4-isoxazole propionate; AMPArkainate. receptors w1,29x. There are numerous reports demonstrating the neuroprotective effects of NMDA antagonists in experimental ischemia models w2,14,21,22,26x. However, most NMDA receptor antagonists do not have a clinical value for stroke because of their side effects, such as psychotomimetic action w11x, learning impairment w15x and neurotoxicity w19,20x. The involvement of AMPA receptors, as well as NMDA receptors, in the development of post-ischemic neuronal )
Corresponding author. Neuroscience Research, Pharmacological Laboratory, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical, 21 Miyukigaoka, Tsukuba, Ibaraki 305, Japan. Fax: q81-298-562515; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 1 0 8 - 5
damage has been demonstrated w4x. The neuroprotective effects of AMPA receptor antagonists, such as 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo ŽF. quinoxaline ŽNBQX., has been shown in various experimental ischemia models w4,5,23,30x. Our previous studies have also demonstrated that 6- Ž1 H-imidazol-1-yl. 7-nitro-2,3 Ž1 H,4 H . quinoxalinedione ŽYM90K., a potent AMPA receptor antagonist w17x, has post-ischemic neuroprotective effects in models of transient global cerebral ischemia and focal cerebral ischemia w10,24,31,32x. These AMPA antagonists are good candidates as therapeutic agents, since they have been effective in these models when administered post-ischemic period w4,8x, and they lack psychotomimetic action and neurotoxicity w9x. Conversely, the drawback of these AMPA receptor antagonists is their low water solubility, which induces the nephrotoxicity w30x. Thus, analogues with better water solubility have been desired. Recently, we discovered a novel AMPA receptor antagonist YM872 Žw2,3-dioxo-7-Ž1 H-imidazol-1-yl.6-nitro1 ,2 ,3 ,4 -te tra h y d ro -1 -q u in o x a lin y l x a c e tic a c id
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Table 1 Physiological parameter in MCA occlusion rats Dose Žmgrkgrh.
Brain temp. Ž8C.
MABP ŽmmHg.
pH
pCO 2 ŽmmHg.
pO 2 ŽmmHg.
Hct Ž%.
Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872
30 min
2h
4h
Ž20. Ž40.
37.5"0.1 37.6"0.1 37.6"0.1
37.4"0.3 37.3"0.2 36.7"0.2
38.8"0.2 38.3"0.2 38.1"0.3
38.7"0.2 38.4"0.2 37.9"0.2
Ž20. Ž40.
37.1"0.3 37.4"0.2 37.2"0.3
36.8"0.6 37.0"0.4 36.7"0.4
37.5"0.6 37.6"0.5 37.9"0.7
37.5"0.4 37.8"0.3 37.1"0.5
Ž20. Ž40.
72"3 87"7 83"2
91"12 98"12 106"9
96"12 105"13 121"12
99"12 91"11 107"11
Ž20. Ž40.
7.39"0.02 7.38"0.01 7.41"0.01
7.50"0.04 7.47"0.02 7.43"0.02
7.48"0.02 7.48"0.02 7.48"0.04
7.47"0.01 7.47"0.01 7.45"0.02
Ž20. Ž40.
47.7"1.3 50.8"1.1 46.6"1.0
36.3"3.1 39.6"2.0 43.7"0.9
34.1"2.0 33.8"1.7 36.2"1.6
32.4"2.5 34.0"0.7 40.2"1.3
Ž20. Ž40.
148.8"2.2 159.2"17.1 144.3"0.3
92.8"3.2 85.6"4.3 137.6"0.6
104.9"13.3 90.1"3.6 111.7"16.3
103.6"14.3 88.5"3.3 96.8"14.4
Ž20. Ž40.
40.7"1.3 40.5"0.1 43.9"1.5
44.3"1.0 44.3"0.6 47.6"1.9
43.0"1.3 43.1"0.7 47.6"1.7
42.0"1.5 42.4"0.8 47.2"1.7
S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
Ža. 4 h infusion study Rectal temp. Ž8C.
pre a
Table 1 Žcontinued. Dose Žmgrkgrh. Žb. 24 h infusion study Rectal temp. Ž8C.
MABP ŽmmHg.
pH
pCO 2 ))ŽmmHg.
pO 2 ŽmmHg.
Hct Ž%.
Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872
1h
3h
12 h
24 h
Ž10. Ž20.
37.7"0.1 37.6"0.1 37.6"0.2
38.6"0.3 37.7"0.3 37.8"0.3
38.3"0.3 37.9"0.3 38.2"0.3
37.6"0.2 37.3"0.4 37.4"0.3
37.6"0.2 37.1"0.3 37.8"0.1
Ž10. Ž20.
37.5"0.1 37.4"0.2 37.6"0.2
38.2"0.3 37.4"0.3 37.7"0.3
37.9"0.3 37.2"0.4 38.0"0.2
37.1"0.2 37.6"0.2 36.9"0.4
36.8"0.2 36.7"0.2 37.4"0.2
Ž10. Ž20.
84"2 80"2 86"4
124"6 115"10 131"5
123"6 123"8 128"2
113"4 113"5 115"3
108"3 112"4 112"6
Ž10. Ž20.
7.45"0.02 7.46"0.01 7.43"0.01
7.54"0.01 7.53"0.01 7.49"0.01
7.53"0.01 7.52"0.02 7.51"0.01
7.52"0.02 7.49"0.01 7.48"0.01
7.53"0.01 7.51"0.02 7.51"0.02
Ž10. Ž20.
44.6"1.3 44.3"1.1 46.9"1.0
32.0"0.8 33.9"1.2 37.3"2.3
29.5"1.2 32.1"0.8 33.4"2.2
28.4"1.2 34.2"1.0 32.7"1.9
31.5"1.3 33.5"1.2 35.5"1.3
Ž10. Ž20.
127.6"8.1 114.0"3.9 132.2"8.3
102.0"7.7 108.7"8.6 90.8"10.0
99.6"6.5 91.1"2.9 92.3"8.1
106.3"7.7 107.3"6.8 111.3"13.6
105.9"7.4 108.2"8.8 88.5"2.1
Ž10. Ž20.
41.6"0.8 40.3"1.1 40.9"0.7
45.2"1.2 44.9"1.4 44.6"1.1
45.5"0.9 43.0"1.9 44.7"1.9
44.0"1.0 40.9"2.0 43.8"1.4
43.5"0.9 41.2"2.1 43.0"1.0
S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
Brain temp. Ž8C.
Saline YM872
pre a
))Asterisk indicates a between-subjects effects Žtreatment factor. was significant Ž p- 0.01. when overall test was done ŽTwo-way, repeated measurement. but the interaction between-group Žtreatment. factor and within-group Žtime. factor was not significant. a The parameters before MCA occlusion Žpre. were obtained under halothane anesthesia.
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S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
monohydrate., which is highly soluble in water w12x. YM872 has a similar affinity for AMPA receptors as YM90K and is approximately 800 times more soluble in pH 7 buffers than those of NBQX and YM90K w12x. It also showed a neuroprotective effect against the neuronal damage induced by focal cerebral ischemia in anesthetized cats w27x. In the present study, we examined the neuroprotective effect of YM872 in focal cerebral ischemia in rats. In addition, we compared the neuroprotective effects between 4 h and 24 h continuous i.v. infusion of YM872 to investigate the optimal duration of administration in this model.
2. Materials and methods The experimental procedures were done in accordance with the guidelines of the Animal Ethical Committee of Yamanouchi Pharmaceutical. Male Sprague–Dawley rats ŽCharles River Japan, Yokohama, Japan. weighing 250– 310 g were used. They were anesthetized with chloral hydrate Ž350 mgrkg i.p.., a guide cannula was implanted into the right striatum for monitoring the brain temperature and a polyethylene tube 50 ŽPE-50. was cannulated into the left jugular vein for continuous administration 2–5 days before ischemia. The animals were anesthetized with 0.5–1.5% halothane in 25% O 2r75% N2 , a PE-50 was inserted into the tail artery for monitoring blood pressure and sampling blood, and the right middle cerebral artery ŽMCA. was permanently occluded using the intraluminal suture occlusion method described by Zea Longa et. al. w33x. Briefly, a 4-0 monofilament nylon suture with its tip rounded by heating near a flame, was introduced through the right common carotid artery ŽCCA. into the internal carotid artery and advanced approximately 17 mm intracranially from the CCA bifurcation. The anesthesia was stopped immediately after MCA occlusion. Rectal temperature was maintained at 37–388C using a heated blanket and an overhead lamp during the surgical procedure and monitored during administration. Brain temperature and physiological parameters were also monitored pre- and post-MCA occlusion period in some of the treated animals Ž n s 4 to 11, per group.. Brain temperature was monitored with a needle-type thermoprobe ŽBAT-12, Physitemp Instrument, NJ, USA. inserted into striatum ŽA: 0.0 mm, L: 2.5 mm, H: 3.5 mm from bregma. through a guide cannula. The implantation of a guide cannula into the striatum was done carefully to minimize the surgical damage. Therefore, the infarct size was not affected by this procedure. Physiological parameters before MCA occlusion were obtained under halothane anesthesia. YM872 Ž10, 20 and 40 mgr3 ml. was dissolved in physiological saline and the solution was adjusted to pH 7.4 with a few drops of 1 N NaOH. The continuous i.v. infusion started 5 min after MCA occlusion. During administration, animals were placed in Bollman Cage
ŽNatsume Seisakusho Tokyo, Japan. for continuous infusion and monitoring physiological parameters. YM872 Ž20 or 40 mgrkgrh, n s 14, n s 12, respectively. or saline vehicle Ž n s 13. were given for 4 h. After administration, animals were allowed to move freely and, after observation of their spontaneous behavior, they were allowed free access to water and food. In a further study, YM872 Ž10 or 20 mgrkgrh, n s 10, n s 11, respectively. or saline Ž n s 12. were given for 24 h. At 24 h after MCA occlusion, animals were reanesthetized with chloral hydrate Ž300 mgrkg i.p.. and decapitated. The kidneys of the animals given YM872 were removed to observe crystal precipitation of YM872 in a segment of the kidney. The brains were removed and inspected to confirm the position of the suture and the absence of subarachnoid hemorrhage andror arterial penetration. The brains were sectioned with a tissue chopper ŽMcllwain Tissue Chopper, Mickle Laboratory Engineering, USA. at 1 mm intervals. The coronal section were stained with 2% solution of 2,3,5-triphenyltetrazolium hydrochloride ŽTTC. and fixed by immersion in 10% phosphate-buffered formalin. The 13 sections per brain were
Fig. 1. Corrected infarct volumes in the cerebral hemisphere ŽH., cortex ŽC. and non-cortical region ŽNC. at 24 h after MCA occlusion in rats. Each value represents the mean"S.E.M. Saline or YM872 was intravenously infused starting from 5 min after the onset of ischemia and continued for 4 h Ža. or 24 h Žb.. ) p- 0.05 vs. the saline group ŽOne-way ANOVA followed by Dunnett multiple range test..
S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
recorded with a CCD color video camera. Areas not stained with TTC, which were considered lesioned, were outlined in a blind fashion. Hemispheric and cortical infarct areas and right and left hemispheric areas were measured by the video image analyzing system ŽNIH image, version 1.52.. The infarct volume and hemispheric volume were calculated by multiplying the areas by the thickness. To compensate for the effect of brain edema, the corrected infarct volume was calculated by the following equation: multiply the infarct volume by the ratio of the left hemispheric volume to right hemispheric volume w7x. Data are represented as the means" S.E.M. To compare infarct volume and physiological parameters among experimental groups, one-way ANOVA followed by Dunnett multiple-range test and two-factor, repeated-measures ANOVA was used, respectively. A p-value - 0.05 was considered significant.
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3.1. NeuroprotectiÕe effect of YM872 in the 4 h i.Õ. infusion study An animal in the saline-treated group died within 24 h. YM872 reduced the infarct volume in a dose-dependent manner ŽFig. 1a.. At a dose of 40 mgrkgrh, YM872 significantly reduced the infarct volume by 43% in the cerebral hemisphere Ž p - 0.05. and 48% in the cortex Ž p - 0.05. as compared with the saline-treated group. At the dose of 20 mgrkgrh, YM872 also reduced the infarct volume of both regions, but the results did not reach the statistically significant level. There was no significant difference in the noncortical infarct volume among the groups.
3.2. NeuroprotectiÕe effect of YM872 in the 24 h i.Õ. infusion study
3. Results Respiratory depression, hypothermia, abnormal behavior such as stereotyped behavior, were not observed in any animals treated with YM872. No crystal precipitation was observed in the renal segments at 24 h after MCA occlusion. There were no significant differences in rectal or brain temperatures, and physiological parameters among the groups. Arterial pCO 2 in the 24 h infusion study was higher in the YM872 groups than in the saline group when the overall difference was tested ŽTable 1b..
An animal in the YM872 Ž20 mgrkgrh.-treated group died within 24 h. The cortical infarct volume in the control group in the 24 h infusion study was smaller and more varied than that in the 4 h infusion study. At a dose of 20 mgrkgrh, YM872 reduced the volume of hemispheric and cortical infarct by 51 and 62%, respectively. The cortical infarct volume in the 20 mgrkgrh-treated rats was significantly smaller than that in the control rats Ž p - 0.05, Fig. 1b, Fig. 2.. At a dose of 10 mgrkgrh, YM872 did not show any neuroprotective effect.
Fig. 2. Photographs show the TTC stained coronal sections at 2, 4, 6, 8, 10 and 12 mm caudal from the frontal pole in representative rats. Left: 24 h infusion of saline. Right: 20 mgrkgrh= 24 h of YM872. Scale bar s 5 mm.
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S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
4. Discussion In the 4 h i.v. infusion study, the novel AMPA antagonist YM872 showed neuroprotective effect in a rat focal cerebral ischemia model. This result is consistent with the efficacy of the other AMPA receptor antagonists in earlier studies w5,4,8,23x. Gill. et. al. w5x reported that NBQX administered as an i.v. bolus of 30 mgrkg followed by an infusion of 10 mgrkgrh for 4 h reduced the infarct volume by 30% at 24 h after a permanent MCA occlusion in rats. YM90K, an AMPA antagonist, showed similar neuroprotective effects at the same dosing regime in our previous study w24x. In addition, NBQX was neuroprotective when administered at 90 min post-occlusion in a permanent and transient MCA-occluded rats w8,30x. In the former study, a 3 h i.v. infusion was performed, and in the latter study, the drug was intraperitoneally given at 90, 120 and 150 min post-occlusion. These results suggest that AMPA receptors play an important role in early time period Žwithin approximately 4 h. of the development of the neuronal damage following focal cerebral ischemia because NBQX has a short biological half life Žapproximately 30 min. in plasma w5x. In addition, YM90K and YM872 are also eliminated rapidly from plasma after termination of i.v. infusion in our previous study Žunpublished data.. In contrast, YM872 at a dose of 20 mgrkgrh did not show significant neuroprotection by the 4 h i.v. infusion, whereas it markedly reduced the infarct volume after 24 h i.v. infusion. This result indicates that the neuroprotective effect of YM872 was enhanced by extending the duration of treatment. It is well known that the elevation of intracellular Ca2q concentration ŽwCa2q xi. following increased glutamate concentration in synaptic clefts plays a critical role in ischemia-induced cell damage w25x. AMPA receptor antagonists are thought to reduce this elevation of wCa2q xi by blocking several pathways mediating the influx of Ca2q w4x. The elevation of wCa2q xi in the early phase Žwithin approximately 4 h. following focal ischemia is the most important factor in neuronal damage, because a 4 h treatment with YM872 of 40 mgrkgrh showed reduction of the infarct volume in this study. The blockade of AMPA receptors by YM872 for 4 h could keep the wCa2q xi below toxic levels in surviving cells within 24 h after focal ischemia. On the other hand, the comparison of the results between the 4 h and 24 h i.v. infusion studies suggests that the sustained elevation of wCa2q xi during the relatively late phase Žmore than 4 h. after focal ischemia also contributes to the extent of neuronal damage, and the prolonged blockade of AMPA receptors by YM872 could also keep the wCa2q xi below toxic level, even though the reduction of wCa2q xi by the treatment with YM872 of 20 mgrkgrh for only 4 h could not be enough to induce a sufficient neuroprotective effect. In other words, the AMPA receptor-related elevation of wCa2q xi after focal cerebral ischemia may reach the neurotoxic level both by an acute
Ca2q influx induced by the increase in glutamate in the early phase and a sustained Ca2q influx in the relatively late phase. Tomabechi et. al. w28x found the sustained accumulation of Ca2q for 24 h in the ischemic brain tissue after focal ischemia in rats. According to their report, the rapid increase in wCa2q xi occurred during the first 5 h after MCA occlusion, and continued more slowly from 5 to 24 h. Konig et. al. w13x reported that long-lasting wCa2q xi ¨ elevation following AMPA exposure was observed in the rat brainstem cells. These findings may support our hypothesis although the time course of the inhibitory effect of YM872 on wCa2q xi levels after ischemic or excitotoxic insults has not been investigated. This hypothesis indicates that prolonged treatment with AMPA antagonists may be useful in the treatment of human stroke, even when the infusion rate is limited due to side effects such as respiratory depression or nephrotoxicity. Since the brain concentration of YM872 reached approximately 100 ngrg at a dose of 20 mgrkgrh in our preliminary study Ždata not shown., the effective brain concentration in this model was estimated to be about 100 ngrg Ž; 0.3 m M.. This is comparable to the concentration of YM872 which selectively inhibits AMPA receptor binding without blocking NMDA as well as other receptors in vitro w12x, and the CSF level at the neuroprotective dose in a cat MCA occlusion model w27x. This indicates that the neuroprotective effect of YM872 in this study is mainly due to the selective blockade of AMPA receptors in the central nervous systems. The brain concentration of YM872 at 10 mgrkgrh may not be enough to extend neuroprotection since no reduction in the infarct volume was observed even by 24 h i.v. infusion. The cortical infarct volume of the saline-treated group in the 24 h i.v. infusion study was relatively smaller and more varied compared with that in the 4 h i.v. infusion study. The reason why this difference occurred remains unknown, but the hemodilution effect might be induced by extending the duration of treatment with saline w18x. Although the decrease in the hematocrit value was not detected in the 24 h infusion study, its possibility may not be excluded completely because it was measured at several timepoints, but not continuously, after MCA occlusion. To clarify this point, further studies are needed to examine whether cerebral blood flow in the MCA territory is affected by saline infusion during the ischemic period. Monitoring of body temperature and physiological parameters, including MABP and blood gases, is important in the evaluation of neuroprotective agents in cerebral ischemia models, since these parameters may affect neuronal damage w3,6,16x. In this study, there were no significant differences in the brain and rectal temperatures, physiological parameters between the corresponding groups except for arterial pCO 2 . In addition, in our preliminary study using anesthetized cats, CBF was not affected by YM872 administration at the effective dose in MCA occluded cats. These results suggest that the neuroprotective
S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
effect of YM872 may not be attributed to non-specific effects such as hypothermia or cerebral hemodynamic effects. Arterial pCO 2 tended to be higher Žcloser to the normal value. in the YM872 groups than in the saline group in the 24 h i.v. infusion study. Spontaneous hyperventilation induced by pronounced ischemic brain damage w14x might be prevented by the administration of YM872.
w12x
w13x
w14x
5. Conclusion The present data provides evidence that YM872, a novel AMPA receptor antagonist, may reduce the neuronal damage in a focal cerebral ischemia model in rats and, therefore, YM872 may be a useful tool to investigate the pathophysiological role of AMPA receptors in models of ischemia. Because the neuroprotective effect of YM872 was enhanced by the prolonged duration of the treatment following focal cerebral ischemia, the long-term administration of YM872 could be useful in the treatment of human stroke.
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w30x D. Xue, Z.G. Huang, K. Barnes, H.J. Lesiuk, K.E. Smith, A.M. Buchan, Delayed treatment with AMPA, but not NMDA, antagonists reduces neocortical infarction, J. Cereb. Blood Flow Metab. 14 Ž1994. 251–261. w31x H. Yao, S. Ibayashi, H. Nakane, H. Cai, H. Uchimura, M. Fujishima, AMPA receptor antagonist, YM90K, reduces infarct volume in thrombotic distal middle cerebral artery occlusion in spontaneously hypertensive rats, Brain Res. 753 Ž1997. 80–85.
w32x S. Yatsugi, M. Takahashi, S. Kawasaki-Yatsugi, K. Koshiya, S. Sakamoto, D. Uematsu, M. Shimizu-Sasamata, Neuroprotective effect of YM90K, a novel AMPArKainate receptor antagonist, in focal ischemia in cats, J. Cereb. Blood Flow Metab. 16 Ž1996. 959–966. w33x E. Zea Longa, P.R. Weinstein, S. Carlson, R. Cummins, Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke 19 Ž1989. 84–91.
Research report
A novel AMPA receptor antagonist, YM872, reduces infarct size after middle cerebral artery occlusion in rats Sachiko Kawasaki-Yatsugi a, ) , Shin-ichi Yatsugi a , Masayasu Takahashi a , Takashi Toya a , Chikako Ichiki a , Masao Shimizu-Sasamata a , Tokio Yamaguchi a , Kazuo Minematsu b a
Neuroscience Research, Pharmacological Laboratory, Institute for Drug DiscoÕery Research, Yamanouchi Pharmaceutical, Tsukuba, Japan b CerebroÕascular DiÕision, Department of Medicine, National CardioÕascular Center, Osaka, Japan Accepted 27 January 1998
Abstract The neuroprotective effect of YM872 Žw2.3-dioxo-7-Ž1 H-imidazol-1-yl. 6-nitro-1,2,3,4-tetrahydro-1-quinoxalinylxacetic acid monohydrate., a novel a-amino-3-hydroxy-5-methyl-4-isoxazole propionate ŽAMPA. receptor antagonist with improved water solubility, was examined in a rat focal cerebral ischemia model. Rats were subjected to permanent middle cerebral artery ŽMCA. occlusion using the intraluminal suture occlusion method for 24 h. YM872 was intravenously infused for 4 h Ž20 and 40 mgrkgrh. or 24 h Ž10 and 20 mgrkgrh., starting 5 min after the MCA occlusion, to investigate the effect of prolonged duration of the treatment on infarct volume. In the 4 h infusion study, YM872 reduced the cortical infarct volume by 48% at a dose of 40 mgrkgrh. YM872 did not significantly reduce the infarct at 20 mgrkgrh for 4 h. In the 24 h infusion study, however, YM872 markedly reduced the cortical infarct volume by 62%, even at 20 mgrkgrh. The present study indicates that the neuroprotective effect of YM872 is enhanced by extending the duration of treatment, and demonstrates the benefit of the prolonged treatment with AMPA antagonists following focal cerebral ischemia. YM872, a highly water soluble compound, is applicable to investigate the role of AMPA receptors in ischemic models without concern about nephrotoxicity and could be useful in the treatment of human stroke. q 1998 Elsevier Science B.V. All rights reserved. Keywords: AMPA antagonist; Focal cerebral ischemia; Rat
1. Introduction Glutamate is thought to mediate the neuronal damage after cerebral ischemia by acting on both N-methyl-Daspartate ŽNMDA. and non-NMDA Ž a-amino-3-hydroxy5-methyl-4-isoxazole propionate; AMPArkainate. receptors w1,29x. There are numerous reports demonstrating the neuroprotective effects of NMDA antagonists in experimental ischemia models w2,14,21,22,26x. However, most NMDA receptor antagonists do not have a clinical value for stroke because of their side effects, such as psychotomimetic action w11x, learning impairment w15x and neurotoxicity w19,20x. The involvement of AMPA receptors, as well as NMDA receptors, in the development of post-ischemic neuronal )
Corresponding author. Neuroscience Research, Pharmacological Laboratory, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical, 21 Miyukigaoka, Tsukuba, Ibaraki 305, Japan. Fax: q81-298-562515; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 1 0 8 - 5
damage has been demonstrated w4x. The neuroprotective effects of AMPA receptor antagonists, such as 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo ŽF. quinoxaline ŽNBQX., has been shown in various experimental ischemia models w4,5,23,30x. Our previous studies have also demonstrated that 6- Ž1 H-imidazol-1-yl. 7-nitro-2,3 Ž1 H,4 H . quinoxalinedione ŽYM90K., a potent AMPA receptor antagonist w17x, has post-ischemic neuroprotective effects in models of transient global cerebral ischemia and focal cerebral ischemia w10,24,31,32x. These AMPA antagonists are good candidates as therapeutic agents, since they have been effective in these models when administered post-ischemic period w4,8x, and they lack psychotomimetic action and neurotoxicity w9x. Conversely, the drawback of these AMPA receptor antagonists is their low water solubility, which induces the nephrotoxicity w30x. Thus, analogues with better water solubility have been desired. Recently, we discovered a novel AMPA receptor antagonist YM872 Žw2,3-dioxo-7-Ž1 H-imidazol-1-yl.6-nitro1 ,2 ,3 ,4 -te tra h y d ro -1 -q u in o x a lin y l x a c e tic a c id
40
Table 1 Physiological parameter in MCA occlusion rats Dose Žmgrkgrh.
Brain temp. Ž8C.
MABP ŽmmHg.
pH
pCO 2 ŽmmHg.
pO 2 ŽmmHg.
Hct Ž%.
Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872
30 min
2h
4h
Ž20. Ž40.
37.5"0.1 37.6"0.1 37.6"0.1
37.4"0.3 37.3"0.2 36.7"0.2
38.8"0.2 38.3"0.2 38.1"0.3
38.7"0.2 38.4"0.2 37.9"0.2
Ž20. Ž40.
37.1"0.3 37.4"0.2 37.2"0.3
36.8"0.6 37.0"0.4 36.7"0.4
37.5"0.6 37.6"0.5 37.9"0.7
37.5"0.4 37.8"0.3 37.1"0.5
Ž20. Ž40.
72"3 87"7 83"2
91"12 98"12 106"9
96"12 105"13 121"12
99"12 91"11 107"11
Ž20. Ž40.
7.39"0.02 7.38"0.01 7.41"0.01
7.50"0.04 7.47"0.02 7.43"0.02
7.48"0.02 7.48"0.02 7.48"0.04
7.47"0.01 7.47"0.01 7.45"0.02
Ž20. Ž40.
47.7"1.3 50.8"1.1 46.6"1.0
36.3"3.1 39.6"2.0 43.7"0.9
34.1"2.0 33.8"1.7 36.2"1.6
32.4"2.5 34.0"0.7 40.2"1.3
Ž20. Ž40.
148.8"2.2 159.2"17.1 144.3"0.3
92.8"3.2 85.6"4.3 137.6"0.6
104.9"13.3 90.1"3.6 111.7"16.3
103.6"14.3 88.5"3.3 96.8"14.4
Ž20. Ž40.
40.7"1.3 40.5"0.1 43.9"1.5
44.3"1.0 44.3"0.6 47.6"1.9
43.0"1.3 43.1"0.7 47.6"1.7
42.0"1.5 42.4"0.8 47.2"1.7
S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
Ža. 4 h infusion study Rectal temp. Ž8C.
pre a
Table 1 Žcontinued. Dose Žmgrkgrh. Žb. 24 h infusion study Rectal temp. Ž8C.
MABP ŽmmHg.
pH
pCO 2 ))ŽmmHg.
pO 2 ŽmmHg.
Hct Ž%.
Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872 Saline YM872
1h
3h
12 h
24 h
Ž10. Ž20.
37.7"0.1 37.6"0.1 37.6"0.2
38.6"0.3 37.7"0.3 37.8"0.3
38.3"0.3 37.9"0.3 38.2"0.3
37.6"0.2 37.3"0.4 37.4"0.3
37.6"0.2 37.1"0.3 37.8"0.1
Ž10. Ž20.
37.5"0.1 37.4"0.2 37.6"0.2
38.2"0.3 37.4"0.3 37.7"0.3
37.9"0.3 37.2"0.4 38.0"0.2
37.1"0.2 37.6"0.2 36.9"0.4
36.8"0.2 36.7"0.2 37.4"0.2
Ž10. Ž20.
84"2 80"2 86"4
124"6 115"10 131"5
123"6 123"8 128"2
113"4 113"5 115"3
108"3 112"4 112"6
Ž10. Ž20.
7.45"0.02 7.46"0.01 7.43"0.01
7.54"0.01 7.53"0.01 7.49"0.01
7.53"0.01 7.52"0.02 7.51"0.01
7.52"0.02 7.49"0.01 7.48"0.01
7.53"0.01 7.51"0.02 7.51"0.02
Ž10. Ž20.
44.6"1.3 44.3"1.1 46.9"1.0
32.0"0.8 33.9"1.2 37.3"2.3
29.5"1.2 32.1"0.8 33.4"2.2
28.4"1.2 34.2"1.0 32.7"1.9
31.5"1.3 33.5"1.2 35.5"1.3
Ž10. Ž20.
127.6"8.1 114.0"3.9 132.2"8.3
102.0"7.7 108.7"8.6 90.8"10.0
99.6"6.5 91.1"2.9 92.3"8.1
106.3"7.7 107.3"6.8 111.3"13.6
105.9"7.4 108.2"8.8 88.5"2.1
Ž10. Ž20.
41.6"0.8 40.3"1.1 40.9"0.7
45.2"1.2 44.9"1.4 44.6"1.1
45.5"0.9 43.0"1.9 44.7"1.9
44.0"1.0 40.9"2.0 43.8"1.4
43.5"0.9 41.2"2.1 43.0"1.0
S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
Brain temp. Ž8C.
Saline YM872
pre a
))Asterisk indicates a between-subjects effects Žtreatment factor. was significant Ž p- 0.01. when overall test was done ŽTwo-way, repeated measurement. but the interaction between-group Žtreatment. factor and within-group Žtime. factor was not significant. a The parameters before MCA occlusion Žpre. were obtained under halothane anesthesia.
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S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
monohydrate., which is highly soluble in water w12x. YM872 has a similar affinity for AMPA receptors as YM90K and is approximately 800 times more soluble in pH 7 buffers than those of NBQX and YM90K w12x. It also showed a neuroprotective effect against the neuronal damage induced by focal cerebral ischemia in anesthetized cats w27x. In the present study, we examined the neuroprotective effect of YM872 in focal cerebral ischemia in rats. In addition, we compared the neuroprotective effects between 4 h and 24 h continuous i.v. infusion of YM872 to investigate the optimal duration of administration in this model.
2. Materials and methods The experimental procedures were done in accordance with the guidelines of the Animal Ethical Committee of Yamanouchi Pharmaceutical. Male Sprague–Dawley rats ŽCharles River Japan, Yokohama, Japan. weighing 250– 310 g were used. They were anesthetized with chloral hydrate Ž350 mgrkg i.p.., a guide cannula was implanted into the right striatum for monitoring the brain temperature and a polyethylene tube 50 ŽPE-50. was cannulated into the left jugular vein for continuous administration 2–5 days before ischemia. The animals were anesthetized with 0.5–1.5% halothane in 25% O 2r75% N2 , a PE-50 was inserted into the tail artery for monitoring blood pressure and sampling blood, and the right middle cerebral artery ŽMCA. was permanently occluded using the intraluminal suture occlusion method described by Zea Longa et. al. w33x. Briefly, a 4-0 monofilament nylon suture with its tip rounded by heating near a flame, was introduced through the right common carotid artery ŽCCA. into the internal carotid artery and advanced approximately 17 mm intracranially from the CCA bifurcation. The anesthesia was stopped immediately after MCA occlusion. Rectal temperature was maintained at 37–388C using a heated blanket and an overhead lamp during the surgical procedure and monitored during administration. Brain temperature and physiological parameters were also monitored pre- and post-MCA occlusion period in some of the treated animals Ž n s 4 to 11, per group.. Brain temperature was monitored with a needle-type thermoprobe ŽBAT-12, Physitemp Instrument, NJ, USA. inserted into striatum ŽA: 0.0 mm, L: 2.5 mm, H: 3.5 mm from bregma. through a guide cannula. The implantation of a guide cannula into the striatum was done carefully to minimize the surgical damage. Therefore, the infarct size was not affected by this procedure. Physiological parameters before MCA occlusion were obtained under halothane anesthesia. YM872 Ž10, 20 and 40 mgr3 ml. was dissolved in physiological saline and the solution was adjusted to pH 7.4 with a few drops of 1 N NaOH. The continuous i.v. infusion started 5 min after MCA occlusion. During administration, animals were placed in Bollman Cage
ŽNatsume Seisakusho Tokyo, Japan. for continuous infusion and monitoring physiological parameters. YM872 Ž20 or 40 mgrkgrh, n s 14, n s 12, respectively. or saline vehicle Ž n s 13. were given for 4 h. After administration, animals were allowed to move freely and, after observation of their spontaneous behavior, they were allowed free access to water and food. In a further study, YM872 Ž10 or 20 mgrkgrh, n s 10, n s 11, respectively. or saline Ž n s 12. were given for 24 h. At 24 h after MCA occlusion, animals were reanesthetized with chloral hydrate Ž300 mgrkg i.p.. and decapitated. The kidneys of the animals given YM872 were removed to observe crystal precipitation of YM872 in a segment of the kidney. The brains were removed and inspected to confirm the position of the suture and the absence of subarachnoid hemorrhage andror arterial penetration. The brains were sectioned with a tissue chopper ŽMcllwain Tissue Chopper, Mickle Laboratory Engineering, USA. at 1 mm intervals. The coronal section were stained with 2% solution of 2,3,5-triphenyltetrazolium hydrochloride ŽTTC. and fixed by immersion in 10% phosphate-buffered formalin. The 13 sections per brain were
Fig. 1. Corrected infarct volumes in the cerebral hemisphere ŽH., cortex ŽC. and non-cortical region ŽNC. at 24 h after MCA occlusion in rats. Each value represents the mean"S.E.M. Saline or YM872 was intravenously infused starting from 5 min after the onset of ischemia and continued for 4 h Ža. or 24 h Žb.. ) p- 0.05 vs. the saline group ŽOne-way ANOVA followed by Dunnett multiple range test..
S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
recorded with a CCD color video camera. Areas not stained with TTC, which were considered lesioned, were outlined in a blind fashion. Hemispheric and cortical infarct areas and right and left hemispheric areas were measured by the video image analyzing system ŽNIH image, version 1.52.. The infarct volume and hemispheric volume were calculated by multiplying the areas by the thickness. To compensate for the effect of brain edema, the corrected infarct volume was calculated by the following equation: multiply the infarct volume by the ratio of the left hemispheric volume to right hemispheric volume w7x. Data are represented as the means" S.E.M. To compare infarct volume and physiological parameters among experimental groups, one-way ANOVA followed by Dunnett multiple-range test and two-factor, repeated-measures ANOVA was used, respectively. A p-value - 0.05 was considered significant.
43
3.1. NeuroprotectiÕe effect of YM872 in the 4 h i.Õ. infusion study An animal in the saline-treated group died within 24 h. YM872 reduced the infarct volume in a dose-dependent manner ŽFig. 1a.. At a dose of 40 mgrkgrh, YM872 significantly reduced the infarct volume by 43% in the cerebral hemisphere Ž p - 0.05. and 48% in the cortex Ž p - 0.05. as compared with the saline-treated group. At the dose of 20 mgrkgrh, YM872 also reduced the infarct volume of both regions, but the results did not reach the statistically significant level. There was no significant difference in the noncortical infarct volume among the groups.
3.2. NeuroprotectiÕe effect of YM872 in the 24 h i.Õ. infusion study
3. Results Respiratory depression, hypothermia, abnormal behavior such as stereotyped behavior, were not observed in any animals treated with YM872. No crystal precipitation was observed in the renal segments at 24 h after MCA occlusion. There were no significant differences in rectal or brain temperatures, and physiological parameters among the groups. Arterial pCO 2 in the 24 h infusion study was higher in the YM872 groups than in the saline group when the overall difference was tested ŽTable 1b..
An animal in the YM872 Ž20 mgrkgrh.-treated group died within 24 h. The cortical infarct volume in the control group in the 24 h infusion study was smaller and more varied than that in the 4 h infusion study. At a dose of 20 mgrkgrh, YM872 reduced the volume of hemispheric and cortical infarct by 51 and 62%, respectively. The cortical infarct volume in the 20 mgrkgrh-treated rats was significantly smaller than that in the control rats Ž p - 0.05, Fig. 1b, Fig. 2.. At a dose of 10 mgrkgrh, YM872 did not show any neuroprotective effect.
Fig. 2. Photographs show the TTC stained coronal sections at 2, 4, 6, 8, 10 and 12 mm caudal from the frontal pole in representative rats. Left: 24 h infusion of saline. Right: 20 mgrkgrh= 24 h of YM872. Scale bar s 5 mm.
44
S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
4. Discussion In the 4 h i.v. infusion study, the novel AMPA antagonist YM872 showed neuroprotective effect in a rat focal cerebral ischemia model. This result is consistent with the efficacy of the other AMPA receptor antagonists in earlier studies w5,4,8,23x. Gill. et. al. w5x reported that NBQX administered as an i.v. bolus of 30 mgrkg followed by an infusion of 10 mgrkgrh for 4 h reduced the infarct volume by 30% at 24 h after a permanent MCA occlusion in rats. YM90K, an AMPA antagonist, showed similar neuroprotective effects at the same dosing regime in our previous study w24x. In addition, NBQX was neuroprotective when administered at 90 min post-occlusion in a permanent and transient MCA-occluded rats w8,30x. In the former study, a 3 h i.v. infusion was performed, and in the latter study, the drug was intraperitoneally given at 90, 120 and 150 min post-occlusion. These results suggest that AMPA receptors play an important role in early time period Žwithin approximately 4 h. of the development of the neuronal damage following focal cerebral ischemia because NBQX has a short biological half life Žapproximately 30 min. in plasma w5x. In addition, YM90K and YM872 are also eliminated rapidly from plasma after termination of i.v. infusion in our previous study Žunpublished data.. In contrast, YM872 at a dose of 20 mgrkgrh did not show significant neuroprotection by the 4 h i.v. infusion, whereas it markedly reduced the infarct volume after 24 h i.v. infusion. This result indicates that the neuroprotective effect of YM872 was enhanced by extending the duration of treatment. It is well known that the elevation of intracellular Ca2q concentration ŽwCa2q xi. following increased glutamate concentration in synaptic clefts plays a critical role in ischemia-induced cell damage w25x. AMPA receptor antagonists are thought to reduce this elevation of wCa2q xi by blocking several pathways mediating the influx of Ca2q w4x. The elevation of wCa2q xi in the early phase Žwithin approximately 4 h. following focal ischemia is the most important factor in neuronal damage, because a 4 h treatment with YM872 of 40 mgrkgrh showed reduction of the infarct volume in this study. The blockade of AMPA receptors by YM872 for 4 h could keep the wCa2q xi below toxic levels in surviving cells within 24 h after focal ischemia. On the other hand, the comparison of the results between the 4 h and 24 h i.v. infusion studies suggests that the sustained elevation of wCa2q xi during the relatively late phase Žmore than 4 h. after focal ischemia also contributes to the extent of neuronal damage, and the prolonged blockade of AMPA receptors by YM872 could also keep the wCa2q xi below toxic level, even though the reduction of wCa2q xi by the treatment with YM872 of 20 mgrkgrh for only 4 h could not be enough to induce a sufficient neuroprotective effect. In other words, the AMPA receptor-related elevation of wCa2q xi after focal cerebral ischemia may reach the neurotoxic level both by an acute
Ca2q influx induced by the increase in glutamate in the early phase and a sustained Ca2q influx in the relatively late phase. Tomabechi et. al. w28x found the sustained accumulation of Ca2q for 24 h in the ischemic brain tissue after focal ischemia in rats. According to their report, the rapid increase in wCa2q xi occurred during the first 5 h after MCA occlusion, and continued more slowly from 5 to 24 h. Konig et. al. w13x reported that long-lasting wCa2q xi ¨ elevation following AMPA exposure was observed in the rat brainstem cells. These findings may support our hypothesis although the time course of the inhibitory effect of YM872 on wCa2q xi levels after ischemic or excitotoxic insults has not been investigated. This hypothesis indicates that prolonged treatment with AMPA antagonists may be useful in the treatment of human stroke, even when the infusion rate is limited due to side effects such as respiratory depression or nephrotoxicity. Since the brain concentration of YM872 reached approximately 100 ngrg at a dose of 20 mgrkgrh in our preliminary study Ždata not shown., the effective brain concentration in this model was estimated to be about 100 ngrg Ž; 0.3 m M.. This is comparable to the concentration of YM872 which selectively inhibits AMPA receptor binding without blocking NMDA as well as other receptors in vitro w12x, and the CSF level at the neuroprotective dose in a cat MCA occlusion model w27x. This indicates that the neuroprotective effect of YM872 in this study is mainly due to the selective blockade of AMPA receptors in the central nervous systems. The brain concentration of YM872 at 10 mgrkgrh may not be enough to extend neuroprotection since no reduction in the infarct volume was observed even by 24 h i.v. infusion. The cortical infarct volume of the saline-treated group in the 24 h i.v. infusion study was relatively smaller and more varied compared with that in the 4 h i.v. infusion study. The reason why this difference occurred remains unknown, but the hemodilution effect might be induced by extending the duration of treatment with saline w18x. Although the decrease in the hematocrit value was not detected in the 24 h infusion study, its possibility may not be excluded completely because it was measured at several timepoints, but not continuously, after MCA occlusion. To clarify this point, further studies are needed to examine whether cerebral blood flow in the MCA territory is affected by saline infusion during the ischemic period. Monitoring of body temperature and physiological parameters, including MABP and blood gases, is important in the evaluation of neuroprotective agents in cerebral ischemia models, since these parameters may affect neuronal damage w3,6,16x. In this study, there were no significant differences in the brain and rectal temperatures, physiological parameters between the corresponding groups except for arterial pCO 2 . In addition, in our preliminary study using anesthetized cats, CBF was not affected by YM872 administration at the effective dose in MCA occluded cats. These results suggest that the neuroprotective
S. Kawasaki-Yatsugi et al.r Brain Research 793 (1998) 39–46
effect of YM872 may not be attributed to non-specific effects such as hypothermia or cerebral hemodynamic effects. Arterial pCO 2 tended to be higher Žcloser to the normal value. in the YM872 groups than in the saline group in the 24 h i.v. infusion study. Spontaneous hyperventilation induced by pronounced ischemic brain damage w14x might be prevented by the administration of YM872.
w12x
w13x
w14x
5. Conclusion The present data provides evidence that YM872, a novel AMPA receptor antagonist, may reduce the neuronal damage in a focal cerebral ischemia model in rats and, therefore, YM872 may be a useful tool to investigate the pathophysiological role of AMPA receptors in models of ischemia. Because the neuroprotective effect of YM872 was enhanced by the prolonged duration of the treatment following focal cerebral ischemia, the long-term administration of YM872 could be useful in the treatment of human stroke.
References
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