臺灣博碩士論文加值系統

研究指出腺苷能調節神經過度的衝動，因此維持腦部高腺苷濃度應能有效治療癲癇。根據先前的研究結果，equilibrative nucleoside transporter 1 (ENT1)的抑制劑，能夠有效地增加細胞外液的腺苷濃度。因此本研究將利用三種癲癇動物模型來評估兩種ENT1抑制劑作為抗癲癇藥物的潛力。首先，代表強直-陣攣型癲癇的maximal electroshock seizure (MES) 動物模型中，兩種測試藥物在高劑量的情況下都能夠有效的縮短發作後的症狀持續時間，其中J7更能夠有效的抑制癲癇發作。其次再以高劑量腹腔注射pentylenetetrazole (PTZ) 所誘發之肌痙攣型癲癇此模型上，兩種藥物在較低劑量都能有效延遲癲癇發作的效果，其中J7的延遲效果十分顯著，並在分析其行為表現後，發現J7能夠有效的抑制癲癇級數，但J4無法在癲癇發作後有效的抑制其行為表現。
最後，我們以低劑量PTZ來造成腦部慢性的永久損傷之動物模型 (Kindling模型) 來評估病發後給予藥物之治療效果，有別於急性動物模型，這個模型透過觀察腦波來評估其癲癇是否有反覆發作的情形，在確認成功誘發後，於每天固定時間給予兩種測試藥物，持續一周後停止藥物治療，並繼續觀察一周評估其治療情況。根據實驗結果，在給予藥物治療期間，J7與J4都能夠有效地降低癲癇發作的時間與次數，其中J7的抑制效果又與藥物carbamazepine (CBZ) 相似。
這些結果顯示，J7及J4在兩種急性與一種慢性癲癇模型下都展現出抗癲癇的效果，而其中又以J7更具發展潛力。

As adenosine can regulate the excitability of neuron cells, it can be effective in the treatment of epilepsy by maintaining high levels of adenosine in the brain. Previous study has shown that, the inhibition of equilibrative nucleoside transporter 1 (ENT1) can increase brain extracellular levels of adenosine. Thus, the present study aimed to assess the effectiveness of ENT1 inhibitors on the treatment of epilepsy using three epilepsy animal models.
First, the maximal electroshock seizure (MES) animal model was used to evaluate the effectiveness of ENT1 inhibitors on tonic-clonic epilepsy. The results showed that high dose of J7 and J4 can effectively decrease the duration of seizure after the seizure occurred. J7 was also effective in inhibiting the occurrence of seizures.
Second, acute epilepsy model established by high-dose intraperitoneal injection of pentylenetetrazole (PTZ) was used to evaluate the effectiveness of ENT1 inhibitors on myoclonic epilepsy. In this model, both test drugs can effectively delay the seizures onset, in which J7, but not J4, was able to comprehensively inhibit the Racine’s score.
Finally, chronic animal model (Kindling model) induced by low-dose PTZ was use to assess the therapeutic effect of these drugs on permanent chronic brain damage. In this model, both J7 and J4 were able to reduce the time and frequency of seizures. The effect of J7 was similar to that of carbamazepine (CBZ).
These findings showed that both J7 and J4 exhibit antiepileptic effects in both acute and chronic epilepsy models. Especially, J7 can be more promosing for further anti- epileptic development.

目錄 I
摘要 III
Abstract IV
圖目錄 VI
表目錄 VII
第一章 緒論 1
1.1 現行癲癇疾病簡介 1
1.2 癲癇藥物簡介 2
1.3 癲癇動物模型與藥物開發之關係 3
1.4 腺苷與癲癇之關係 4
第二章 研究動機與目的 12
第三章 實驗方法與材料 13
3.1 實驗材料 13
3.2 實驗動物 14
3.3 動物行為檢測 14
3.4 急性動物模型 14
3.5 慢性動物模型 15
3.5.1 電擊手術操作 15
3.5.2 低劑量PTZ誘發之Kindling模型 15
3.6小鼠腦部微透析實驗 16
3.6.1 體外回收率測試 16
3.6.2 腦部定位並埋入探針外管 16
3.6.3清醒小鼠腦部微透析 16
3.7 線嘌呤核苷及核苷酸螢光衍生化及極致層相層析 17
3.7.1 螢光衍生化 17
3.7.2 高效能液相層析 17
3.8實驗數據分析 18
第四章 實驗結果 19
4.1 數字系利於MES誘發癲癇模型之藥物評估 19
4.1.1 MES模型之建立 19
4.1.2 J7於MES誘發癲癇模型藥效評估 19
4.1.3 J4於MES誘發癲癇模型藥效評估 20
4.2 數字系列於高劑量PTZ誘發癲癇模型之藥物評估 20
4.2.1 高劑量PTZ模型之建立 20
4.2.2 J7對高劑量PTZ誘發的癲癇模型藥效評估 21
4.2.3 J4對高劑量PTZ誘發的癲癇模型藥效評估 21
4.3 數字系列對於慢性癲癇點燃模型之藥效評估 22
4.3.1 癲癇點燃模型之建立 22
4.3.2 J7與J4對點燃癲癇模型藥效評估 22
4.3.3 癲癇小鼠腦內腺苷評估 23
4.4 透過電生理分析藥物效果 23
第五章 討論 36
第六章 參考資料 41

1.Simonato, M., French, J. A., Galanopoulou, A. S., and O''Brien, T. J. (2013) Issues for new antiepilepsy drug development. Curr Opin Neurol 26, 195-200
2.Castel-Branco, M. M., Alves, G. L., Figueiredo, I. V., Falcao, A. C., and Caramona, M. M. (2009) The maximal electroshock seizure (MES) model in the preclinical assessment of potential new antiepileptic drugs. Methods Find Exp Clin Pharmacol 31, 101-106
3.Rubio, C., Rubio-Osornio, M., Retana-Marquez, S., Veronica Custodio, M. L., and Paz, C. (2010) In vivo experimental models of epilepsy. Cent Nerv Syst Agents Med Chem 10, 298-309
4.Dhir, A. (2012) Pentylenetetrazol (PTZ) kindling model of epilepsy. Curr Protoc Neurosci Chapter 9, Unit9 37
5.Boison, D. (2016) Adenosinergic signaling in epilepsy. Neuropharmacology 104, 131-139
6.Swiader, M. J., Kotowski, J., and Luszczki, J. J. (2014) Modulation of adenosinergic system and its application for the treatment of epilepsy. Pharmacol Rep 66, 335-342
7.Glass, M., Faull, R. L., Bullock, J. Y., Jansen, K., Mee, E. W., Walker, E. B., Synek, B. J., and Dragunow, M. (1996) Loss of A1 adenosine receptors in human temporal lobe epilepsy. Brain Res 710, 56-68
8.Thomas, T. P., and Shih, T. M. (2014) Stimulation of central A1 adenosine receptors suppresses seizure and neuropathology in a soman nerve agent seizure rat model. Toxicol Mech Methods 24, 385-395
9.D''Alimonte, I., D''Auro, M., Citraro, R., Biagioni, F., Jiang, S., Nargi, E., Buccella, S., Di Iorio, P., Giuliani, P., Ballerini, P., Caciagli, F., Russo, E., De Sarro, G., and Ciccarelli, R. (2009) Altered distribution and function of A2A adenosine receptors in the brain of WAG/Rij rats with genetic absence epilepsy, before and after appearance of the disease. Eur J Neurosci 30, 1023-1035
10.Moezi, L., Mansoori, E., Niknahad, H., and Shafaroodi, H. (2014) The role of alpha-2 adrenoceptors in the anticonvulsant effects of adenosine on pentylenetetrazole-induced seizure threshold in mice. Pharmacol Biochem Behav 126, 36-42
11.Moschovos, C., Kostopoulos, G., and Papatheodoropoulos, C. (2012) Endogenous adenosine induces NMDA receptor-independent persistent epileptiform discharges in dorsal and ventral hippocampus via activation of A2 receptors. Epilepsy Res 100, 157-167
12.Xu, Z., Xu, P., Chen, Y., Liu, J., Zhang, Y., Lv, Y., Luo, J., Fang, M., Zhang, J., Wang, J., Wang, K., Wang, X., and Chen, G. (2015) ENT1 inhibition attenuates epileptic seizure severity via regulation of glutamatergic neurotransmission. Neuromolecular Med 17, 1-11
13.Shih, J. J., Tatum, W. O., and Rudzinski, L. A. (2013) New drug classes for the treatment of partial onset epilepsy: focus on perampanel. Ther Clin Risk Manag 9, 285-293
14.Ham, J., and Evans, B. A. (2012) An emerging role for adenosine and its receptors in bone homeostasis. Front Endocrinol (Lausanne) 3, 113
15.Racine, R. J. (1972) Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 32, 281-294
16.Bhatt, D. P., Chen, X., Geiger, J. D., and Rosenberger, T. A. (2012) A sensitive HPLC-based method to quantify adenine nucleotides in primary astrocyte cell cultures. J Chromatogr B Analyt Technol Biomed Life Sci 889-890, 110-115
17.Locharernkul, C., Loplumlert, J., Limotai, C., Korkij, W., Desudchit, T., Tongkobpetch, S., Kangwanshiratada, O., Hirankarn, N., Suphapeetiporn, K., and Shotelersuk, V. (2008) Carbamazepine and phenytoin induced Stevens-Johnson syndrome is associated with HLA-B*1502 allele in Thai population. Epilepsia 49, 2087-2091
18.Livingston, S., Rodriguez, H., Greene, C. A., and Pauli, L. L. (1968) Systemic lupus erythematosus. Occurrence in association with ethosuximide therapy. JAMA 204, 731-732
19.Sinclair, C. J., Krizanac-Bengez, L., Stanness, K. A., Janigro, D., and Parkinson, F. E. (2001) Adenosine permeation of a dynamic in vitro blood-brain barrier inhibited by dipyridamole. Brain Res 898, 122-125
20.Schrader, J., Wahl, M., Kuschinsky, W., and Kreutzberg, G. W. (1980) Increase of adenosine content in cerebral cortex of the cat during bicuculline-induced seizure. Pflugers Arch 387, 245-251