1328613 九、發明說明： 【發明所屬之技術領域】 本發明係關於使用SIMP活化蛋白質激酶抑制劑來治療神 經退化疾病。 【先前技術】 神經退化疾病是一種逐步喪失神經細胞的病症。這類疾病的 例子包含阿茲海默氏症（Alzheimer’s disease)、帕金森氏症 (Parkinson’s disease)及漢丁頓舞蹈症（Huntington’s disease)，其中 漢丁頓舞蹈症是一種體染色體顯性缺陷（autosomal dominant disorder)的神經退化疾病，係因Huntingtin (Htt)基因第一表現子 上出現多餘的CAG三核苷酸序列重複片段而造成該疾病。相關 文獻例如可參考 Perutz et al.，Trends Biochem. Sci. 1999;24:56-63 及 Rubinsztein et al·, J. Med. Genet. 1999; 36:265-270.。漢 丁頓舞 蹈症病人會有異常的身體動作、痴呆及精神方面的問題。醫療藥 品，如多巴胺阻斷劑（dopamine blocker)，可減少異常的動作及行 為，但無法阻止神經退化。因此，需要一種可有效治療漢丁頓舞 蹈症及其他神經退化疾病的新的藥物。 【發明内容】 本發明係關於使用5’AMP活化蛋白質激酶抑制劑來治療神 經退化疾病。 在一方面，本發明係關於一種辨識用於治療神經退化疾病（例 如’漢丁頓舞蹈症）之化合物的方法。該方法包含：使表現5 ’AMP 活化蛋白質激酶(AMPK)的第一細胞與一化合物接觸；及決定該 5’AMP活化蛋白質激酶的表現量、填酸化程度或酵素活性。相較 於不與前述化合物接觸之第二細胞，如果第一細胞的5 ’AMP活化 蛋白質激酶表現量、鱗酸化程度或酵素活性低於以相同方法決定 的第二細胞5’AMP活化蛋白質酵素表現量、鱗酸化程度或酵素活 5 1328613 性，則該化合物係為可有效治療神經退化疾病之化合物。第一及 第二細胞的例子包含神經膠細胞及神經元細胞。本發明之辨識方 法係可在體内或體外進行。在一體内實施態樣中，第一及第二細 胞係存在於非人類動物中，例如，存在於非人類動物的紋狀體 (striatum)中。適當的非人類動物係為老鼠，例如R6/2老鼠。 前述5’AMP活化蛋白質激酶係指一全長5’AMP活化蛋白質 激酶多胜肽（例如Carling等人所描述的哺乳動物、酵母菌或植物 AMP 活化蛋白質酵素，1994, J. Biol. Chem. 269:11442-11448)或 具有相同功能的等同物（equivalent)。相同功能的等同物係指 5’AMP活化蛋白質激酶蛋白質衍生之多胜肽，例如，融合多胜 · 肽、具有一或多個突變、插入、刪除、截斷之多胜肽或其結合。 該多胜肽大致上可保有5’AMP活化蛋白質激酶之酵素活性，換言 之，可保有使目標蛋白質磷酸化之能力，例如下列參考文獻中所 描述者：Lee et al·，J. Biol. chem. 2003;278:39653-39661; Hong et al·，J. Biol Chem. 2003; 278:27495-27501;及 Fryer et al，J. Biol.1328613 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to the use of SIMP to activate protein kinase inhibitors for the treatment of degenerative diseases. [Prior Art] A neurodegenerative disease is a condition in which nerve cells are gradually lost. Examples of such diseases include Alzheimer's disease, Parkinson's disease, and Huntington's disease, in which Handington's disease is a somatic chromosome dominant defect ( Autosomal dominant disorder is a neurodegenerative disease caused by the appearance of redundant CAG trinucleotide repeats on the first expression of the Huntingtin (Htt) gene. For related literature, for example, see Perutz et al., Trends Biochem. Sci. 1999; 24:56-63 and Rubinsztein et al., J. Med. Genet. 1999; 36:265-270. Handington's patients have abnormal physical movements, dementia and mental problems. Medical drugs, such as dopamine blockers, reduce abnormal movements and behavior, but do not prevent neurodegeneration. Therefore, there is a need for a new drug that is effective in treating Huntington's disease and other neurodegenerative diseases. SUMMARY OF THE INVENTION The present invention relates to the use of 5'AMP-activated protein kinase inhibitors to treat neurodegenerative diseases. In one aspect, the invention relates to a method of identifying a compound for use in the treatment of a neurodegenerative disease, such as 'Hantington's disease. The method comprises: contacting a first cell expressing 5' AMP-activated protein kinase (AMPK) with a compound; and determining the amount of expression, acidification, or enzyme activity of the 5'AMP-activated protein kinase. Compared to the second cell not in contact with the aforementioned compound, if the 5 'AMP-activated protein kinase, the degree of scalification or the enzyme activity of the first cell is lower than that of the second cell 5'AMP-activated proteinase determined by the same method The amount, the degree of squamization or the enzyme activity 5 1328613, the compound is a compound effective for treating neurodegenerative diseases. Examples of the first and second cells include glial cells and neuronal cells. The identification method of the present invention can be carried out in vivo or in vitro. In an integrated embodiment, the first and second cell lines are present in a non-human animal, for example, in a striatum of a non-human animal. Suitable non-human animal lines are mice, such as R6/2 mice. The aforementioned 5'AMP-activated protein kinase refers to a full-length 5'AMP-activated protein kinase polypeptide (for example, mammalian, yeast or plant AMP-activated proteinase as described by Carling et al., 1994, J. Biol. Chem. 269: 11442-11448) or equivalent with the same function. The equivalent of the same function refers to a 5'AMP-activated protein kinase protein-derived multi-peptide, for example, a fusion poly-peptide, a multi-peptide having one or more mutations, insertions, deletions, truncations, or a combination thereof. The multi-peptide is substantially capable of retaining the enzyme activity of the 5'AMP-activated protein kinase, in other words, retaining the ability to phosphorylate the target protein, as described in the following references: Lee et al., J. Biol. chem. 2003; 278:39653-39661; Hong et al., J. Biol Chem. 2003; 278:27495-27501; and Fryer et al, J. Biol.
Chem. 2002;277: 25226-25232 « 另一方面’本發明係關於一種治療神經退化疾病（例如，漢丁 頓舞蹈症）的方法。該方法包含辨識對象已罹患神經退化疾病或存 在發展成神經退化疾病的風險，並提供對象有效劑量之5’AMP # 活化蛋白質激酶抑制劑《名詞”抑制劑”係指具有可抑制5，AMP活 化蛋白質激酶基因表現量、後轉譯修飾（例如，磷酸化）或酵素活 性能力的化合物，且該抑制具有統計上的意義。這些試劑包含， 但不限於，非有機小分子、有機小分子、胜肽、抗體及核酸（例如， 反義RNA、核醣酵素或RNA干擾試劑）。例如，有機小分子可為 · 下述實施方式及實施例中的CGS21680。 另一方面，本發明係關於一種治療5’AMP活化蛋白質激酶相 關疾病的方法。該方法包含辨識對象已罹患5’AMP活化蛋白質酵 6 1328613Chem. 2002; 277: 25226-25232 «On the other hand, the present invention relates to a method of treating a neurodegenerative disease (e.g., Huntington's disease). The method comprises identifying that the subject is at risk of developing a neurodegenerative disease or developing a neurodegenerative disease, and providing an effective dose of the subject 5' AMP #activated protein kinase inhibitor "noun" inhibitor" means inhibiting 5, AMP activation A protein kinase gene expresses a compound that is post-translationally modified (eg, phosphorylated) or enzymatically active, and the inhibition is statistically significant. These agents include, but are not limited to, non-organic small molecules, small organic molecules, peptides, antibodies, and nucleic acids (eg, antisense RNA, ribozyme, or RNA interference reagents). For example, the organic small molecule may be CGS21680 in the following embodiments and examples. In another aspect, the invention features a method of treating a 5'AMP-activated protein kinase-associated disease. The method comprises identifying that the subject has suffered from 5'AMP-activated protein leaven 6 1328613
素相關疾病或存在發M ^廿捍祝财么 展成5，AMP活化蛋白質激酶相關疾病的風 有致劑量之前述__。“價P活化蛋白質 酵素相關疾病’’係指邀 Λ ^ 、5 AMP活化蛋白質激酶基因表現、磷酸化 程度或酵素活性提昇古 有關之病症。這類疾病包含，但不限於，糖 尿病及肥胖症。 圍亦包含兩種組裝產品（package product)。一組 裝產品包含一容考、—士 ° 有效量之5’AMP活化蛋白質激酶抑制劑及 —附於谷器之說明，兮％ 邊説明指出提供5’AMP活化蛋白質激酶抑制 劑係用於/α療已罹患神經退化疾病或存在發展成神經退化疾病 風險的對f另—組裝產品包含-容器、-有效量之CGS21680 及附，於令器之說明’該說明指出提供CGS21680係用於治療已 羅心’ 5 AMP ’g·化蛋白質激酶相關疾病或存在發展$ 颜p活化 蛋白質激酶相關疾病風險的對象。 本發明之-個或多個實施態樣係詳述如下，本發明之特色、 目的及優點皆描述於實施方式及中請專利範圍中。 【實施方式】 本發明係根據-項超出預期的發現，ΑΜρκ抑制劑，例如 CGS2168G，·可有效改善R6/2老鼠模型的數種主要漢丁頓舞蹈症 病理特徵。③些AMPK抑制财用來治療漢了頓舞蹈症及其他神 經退疾病6 SUb >本發明係’關於—種辨㈣於治療神經退化疾病之 AMPK抑制劑的方法。ΑΜρκ抑制劑可自市面購得或依循下述方 法或其他本技術領域已知的方法來辨識。 候選化合物（例如，蛋白質、胜肽、模擬胜肽 (PePtld〇mimetlC)、甘胺酸胺基取代的陽離子募聚合物 (peptcuds)、抗體、小分子或其他藥物）可使用本技術領域已知 的組合資料庫方法中的任何方法獲得。這些資料庫包含：胜肽資 1328613 料庫、peptoid資料庫（骨幹為一新穎、非胜肽骨幹，但具有胜肽 功能並能抑制酵素降解的分子資料庫）；空間定位（spatial ly addressable)平行固相或液相資料庫；藉由去迴旋 (deconvolutiuon)或親和性色層分析選擇獲得之合成資料庫；及” 一珠粒一化合物”資料庫。例如可參考Zuckermann et al. 1994, J. Med. Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12:145.。合成資料庫的合成方法例子可參考下列文獻，例 如，DeWitt et al. , 1993，PNAS USA 90:6909; Erb et al.，1994, PNAS USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al. , 1993, Science 261 : 1303; Carrel 1 et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al.，1994 J. Med. Chem. 37:1233.。這些化合物資料庫可出現 在溶液中（例如，Houghten，1992，Biotechniques 13:412-421)， 或珠粒上（Lam, 1991，Nature 354:82-84)、晶片（Fodor，1993, Nature 364:555-556)、細菌（美國專利第5, 223, 409號）、種子（美 國專利第 5,223,409 號）、質體（Cull et al·，1992，PNAS USA 89:1865-1869)、或嗟菌體（Scott and Smith 1990，Science 249:386-390； Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, PNAS USA 87:6378-6382; Felici 1991, J. Mol. Biol. 222:30卜310; and U.S. Patent No. 5,223,409)。 為了辨識AMPK抑制劑，可使候選化合物與一包含AMPK的系 統接觸；該系統可為不含細胞的系統或包含細胞的系統，例如， 體外細胞株模型或體内動物模型。在包含細胞的系統中，細胞可 自然地表現AMPK基因，或被修飾來表現重組核酸。前述重組核 酸可包含：融合於異源啟動子之AMPK基因密碼區或融合於報導 基因之AMPK基因啟動子序列。然後量測AMPK的表現量、磷酸化 1328613 能力或酵素活性。 表現量可藉由mRNA表現量或蛋白質量來決定。量測組織或 體液樣本中mRNA表現量的方法已是本技術領域熟悉的技術。為 了量測mRNA表現量，可溶解細胞，然後決定細胞溶解物、或細 胞溶解物中純化或半純化RNA中的mRNA量，測量mRNA量的方法 可為’例如’雜父檢測（hybridizaiton assay)(使用可偵測之標 記基因專一 DNA或RNA探針）及定量或半定量rT_pcr(使用適當的 基因專一引子）。或者，可使用組織切片或未水解細胞懸浮液及 可偵測的（例如，螢光或酵素）已標記DNA或RNA探針來執行定量 或半定量原位雜交檢測。其他mRNA定量方法包含RNA保護檢測 (RPA)及 SAGE。 量測組織或體液樣本中蛋白質表現量的方法也已是本技術 領域熟悉的技術。這些量測方法中有多種方法使用可與目標蛋白 質專一性結合的抗體（例如’單株抗體或多株抗體）。在這些檢測 中’抗體本身或與其結合的第二抗體可被標記以供偵測。或者， 這些抗體可與生物素結合’並可使用具可偵測標記之抗生物素蛋 白（可與生物素結合的多胜肽）來偵測該生物素化抗體的存在。 結合前述方法（包含’’多層三明治”檢測）可提升這些方法的靈 敏度。部分蛋白質檢測方法（例如，ELISA或西方墨點法）可用於 檢測體液或細胞溶胞產物，其他方法（例如，免疫組織方法或營 光流式細胞儀）則可用於檢測組織切片或未水解之細胞懸浮液。 量測標記物質數量的方法係根據該標記物質的特性，這些方法已 是本技術領域熟悉的技術。適當的標記物質包含放射性同位素 (例如，125I、131I、35S、3H、或32P)、酵素（例如，鹼性磷酸酵素 (alkaline phosphatase)、山葵過氧化酵素（horseradish peroxidase)、冷光素（luciferase)、或β-半乳糖酵素 (β-galactosidase)、螢光物質（例如：螢光素（fluorescein)、 1328613 羅單明（rhodamine)、藻紅素（phycoerythrin)、綠色螢光蛋白 (GFP)或藍色螢光蛋白（BFP))或發光物質（例如：QdotT!i奈米粒 子，由 Quantum Dot Corporation, Palo Alto，CA 所提供）；其 他可應用的檢測法包含定量免疫沈激法（immunoprecipitation) 或補體結合試驗（complement fixation assay)。 AMPK的磷酸化能力及酵素活性可藉由實施例甲所述的方法 量測，或使用本技術領域已知的方法，例如下列參考資料中所述 的方法：Rutteret al.，2003，Biochem. J. ; 375:1-16; Lee et al., J. Biol. Chem. 2003; 278:39653-39661 ； Hong et al., J. Biol. Chem. 2003; 278:27495-27501 ;及 Fryer etal·，J. Biol. Chem. 2002; 277:25226-25232.。 為了決定候選化合物抑制AMPK的能力，比較存在候選化合 物之實驗組及缺乏候選化合物之控制組的AMPK磷酸化程度及酵 素活性’測量方法係使用前述方法。如果實驗組的磷酸化程度或 酵素活性低於控制組，則該候選化合物可被視為具備有效治療神 經退化疾病的能力。 進一步可使用動物模型來驗證前述化合物的功效。例如，可 利用基因轉殖R6/2老鼠模型來驗證化合物對漢丁頓舞蹈症的療 效（Mangiarini etal.，1996，Celll; 87:493-506) °R6/2 小鼠 會表現人類Huntingtin(Htt)基因第一表現子，具有122個或更 多的CAG三核芬酸序列重複片段，牠們會逐漸表現出漢丁頓舞蹈 症神經學上的顯性特徵，包含類舞蹈動作、非自願的固定動作、 震顔、癲癇發作及非動作異常的症狀。提供前述候選化合物給 R6/2小鼠並依下列實施例所述的方法或其他標準方法進行試 驗。R6/2小鼠精神狀況任何統計學上有意義的改善都顯示該化合 物係為治療漢丁頓舞蹈症的候選化合物。 本發明亦關於一種治療神經退化疾病的方法。被處理的對象 1328613 =猎=經退化疾病（例如，漢丁頓舞蹈症）的標準診斷方 理對象也可選擇性地藉由前述方法檢測其順基因表 性;如果該對象樣本中的基因表現或酵素活性程度較 候=本^則該對象係為施予有效劑量之綱抑制劑治療的 化广療，ting)”係指為提供一化合物予罹患神經退 ^調_、緩和、醫治、預防，或改 。^失調症狀、續發失調疾病體f或後天失調。，，有效量 =產生預期醫療效果之化合物劑量，例如：前述料—受測對 方^量。治Ϊ方法係以活體内（in viv〇)或活體外(ex vivo)的 方式把仃，可卒獨或與其他藥物或治療方式合併使用。 〜療方法中，係提供職抑制劑給對象。一般而 :该化。物懸洋於一藥物學上可接受之載體(例 水），亚以口服方式提供，或用靜脈注入、注射，或皮下、肌t 鞘内、腹膜内、直腸内、陰㈣、# : 、 直接運送至紋狀體，換言之，芦由纹莫^貝舞心症），該化合物可 提供該化合物。 胃⑽㈣（齡astriatal)注射 質，^:^大=選重^^路#%配方性質，病人疾病性 的藥物，及醫師的判斷來決定。適當積的二二別：被提供 大的調整範圍’例如：口服方式需要比靜脈:主射：ί二:：有很 藥劑量的變動可根攄木Λ靜脈射更冋的劑置。用 來調整1用適當的運送料(例如被瞭解的最佳標準經驗法則 包覆藥物能增加運送效率，特別是；：或可植入裝置） 此外了提供—含有順抑制劑編碼核酸序列之聚核普酸 1328613 給對象。前述核酸序列的編碼包含可辨識ampk並抑制其表現或 酵素活性之抗AMPK抗體、反義RNA(anti-sense RNA)或小型干擾 RNA ( small interference RNA)。前述聚核苷酸可使用本技術領 域已知的高分子、生物可降解微粒或微膠囊輸送裝置來提供給對 象。另一種達到攝取核酸的方法係使用標準程序製備的微脂粒 (liposome)。前述聚核苷酸係可單獨或與組織專一抗體一起併入 運輸載具（delivery vehicle)中。並且也可視需要選擇性地製備 由質體或其他藉由靜電力或共價鍵連結在聚-L-賴胺酸 (poly-L-lysine)之載體組成的分子共軛化合物，聚-L-賴胺酸與 一可與目標細胞上受體結合之配體（ligand)鍵結（Cristiano，et al. (1995) J. Mol. Med. 73:479)。再者，係可視需要選擇性 地藉由本技術領域所熟悉的組織特異性轉錄調節元件 (transcriptional regulatory elements，TRE)來標定特定組 織。傳送”裸露DNA(naked DNA)”（換言之，不需運輸載具）到肌 肉、皮内、皮下處係為另一種達到體内表現的方法。 在前述的聚核苷酸中，例如，表現載體，編碼AMPK抑制劑 之核酸序列係與啟動子或增強子-啟動子組合相連結。合適的表 現載體包含質體和病毒載體，例如：泡療病毒（herpes viruses)、 反轉錄病毒（retroviruses)、牛痕病毒（vaccinia viruses)、減 毒牛殖疫苗（attenuated vaccinia viruses)、金絲雀痕病毒 (canary pox viruses)、腺病毒（adenoviruses)及腺病毒之相關 病毒（adeno-associated viruses) ° 如醫藥領域人士所熟知，病人所需之藥劑量係根據許多因素 決定，包含：病人體重、身體表面積、年齡、所施予藥物的特殊 性、性別、時間、給藥途徑、一般健康狀態以及是否有其他同時 施用之藥物。劑量可以改變，但施予聚核苷酸之較佳劑量係為 106-1012個聚核苷酸分子。如果需要，可重複施予該劑量，給藥 12 1328613 途徑可為前述之任何途徑。 以活體外方式治療對象之神經退化疾病可包含轉染 (transfecting)或轉導（transducing)可編碼AMPK抑制劑之聚核 苷酸至受測對象之細胞。另外，亦可設計載體，使其於活體外轉 染細胞，以便藉由同源重組（homologous recombination)的方 式，於細胞基因組中内源性AMPK抑制劑之轉錄起始位置前插入 一段新的、具活性的啟動子。經挑選及放大該可表現所需量之 AMPK抑制劑之細胞後，將前述經轉染或轉導細胞送回受測對象體 内。這些細胞包含神經細胞（neural cel Is )、造血細胞 (hemopoietic cel Is)(例如：骨髓細胞（bone marrow cel Is)、 巨嗟細胞（macrophages )、單核白血球（monocytes)、樹突細胞 (dendritic cells)、T 細胞（T cells)或 B 細胞（B cells))、 纖維細胞（fibroblasts)、上皮細胞（epithelial cells)、内 皮細胞（endothelial cells)、角質細胞（keratinocytes)或 肌肉細胞（muse 1 e ce 11 s )。前述經轉染或轉導細胞只要存活於 受測對象體内即可作為AMPK抑制劑之來源。 前述活體外方法之步驟係包含：從對象獲取細胞；培養細 胞；轉導表現載體於前述細胞；及維持細胞生長於可表現AMPK 抑制劑之適當環境。轉導作用係可藉由任何活體外基因治療之標 準方法實施，包含：破酸約（calcium phosphate )、微脂粒感染 (lipofection)、電穿孔（electroporation)、病毒感染以及基因 搶（biolistic)基因轉殖，亦可視需要選擇使用微脂粒 (liposomes)或高分子微粒（polymeric microparticles);細 胞成功轉殖基因後，接著被篩選出來以表現轉殖之基因，例如表 現AMPK抑制劑；然後，該成功轉殖基因之細胞以注入或植入方 式送回到對象體内。 本發明之目的進一步包含一種組裝產品，包含：一容器、一 13 1328613 有效量之AMPK抑制劑及一附於容器之說明，該說明指出提供ΑΜρκ 抑制劑用於治療已罹患神經退化疾病或存在發展成神經退化疾 病風險的對象。前述抑制劑可與醫藥可接受性之載體混合，係包 含：溶劑、分散媒介（dispersion medium)、包覆外衣（coating)、 抗細菌（antibacterial)及抗黴菌（antifungal)試劑、及等 滲透壓之延遲吸收試劑（absorption delaying agent)。 前述抑制劑可使用傳統方法製成不同施藥途徑的藥劑配方 令’例如·可製成膠囊、密封凝膠（gel seal)或口服錠劑中；膠 囊可包含任何醫藥可接受之材料，例如：明膠（gelatin)或纖 維素（cellulose);錠劑可依照傳統的程序配製，係藉由壓縮抑 制劑、固悲載體及潤滑劑而製成’固態載體包含，例如：殿粉 (starch)和糖膨潤土（sugar bentonite);該抑制劑亦可以一 含有黏結劑（binder )(例如：乳糖（iactose )或甘露醇 (mannitol))、傳統常用的填充物及製錠試劑（tabletingagent) 之硬设旋劑或膠囊形式施用。前述抑制劑可經由非腸胃途徑服 用，非腸胃藥劑形式包括，例如：水溶液、等張性生理食鹽水、 含5%葡萄糖之活性試劑、或其他習知醫藥可接受之賦形劑 (excipient )，環糊精（cyclodextrins)或其他本技術領域所熟 悉的助溶劑（solubilizing agent)可作為藥物賦形劑以運送治療 藥劑。此外，前述抑制劑可更進一步經由大腦手術直接注入紋狀 體。 抑制劑的功效可經由活體内和活體外（in vi tr〇)的方式評 估，例如：該抑制劑可在活體外測試其抑制ΑΜρκ基因表現或酵 素活性之能力；在活體内研究中，可將該抑制劑注射至一動物體 内（例如：一動物模型），並獲得其對神經退化疾病之功效。根據 前述結果，可決定適當的劑量範圍及藥物施用途徑。 前述ΑΜΡΚ抑制劑，例如CGS21680，可用於治療其他與ΑΜΡΚ 1328613 基因表現量或酵素活性異常高有關的疾病（例如，糖尿病或肥胖 症）。被處理對象也可選擇性地藉由前述方法或檢測對象樣本中 AMPK多胜肽基因表現或酵素活性來辨識；如果該對象樣本中的 AMPK多胜肽的基因表現或酵素活性程度較正常樣本高，則該對象 係為施予有效量之AMPK抑制劑治療的候選者。 下列實施例係僅用於說明本發明，非用於限制本說明書所揭 露之内容，任何熟習本技術領域的人士，皆可根據本說明書中揭 露之内容，進一步做任何可能的更動與潤飾以達到最大功效，所 有詳列於本說明書之著作係以全文引入作為參考資料。 實施例一 在本實施例中使用R6/2基因轉殖小鼠來研究CGS21680對 AMPK 的功效。來自 Jackson Laboratories(BarHarbor, Me, USA) 的雄性R6/2小鼠與同窩出生之控制組小鼠，與雌性控制組小鼠 (B6CBAFI//)交配。為確保其後代的CAG片段保持在約150個重 複長度，自鼠尾組織萃取基因組DNA ( genomic DNA)，並利用兩 段位於轉基因（transgene)上的引子（primer) (5’-ATGAAGGCCT TCGAG TCCCT CAAGT CCTTC-3’ ，及 5’-CTCAC GGTCG GTGCA GCGGC TCCTC AGC-3’）來進行 PCR 基因定性分析（PCR genotyping ) (Hogan # et al. (1994)In: Manipulating the mouse embryo: a laboratory manual, Ed 2. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory)。動物實驗係遵循台灣中央研究院動物保護 及利用委員會所認證的規範與步驟進行。 CGS21680 (Research Biochemicals, Natick, MA)溶解於含 1% DMS0的食鹽水溶液中，然後提供給小鼠。更具體而言，將16 隻7週大的R6/2小鼠分為兩組（每組8隻），然後分別從腹臈内 注射CGS21680(5pg/g體重）及同樣劑量之載體溶劑，每天注射一 15 1328613 次持續3. 5週。 之後，收集每隻老鼠的紋狀體組織細胞溶解碎片，並使用 Rutter等人在Biochem. J. 2003;375:1-16.中描述的標準西方 墨點法進行分析。更具體而言，膜碎片與包含125 mMTris-HCl(pH 6. 8)、20%甘油、1% SDS、15% 2-乙基硫醇（2-mercaptoethanol)、 200 mM二硫秀克糖醇（dithiothreitol)及0.01%溴盼藍 (bromphenol blue)的2X樣本緩衝液混合，並沸騰5分鐘；離心 移除不溶解的物質；並在8%分離凝膠上分離。在完成電泳分析之 後，將蛋白質轉移至聚乙二烯二氟薄膜上，以5%脫脂牛奶磷酸緩 衝液封鎖，並與抗AMPK抗血清（1:1〇〇〇，Abeam Limited, Cambridge, UK)或抗 AMPKP 抗血清（1:1〇〇〇，Cell Signaling Technology, Beverly，MA，USA)於 4°C 隔夜培養。之後以 PBS 清 洗三次，每次5分鐘；該薄膜自室溫下於過氧酵素-驢抗兔子免 疫球蛋白(peroxidase-conjugated donkey anti-rabbit IgG)(l :5000，Amersham，UK)中培養1小時，然後以PBS清洗三 次。免疫反應帶使用非放射性發光方法（light emitting nonradioactive method)(ECL, Amersham，UK)顯現。AMPK 在 Thrl72 中的構酸化程度係使用 ImageQuant v. 3. 15(Moiecular dynamics)密度檢測儀檢測抗AMPK-P免疫反應帶來定量。使用相 同的方法檢測未提供CGS21680之外的其他同窩出生的野生型小 鼠。 結果發現三組小鼠的AMPK蛋白質量都大致相同，但僅提供 載體的R6/2小鼠的磷酸化程度（超過1〇〇%)高於控制組小鼠，顯 示高AMPK蛋白質磷酸化程度與顯性漢丁頓舞蹈症有關。並且， 提供CGS21680可顯著降低R6/2小鼠約35%的AMPK磷酸化程度。 因為AMPK磷酸化程度反應出其活化程度，此一結果顯示CGS21680 可以抑制AMPK的酵素活性。 1328613 實施例二 R6/2小鼠顯示受損的行動能力，包含類舞蹈動作、非自願的 僵硬動作、震顫及癲癇發作。在本實施例中研究CGS21680對R6/2 小鼠這些行動能力的影響。 兩組R6/2小鼠（每組8隻）依實施例一所述之相同方法，分別 持續5週提供CGS21680及載體溶劑。在每次供藥24小時後，如 文獻所述量測行動能力1〇分鐘（Lee et al. Chin. J.The related diseases or the presence of M ^ 廿捍 财 么 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展 展"Pat P-activated proteinase-associated disease" refers to the invocation of AMP, 5 AMP-activated protein kinase gene expression, phosphorylation, or enzyme activity to enhance ancient related disorders. Such diseases include, but are not limited to, diabetes and obesity. The package also contains two package products. One assembly product contains a test, an effective amount of 5'AMP-activated protein kinase inhibitor and a description attached to the barn. 'AMP-activated protein kinase inhibitors are used in /alpha therapy for neurodegenerative diseases or in the presence of a neurodegenerative disease. The other assembly products contain - containers, - effective amounts of CGS21680 and attached, instructions for the device 'This note indicates that the CGS21680 line is provided for the treatment of a subject with a '5 AMP 'g-protein kinase-related disease or a risk of developing a disease-activated protein kinase-associated disease. One or more embodiments of the invention DETAILED DESCRIPTION OF THE INVENTION The features, objects, and advantages of the present invention are described in the embodiments and the scope of the claims. - Items beyond expectations, ΑΜρκ inhibitors, such as CGS2168G, can effectively improve the pathological features of several major Hamiltonian choreas in the R6/2 mouse model. 3 AMPK inhibitors are used to treat Hantonian chorea and others. Neurodegenerative Disease 6 SUb> The present invention is a method for the treatment of AMPK inhibitors of neurodegenerative diseases. The ΑΜρκ inhibitors are commercially available or follow the methods described below or other methods known in the art. Can be identified. Candidate compounds (eg, proteins, peptides, peptides (PePtld〇mimetlC), glycine acid-substituted cationic polymer (peptcuds), antibodies, small molecules or other drugs) can be used in the art. Any of the known combinatorial library methods are available. These libraries contain: the peptide 1328613 library, the peptoid database (the backbone is a novel, non-peptide peptide backbone, but with peptide function and inhibits enzyme degradation) Molecular database); spatially ly addressable parallel solid or liquid database; selected by deconvolutiuon or affinity chromatography A synthetic library of choices; and a "bead-one compound" database. See, for example, Zuckermann et al. 1994, J. Med. Chem. 37:2678-2685; and Lam, 1997, Anticancer Drug Des. 12: 145. Examples of synthetic methods for synthetic databases can be found in, for example, DeWitt et al., 1993, PNAS USA 90:6909; Erb et al., 1994, PNAS USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678; Cho et al., 1993, Science 261: 1303; Carrel 1 et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994 , Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al., 1994 J. Med. Chem. 37:1233. These compound libraries can be found in solution (for example, Houghten, 1992, Biotechniques 13: 412-421), or on beads (Lam, 1991, Nature 354: 82-84), wafers (Fodor, 1993, Nature 364: 555-556), bacteria (U.S. Patent No. 5,223,409), seeds (U.S. Patent No. 5,223,409), plastids (Cull et al., 1992, PNAS USA 89:1865-1869), or bacteriophage (Scott and Smith 1990, Science 249: 386-390; Devlin, 1990, Science 249: 404-406; Cwirla et al., 1990, PNAS USA 87: 6378-6382; Felici 1991, J. Mol. Biol. 222: 30 卜 310; and US Patent No. 5,223,409). To identify an AMPK inhibitor, the candidate compound can be contacted with a system comprising AMPK; the system can be a cell-free system or a system comprising cells, for example, an in vitro cell strain model or an in vivo animal model. In a system comprising cells, the cells can naturally express the AMPK gene or be modified to express the recombinant nucleic acid. The aforementioned recombinant nucleic acid may comprise: an AMPK gene cryptodomain fused to a heterologous promoter or an AMPK gene promoter sequence fused to a reporter gene. Then measure the amount of AMPK, phosphorylation 1328613 or enzyme activity. The amount of expression can be determined by the amount of mRNA expression or the amount of protein. Methods for measuring mRNA expression in tissue or body fluid samples are well known in the art. In order to measure the amount of mRNA expression, the cells can be lysed, and then the amount of mRNA in the purified or semi-purified RNA in the cell lysate or in the cell lysate can be determined, and the method of measuring the amount of mRNA can be, for example, a hybridizaiton assay (hybridizaiton assay) Use detectable marker gene-specific DNA or RNA probes) and quantitative or semi-quantitative rT_pcr (using appropriate gene-specific primers). Alternatively, quantitative or semi-quantitative in situ hybridization assays can be performed using tissue sections or unhydrolyzed cell suspensions and detectable (e. g., fluorescent or enzymatic) labeled DNA or RNA probes. Other mRNA quantification methods include RNA protection assay (RPA) and SAGE. Methods for measuring protein expression in tissue or body fluid samples are also well known in the art. Among these methods, there are various methods using an antibody (e.g., 'mono antibody or multiple antibodies') that specifically binds to a target protein. In these assays, the antibody itself or a second antibody that binds thereto can be labeled for detection. Alternatively, these antibodies can bind to biotin' and the presence of the biotinylated antibody can be detected using a detectably labeled avidin (a multi-peptide that binds to biotin). Combining the aforementioned methods (including ''multilayer sandwich' detection) can increase the sensitivity of these methods. Some protein detection methods (eg, ELISA or Western blotting) can be used to detect body fluids or cell lysates, other methods (eg, immune tissue) The method or camp optical flow cytometer can be used to detect tissue sections or unhydrolyzed cell suspensions. The method of measuring the amount of labeling substances is based on the characteristics of the labeling substances, and these methods are well known in the art. The labeling substance comprises a radioactive isotope (for example, 125I, 131I, 35S, 3H, or 32P), an enzyme (for example, alkaline phosphatase, horseradish peroxidase, luciferase, or --galactosidase, fluorescent substances (eg fluorescein, 1328613 rhodamine, phycoerythrin, green fluorescent protein (GFP) or blue fluorescent protein (BFP)) or luminescent material (eg QdotT!i nanoparticle, by Quantum Dot Corporation, Palo Alto, CA Other applicable assays include quantitative immunoprecipitation or complement fixation assay. Phosphorylation and enzyme activity of AMPK can be measured by the method described in Example A, or using this Methods known in the art, such as those described in the following references: Rutter et al., 2003, Biochem. J.; 375: 1-16; Lee et al., J. Biol. Chem. 2003; 278:39653 -39661; Hong et al., J. Biol. Chem. 2003; 278:27495-27501; and Fryer et al., J. Biol. Chem. 2002; 277:25226-25232. In order to determine the ability of candidate compounds to inhibit AMPK The method for measuring the degree of AMPK phosphorylation and the activity of the enzyme in the control group in which the candidate compound is present and the control group lacking the candidate compound is used. If the phosphorylation degree or the enzyme activity of the experimental group is lower than the control group, the candidate compound is used. It can be considered as having the ability to effectively treat neurodegenerative diseases. Animal models can be further used to verify the efficacy of the aforementioned compounds. For example, the gene can be verified by the gene transfer R6/2 mouse model. The effect of the compound on Huntington's disease (Mangiarini et al., 1996, Celll; 87: 493-506) °R6/2 mice will express the first expression of the human Huntingtin (Htt) gene, with 122 or more CAG trinuclear acid sequence repeats, which gradually exhibit neurologically dominant features of Huntington's disease, including dance-like movements, involuntary fixations, seizures, seizures, and symptoms of non-action abnormalities. . The aforementioned candidate compounds were supplied to R6/2 mice and tested according to the methods described in the following examples or other standard methods. Any statistically significant improvement in mental status of R6/2 mice indicates that the compound is a candidate compound for the treatment of Huntington's disease. The invention also relates to a method of treating a neurodegenerative disease. The object to be treated 1328613 = Hunting = standard diagnostic object of degenerative disease (eg, Huntington's disease) can also be selectively detected by the aforementioned method; if the gene expression in the sample of the subject Or the degree of enzyme activity is lower than the current = this is the treatment of the effective dose of the class of agents for the treatment of chemotherapy, ting)" refers to the provision of a compound to the neurological retreat _, palliative, treatment, prevention , or change. ^ dysregulated symptoms, continually dysfunctional disease f or acquired disorders., effective amount = the dose of the compound that produces the expected medical effect, such as: the above-mentioned materials - the amount of the other side tested. The method of treatment is in vivo ( In viv〇) or in vitro (ex vivo) method, can be used alone or in combination with other drugs or treatments. In the treatment method, the department provides the agent to the subject. Generally: the chemical. In a pharmaceutically acceptable carrier (eg water), subcutaneously provided by intravenous injection, injection, or subcutaneous, intramuscular, intrathecal, intraperitoneal, intrarectal, vaginal (four), #:, direct delivery To the striatum, in other words The compound can provide the compound. Stomach (10) (four) (age astriatal) injection quality, ^: ^ large = selection weight ^ ^ road #% formulation properties, patient disease drugs, and Physician's judgment to decide. Appropriate product of the second two: is provided with a large adjustment range 'Example: Oral method needs to be more than vein: main shot: ί二:: There is a very dose change can be rooted in the raft venous shot more 冋The agent is used to adjust 1 with the appropriate carrier material (for example, the best standard rule of thumb is known to coat the drug to increase transport efficiency, especially; or implantable devices) The nucleic acid sequence of polynucleotide 1328613 is administered to a subject. The encoding of the aforementioned nucleic acid sequence comprises an anti-AMPK antibody, an anti-sense RNA or a small interference RNA which can recognize ampk and inhibit its expression or enzyme activity. The aforementioned polynucleotides can be provided to a subject using polymeric, biodegradable microparticles or microcapsule delivery devices known in the art. Another method of achieving nucleic acid uptake is the use of standard procedures for preparing vesicles. (liposome) The aforementioned polynucleotides may be incorporated into a delivery vehicle either alone or in combination with a tissue-specific antibody, and may optionally be prepared by plastids or other means by electrostatic or covalent bonds. A molecular conjugated compound linked to a poly-L-lysine carrier, a poly-L-lysine coupled to a ligand that binds to a receptor on a target cell (Cristiano, et al. (1995) J. Mol. Med. 73: 479). Further, optionally, tissue-specific transcriptional regulatory elements (TRE), which are familiar in the art, can be used as needed. Calibrate a specific organization. Transferring "naked DNA" (in other words, without transporting the carrier) to the muscle, intradermal, and subcutaneous is another way to achieve in vivo performance. In the aforementioned polynucleotide, for example, a expression vector, a nucleic acid sequence encoding an AMPK inhibitor is linked to a promoter or an enhancer-promoter combination. Suitable expression vectors include plastid and viral vectors, such as: herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canaries Canary pox viruses, adenoviruses, and adeno-associated viruses ° As is well known in the medical field, the amount of medication required by a patient is determined by a number of factors, including: patient weight, Body surface area, age, specificity of the drug administered, sex, time, route of administration, general state of health, and whether there are other drugs that are administered simultaneously. The dosage can vary, but a preferred dosage for administration of the polynucleotide is from 106 to 1012 polynucleotide molecules. The dose can be administered repeatedly if desired, and the route of administration 12 1328613 can be any of the foregoing routes. A neurodegenerative disease that treats a subject in an in vitro manner can include transfecting or transducing a polynucleotide encoding an AMPK inhibitor to a subject. Alternatively, vectors can be designed to transfect cells in vitro to insert a new, pre-transcriptional position of the endogenous AMPK inhibitor in the genome of the cell by homologous recombination. Active promoter. After selecting and amplifying the cells which can express the desired amount of the AMPK inhibitor, the transfected or transduced cells are returned to the subject. These cells contain neural cel Is and hemophoietic cel Is (eg, bone marrow cel Is, macrophages, monocytes, dendritic cells). ), T cells or B cells, fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells (muse 1 e ce 11 s ). The aforementioned transfected or transduced cells can be used as a source of AMPK inhibitors as long as they survive in the subject. The steps of the aforementioned in vitro method comprise: obtaining cells from a subject; culturing the cells; transducing the expression vector to the aforementioned cells; and maintaining the cells in an appropriate environment that exhibits an AMPK inhibitor. Transduction can be performed by any standard method of in vitro gene therapy, including: calcium phosphate, lipofection, electroporation, viral infection, and biolistic genes. For transfer, liposomes or polymeric microparticles may be selected as needed; after successful cell transfer, the cells are screened to express the transgene, such as an AMPK inhibitor; Cells that successfully transfect the gene are returned to the subject by injection or implantation. The object of the present invention further comprises an assembled product comprising: a container, a 13 1328613 effective amount of an AMPK inhibitor, and a description attached to the container, the instructions indicating the provision of a ΑΜρκ inhibitor for treating a neurodegenerative disease or a presence The subject of neurodegenerative disease risk. The aforementioned inhibitor may be mixed with a pharmaceutically acceptable carrier, including: a solvent, a dispersion medium, a coating, an antibacterial and an antifungal agent, and an isotonic pressure. Delay absorption agent. The aforementioned inhibitors can be formulated into a capsule, a gel seal or an oral lozenge using conventional methods for making a different application route; the capsules can comprise any pharmaceutically acceptable material, for example: Gelatin or cellulose; tablets can be formulated according to conventional procedures, made by compression inhibitors, solid carrier and lubricants. 'Solid carrier contains, for example, starch and sugar Suction bentonite; the inhibitor may also be a hardener containing a binder (eg, lactose or mannitol), conventionally used fillers, and tableting agents. Or in the form of a capsule. The aforementioned inhibitor may be administered via a parenteral route, such as an aqueous solution, isotonic saline, an active agent containing 5% dextrose, or other conventional pharmaceutically acceptable excipients, Cyclodextrins or other solubilizing agents known in the art can be used as pharmaceutical excipients to deliver therapeutic agents. In addition, the aforementioned inhibitors can be further directly injected into the striatum via brain surgery. The efficacy of the inhibitor can be assessed in vivo and in vitro (in vi tr〇), for example, the inhibitor can be tested in vitro for its ability to inhibit ΑΜρκ gene expression or enzyme activity; in in vivo studies, The inhibitor is injected into an animal (for example, an animal model) and its effect on neurodegenerative diseases is obtained. Based on the foregoing results, the appropriate dosage range and route of administration of the drug can be determined. The aforementioned guanidine inhibitors, such as CGS21680, can be used to treat other diseases (e.g., diabetes or obesity) that are associated with an abnormal amount of ΑΜΡΚ 1328613 gene expression or enzyme activity. The treated subject can also be selectively identified by the aforementioned method or by detecting the AMPK polypeptide gene expression or enzyme activity in the sample of the subject; if the AMPK polypeptide in the subject sample has a higher gene expression or enzyme activity than the normal sample The subject is a candidate for the administration of an effective amount of an AMPK inhibitor treatment. The following examples are only intended to illustrate the invention, and are not intended to limit the disclosure of the present specification. Any person skilled in the art can further make any possible changes and retouching according to the contents disclosed in the specification. Maximum efficacy, all the works detailed in this manual are introduced as a reference in full text. Example 1 In this example, R6/2 gene-transferred mice were used to study the efficacy of CGS21680 on AMPK. Male R6/2 mice from Jackson Laboratories (Bar Harbor, Me, USA) were co-administered with control group mice from the control group (B6CBAFI//). To ensure that the CAG fragment of its progeny remains at approximately 150 repeat lengths, genomic DNA was extracted from the rat tail tissue and two primers located on the transgene (5'-ATGAAGGCCT TCGAG TCCCT CAAGT) were utilized. CCTTC-3', and 5'-CTCAC GGTCG GTGCA GCGGC TCCTC AGC-3') for PCR genotyping (Hogan # et al. (1994) In: Manipulating the mouse embryo: a laboratory manual, Ed 2. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory). The animal experiment department follows the specifications and procedures certified by the Animal Protection and Utilization Committee of the Central Research Institute of Taiwan. CGS21680 (Research Biochemicals, Natick, MA) was dissolved in a saline solution containing 1% DMS0 and then supplied to mice. More specifically, 16 7-week-old R6/2 mice were divided into two groups (8 in each group), and then CGS21680 (5 pg/g body weight) and the same dose of carrier solvent were injected from the intraperitoneal cavity, respectively. Injection of a 15 1328613 times lasted for 3.5 weeks. Thereafter, striatum tissue lysis fragments of each mouse were collected and analyzed using the standard Western blot method described by Rutter et al., Biochem. J. 2003; 375: 1-16. More specifically, the membrane fragments contain 125 mMTris-HCl (pH 6.8), 20% glycerol, 1% SDS, 15% 2-mercaptoethanol, 200 mM dithioxitol (dithiothreitol) and 0.01% bromphenol blue in 2X sample buffer were mixed and boiled for 5 minutes; the insoluble material was removed by centrifugation; and separated on an 8% separation gel. After completion of the electrophoretic analysis, the protein was transferred to a polydiene difluoro film, blocked with 5% skim milk phosphate buffer, and anti-AMPK antiserum (1:1, Abeam Limited, Cambridge, UK) Or anti-AMPKP antiserum (1:1 〇〇〇, Cell Signaling Technology, Beverly, MA, USA) was incubated overnight at 4 °C. Thereafter, it was washed three times with PBS for 5 minutes each time; the film was cultured for 1 hour at room temperature in peroxidase-conjugated donkey anti-rabbit IgG (1:5000, Amersham, UK). Then washed three times with PBS. The immunoreactive bands were visualized using a light emitting nonradioactive method (ECL, Amersham, UK). The degree of acidification of AMPK in Thrl72 was quantified using the ImageQuant v. 3. 15 (Moiecular dynamics) density detector to detect anti-AMPK-P immune responses. The same method was used to detect wild-type mice born in littermates other than CGS21680. The results showed that the amount of AMPK protein in the three groups of mice was almost the same, but the degree of phosphorylation of R6/2 mice (only more than 1%) was higher than that of the control group, indicating a high degree of phosphorylation of AMPK protein. It is related to dominant Handington's disease. Moreover, the provision of CGS21680 significantly reduced the degree of AMPK phosphorylation in R6/2 mice by approximately 35%. Since the degree of phosphorylation of AMPK reflects its degree of activation, this result indicates that CGS21680 can inhibit the enzyme activity of AMPK. 1328613 Example 2 R6/2 mice show impaired mobility, including dance-like movements, involuntary stiff movements, tremors, and seizures. In this example, the effect of CGS21680 on these mobility capabilities in R6/2 mice was investigated. Two groups of R6/2 mice (8 per group) were provided with CGS21680 and vehicle solvent for 5 weeks in the same manner as described in Example 1. After 24 hours of each drug delivery, measure the action capacity as described in the literature for 1 minute (Lee et al. Chin. J.
PhysioU992;35:317-336)。簡言之，將動物置於一具有16x16水 平感測器之行動監視器中（Coulbourn Instrument, Allentown, PA， USA)’這些感測器係用於定位動物之平面位置，行動能力係藉由 每10分鐘於X-Y平面上記錄到的光束打斷之全部次數來量測。 結果顯示：在供藥2週後，僅供給載體的小鼠表現出典型的 行動能力退化。相對地，供給CGS21680的小鼠則未表現出這些 退化行為’並且行動能力受到改善。這些存在這兩組小鼠間的差 異具有統計學上的意義（根據Student’s t-test)»此一結果說明提供 CGS21680可改善R6/2小鼠的行動能力。 每隻老鼠的動作協調度可使用標準滚輪實驗（rotarod performace)來檢測。檢測結果沒有發現差異。R6/2老鼠的動作協 調度在其4週大時開始退化，比提供CGS21680的R6/2老鼠開始 退化的時間早了 3週。因此，預期在老鼠4週大之前提供 CGS21680可改善R6/2老鼠的動作協調能力。 實施例三 在本實施例中檢測CGS21680在神經化學上對R6/2小鼠的的 影響。漢丁頓舞蹈症的病人其神經化學物質會發生變化，例如含 膽驗（choline)的化合物及N-乙酿天門東酸（N-acetylaspartate， 17 1328613 NAA)。含膽鹼(choline)化合物的增加及NAA的減少與神經創傷 及軸索（axonal)官能退化或喪失有關。例如參考…以“ et &1， biochem. Pharmacol. 2002;64:67-77;及 jenkins et al，j Neur〇chem 2000;75:2108-2119。因此，可使用標準方法來檢測R6/2小鼠令的 含膽鹼化合物及NAA量。 兩組R6/2小鼠（母組6隻）分別依前述方法提供cgs21680及 載體浴劑’另一組包含6隻野生型同窩出生小鼠亦提供載體溶 劑。在供藥2週後’利用體内質子定位磁譜分析儀（iH_MRS)分析 小鼠的神經化學改變。 更具體而言’利用腹腔注射水合氯醛（chl〇ral hydrate)(4. 088毫克/10克體重）麻醉動物。MRS係在一具有500 us 内 6. 5G/cm 主動遮蔽梯度（active shielding gradient)之 動物體内顯像儀（Biospec 4.7T spectrometer)中進行。小鼠置 於一具有頭部固定器之俯臥位置，一 20公分鳥籠狀線圈用於提 供RF射頻電波刺激訊號，一直徑2cm之表面線圈直接置於頭部 上方以接收訊號，欲使用1H-MRS量測的紋狀體上方之欲測目標容 積（volume of interest, V0I)係依據冠切擴散加權影像（coronal diffusion-weighted image)來選擇，該冠切擴散加權影像係使 用脈衝梯度迴聲擴散方法（pulse gradient spin-echo diffusion inethod)，其重複時間（repet it ion t ime，TR)為 1500 毫秒，迴 聲時間（echo time，TE)為62毫秒，可見區3公分x 3公分， 切片厚度為1毫米，b值1300秒/平方毫米，平均數2，一 256 X 128矩陣零填滿至256 X 156。擴散敏感梯度在重調焦距脈衝 波（refocusing pulse)之前或之後應用於讀取X方向。使用連續 三次化學位移選擇性飽和（chemical shift selective saturation，CHESS)脈衝來抑水，然後使用點解析波譜（PRESS) 序列來定位3.5 X 3.5 X 3. 5立方毫米（_3)紋狀體波睹體積 1328613 像素（voxel) ’ 光譜寬（Spectrai width，sw) 4000 赫茲（Hz)， TR: 3_5秒（s)，TE: 136毫秒，平均訊號256，及全掃瞄時間 8分32秒。辨識NAA、膽驗（Choi ine)及肌if (Creatine)的波峰 區面積’統計分析紋狀體代謝物及膽鹼相對於肌酐之比率。 結果顯示：提供載體之R6/2小鼠的平均膽鹼/肌酐比率遠高 於野生型小鼠（1. 74 ± 0. 12相對於1. 25± 0. 05)。相反地，提供 CGS21680可改變升高的R6/2小鼠的膽鹼/肌酐比率（分別為1.74 ± 0. 12及1. 44 ± 〇. 〇8，p = 〇· 023)。當含膽鹼之化合物含量的 改變影響到細胞膜時’可能會改變細胞膜的電生理活性及相關的 訊號傳遞（Gopalakrishna et al.， J. Cell. Biochem. 2000;77:517-528 及 Shander et al.， J. Mol. Cell. Cardiol. 1996;28:743-753.)。更明確而言，R6/2小鼠在其電生理特性上 顯示出明顯的改變（Klapstein et al.， J Neurophysiol. 2001;86:2667-2677)。因此，前述結果顯示CGS21680可降低膽 鹼含量’使膽鹼含量與野生型小鼠相似，進而改變R6/2小鼠的 病理發展β 小鼠的ΝΑΑ含量可藉由1H-MRS檢測。提供載體溶劑的R6/2 小鼠其平均ΝΑΑ/肌酐比率遠低於野生型小鼠（0.67 ± 0.02相對 於1· 12 ± 0. 04) ’顯示R6/2小鼠存在顯著的神經傷害。然而， 提供CGS21680不會影響R6/2小鼠的ΝΑΑ/肌酐比率（未提供及提 供00521680小鼠之^八/肌酐比率分別為〇.67±0.02及0.69± 〇. 0 ’ ρ = 0. 691)。由於R6/2小鼠的ΝΑΑ含量在其4週大時開 始減少，在其7週時才開始提供CGS21680無法改變神經損害及 降低ΝΑΑ含量。因此，早期提供CGS21680C例如，4週大時）可能 可以增加ΝΑΑ含量。 實施例四 在本實施例中研究CGS21680紋狀體萎縮。紋狀體萎縮是漢 19 1328613 丁頓舞蹈症的主要特徵之一。在R6/2小鼠中，紋狀體顯著的萎 縮發展係在3至13週時發生（Ferrante et al.，J. Neurosci. 2000;20:4389-4397)。因此，檢測前述提供CGS21680及載體之 R6/2小鼠的紋狀體萎縮。 使用戊基巴比特魯納（sodium pentobarbital)(100微克/克） 深度麻醉動物，使溶於〇. 1莫耳濃度之磷酸鹽緩衝液（PB, pH7. 4) 之4%多聚甲搭（paraformaldehyde)灌注心臟，小心取出大腦， 以4%多聚甲醛/0.1莫耳濃度之pb後固定2-5小時，之後浸於 30%甘油（溶於〇. 1莫耳濃度pB)中，使用冷凍組織切片機（CM3050,PhysioU992; 35:317-336). Briefly, animals were placed in a motion monitor with a 16x16 level sensor (Coulbourn Instrument, Allentown, PA, USA). These sensors are used to locate the planar position of the animal. The total number of times the beam was recorded on the XY plane was measured for 10 minutes. The results showed that only the vehicle-supplying mice showed a typical deterioration in mobility after 2 weeks of administration. In contrast, mice fed CGS21680 did not exhibit these degenerative behaviors' and their mobility was improved. The difference between these two groups of mice was statistically significant (according to Student's t-test)» This result indicates that providing CGS21680 improves the mobility of R6/2 mice. The coordinate coordination of each mouse can be detected using a rotarod performace. No difference was found in the test results. The R6/2 mouse's motor coordination schedule began to degenerate at 4 weeks of age, three weeks earlier than the time when the R6/2 mice that provided CGS21680 began to degenerate. Therefore, it is expected that CGS21680 will improve the motor coordination ability of R6/2 mice before the mice are 4 weeks old. Example 3 In this example, the effect of CGS21680 on neurochemically in R6/2 mice was examined. Patients with Huntington's disease have neurochemical changes, such as choline-containing compounds and N-acetylaspartate (17 1328613 NAA). An increase in choline-containing compounds and a decrease in NAA are associated with neurological trauma and axonal functional degradation or loss. For example, reference is made to "et & 1, biochem. Pharmacol. 2002; 64: 67-77; and Jenkins et al, j Neur〇chem 2000; 75: 2108-2119. Therefore, standard methods can be used to detect R6/2 The amount of choline-containing compound and NAA in mice. Two groups of R6/2 mice (6 in the mother group) provided cgs21680 and carrier bath respectively according to the above method. The other group contained 6 wild-type littermates. The carrier solvent was provided. After 2 weeks of drug administration, the neurochemical changes of the mice were analyzed using an in vivo proton localization magnetic spectroscopy analyzer (iH_MRS). More specifically, 'intraperitoneal injection of chloral hydrate (chl〇ral hydrate) (4) 088 mg/10 g body weight) Anesthetized animals. MRS was performed in an animal in vivo imager (Biospec 4.7T spectrometer) with an active shielding gradient of 6.5 G/cm within 500 us. Placed in a prone position with a head holder, a 20 cm birdcage coil is used to provide RF radio frequency stimulation signals, a 2 cm diameter surface coil is placed directly above the head to receive signals, and 1H-MRS is used. The measured target volume above the striatum (volume The interest, V0I) is selected based on a coronal diffusion-weighted image using a pulse gradient spin-echo diffusion inethod, the repetition time (repet It ion t ime, TR) is 1500 milliseconds, echo time (TE) is 62 milliseconds, visible area is 3 cm x 3 cm, slice thickness is 1 mm, b value is 1300 seconds/mm 2 , average is 2, one The 256 X 128 matrix is zero-filled to 256 X 156. The diffusion-sensitive gradient is applied to read the X direction before or after refocusing the refocusing pulse. Three consecutive chemical shift selective saturation (CHESS) Pulse to suppress water, then use the point resolution spectrum (PRESS) sequence to locate 3.5 X 3.5 X 3. 5 cubic millimeters (_3) striatum volume 1328613 pixels (voxel) 'Spectrai width, sw 4000 Hertz (Hz), TR: 3_5 seconds (s), TE: 136 milliseconds, average signal 256, and full scan time 8 minutes and 32 seconds. The area of the peak area of NAA, Choi ine, and muscle if (Creatine) was identified as a statistical analysis of the ratio of striatal metabolites to choline to creatinine. The results showed that the average choline/creatinine ratio of the R6/2 mice providing the vector was much higher than that of the wild type mice (1. 74 ± 0.12 vs. 1. 25 ± 0.05). Conversely, the provision of CGS21680 altered the choline/creatinine ratio of elevated R6/2 mice (1.74 ± 0.12 and 1.44 ± 〇. 〇8, p = 〇·023, respectively). When changes in the content of choline-containing compounds affect the cell membrane, 'the electrophysiological activity of the cell membrane and related signal transmission may be altered (Gopalakrishna et al., J. Cell. Biochem. 2000; 77:517-528 and Shander et al). J. Mol. Cell. Cardiol. 1996; 28: 743-753.). More specifically, R6/2 mice showed significant changes in their electrophysiological properties (Klapstein et al., J Neurophysiol. 2001; 86: 2667-2677). Therefore, the foregoing results show that CGS21680 can reduce the choline content, which makes the choline content similar to that of wild-type mice, thereby changing the pathological development of R6/2 mice. The sputum content of β mice can be detected by 1H-MRS. The average sputum/creatinine ratio of R6/2 mice providing vehicle solvent was much lower than that of wild-type mice (0.67 ± 0.02 vs. 1.12 ± 0.04), indicating that there was significant neurological damage in R6/2 mice. However, the provision of CGS21680 did not affect the sputum/creatinine ratio of R6/2 mice (not provided and provided the ratio of VIII/creatinine for 00521680 mice was 67.67±0.02 and 0.69± 〇. 0 ' ρ = 0. 691 ). Since the sputum content of R6/2 mice began to decrease at 4 weeks of age, the provision of CGS21680 at 7 weeks did not alter neurological damage and reduced sputum content. Therefore, the early supply of CGS21680C, for example, at 4 weeks of age, may increase the strontium content. Example 4 In this example, CGS21680 striatum atrophy was studied. Striatum atrophy is one of the main features of Han 19 1328613 Dyton's disease. Significant atrophic development of the striatum occurred in R6/2 mice at 3 to 13 weeks (Ferrante et al., J. Neurosci. 2000; 20: 4389-4397). Therefore, the striatum atrophy of the aforementioned R6/2 mouse providing CGS21680 and the vector was examined. The animals were deeply anesthetized with sodium pentobarbital (100 μg/g) to make a 4% polymethacrylate (PB, pH 7.4) dissolved in 莫. 1 molar concentration (PB, pH 7.4) Paraformaldehyde) perfused the heart, carefully remove the brain, fix it with 4% paraformaldehyde / 0.1 molar concentration of pb, then immerse in 30% glycerol (dissolved in 〇. 1 molar concentration pB), use frozen Tissue microtome (CM3050,
Leica Microsystems Nussloch GmbH, Nussloch，Germany)將組 織切成20微米厚度。從新紋狀體（neostriatum)頭端（rostral) 至前聯合（anterior commissure)(耳間（interaural) 5. 34mm/ 前囪（bregma) 1.54mm至耳間3. 7_/前囪〇.1_)連續冠切組織 切片以定義紋狀體萎縮及側腦室（laterai ventricle )之擴大 處。紋狀體及側腦室的所有區域皆使用NIH Image 1.62 Nissl 影像軟體來量測’並且GFAP染色之切片係藉由計算顯微攝影定 義區域内的相同種類細胞來定量，該顯微攝影照片係透過相位差 (phase contrast)顯微鏡拍攝。 提供CGS21680可減少12週大R6/2小鼠側腦室擴大處的大 小（提供載體溶液及CGS21680之R6/2小鼠分別為1.63 ±0.14及 0.91 ± 0.14 mm2，p < 0.001，„ = 5)。此一結果顯示提供 CGS21680 可改變漢丁頓舞蹈症的紋狀體萎縮。 實施例五 在本實施例中測試CGS21680對腺苷酸受體的效用。腺普酸 (Adenosine)是一種可調節不同生理功能的重要因子，包含四種 不同的腺苷酸受體次型（Αι、Αςα、An及A3)。以Ak-R腺苦酸受體 為例’ Au-R腺苷酸受體刺激可延遲人類嗜中性白血球的瑪亡，在 20 1328613 組織缺氧時增強細胞的存活能力。相關文獻可參考，例如，Walker et al., J. Immunol. 1997; 158:2926-2931; Jones et al., Neuroscience 1998;85:229-237; and Kobayashi et al., J. Biol,The tissue was cut to a thickness of 20 microns by Leica Microsystems Nussloch GmbH, Nussloch, Germany. From the neostriatum head (rostral) to the anterior commissure (interaural) 5. 34mm / bregma 1.54mm to the ear 3. 7_ / front 〇.1_) continuous The tissue section was cut to define the atrophy of the striatum and the enlargement of the lateral ventricle. All areas of the striatum and lateral ventricles were measured using the NIH Image 1.62 Nissl imaging software and the GFAP-stained sections were quantified by calculating the same type of cells in the defined area of the photomicrograph. Phase contrast microscope shot. Providing CGS21680 reduced the size of the lateral ventricle enlargement in 12-week-old R6/2 mice (R6/2 mice providing vector solution and CGS21680 were 1.63 ± 0.14 and 0.91 ± 0.14 mm2, respectively, p < 0.001, „ = 5) This result shows that providing CGS21680 can alter the striatum atrophy of Huntington's disease. Example 5 In this example, the effect of CGS21680 on adenylate receptor was tested. Adenosine is an adjustable difference. An important factor in physiological function, including four different adenosine receptor subtypes (Αι, Αςα, An, and A3). Taking Ak-R adenosine receptor as an example' Au-R adenosine receptor stimulation Delaying the death of human neutrophils, and enhancing the viability of cells in hypoxia under 20 1328613. For related literature, for example, Walker et al., J. Immunol. 1997; 158: 2926-2931; Jones et al Neuroscience 1998;85:229-237; and Kobayashi et al., J. Biol,
Chem· 1999;274:20358-65.。此外，當 NGF 引起的 MAPK 下游蛋 白（cascade)表現受到抑制時，A2a-R刺激可恢復受到阻礙的NGF-誘發之神經發展。在中央神經系統中，A2A-R基因會在γ-氨基丁 酸紋狀體皮質神經（GABAergic striopal 1 idal neurons)中高度 · 表現，γ-氨基丁酸紋狀體皮質神經在漢丁頓舞蹈症病狀發展中會 選擇性退化（Glass etal.，Neuroscience 2000;97:505-519)。 紋狀體A2a-R之表現在HD病變過程中明顯降低，而此一現象可能 · 歸因於γ-氣基丁酸神經細胞之喪失（Sapp etal·，Neuroscience 1995:64: :397-4040)。因此，在本實驗中測試前述R6/2小鼠紋 狀體A2A-R的表現及功能，例如檢測腺苷酸環化酵素（adenylyl ’ cyalase)活性（Lai etal·，Mol. Pharmacol. 1999;56:644-650)。 以西方墨點法測試A2a-R的蛋白質表現量，步驟係如實施例 一所述。使用抗 Gsa 抗體（1:2000; DuPont New england Nuclear， Wilmington，DE，USA)及抗 AC5N(1:5000)抗體為一抗，其中抗 AC5N抗體會與包含第五型腺苷酸環化酵素（ACV，主要的紋狀體 AC)之1至240個胺基酸的重組蛋白質反應。 肇 檢測結果發現：R6/2小鼠的紋狀體A2a-R平均蛋白質量明顯 低於野生型小鼠，並發現R6/2小鼠的ACV表現量及短鏈（short form)Gsot蛋白質（GsaS)明顯降低。此發現與錦紫蘇（forskolin) 引起的腺苷酸環化酵素活性降低結果一致。相對地，R6/2小鼠中 長鏈（long form)Gsa蛋白質（GsaS)表現量上升，顯示伴隨大量表 現polyQ的突變Htt會分別調控不同訊號分子的表現。 腺苷酸環化酵素（Adenylyl cyclase，AC)活性係使用習知 檢測方法檢測（Chern et al· (1993) Mol. Pharmacol. 21 1328613 44:950-958)。簡述如下，先從小鼠身上取得紋狀體組織，然後 在水解緩衝液（0.4 mM EDTA，1 mM EGTA, 25 mM Tris-HCl，250 嚴糖，〇. 1 mM 留培丁（ leupeptin),及 40 μΜ PMSF，pH 7_ 4) 中以 W3-80 超音波震盈器（Ultrasonics Farmingadale，NY, USA) 震盪45秒，功率輸出設定為20%。然後以50, OOOg離心前述震盪 後的均質產物（homogenate) 30分鐘，並收集P1模碎片及溶解 部分。AC活性檢測係在37°C下與400微升之反應混合物作用10 分鐘，前述反應混合物包含1毫莫耳濃度（mM)之三磷酸腺苷 (ATP) ’ 1〇〇毫莫耳濃度氣化鈉（NaCl)，50毫莫耳濃度N-2-羥乙 基對二氮己瓖（Hepes)，0. 2毫莫耳濃度EGTA，0. 5毫莫耳濃度 3-異丁基-1-甲基黃嗓吟（3-isobutyl-l-methylxanthine)，6 毫 莫耳濃度氣化鎂（MgC12)，1微莫耳濃度（# M)鳥嘌呤三磷酸 (GTP)，及20微克（yg)之膜蛋白；並藉由加入0.6毫升（ml) 10% 三氣乙酸（TCA)來終止反應；cAMP以Dowex色層分析儀（Sigma, St. Louis, MO，USA)分離，並使用習知方法檢測（Chern et al. (1993) Mol· Pharmacol. 44:950-958)。酵素反應在3〇分鐘内與4〇微 升以内的膜蛋白呈現線性關係。 超出預期之外，測試結果發現：在R6/2小鼠中，CGS21680 所誘發的腺苷酸環化酵素活性遠高於野生型小鼠^相反地，在 R6/2小鼠中’由一般腺苷酸環化酵素活化劑錦紫蘇（sigma, St. Louis，M0)所誘發的腺苷酸環化酵素活性遠低於野生型小鼠。並 且’提供CGS21680給R6/2小鼠兩星期並不會明顯影響蛋白質表 現量或A2a-R活性，此結果說明緩慢提供CGS21680給R6/2小鼠 並不會使A2A-R失去敏感度。 前述結果顯示：雖然R6/2小鼠的a2a-R表現量降低了，但 刺激A2a-R可有效使cAMP含量提昇，使CAMP含量不低於野生型 小鼠。 22 1328613 實施例六 在漢丁頓舞蹈症病人及R6/2小鼠中都可發現血糖過高的現 象。因此，在本實施例中測試CGS21680對R6/2小鼠高血糖症狀 的影響。將前述R6/2小鼠及同窩出生的野生型小鼠斷頭，並使 用標準方法收集每隻小鼠的血液樣本（1至1.51)。使用ABBOTT Alcyon 300i(ABBOTT Labs，USA)測量血糖值。 檢測結果發現：未提供CGS21680的R6/2小鼠的血糖值比 野生型小鼠高出將近1倍，相反地，長期提供CGS21680可使R6/2 小鼠異常升高的血糖值降低至與野生型小鼠相近。此一結果顯示 CGS21680具有下列兩種功能：（1)可改善R6/2小鼠血糖調節及能 量代謝’及（2)可用於治療糖尿病或肥胖症。 其他實施態樣 在本說明書中所揭露的所有特徵都可能與其他方法結合，本 說明書中所揭露的每一個特徵都可能選擇性的以相同、相等或相 似目的特徵所取代’因此，除了特別顯著的特徵之外，所有的本 說明書所揭露的特徵僅是相等的基因序列或相似特徵中的一個 例子。 根據本說明書所揭露的内容，任何熟習本技術領域之人士 皆可基與本發明之特色’在不麟本發明精神與目的下，對本發 明做不同的更動與修飾’使其適用於不同的情況與對象，因此， 其他實施態樣也包含在本發明之申請專利範圍内。 23Chem. 1999; 274: 20358-65. In addition, A2a-R stimulation restores impaired NGF-induced neural development when NGF-induced MAPK downstream cascade expression is inhibited. In the central nervous system, the A2A-R gene is highly expressed in the GABAergic striopal 1 idal neurons, and the gamma-aminobutyric acid striatum cortical nerves are in the Huntington's disease. It is selectively degraded in the development of the disease (Glass et al., Neuroscience 2000; 97: 505-519). The performance of striatum A2a-R is significantly reduced during HD lesions, and this phenomenon may be attributed to the loss of gamma-aerobic butyric nerve cells (Sapp et al, Neuroscience 1995:64: :397-4040) . Therefore, the performance and function of the aforementioned R6/2 mouse striatum A2A-R were tested in this experiment, for example, detection of adenylyl cyclase (Lai et al., Mol. Pharmacol. 1999; 56 :644-650). The protein expression of A2a-R was tested by Western blotting method as described in Example 1. An anti-Gsa antibody (1:2000; DuPont New england Nuclear, Wilmington, DE, USA) and an anti-AC5N (1:5000) antibody were used as primary antibodies, wherein the anti-AC5N antibody would be associated with a fifth type of adenylate cyclase ( Recombinant protein reaction of 1 to 240 amino acids of ACV, the main striatum AC). The 肇 test results showed that the average protein content of striatum A2a-R in R6/2 mice was significantly lower than that in wild-type mice, and the ACV expression and short form Gsot protein (GsaS) of R6/2 mice were found. )obviously decased. This finding is consistent with a decrease in adenylate cyclase activity caused by forskolin. In contrast, the expression of long form Gsa protein (GsaS) in R6/2 mice increased, indicating that Htt, which is accompanied by a large number of polyQs, regulates the expression of different signal molecules. Adenylyl cyclase (AC) activity is detected using a conventional detection method (Chern et al. (1993) Mol. Pharmacol. 21 1328613 44:950-958). Briefly, the striatum is obtained from mice first, then in hydrolysis buffer (0.4 mM EDTA, 1 mM EGTA, 25 mM Tris-HCl, 250 rigose, 〇. 1 mM leupeptin, and 40 μΜ PMSF, pH 7_ 4) with a W3-80 ultrasonic oscillator (Ultrasonics Farmingadale, NY, USA) oscillated for 45 seconds with a power output set at 20%. The oscillating homogenate was then centrifuged at 50, OOOg for 30 minutes, and the P1 mold fragments and dissolved fractions were collected. The AC activity assay was performed with a 400 microliter reaction mixture at 37 ° C for 10 minutes. The reaction mixture contained 1 millimolar (mM) adenosine triphosphate (ATP) ' 1 〇〇 millimolar sodium carbonate (NaCl) ), 50 mM concentration of N-2-hydroxyethyl p-dinitroxanthene (Hepes), 0.2 mmol of EGTA, 0.5 mmol of 3-isobutyl-1-methyl yellow 3-isobutyl-l-methylxanthine, 6 millimolar concentration of magnesium (MgC12), 1 micromolar (# M) guanine triphosphate (GTP), and 20 micrograms (yg) of membrane protein And quenching by adding 0.6 ml (ml) of 10% tri-glycolic acid (TCA); cAMP was separated by Dowex chromatography (Sigma, St. Louis, MO, USA) and detected using conventional methods (Chern Et al. (1993) Mol. Pharmacol. 44:950-958). The enzyme reaction showed a linear relationship with membrane proteins within 4 μL within 3 min. Beyond expectations, the test found that CGS21680-induced adenylate cyclase activity was much higher in R6/2 mice than in wild-type mice. In contrast, in R6/2 mice, 'from the general gland The adenylate cyclase activity induced by the nucleoside cyclase activator, sigma (St. Louis, M0), is much lower than that of wild-type mice. Moreover, the provision of CGS21680 to R6/2 mice for two weeks did not significantly affect protein expression or A2a-R activity. This result indicates that slow delivery of CGS21680 to R6/2 mice does not destabilize A2A-R. The above results showed that although the expression of a2a-R in R6/2 mice was decreased, stimulation of A2a-R was effective in increasing cAMP content, and the CAMP content was not lower than that in wild type mice. 22 1328613 Example 6 Hyperglycemia can be found in both Huntington's chorea and R6/2 mice. Therefore, the effect of CGS21680 on hyperglycemia symptoms in R6/2 mice was tested in this example. The aforementioned R6/2 mice and littermate-born wild type mice were decapitated, and blood samples (1 to 1.51) of each mouse were collected by standard methods. Blood glucose values were measured using ABBOTT Alcyon 300i (ABBOTT Labs, USA). The results showed that the blood glucose level of R6/2 mice without CGS21680 was nearly 1 times higher than that of wild-type mice. Conversely, long-term supply of CGS21680 reduced the abnormally elevated blood glucose level of R6/2 mice to wild. Type mice are similar. This result shows that CGS21680 has the following two functions: (1) improved blood glucose regulation and energy metabolism in R6/2 mice' and (2) can be used to treat diabetes or obesity. Other Embodiments All of the features disclosed in this specification may be combined with other methods, and each of the features disclosed in this specification may be selectively replaced with the same, equal or similar purpose features. In addition to the features of the present specification, all of the features disclosed in this specification are only one of the equivalent gene sequences or similar features. In accordance with the teachings of the present disclosure, any person skilled in the art can make various changes and modifications to the present invention in the context of the present invention. And other objects, therefore, other embodiments are also included in the scope of the patent application of the present invention. twenty three