TWI626051B - 抗癌組合物的用途 - Google Patents
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- TWI626051B TWI626051B TW105111714A TW105111714A TWI626051B TW I626051 B TWI626051 B TW I626051B TW 105111714 A TW105111714 A TW 105111714A TW 105111714 A TW105111714 A TW 105111714A TW I626051 B TWI626051 B TW I626051B
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Abstract
本發明揭示一種抗癌組合物及其用途，該組合物具有抑制致癌物的作用。
Description
本發明揭示一種抗癌組合物及其用途，特別涉及經由五没食子醯吡喃葡糖苷(PGG)，或是其與sorafenib組合治療呈現生物活性。
甘氨酸N-甲基轉移酶(Glycine N-methyl transferase，GNMT)擁有多個活性，其中之一為肝細胞癌的腫瘤抑制基因(tumor suppressor gene)。當暴露于如黃麴黴毒素(aflatoxin)、多環芳香烴碳氫化合物(ployaromatic hydrocarbons)等致癌物質，該酶可經由降低此等致癌物質誘發DNA加合物的形成(DNA adduct formation)和細胞毒性而呈現保護效應。一些研究推定，甘氨酸N-甲基轉移酶會參與肝臟排毒途徑(hepatic detoxification pathways)的啟動。亦有研究發現，超過80%以上的肝細胞癌病患其組織內該酶之表現量降低，發展成肝硬化患者的危險率增高。不論雄性或是雌性小鼠，經敲除甘氨酸N-甲基轉移酶基因，自然地發展成肝細胞癌比率增高。轉殖人類甘氨酸N-甲基轉移酶基因小鼠其肝臟過度表達，呈現防止黃麴黴毒素B1誘導肝細胞癌(aflatoxin B1-induced liver tumorigenesis)的現象。DebRoy,S.等人於2013年PloS one第8卷，e70062表明類似現象，當甘氨酸N-甲基轉移酶過度表達，可抑制癌細胞增生。
多年來，天然物(natural products)已被視為研發藥物的重
要資源。據估計到1990年，將近80%的藥品從天然物或天然物類似物(natural products analogs)衍生。中藥係天然物的一種獨特來源，不僅涵蓋著種類繁多的植物種類，更為人們長時間使用的一些藥物。
本案申請人鑑於習知技術中的不足，經過悉心試驗與研究，並一本鍥而不捨之精神，終構思出本案發明「抗癌組合物及其用途」，且能夠克服先前技術的不足，以下為本案之簡要說明。
本案主要可區分為兩部份，其一在於建立藥物篩選平台，並證實此平台能夠協助找到抗肝臟疾病的潛力藥物。第二，經由此平台尋找到兩種潛力藥物，並針對其一藥物，在小鼠模式得到驗證。
而此兩種藥物，皆具有肝臟保護作用，且可應用於肝臟疾病，如：脂肪肝、肝纖維化/硬化、肝癌的預防與治療。
為了克服先前技術的缺點，本發明經由實驗，探討五没食子醯吡喃葡糖苷的活性，或是與sorafenib組合之醫藥品及或/食品用途。
本發明之目的即在於提供一種多重組合投與方式，以謀求醫藥品及或/食品用途的多樣化。
根據本發明，於適當實施例，五没食子醯吡喃葡糖苷(PGG)可經由芍藥(Paeonia lactiflora Pall.)或五倍子(Galla Chinesis)中藥萃取。
圖1 pLV-GNMTpLuc質體的架構
圖2 為篩選平臺的螢光素酶活性
1....Huh7原細胞
2....H7GPL
圖3 為篩選平臺對不同藥物的反應結果
1....二甲基亞碸 2....0.1μM SAHA 3....1.0μM SAHA
4....10μM SAHA
圖4 篩選中藥資料庫
圖5 芍藥萃取物具最顯著的螢光素酶活性
圖6 芍藥的活性與劑量關係
圖7 流程1
圖8 劃分F1-F8的活性
圖9 劃分F3,F3-1到F3-6的活性
圖10 劃分F3-6活性的劑量依賴模式
圖11A Huh7細胞的GNMT mRNA的表現量
圖11B Huh7細胞的GNMT蛋白質的表現量
圖12 劃分F3-6抑制Huh7細胞的增生
圖13 腫瘤形成率
1....溶媒 2....25毫克/公斤劃分F3-6
3....300毫克/公斤劃分F3-6
圖14 腫瘤大小與劑量
1....溶媒 2....25毫克/公斤 3....300毫克/公斤
4....劃分F3-6
圖15 1,2,3,4,6-五没食子醯吡喃葡糖苷的結構式
圖16 劃分F3-6活性的劑量依賴模式
1....溶酶 2....F3-6 3....PGG(從F3-6劃分萃取) 4....PGG
(Sigma) 5...沒食子酸 6....葡萄糖
圖17A PGG(0.1mg/ml)誘導Huh7細胞的GNMT mRNA的表達量
圖17B PGG(0.1mg/ml)誘導Huh7細胞的GNMT蛋白質的表達量
圖18 PGG影響不同細胞株的增生
1....Huh7 2....Hep3B 3....SK-HEP-1
4....Mahlavu 5....HepG2
圖19 Huh7細胞群落形成
圖20 腫瘤的體積
1....溶媒 2....300毫克/公斤PGG
圖21 腫瘤組織中GNMT/actin蛋白質量的表現
圖22 sub-G1週期的細胞數量
圖23 Huh7細胞染Annexin-V的螢光強度
圖24 PGG誘導Huh7細胞的蛋白酶3/7活性表達
圖25 索拉非尼的結構式
圖26 索拉非尼化合物對細胞增生的影響
1....Huh7-GFP細胞 2....Huh7-GNMT
圖27 腫瘤細胞投予索拉非尼化合物進行治療
1....植入Huh7-GFP細胞並投予生理食鹽水
2....植入Huh7-GNMT細胞並投予生理食鹽水
3....植入Huh7-GFP細胞並投予10毫克/公斤索拉非尼
4....植入Huh7-GNMT細胞並投予10毫克/公斤索拉非尼
圖28 PGG與索拉非尼影響細胞的存活率
圖29 腫瘤的體積
1....生理食鹽水 2....25毫克/公斤劃分F3-6
3....300毫克/公斤劃分F3-6 4....10毫克/公斤索拉非尼
5....25毫克/公斤劃分F3-6+10毫克/公斤索拉非尼
6....25毫克/公斤PGG+10毫克/公斤索拉非尼
圖30 腫瘤中GNMT mRNA的表達量
1....生理食鹽水 2....25毫克/公斤劃分F3-6
3....300毫克/公斤劃分F3-6 4....10毫克/公斤索拉非尼
5....25毫克/公斤劃分F3-6+10毫克/公斤索拉非尼
6....25毫克/公斤PGG+10毫克/公斤索拉非尼
圖31 細胞中c-Myc mRNA相對表達量
1....溶媒 2....PGG 3....Huh7
4....HepG2
圖32A PGG抑制Huh7細胞的c-Myc蛋白質表達(劑量依賴)
圖32B PGG抑制HepG2細胞的c-Myc蛋白質表達(劑量依賴)
圖33A PGG抑制Huh7細胞的c-Myc蛋白質表達(時間依賴)
圖33B PGG抑制HepG2細胞的c-Myc蛋白質表達(時間依賴)
圖34 腫瘤中c-Myc/actin蛋白質含量比 1....溶媒
2....PGG
圖35 腫瘤中c-Myc m RNA表達
1....溶媒 2....25毫克/公斤劃分F3-6
3....300毫克/公斤劃分F3-6
圖36 GNMT啟動子活性相對表現量
圖37 mRNA的相對表現量
1....c-Myc 2....GNMT 3....shLacZ 4....shMyc
圖38 c-Myc過量表達抑制GNMT基因啟動子驅動螢光素酶活性
圖39 PGG與c-Myc過量表達對GNMT螢光素酶報導的影響
1....溶媒 2....PGG 3....shLacZ 4....shMyc
圖40 PGG影響不同細胞株的存活率
1....shLacZ 2....shGNMT-1 3....shGNMT-2
圖41 Huh7細胞的存活率
1....載體 2....c-Myc 3….溶媒 4….PGG
圖42 PGG對Huh7細胞c-Myc mRNA表達的影響
1....載體 2....c-Myc 3….溶媒 4….PGG
圖43 投予環己烯亞胺後的c-Myc蛋白質信號強度
1....溶媒 2....PGG
圖44 Huh7細胞的存活率
1....shLacZ 2....shMyc
圖45 c-Myc的蛋白質表現量
1...N-[(苄氧基)羰基]-L-亮氨醯-N-[(1S)-1-甲醯基-3-甲基丁
基]-L-亮氨醯胺(Z-LEU-LEU-LEU-CHO，MG132) 2...乙二胺
四乙酸(ethylenediamine-tetraacetic acid，EDTA)
3...氯化銨(NH4Cl)
4...氯化喹啉二磷酸鹽(chloroquine diphosphate)
5...N-苄氧羰基-纈胺酸-丙胺酸-天門冬胺酸(甲氧基)氟甲基酮(N-Benzyloxycarbonyl-Val-Ala-Asp(Ome)fluoromethyl ketone，Z-VAD-FMK)
6...N-苄氧羰基-L-亮氨酸-L-norleucinal
(N-Benzyloxy-carbonyl-L-leucyl-L-norleucinal，calpeptin)
圖46 PGG抑制HL60細胞c-Myc的蛋白質表現量
圖47 PGG抑制PC-3細胞c-Myc的蛋白質表現量
本案將可由以下的實施例說明而得到充分瞭解，使得熟習本技藝之人士可以據以完成之，然而本案之實施並非可由下列實施例而被限制其實施型態，熟習本技藝之人士仍可依據除既揭露之實施例的精神推演出其他實施例，該等實施例皆當屬於本發明之範圍。
就全球而言，肝細胞癌(hepatocellular carcinoma，HCC)是一種極普遍的惡性腫瘤，第三高死亡率的常見癌症。每年被診斷的新病例，大約超過五百萬個。由於診斷不易、治療方案不良、以及高復發率，因而罹患率幾乎等於死亡率。目前僅有商品名為Nexavar的索拉非尼(sorafenib)獲得美國食品和藥物管理局(Food and Drug Administration,FDA)於2007年核准，為治療肝細胞癌之藥物。雖然該藥物的副作用輕微，然而服用sorafenib的病患，卻易於產生抗藥性。鑒於肝細胞癌的治療存在著些許限度，企需研發預防性藥物、新治療藥物或藥物組合方案之策略，以擴大患者之生存率。
建立以細胞模式為基礎的甘氨酸N-甲基轉移酶基因表達藥物篩選平臺(Establishment of a GNMT expression oriented cell-based drug screening platform)
在克隆甘氨酸N-甲基轉移酶(Glycine N-methyl-transferase，GNMT)啟動子區後，將螢火蟲螢光素酶(firefly luciferase，LUC)報導基因置入慢病毒載體-pLKO.1(lentiviral vector-pLKO.1)。由產生的VSV-G包膜表達的質體-pMD.G
(plasmid-pMD.G)和如圖1所示pLV-GNMTpLuc，以挑選的質體-pCMV-△R8.91(plasmid-pCMV-△R8.91)進行人類胚胎腎細胞株HEK293T(ATCC CRL-11268)的共轉染(cotransfection)。挑選所獲得的細胞株，經放大且鑑定為Huh-7-GNMT啟動子-螢光素酶(Huh7-GNMT promoter-Luciferase，H7GPL)。
經由「螢光素酶活性/螢光偵測染劑(alamarBlue)」螢光強度的比值，計算相對螢光素酶活性(relative luciferase activity，RLA)。而以「藥物的相對螢光素酶活性/溶劑對照組的相對螢光素酶活性」的比值計算公式，評估苷胺酸甲基轉移酶(GNMT)啟動子被誘導後的表現差異(fold changes)。
以所建立的穩定細胞系Huh7-GNMT啟動子-螢光素酶細胞，激活甘氨酸N-甲基轉移酶啟動子(human glycine N-methyltransferase promoter)的化合物，作為藥物篩選平臺。如圖2所示經由螢光素酶活性測試，證實該細胞擁有報導基因的片段。Chen,M.H.等人於2011年PLoS One第6(11)卷e2718表明，26個美國食品藥品管理局(Food and Drug Administration，FDA)所批准的藥物，已經確定能扭轉肝癌的基因表達特徵。將Huh-7-GNMT啟動子-螢光素酶(H7GPL)細胞置入培養，投予上述26種藥物和溶劑對照組，發現其中有23%可增高細胞螢光素酶活性。
如表1所示從該26種藥物選出包含環吡酮胺(ciclopirox)、氯碘喹啉(Clioquinol)、芹菜素(apigenin)、木犀草素(luteolin)、辛二醯苯胺異羥肟酸(suberoylanilide hydroxamic acid，SAHA)、曲古菌素A(trichostatin A，TSA)等6種可以提高GNMT啟動子活性。此結果表明該平臺，可用於肝細胞癌治療藥物的識別。
辛二醯苯胺異羥肟酸(SAHA)係組蛋白去乙醯化酶(histone deacetylase)抑制劑，已知具備苷胺酸甲基轉移酶(GNMT)誘導活性。以辛二醯苯胺異羥肟酸測試Huh7-GNMT啟動子-螢光素酶細胞，進行高通量藥物篩選平臺(high-throughput screening platform)的品質。如圖3所示，以0.1μM、1.0μM、10μM等不同濃度的辛二醯苯胺異羥肟酸和二甲基亞碸(DMSO)的溶劑對照
組，培養Huh7-GNMT啟動子-螢光素酶細胞呈現極小井間差異(inter-well variation)的治療劑量反應。從平臺的品質與統計參數(Statistical parameters)相關聯的信號/背景(Signal/Background，S/B)、信號/雜訊(Signal/Noise，S/N)和Z'係數(Z’factor)三個因素，進行篩選平臺品質的評估。
篩選中藥資料庫(Screening a traditional Chinese medicine drug library)
衛生福利部國家中醫藥研究所(National research institute of Chinese medicine NRICM)的中藥資料庫(traditional Chinese medicine drug library)包含著中草藥萃取的324種純化合物和480種天然萃取物，運用上述平臺進行該中藥資料庫的篩選。於初步篩選，當[(個別螢光素酶活性-所有檢品平均螢光素酶活性)]/所有樣品標準差的Z值(Z score)大於1.5，顯示該化合物能提高苷胺酸甲基轉移酶報導活性(GNMT reporter activity)。如圖4所示，包含著13種純化合物和13種中草藥萃取物共26種檢品(hits)，其Z值大於1.5，擁有提高苷胺酸甲基轉移酶螢光素酶活性能力。
芍藥萃取物的抗肝細胞癌活性(The anti-HCC effect of Paeonia lactiflora Pall.extract derived bioactive fraction)
將採購的市售芍藥藥材，經製備萃取物，並加以確認芍藥的活性部位，而製備的甲醇萃取物的誘導苷胺酸甲基轉移酶啟動子活性(GNMT promoter activity)，如圖6所示，呈現著劑量依賴模式。經由生物活性導向分離(bioassay guided fractionation)的方法，鑑定芍藥(Paeonia lactiflora Pall.)乙醇萃取物的活性。係將芍藥的甲醇萃取物以乙酸乙酯加以分配(partitioned)後，將其有機層置入矽凝膠(silica gel)運用二氯甲烷(Dichloromethane)與甲醇的梯度
(gradient conditions)溶媒作為流動相進行中低壓製備液相色譜法(Medium Pressure Liquid chromatography，MPLC)分離並產生8個不同劃分(F1-F8)。在測試GNMT啟動子活性後，如圖8所示，發現其F3劃分呈現最具增強GNMT啟動子活性的潛力並進一步用葡聚糖凝膠LH 20(Sephadex LH-20)管柱進行滲透層析儀(permeation chromatography)的純化。經報導基因(reporter assay)測試，如圖9顯示F3-6劃分具有超過4倍的活性潛力。此外如圖10顯示該劃分具有誘導螢光素酶活性(induced luciferase activity)。對於內源性GNMT表達，如圖11圖顯示F3-6劃分呈現上調Huh7細胞的mRNA和蛋白質的活性。後續於測試F3-6劃分抗肝癌的療效，如圖12顯示F3-6劃分抑制Huh7細胞的增生呈現劑量依賴性。
對於異種移植(xenograft assay)測定，如圖13顯示，與溶媒對照組相比較投予F3-6劃分明顯降低小鼠腫瘤形成率tumor incidence rate)。當接種Huh7細胞20天後，對照組非肥胖糖尿病/重症联合免疫缺陷(NOD-SCID)裸鼠開始增生腫瘤，而在8周內增生腫瘤的小鼠有8隻達80%。相對地以小鼠體重投予25毫克/公斤(mg/kg)的F3-6劃分治療30天的組別，其腫瘤的發生率降至33%(3/9隻)。而在實驗期間以300毫克/公斤劑量F3-6劃分治療的組別，並未觀察到小鼠增生腫瘤。與溶媒對照組相比較，如圖14顯示投予25毫克/公斤劑量F3-6劃分治療的組別，腫瘤的平均體積顯著地偏小。
F3-6劃分的活性成分PGG化合物，於體外和體內均能抑制肝癌細胞的生長(PGG,the active component in F3-6,inhibits liver cancer cells growth both in vitro and in vivo)
以高效液相色譜法(HPLC)再純化F3-6劃分，經鑑定為純
度>98%的有效成分，如圖15所示1,2,3,4,6-五没食子醯吡喃葡糖苷(1,2,3,4,6-penta-O-galloyl-Beta-D-gluco-pyranoside，PGG)化合物的結構式。從F3-6劃分所分離純化的化合物與西格瑪(Sigma)公司購買的化合物進行比較，確認兩種1,2,3,4,6-五没食子醯吡喃葡糖苷(PGGs)，如圖16所示誘導GNMT效果相類似。雖然結構上由葡萄糖基團和沒食子酸基團組成，1,2,3,4,6-五没食子醯吡喃葡糖苷化合物。如圖16所示，葡萄糖基團和沒食子酸基團的其中任一者不足以影響GNMT啟動子活性，亦無法誘導GNMT啟動子的活性。
此外經由GNMT的免疫印跡(immunoblot)和即時聚合酶鍊式反應測試，更證實PGG誘導GNMT mRNA表達，如圖17證實PGG誘導Huh7細胞的GNMT蛋白的表達，呈現時間依賴性投予後48小時達到最大的治療作用。對於人類肝細胞株(human hepatoma cell line)的中科院细胞的Huh7、Hep3B(ATCC-No 8064)、HepG2(ATCC HB-8065)、SK-HEP-1(ATCC HTB-52)和Mahlavu惡性肝癌細胞株等不同細胞株，分析其細胞增生的影響。
經由螢光偵測染劑alamarBlue®螢光強度分析細胞存活率，如圖18所示。PGG對於不同肝癌細胞增生呈現劑量依賴性，其半數抑制濃度(IC50)，Huh7細胞株為0.03mg/mL、Hep3B細胞株為0.07mg/mL，而SK-HEP-1、Mahlavu和Hep G2細胞株皆為0.10mg/mL。探討於Huh7細胞投予PGG的治療效果，治療7天後分析細胞群落形成能力(colony formation capacity)。PGG能降低Huh7細胞群落的形成，呈劑量依賴性，如圖19所示，在0.05毫克/毫升以上較高濃度其抑制達95%。在裸鼠皮下接種Huh7細胞，以評估PGG體內抗腫瘤作用。當裸鼠的腫瘤
成長到足以明顯辨識，將小鼠隨機區分為2組，分別投予PGG和溶媒觀察10天。與溶媒對照組相比較，如圖20所示300毫克/公斤PGG治療組可觀察到顯著抑制腫瘤的生長現象。運用免疫印跡分析腫瘤檢品，如圖21所示PGG治療組顯示GNMT的誘導作用。
PGG誘導Huh7細胞的凋亡(PGG treatment induces apoptosis in Huh7 cells)
運用流式細胞分析法(flow cytometry assay)，進行PGG影響肝癌細胞週期的分析，以評估PGG抑制肝癌細胞增生的機制。投予PGG治療Huh7細胞24小時，如圖22所示sub-G1週期凋亡細胞數量顯著增加。投予PGG治療的Huh7細胞，經由凋亡標記膜聯蛋白V(Annexin V)染色，如圖23所示，PGG可顯著地誘導凋亡。
投予PGG治療12小時與上述結果相似，如圖24所示可觀察到半胱氨酸蛋白酶(caspase)3/7活性增加5倍。免疫印跡法更證實，投予PGG治療，於半胱氨酸蛋白酶3片段的含量隨著劑量從0.01增加呈現依賴性。綜上所述，投予PGG治療Huh7細胞，能誘導細胞的凋亡。
PGG敏化Huh7細胞影響投予sorafenib的療效(PGG sensitizes Huh7 cells to sorafenib treatment)
雖然目前索拉非尼(sorafenib)仍為治療肝癌的首選藥物，基於其副作用存在著關鍵問題，導致其臨床療效有限。該sorafenib結構式如圖25所示，其化學名為4-(4-{3-[4-氯-3-(三氟甲基)苯]酶基}苯氧基)-N2-甲基酶啶-2-氨甲醯-4-甲基苯磺酸鹽(4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]phenoxy]-N-methyl-pyridine-2-carbox-amide))。
如上所述，GNMT具有抑制肝細胞癌腫瘤的活性。為評估PGG，能否成為GNMT的誘導劑。因此，投予GNMT或PGG進行治療，觀察是否該化合物的過度表達足以敏化肝癌細胞對於索拉非尼的治療效果。經由增加索拉非尼濃度以處理包含表現綠色螢光蛋白(Green fluorescent protein，GFP)的Huh7-GFP和Huh7-GNMT細胞後，進行上述細胞存活率的分析。雖然索拉非尼於抑制Huh7-GFP和Huh7-GNMT細胞增殖呈現劑量依賴性，然而以索拉非尼(sorafenib)治療，對於GNMT過度表達的Huh7細胞，比Huh7-GFP細胞更為敏感。如圖26所示，以2.5μM的索拉非尼處理，Huh7-GNMT細胞的存活率與Huh7-GFP細胞相比較，呈現明顯下降現象。
為評估體內添加GNMT對於索拉非尼治療的影響，將Huh7-GNMT和Huh7-GFP細胞經由皮下植入非肥胖糖尿病/重症联合免疫缺陷(NOD-SCID)無胸腺雌裸鼠。即刻將小鼠隨機分組，投予索拉非尼或生理食鹽水治療。如上所述與Huh7-GFP細胞形成腫瘤相比較，GNMT過度表達可減少腫瘤的生長。以口服10毫克/公斤，每週3天索拉非尼處理小鼠或生理食鹽水治療，可觀察到索拉非尼呈現顯著抑制腫瘤生長的現象。和所有其他組別相比較，植入Huh7-GNMT細胞再投予索拉非尼治療腫瘤的重要成果，如圖27所示，其降低腫瘤大小呈現顯著的統計意義。以此等結果作為基礎，探討PGG作為與索拉非尼組合治療(combination therapy)肝癌的療效。
於Huh7細胞的細胞毒試驗，以評估索拉非尼與PGG的組合治療。如圖28所示投予PGG經72小時頗能加強索拉非尼療效，甚至在索拉非尼的較高劑量，仍能影響Huh7細胞的存活率。對於NOD-SCID小鼠，經由皮下進行Huh7細胞的異種移植。不
論單獨投予25、300毫克/公斤劃分F3-6或低劑量10毫克/公斤索拉非尼的治療，與對照組相比較均能呈現導致小鼠減少腫瘤大小的現象。如圖29所示與任一單用藥相比較，索拉非尼與25毫克/公斤F3-6劃分或25毫克/公斤PGG進行組合治療，更能削減腫瘤生長的誘發。此外於異種移植腫瘤的小鼠，與對照組相比較，如圖30所示投予F3-6劃分與PGG進行治療，可觀察到GNMT的mRNA呈現增加現象。
PGG經由c-Myc的抑制而增強GNMT啟動子活性(PGG enhances GNMT promoter activity via suppression of c-Myc)
運用微陣列分析(microarray analysis)，評估PGG影響GNMT表達的機制。以PGG治療Huh7細胞的第6小時和第48小時運用途徑富集分析(pathway enrichment analysis)，發現169基因於該選定的時段發生明顯改變。此結果表明，以下途徑均受到影響：轉化生長因子-β(transforming growth factor-β,TGF-β)訊息傳遞途徑、癌症的途徑、表皮生長因子(epidermal growth factor receptor，ErbB)訊息傳遞途徑、細胞週期和急性髓系白血病途徑(acute myeloid leukemia)。如表2所示，所有參與途徑都有c-Myc基因。經運用即時定量聚合酶連鎖反應(Real-time Quantitative
Polymerase.Chain Reaction，qRT-PCR，Q-PCR)的證實，如圖31所示PGG對c-Myc表達確實具有影響力。於Huh7和Hep G2細胞的免疫印跡分析，顯示PGG抑制c-Myc基因表達，如圖32、圖33，呈現著劑量和時間的依賴性。與體外結果呈現一致的結果，在異種移植腫瘤小鼠投予PGG或F3-6劃分的治療，如圖34、圖35所示，可降低c-Myc的表達。
由於PGG採取往下調降(downregulate)c-Myc方式而影響GNMT啟動子，則運用小髮夾型核醣核酸(short hairpin RNA，shRNA)敲除c-Myc基因，推斷將可呈現類似PGG治療的效果。以GNMT啟動子報導基因質體(promoter reporter plasmid)、胸苷激酶(thymidine kinase，TK)啟動子驅動海腎螢光素酶質體(renilla luciferase plasmid)、shMyc質體或shLacZ質體共轉染(co-transfected)Huh7細胞72小時，經由雙螢光素酶報導基因分析(dual luciferase reporter assays)，進行GNMT啟動子活性的測試。可發現shRNA調控著c-Myc令其無表達，與shLacZ對照組相比較，如圖36所示，造成GNMT啟動子活性將近5倍的誘導作用。經由改造Huh7-shMyc和Huh7-shLacZ穩定細胞，以敲除c-Myc基因，進行GNMTmRNA的表達測定。與對照組Huh7-shLacZ細胞相比較，如圖37所示，更顯著發現Huh7-shMyc細胞內源性GNMT mRNA的含量獲得提高。經由GNMT啟動子報導基因與胸苷激酶海腎報導基因(TK renilla reporter)以測試GNMT啟動子活性，評估Huh7細胞能否異位高度表達(ectopically overexpressed)c-Myc，以驗證c-Myc對於GNMT啟動子的影響。
如圖38所示，可觀察到c-Myc的過度表達，顯著地抑制GNMT基因啟動子驅動螢光素酶活性，以及內源性GNMT蛋白
的表達。此結果顯示c-Myc調控減少GNMT表達，而c-Myc的抑制現象頗類似PGG的治療效果。
經敲除c-Myc基因再投予PGG治療，評估GNMT啟動子活性能否受損害，以確認抑制c-Myc的表達導致影響PGG誘發GNMT啟動子。如圖39所示，經由PGG誘發GNMT啟動子的作用，於敲除c-Myc的細胞中呈現顯著地降低。綜合此資料，可表明c-Myc的往下調降，在PGG誘導Huh7細胞內GNMT啟動子表達，擔負著主導角色。
PGG抗肝癌效應與c-Myc抑制作用具相關聯性(The anti-HCC effect of PGG is associated with c-Myc suppression)
對於PGG如何抑制肝癌細胞生長的機制，推論PGG憑藉著誘導GNMT表達而影響肝癌細胞，預期敲除GNMT可聲援PGG的治療效果。以改造的Huh7細胞，針對著GNMT令其能穩定表達shRNAs，進行評估。結果如圖40所示，對於PGG所導致的細胞毒性，即使敲除GNMT並無重大的改善效果。由於c-Myc係一已知的致癌基因(oncogene)，因而轉為測試c-Myc往下調降的功能，可否影響PGG的抗肝癌作用。
於c-Myc的異位表達能否減少PGG誘導的細胞毒性，而影響肝癌細胞？然而c-Myc的過度表達，如圖41所示Huh7細胞無法防止PGG的影響，仍然呈現細胞毒性。此外c-Myc的異位表達，如圖42所示可削減PGG誘導c-Myc mRNA的抑制現象，但無法防止Huh7細胞受PGG誘導所產生c-Myc蛋白質的消耗，顯示PGG可降低c-Myc蛋白質的穩定性。以環己烯亞胺(cycloheximide，CHX)進行放线菌酮追蹤測定(cycloheximide chase assay)，如圖43所示，經PGG治療後，於蛋白半衰期測定確定c-Myc的降解動力學(degradation kinetics)，顯示PGG，可明
顯地減少c-Myc蛋白的半衰期。
如圖43所示，證實PGG係經由增強蛋白的降解，導致c-Myc蛋白質的表現量往下調降。基於PGG干擾著c-Myc蛋白質的穩定性，因而在PGG誘導細胞毒性方面，運用敲除系統足以發揮c-Myc的作用。與Huh7 shLacZ細胞相比較，如圖44所示，PGG僅能呈現少許降低Huh7-shMyc細胞的存活率。而敲除c-Myc細胞左右著PGG殺傷細胞的誘導作用，顯示經由c-Myc往下調降的作用，方為PGG影響Huh7細胞生長活性的重大主軸。
為確定蛋白質降解途徑(proteolytic pathway)與PGG誘導c-Myc的降解有關聯性，以各種蛋白質降解抑制劑，評估PGG調控著c-Myc的降解作用。如圖45所示，可發現蛋白酶抑制劑如氯化銨(NH4Cl)和N-[(苄氧基)羰基]-L-亮氨醯-N-[(1S)-1-甲醯基-3-甲基丁基]-L-亮氨醯胺(Z-LEU-LEU-LEU-CHO，MG132)並無法防止PGG誘導所造成的c-Myc降解；氯化喹啉二磷酸鹽(chloroquine diphosphate)、乙二胺四乙酸(ethylenediamine-tetraacetic acid，EDTA)、N-苄氧羰基-L-亮氨酸-L-norleucinal (N-Benzyloxy-carbonyl-L-leucyl-L-norleucinal，calpeptin)和細胞凋亡抑制劑N-苄氧羰基-纈胺酸-丙胺酸-天門冬胺酸(甲氧基)氟甲基酮(N-Benzyloxycarbonyl-Val-Ala-Asp(Ome)fluoromethyl ketone，Z-VAD-FMK)能阻止PGG誘導所造成c-Myc蛋白質表現的抑制作用。
該等結果表明PGG在Huh7細胞，經由蛋白酶體獨立機制(proteasome independent mechanism)誘導c-Myc的降解作用。為確定PGG誘導c-Myc的抑制現象，是否屬於腫瘤細胞內普遍存在的現象。運用免疫印跡分析如圖46所示，表明投予PGG的治療，亦可在其他人類腫瘤包括人類前列腺癌(prostate cancer)
PC-3(ATCC CRL-1435)和人類早幼粒細胞白血病(promyelocytic leukemia)細胞株HL-60(ATCC CCL-240)等細胞株，呈現減少c-Myc表達的現象。
術語“治療”、“治療中”及其類似術語係指緩減、改善、消減或消除目前患者所處病症或該病症相關之任何症狀的方法，以及預防該病症或其他涉及該症狀的方法。而術語『有效劑量(therapeutically effective amount)』係代表足以改善或防止醫學症狀或生物體狀態惡化之劑量。有效地劑量亦說明投與化合物之劑量足供用於診斷之劑量。除非說明書另有敘述，否則『活性化合物』以及『醫藥活性化合物』於此均可替換使用，係指稱一具有製藥學、藥理學或治療效果之物質。
術語『賦形劑(excipients)』或稱為『藥學上可接受之載體或賦形劑』、『生物可利用之載體或賦形劑』，係包括溶媒、分散劑、包衣、抗菌或抗真菌劑，保存或延緩吸收劑等任何用於製備成劑型之習知適當化合物。通常此類載體或賦形劑，本身不具備治療疾病之活性，且將本技術所揭示之衍生物，搭配選用各種藥學上可接受之載體或賦形劑，製備成各劑型，投與動物或人類亦不致於造成不良反應、過敏或其它不適當反應。因而本技術所揭示之衍生物，搭配藥學上可接受之載體或賦形劑，係適用於臨床及人類。
術語“藥學上可接受之賦形劑”，包括但不限於，聚合物、樹脂、增塑劑、填料、潤滑劑、稀釋劑、黏合劑、崩解劑、溶劑、共一溶劑、界面活性劑、防腐劑、甜味劑、調味劑、藥學級的染料或顏料、及黏度劑至少一者。
術語“醫藥組成物(pharmaceutical composition)”為一種固體或液體組成物，其形式、濃度和純度程度適合投予給病患(如人類或動物病患)，在投予之後，其可誘發所欲生理變化。醫藥組成物
典型地為無菌及/或非發熱性者(non-pyrogenic)。
於適當實施例，對患者運用組合療法(combination therapies)可採取投與含有五没食子醯吡喃葡糖苷(1,2,3,4,6-pentagalloyl-D-glucose，PGG)化合物成分，所製備的單一劑量口服組成物，如片劑、膠囊，或口服含有五没食子醯吡喃葡糖苷(PGG)化合物的食品。再投與含有索拉非尼(sorafenib)化合物的不同劑型或相同劑型的組合療法。該組合療法，亦可採取先後依序投與或是間隔短暫時間方式。此等組合療法，可隨病患的情況和年齡變化，而選用各種劑型，例如舌下或口腔含片之口服劑型，直腸給藥，鼻腔用藥、乾燥粉末或噴霧之吸入劑，甚至於陰道給藥，皮下、肌肉、靜脈和皮內之注射劑，局部給藥(topical)等等，例如護手霜、噴霧(mist)、溶液劑(solution)、乳液(lotion)、膠凍(gel)、乳霜(cream)、軟膏、糊劑(paste)、油膏(unguent)、乳液霜(emulsion)和懸浮劑，更可包括口服或注射投藥。間隔短暫時間方式投與，通常以前後3小時內完成投與。
本說明書所敘述之所有技術性及科學術語，除非另外有所定義，皆隸屬於該所屬領域之具有通常技藝者可共同瞭解的意義。
實驗方法(Materials and methods)
細胞培養與藥物(Cell culture and Reagents)
人類肝癌細胞系Huh7、Hep G2、Hep 3B、SK HEP 1、Mahlavu，以及人類胚胎腎細胞株HEK-293T均以Gibco BRL公司(Gibco BRL,Grand Island,NY)的Dulbecco改良的Eagle培養基(Dulbecco’s modified Eagle’s medium，DMEM)進行培養。該培養基含10%熱去活化胎牛血清(heat-inactivated fetal bovine serum)100U/mL青黴素(penicillin)，100μg/ml)鏈黴素(streptomycin)，0.1mm/L非必需氨基酸(nonessential amino acids)和2毫米/L L-
谷氨醯胺(L-glutamine)，組裝於5%二氧化羰濕潤的培養箱。人類前列腺癌細胞株PC-3和人早幼粒細胞白血病細胞株HL-60在Gibco BRL公司RPMI(Roswell Park Memorial Institute)1640培養基進行培養。穩定的細胞包括Huh7-GFP、Huh7-GNMT、Huh7-shGNMT、Huh7-shLacZ、Huh7-shMyc係經由慢病毒(lentiviral)系統建立，而置入含有1μg/ml嘌呤黴素(puromycin)的Dulbecco改良的Eagle培養基(DMEM)。
化合物3-甲基腺嘌呤(3-methyladenine，3-MA)、N-[(苄氧基)羰基]-L-亮氨醯-N-[(1S)-1-甲醯基-3-甲基丁基]-L-亮氨醯胺(Z-LEU-LEU-LEU-CHO，MG132)、氯化喹啉二磷酸鹽(chloroquine diphosphate)、N-(反式-環氧丁二醯基)-L-亮氨酸-4-胍基丁基醯胺((1S,2S)-2-(((S)-1-((4-Guanidinobutyl)amino)-4-methyl-1-oxopentan-2-yl)carbamoyl)cyclopropanecarboxylic acid，E-64)、環己烯亞胺(cycloheximide，CHX)、抑肽素(N-isovaleryl-L-valyl-L-valyl-3-hydroxy-6-methyl-g-aminoheptanoyl-L-alanyl-3-hydroxyl-6-methyl-g-aminoheptanoic acid，pepstatin A)與碘化丙啶(propidium iodide)均購自Sigma-Aldrich公司(St Louis,MO,USA)。N-苄氧羰基-纈胺酸-丙胺酸-天門冬胺酸(甲氧基)氟甲基酮(N-Benzyloxycarbonyl-Val-Ala-Asp(Ome)fluoromethyl ketone，Z-VAD-FMK)購自Selleckchem.com公司，而N-苄氧羰基-L-亮氨酸-L-norleucinal(N-Benzyloxy-carbonyl-L-leucyl-L-norleucinal，calpeptin)購自Millipore公司(Billerica,MA,.USA)。
質體與轉染(Plasmids and transfections)
經慢病毒(lentivirus)產生的pCMV-△R8.91、pMD.G和pLKO.1質體，以及c-Myc、GNMT的小髮夾型核醣核酸(short hairpin RNA，shRNA)和LacZ基因編码的β-半乳糖苷酶，皆從中
央研究院(Academia Sinica)的RNAi核心實驗室(National RNAi Core Facility)獲得。運用已知方法克隆甘氨酸N-甲基轉移酶(Glycine N-methyl-transferase，GNMT)啟動子區-1812~+14，再將螢火蟲螢光素酶(firefly luciferase，LUC)報導基因置入慢病毒載體-pLKO.1(lentiviral vector-pLKO.1)，構建GNMT啟動子螢光素酶質體(GNMT promoter-luciferase plasmid)。pcDNA-c-Myc質體由中國醫藥大學廖世平博士提供。而轉染(transfection)，係細胞被植入而獲得70%~90%的融合度(confluence)，根據操作手冊進行轉染以及運用Fermentas公司(漢諾威，MD)轉染試劑(TurboFect Reagent)和Life Technologies公司(Mulgrave,Australia)脂質體3000(Lipofectamine 3000)，可顯示質體DNAs已被轉染。
建立甘氨酸N-甲基轉移酶基因表達之高通量藥物篩選平臺(Establishment of GNMT gene expression-oriented platform for high-throughput screening)
將購自RNAi核心實驗室(National RNAi Core Facility)的質體-pLKO.1(plasmid-pLKO.1)，於pLKO.1含有ClaI和EcoRI酶切點的U6啟動子片段(promoter fragment)，以ClaI-BamHI-EcoRI(CBE)連接子加以取代。運用2個黏合(anneal)的5’-CGATATCGGATCCGTCGACG(SEQ ID NO.1)以及5’-AATTCGTCGACGGATCCGATAT(SEQ ID NO.2)寡核苷酸作為引物(primers)，加以生成CBE連接子(CBE linker)。所產生雙股的DNA片段其ClaI的5'突出端(overhang)緊接著EcoRV酶切點，BamH I酶切點、Sal I酶切點、以及EcoR I的5’突出端。運用CBE連結子連結於載體pLKO.1的ClaI和EcoRI，而生成pIV-CBE質體。包含從Promega公司(Promega,Madison,WI,USA)購買的pGL3啟動子載體，約~4kb片段合成poly(A)信
號加尾元件(polyadenylation signal,PAS)，將GNMT啟動子、螢火蟲螢光素酶報告基因和SV40後方poly(A)信號加尾元件生成擁有SspI、SalI酶切點，-1812/+14啟動子區的GNMT啟動子螢火蟲螢光素酶質體(GNMT promoter-luciferase plasmid)。經EcoRV和Sal I酶切該pLV-CBE質體，產生的片段加以回收，產生含有GNMT啟動子的pLV-GNMTpLuc質體，用於形成慢病毒。可概述為，經由TurboFectTM試劑(Fermentas，漢諾威，MD)令VSV-G包膜表達的質體-pMD.G(plasmid-pMD.G)和pLV-GNMTpLuc，以挑選的包裝質體-pCMV-△R8.91(plasmid-pCMV-△R8.91)進行HEK293T細胞株(ATCC No.CRL-11268)的共轉染(cotransfected)。根據網站(HTTP：//rnai.genmed.sinica.edu.tw/)的實驗方法，可從上清液獲得慢病毒。
讓Huh7細胞感染慢病毒病毒後，選用含有嘌呤黴素(puromycin)培養基進行培養。挑選所獲得的細胞株，經放大且鑑定為Huh-7-GNMT啟動子-螢光素酶(Huh-7-GNMT promoter-Luciferase，H7GPL)細胞。以Promega公司的螢光素酶檢測系統(Luciferase Assay System)測定H7GPL的細胞裂解物的螢光素酶活性，作為篩選藥物平台的評估依據。
運用Zhang,J.H.等人於1999年Journal of biomolecular screening第4期，第67-73頁Z'係數公式，以諸如信號/背景(Signal/Background，S/B)、信號/雜訊(Signal/Noise，S/N)和Z'係數(Z’factor)三個因素的統計參數(Statistical parameters)比較數值，作為篩選平臺品質的評估依據。
藥物篩選(Drug screening)
衛生福利部國家中醫藥研究所(National research institute of Chinese medicine NRICM)的中藥資料庫(traditional Chinese
medicine library)，包含著中草藥萃取的324種純化合物和480種天然萃取物。將H7GPL細胞置入96孔培養板，分別將324純化合物，溶解在二甲基亞碸(DMSO)其濃度為20毫克/毫升，而480粗提取物溶解在二甲基亞碸製成200毫克/毫升濃度。令2毫克/毫升濃度的粗提物，0.2毫克/毫升濃度的純化合物，分別投予H7GPL細胞培養24小時，進行初步篩選。再以Promega公司的螢光素酶檢測系統(Luciferase Assay System)，裂解細胞，測定其螢光素酶活性。在每個培養板，6孔置入最終濃度為1%的二甲基亞碸溶劑作為控制組。所有檢測數據轉換為Z值，獲得每一孔內報導基因的活性。
然後挑選初篩Z值1.5的檢品(hit)，以上述相同平臺進行第二次篩選。再置入H7GPL細胞於上述的複製培養板，而以該等檢品處置20小時後，添加AbD serotec公司alamarBlue ®試劑(Oxford,UK)，再進行培養4小時。根據實驗操作手冊的方法，進行細胞毒性測定，以評估報導基因。誘導GNMT啟動子活性1.5倍的藥物，視為二次篩選所得擁有提高苷胺酸甲基轉移酶螢光素酶活性能力的藥物。
定量即時聚合酶連鎖反應(Quantitative Real-time Polymerase.Chain Reaction，qRT-PCR，Q-PCR)
以Sigma-Aldrich公司的Tri Reagent將細胞溶解均質化製成總核酸(Total RNA)，再運用Invitrogen公司(Carlsbad,CA,USA)反轉錄酶試劑(Super Script II Reverse Transcriptase Kit)進行反轉錄成cDNA。以Applied Biosystems公司(Foster City,CA)螢光錠量裝置(ABI StepOne Plus System)，使用Roche Diagnostics公司(Basel,Switzerland)的螢光標記試劑(LightCycler® First Start DNA Master SYBR Green I reagent)令PCR呈色，執行PCR定
量。使用TATA盒結合蛋白(TATA-box binding protein，TBP)以其mRNA含量作為標準值，讓mRNA含量正常化。以下引物：GNMT上游引物，ACTGGATGACTCTGGACAA(SEQ ID NO.3)和下游引物，ACTGAGGATGTGGTCGT(SEQ ID NO.4)；TBP上游引物、TGCACAGGAGCCAAGAGTGAA(SEQ ID NO.5)和下游引物，CACATCACAGCTCCCCACCA(SEQ ID NO,6)；c-Myc上游引物、TTCGGTTGTTGCTGATCTGTCT(SEQ ID NO.7)和下游引物，CCCTCCACTCGGAAGGACTAT(SEQ ID NO.8)。
免疫印跡法(Immunoblotting)
以RIPA細胞裂解緩衝液(RIPA lysis buffer)獲得細胞或異種移植腫瘤(xenograft tumors)。RIPA裂解緩衝液組成為50mM的三羥甲基氨基甲烷(2-Amino-2-hydroxyl-methyl-propane-1,3-diol，Tris)其pH 7.5、150mM的氯化鈉、1%的Triton X 100、0.5%去氧膽酸鈉(sodium deoxycholate)、0.1%十二烷基硫酸鈉(SDS)、添加輔以蛋白酶和磷酸酶抑制劑。而磷酸酶抑制劑組成為1mM苯甲基磺醯氟(Phenylmethane-sulfonyl fluoride，PMSF)、10μg/ml亮胰酶肽(Leupeptin)、50μg/ml對甲苯磺醯-L-賴氨酸氯甲基酮鹽酸鹽(Tosyl-L-lysyl-chloromethane hydrochloride，TLCK)、50μg/ml甲苯磺醯苯丙氨醯氯甲酮(tosylphenyl-alanine chloromethyl ketone，TPCK)、1μg/ml抑肽酶(Aprotinin)、1毫米氟化鈉、5mM焦磷酸鈉(sodium pyrophosphate，NaPPi)和10mM釩酸鈉(Na3VO4)。運用已知方法將蛋白裂解、量化，而後在十二烷基硫酸鈉聚丙烯酰胺凝膠電泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE)凝膠上進行分離，以及進行免疫印跡法實驗。運用的抗體：抗-GNMT單株抗體14-1，懷麥克生技有限公司(YMAC Bio Tech公司，臺灣)，抗-半胱氨酸蛋白酶-3
(9H19L2，Thermo scientific公司)，抗-c-Myc(兔單株抗體D84C12，cell signaling Technonogy公司和抗體46-0603，Invitrogen公司)和抗-β-肌動蛋白(AC-15，sigma-aldrich公司)。
細胞存活的分析(Cell viability and colony formation assay)
根據操作手冊以AbD serotec公司的螢光偵測染劑AlamarBlue ®法和Promega公司的多重複合細胞毒性檢測法(MultiTox-Fluor multiplex cytotoxicity assay)，評估待測藥物對於細胞毒性的影響。將Huh7細胞接種在1×104細胞/孔的6孔培養板。
過夜培養後，分別以特定濃度的藥物處理細胞7天。以結晶紫染色存活細胞，經由量化的OpenCFU細胞株計數軟體(OpenCFU colony counting software)，分析該細胞株數量。
萃取、分離以及增強苷胺酸甲基轉移酶啟動子活性化合物的確認(Extraction,isolation and identification of GNMT enhancer Compounds)
購自台北市中藥店(Taipei,Taiwan)的市售白芍藥根部藥材(Paeoniae radix alba)150公克，確認為白芍藥(Paeonia lactiflora Pall.)。如流程1(Scheme 1)所示經0.6公升50%含水甲醇迴流萃取(reflux extracted)兩次(88-90℃)，每次1小時。上清液經濾紙過濾，合併其濾液，以0.7公升乙酸乙酯加以分配3次。將該乙酸乙酯分配層減壓揮除溶媒後，獲得粗萃取物24.9公克，置入充填默克公司40-63微米矽膠(德國)，300×30毫米管柱的中低壓製備液相層析儀(MPLC)，以二氯甲烷(Dichloromethane)和甲醇作為流動相溶媒進行分離。流動相的梯度條件，由100%二氯甲烷至60%二氯甲烷/40%甲醇直線梯度沖提1小時，隨後由40%甲醇至100%甲醇直線梯度沖提20分鐘，最後以100%甲醇沖提20分鐘，流速18毫升/分鐘。
收集的劃分沖提物(eluents)，分別減壓揮除溶媒後，置於已經吸附默克公司矽膠60 F254(德國)的薄層片，經由乙酸乙酯、甲醇和0.1%乙酸(15：2：0.5)的混合溶媒作為流動相，進行薄層色譜法的測定。其結果以波長254nm，噴塗香蘭素硫酸(vanillin/sulfuric acid)試劑進行分析，將獲得的8個不同劃分(F1~F8)，測試GNMT啟動子活性發現最具活性的F3劃分。將該F3劃分揮除溶媒後，安置於充填著葡聚糖凝膠LH 20(Sephadex LH-20)管柱，以甲醇為流動相進行沖提，並將沖提液分別減壓揮除溶媒後，於測試GNMT啟動子活性獲得生物活性F3-6的劃分。
以裝置著RP-18管柱(Cosmosil，250×10，5μm；nacalai，日本)，安捷倫1100型(Agilent 1100)二極體陣列檢測器(Photodiode Array Detector)的高效液相層析(high performance liquid chromatography，HPLC)系統，進行F3-6劃分的再純化。兩種流動相分別為含有0.1%乙酸的水和乙腈，其直線梯度沖提條件為5%乙腈溶液20分鐘，到100%乙腈，3毫升/分鐘的流速。以203nm加以監測，樣品濃度：440mg/mL，注入體積：10-50uL。而獲得褐色無定形粉末。其UV值為211、231和278nm。將該粉末稀釋在甲醇後，直接注入電噴霧質譜分析(MATLCQ ESI-MS)進行質譜分析。質譜記錄，顯示m/z值分別為963.02[M+Na]+和939.03[M-H]-。以氘代甲醇(CD3OD)溶劑的VNMRS 600核磁共振譜儀(Varian,PaloAlto,CA)，其記錄與文獻光譜資料比對。該淡褐色無定形粉末，經鑑定如圖15所示，該粉末為1,2,3,4,6-五没食子醯吡喃葡糖苷(1,2,3,4,6-penta-O-galloyl-Beta-D-glucopyranoside，PGG)化合物。五没食子醯吡喃葡糖苷(PGG)進行細胞相關的檢測，以磷酸鹽緩衝生理鹽水(PBS)作為溶媒加以溶解。
流式細胞分析法(flow cytometry assay)
將已成長的細胞，置入冰冷的75%乙醇過夜加以固定，添加1毫克/毫升核糖核酸酶(RNase A)並在37℃以10μg/ml碘化丙啶(ropidium iodide)染色30分鐘。然後置入BD生物科學公司的Accuri C6(BD Biosciences，San Jose)流式細胞儀進行分析。根據eBioscience公司(eBioscience，San Diego，美國)的實驗手冊，以膜聯蛋白annexin V-APC Apoptosis Detection Kit進行染色。
半胱氨酸蛋白酶-3和半胱氨酸蛋白酶-7活性分析(Caspase 3/7 activity assay)
將Huh7細胞置入96孔培養板，分別以溶媒或PGG培養12小時。根據Promega Technical公司Bulletin-Apo-ONE® Homogeneous Caspase-3/7的實驗手冊，進行半胱氨酸蛋白酶-3和半胱氨酸蛋白酶-7(Caspase 3/7)活性的量測。
微陣列分析(Microarray analysis)
根據Affymetrix基因晶片表達分析操作手冊，進行陣列雜交(Array hybridization)。運用Affymetrix公司(美國)Exprssion ConsoleTM的軟體，將原始資料標準化處理。採用t檢定統計量(t-test statistic)測定治療組以及控制組，其顯著差異探針組(Significant changed probe sets)q值為<0.005，平均數差異(mean-diff)q值為>1.5。經選定差異表達的基因(differentially expressed genes)探針組，運用DAVID公司(Database for Annotation,Visualization and Integrated Discovery；http：//david.abcc.ncifcrf.gov/)的DAVID生物資訊學線上工具(DAVID Bioinformatics online tools)進行標記(annotation)和途徑富集分析(pathway enrichment analysis)。
體內腫瘤的動物實驗(In vivo tumor models)
所有的動物實驗，均遵守高雄醫學大學的實驗動物照顧與使用委員會的相關規定，且獲得委員會的核准。5到6周齡非肥胖糖尿病/重症联合免疫缺陷(non-obese diabetic-severe combined immunodeficiency，NOD-SCID)無胸腺雌裸鼠，於右肢皮下注射1×106的Huh7細胞，進行腫瘤發生率的測定(tumor incidence assay)。移植5天後，將小鼠隨機區分為3組，每週3次分別經由腹腔(i.p)注射25毫克/公斤劃分F3-6、口服300毫克/公斤劃分F3-6和的生理食鹽水。(如圖13、14所示)
另外為評估GNMT和索拉非尼的組合效果(combinatory effect)，比照上述方法於非肥胖糖尿病/重症联合免疫缺陷(NOD-SCID)無胸腺雌裸鼠皮下，分別植入2×106的Huh7-GFP細胞或是Huh7-GNMT細胞。當小鼠腫瘤被檢測到明顯的成長狀態，顯示為相同的異種移植，將小鼠隨機區分為2組，每週3次分別投予10毫克/公斤LC公司(LC Laboratories)的索拉非尼或生理食鹽水。(如圖27所示)
比照上述方法，於Balb/c裸鼠皮下植入2×106的Huh7細胞，以測試PGG的體內抗腫瘤作用。當小鼠被檢測到明顯的腫瘤成長狀態，將小鼠隨機分組，每天1次共10天分別投予PGG和生理食鹽水，並每天進行體內抗腫瘤作用的測試。如圖20所示。
比照上述方法，將Huh7細胞(2×106)植入非肥胖糖尿病/重症联合免疫缺陷(NOD-SCID)無胸腺雌裸鼠，以評估藥物組合效果。檢測到成長到足以明顯辨識的腫瘤，將小鼠隨機區分為6組，分別投予生理食鹽水、25毫克/公斤劃分F3-6、300毫克/公斤劃分F3-6、10毫克/公斤索拉非尼、25毫克/公斤劃分F3-6+10毫克/公斤索拉非尼和300毫克/公斤劃分F3-6+10毫克/公斤索拉非尼。小鼠每週3次分別投予25毫克/公斤和300毫克/公斤的F3-6
劃分或25毫克/公斤PGG，以及每週2次10毫克/公斤索拉非尼。(如圖29所示)
每天或每週3次運用游標尺(Vernier calliper measurement)，測量腫瘤的長度(L)和寬度(W)，以監測腫瘤的生長。而腫瘤體積(Tumor volume，TV)，腫瘤體積=(L×W2)/2公式加以計算。劃分F3-6溶於磷酸鹽緩衝生理鹽水(Phosphate buffered saline，PBS)，PGG溶於磷酸鹽緩衝生理鹽水或0.5%羧甲基纖維素鈉鹽(carboxymethyl cellulose sodium salt)而索拉非尼溶於蓖麻油聚氧乙烯醚(Polyoxyethylene castor oil，Cremophor EL)/乙醇溶液。
統計分析(Statistical analysis)
實驗結果，皆以平均值加減標準誤(Mean±SEM)表示。統計間的差異，在非配對及配對樣本中分別採用非相依性的Student’s t-test。當多個治療組與對照組相比較，採用單因子變異數分析(one way ANOVA)，或雙因子重複測量變異數分析(two way repeated measures ANOVA)。當變異數分析(Analysis of variance，ANOVA)呈現統計學差異時，採用Dunnett's或Student-Newman-Keuls test。P值小於0.05，表示實驗值具有統計學上顯著性差異。資料和圖面的分析，在IBM電腦運用SigmaPlot軟體(版本8.0，Chicago,IL,U.S.A.)和SigmaStat(版本2.03，Chicago,IL,U.S.A.)。
本發明係以下面的實施例予以示範闡明，但本發明不受下述實施例所限制。本發明所用之藥物、生物材料皆市售易於取得，下列僅為示例可取得之管道。
實施例1 白芍藥活性劃分的製備
購自台北市中藥店的市售白芍藥根部藥材(Paeoniae alba
radix)150公克，確認為白芍藥(Paeonia lactiflora Pall.)。如流程1(Scheme 1)所示經0.6公升50%含水甲醇迴流萃取兩次(88-90℃)，每次1小時。上清液經濾紙過濾，合併其濾液，以0.7公升乙酸乙酯加以分配3次。將該乙酸乙酯分配層減壓揮除溶媒後，置入充填默克公司40-63微米矽膠(德國)，300×30毫米管柱的中低壓製備液相層析儀(MPLC)，以二氯甲烷(Dichloromethane)和甲醇作為流動相溶媒進行分離。流動相的梯度條件，由100%二氯甲烷至60%二氯甲烷/40%甲醇直線梯度沖提1小時，隨後由40%甲醇至100%甲醇直線梯度沖提20分鐘，最後以100%甲醇沖提20分鐘，流速18毫升/分鐘。
收集的劃分沖提物(eluent)，分別減壓揮除溶媒後，置於已經吸附默克公司(德國)矽膠60 F254的薄層片，經由乙酸乙酯、甲醇和0.1%乙酸(15：2：0.5)的混合溶媒作為流動相，進行薄層色譜法的測定。其結果以波長254nm，噴塗香蘭素硫酸(vanillin/sulfuric acid)試劑進行分析，將獲得的8個不同劃分(F1~F8)，測試GNMT啟動子活性發現最具活性的F3劃分。將該F3劃分揮除溶媒後，安置於充填著葡聚糖凝膠LH 20(Sephadex LH-20)管柱，以甲醇為流動相進行沖提，_將沖提液分別減壓揮除溶媒後，於測試GNMT啟動子活性獲得生物活性F3-6的劃分。
實施例2 1,2,3,4,6-五没食子醯吡喃葡糖苷(PGG)化合物的製備
以裝置著RP-18管柱(Cosmosil，250×10，5μm；nacalai，日本)，安捷倫1100型(Agilent 1100)二極體陣列檢測器(Photodiode Array Detector)的高效液相層析(high performance liquid chromatography，HPLC)系統，在室溫進行F3-6劃分的再純化。兩種流動相分別為含有0.1%乙酸的水和乙腈，直線梯度沖
提條件為5%乙腈溶液20分鐘，到100%乙腈，3毫升/分鐘的流速。以203nm加以監測，而獲得褐色無定形粉末。其UV值為211、231和278nm。質譜分析。質譜記錄，顯示m/z值分別為963.02[M+Na]+和939.03[M-H]-。以氘代甲醇(CD3OD)溶劑的VNMRS600核磁共振譜儀(Varian,PaloAlto,CA)，其記錄與文獻光譜資料比對。該淡褐色無定形粉末，經鑑定如圖15所示，該粉末為1,2,3,4,6-五没食子醯吡喃葡糖苷(1,2,3,4,6-penta-O-galloyl-Beta-D-glucopyranoside，PGG)化合物。五没食子醯吡喃葡糖苷(PGG)進行細胞相關的檢測，以磷酸鹽緩衝生理鹽水(PBS)作為溶媒加以溶解。
實施例3 五倍子劃分的製備
將從中藥店購買的五倍子(Galla Chinesis)105公克。比照流程1所示萃取方法，以0.6L的50%MeOH進行回流萃取2次，每次1小時。經濾紙過濾上清液、加以收集，以減壓揮除溶媒乾燥，獲得粗萃取物90.6公克。置入充填450公克默克公司(Merck)40-63微米矽膠管柱的中低壓製備液相層析儀(MPLC)。二氯甲烷：甲醇=90：10沖提，獲得劃分E1。乙酸乙酯：甲醇：0.1%醋酸=15：2：0.5沖提物，為劃分E2。100%甲醇沖提物為劃分E3。
將收集的劃分沖提物(eluents)，分別減壓揮除溶媒後，置於已經吸附默克公司(德國)矽膠60 F254的薄層片，經由乙酸乙酯、甲醇和0.1%乙酸(15：2：0.5)的混合溶媒作為流動相，進行薄層色譜法的測定。其結果以波長254nm以及360nm，噴塗香蘭素硫酸(vanillin/sulfuric acid)試劑進行分析。進一步純化最具活性的劃分E2，將該E2劃分揮除溶媒後安置於充填著葡聚糖凝膠LH 20(Sephadex LH-20)管柱，以甲醇為流動相進行沖提，將沖提液分別減壓揮除溶媒後，獲得含有PGG的E2-6劃分。
實施例4 五倍子劃分內PGG化合物的分析
以裝置著Thermo Syncronis公司RP-18管柱(100 x 2.1，1.7μm)，Waters公司二極體陣列檢測器的超極致效能液相層析色譜(UltraPerformance Liquid Chromatography，UPLC)系統，在35℃進行E2-6劃分的分析。流動相分別以乙腈溶液和0.1%磷酸鹽緩衝液，其梯度條件以2%乙腈溶液沖提1分鐘，其次2-10%乙腈溶液線性梯度沖提(linear gradient)1-2分鐘；10%乙腈溶液沖提2分鐘，其次10-25%乙腈線性梯度沖提4-10分鐘；25-50%乙腈溶液線性梯度沖提10-14分鐘。在每個檢品注射前，以1分鐘100%乙腈溶液淨化，5分鐘的平衡期。溶劑的流量保持在每分鐘0.4毫升和1μL注射量，而以203nm進行波峰監測。
本發明實屬難能的創新發明，本發明建立有關人類甘氨酸N-甲基轉移酶啟動子驅動之報告檢測平臺(promoter driven reporter assay platform)，以標識甘氨酸N-甲基轉移酶啟動子之活性化合物。可發現天然之五沒食子醯葡萄糖(Penta-o glloyl-beta-D-glucose,PGG)，為具潛力之候選化合物。此外五沒食子醯葡萄糖經實驗證實，於體外以組合療法投予五沒食子醯葡萄糖與商品名為Nexavar之sorafenib導致加強sorafenib對於Huh7細胞的毒殺作用呈現加乘效果，於體內則導致延遲腫瘤生長之現象。此外，五沒食子醯葡萄糖不僅抑制c-Myc基因的表達，亦可誘導c-Myc蛋白質的降解。顯然五沒食子醯葡萄糖是一種新型的c-Myc抑制劑和肝細胞癌的良好的藥物。深具產業價值，援依法提出申請。此外，本發明可以由本領域技術人員做任何修改，但不脫離如所附申請專利範圍所要保護的範圍。
<110> 高雄醫學大學，國立中國醫藥研究所
<120> 抗癌組合物及其用途
<150> US 62/148,238
<151> 2015-04-16
<160> 8
<170> PatentIn Version 3.3
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> CBE上游連接子
<400> 1
<210> 2
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> CBE下游連接子
<400> 2
<210> 3
<211> 19
<212> DNA
<213> 人工序列
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<223> GNMT上游引物
<400> 3
<210> 4
<211> 17
<212> DNA
<213> 人工序列
<220>
<223> GNMT下游引物
<400> 4
<210> 5
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> TBP上游引物
<400> 5
<210> 6
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> TBP下游引物
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<211> 22
<212> DNA
<213> 人工序列
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<223> c-Myc上游引物
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<210> 8
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<223> c-Myc下游引物
<400> 8
Claims (4)
- 一種組合物之用途，其係用於製備一涉及治療癌症之組合物，該組合物包含一五没食子醯吡喃葡糖苷(1,2,3,4,6-penta-O-galloyl-Beta-D-glucopyranoside，PGG)化合物，其中該組合物與索拉非尼(sorafenib)化合物結合，採取多重組合投與方式(multi-administered method)投予一受試者。
- 如申請專利範圍第1項所述之用途，其中該涉及治療癌症之組合物為醫藥品。
- 如申請專利範圍第1項所述之用途，其中該涉及治療癌症之組合物係用於治療或保健肝臟之不適、前列腺癌及人類早幼粒細胞白血病至少其中之一。
- 如申請專利範圍第1項所述之用途，其中該五没食子醯吡喃葡糖苷(PGG)化合物經由芍藥(Paeonia lactiflora Pall.)或五倍子(Galla Chinesis)萃取。
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| KR20210080349A (ko) | 2018-08-03 | 2021-06-30 | 넥테로 메디칼, 인크. | 정제된 펜타갈로일 글루코스 및 전달용 장치 |
| CN112444590B (zh) * | 2019-09-02 | 2022-12-02 | 江阴天江药业有限公司 | 一种五倍子药材的uplc指纹图谱的构建方法、通过该方法构建的指纹图谱及其应用 |
| CN112807318B (zh) * | 2021-02-22 | 2022-08-02 | 广东盛普生命科技有限公司 | 1,2,3,4,6-o-五没食子酰葡萄糖在制备预防和/或治疗肺纤维化药物中的应用 |
| CN113823364B (zh) * | 2021-10-08 | 2023-08-01 | 武汉轻工大学 | 一种基于网络药理学分析1,2,3,4,6-o-五没食子酰葡萄糖治疗肝癌机制的方法 |
| CN116144746A (zh) * | 2022-12-28 | 2023-05-23 | 北京博奥晶方生物科技有限公司 | 一种药物心毒性预测方法、装置、系统和介质 |
| CN118286235B (zh) * | 2024-04-06 | 2025-05-30 | 兰州大学第二医院 | 化合物pgg作为抗癌药物协同增效剂的应用及包括pgg的药物组合物 |
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