TWI240629B - Hematopoietic arabinogalactan composition - Google Patents
Hematopoietic arabinogalactan composition Download PDFInfo
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- TWI240629B TWI240629B TW089112971A TW89112971A TWI240629B TW I240629 B TWI240629 B TW I240629B TW 089112971 A TW089112971 A TW 089112971A TW 89112971 A TW89112971 A TW 89112971A TW I240629 B TWI240629 B TW I240629B
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Description
1240629 A7 B7 五、發明說明(/ ) 發朋領域· 本發明係關於一種***糖半乳聚糖。特別言之,本 發明係關於一種分離自頁寅根部 之***糖半乳聚糖,及平均分子量至少在1〇〇 KD,且分 離自這些***糖半乳聚糖組成物或其中間物之***糖 半乳聚糖蛋白質組成物。 發明背景 頁蔑(Radix Astragali) ’ 乃是 dwraga/⑽ membra而cews Bge. Var. mongholicus (Bge) Hsiao 或 A. membranaceus (Fisch) Bge· (Fabaceae)曬乾後之根部。黃茛乃是一種眾人 熟知的古老中國草藥。被正式記載於中國藥典中,且主要 當作一種強狀劑,用來治療腎臟炎及糖尿病。通常當作煎 劑或單獨當作”茶”來使用,或是在傳統草藥西加龍(shi-ka-ron)(由紫草(Lithospermum erythrorhizon)及川写 (Ligusticum wallachii)兩種草藥所組成)及人參養容湯(Ren-shen-yang-rong-tang)(其係由包括黃其(Radix Astragali)在內 的十二種草藥所組成)中與其他植物合倂使用。[參閱由W。 Tang 及 G· Eisenbrand 所編輯的”中國草藥(Chinese Drugs of Plant Origin)” 第 26 小節,第 19M97 頁,標題 ”Astragalus membranaceus (Fisch) Bge.’’ ,Springer Verlag, Berlin, 1992] ° 已有人以黃芪煎劑,及由煎劑之酒精沉澱物所製備而 成的溶液來進行注射,並已知其能改善胃及十二指腸潰瘍 的症狀,且能增加慢性白血球過少症患者的白血球指數[參 4 · —— !卜-----勢 (請先閱讀背面之注意事項再填寫本頁) --訂------ •線参· 經濟部智慧財產局員工消費合作社印製 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ' 經濟部智慧財產局員工消費合作社印製 1240629 A7 ___B7 五、發明說明(> ) 閱由Η.-Μ. Chang及Ρ_ Ρ·-Η· But等人所編輯的”中國草藥 之藥理及應用(Pharmacology and Applications of Chinese material Medica),’第 1041-1046 頁,標題”黃其”,World Scientific Publishing Co·,Singapore,1987]。已知黃民煎劑 ,純化之低分子量部分(即’分子量在25,000-35,000之部 分),及內含黃芪之藥草混合物之煎劑’也可恢復局部免疫 反應系統因外來肢體移植所致之免疫反應活性(D._T. chu et al·,“Immunotherapy with Chinese medicinal herbs. I···’’,J. Clin. Lab. Immunol.,25, 1 19-123 (1988)),並逆轉因環磷醯 胺所誘發之免疫抑制反應(D.-T· Chu et al·,“Immunotherapy with Chinese medicinal herbs· II···”,J· Clin. Lab· Immunol·, 25,125-129 (1988)),促進LAK細胞中由rIL-2所致之細 胞毒性(D.-T. Chu et al.,“Fractionated extract of dWraga/w membranaceus'\ J. Clin. Lab. Immunol., 25, 183-187 (1988)) ,促進免疫抑制性老鼠之免疫反應[K.S. Zhao et al·, ''enhancement of the immune response in mice by Astragalus membranaceus extracts’’,Immunopharmacology, 20, 225-234 (1990)],刺激單核細胞之反應[Y. sun et al·,“preliminary observation on the effects of the Chinese medicinal herbs… J. Biol. Response Modifiers,2,227-237 (1983)],及刺激老 鼠之骨髓造血能力[M. Rou et al.,“The effect of Radix astragali on mouse marrow hemopoiesis’’,J. Trad. Chin. Med·,3(3),199-204 (1983); S.-I. Miura et al·,“Effect of a traditional Chinese herbal medicine …Int. J. 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) . ----- (請先閱讀背面之注意事項再填寫本頁) 訂----- 1240629 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明(;)1240629 A7 B7 V. Description of the Invention (/) Fapon Field · The present invention relates to an arabinose galactan. In particular, the present invention relates to an arabinose galactan isolated from the roots of P. chinensis and an arabinose galactan having an average molecular weight of at least 100 KD and isolated from these arabinose galactan compositions or intermediates thereof. Sugar galactan protein composition. BACKGROUND OF THE INVENTION Radix Astragali is the root of dwraga / ⑽ membra and cews Bge. Var. Mongholicus (Bge) Hsiao or A. membranaceus (Fisch) Bge · (Fabaceae) after drying. Yellow buttercup is a well-known ancient Chinese herb. It is officially recorded in the Chinese Pharmacopoeia and is mainly used as a strong agent for the treatment of nephritis and diabetes. Usually used as a decoction or as "tea" alone, or in the traditional herbal shi-ka-ron (composed of Lithospermum erythrorhizon and Ligusticum wallachii) Ren-shen-yang-rong-tang (which consists of twelve herbs including Radix Astragali) is used in combination with other plants. [See by W. "Chinese Drugs of Plant Origin" edited by Tang and G · Eisenbrand, Section 26, page 19M97, titled "Astragalus membranaceus (Fisch) Bge. '', Springer Verlag, Berlin, 1992] ° Astragalus decoction, and a solution prepared from the alcoholic precipitate of the decoction, are injected. It is known to improve the symptoms of gastric and duodenal ulcers and increase the white blood cell index of patients with chronic hypoleukocytosis. ——! ----- Potential (please read the precautions on the back before filling this page) --Order ------ • Line reference · Printed by the staff of the Intellectual Property Bureau of the Ministry of Economic Affairs Consumer Cooperatives This paper is applicable to the paper China National Standard (CNS) A4 specification (210 X 297 mm) '' Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economy 1240629 A7 ___B7 V. Description of the invention (>) Read by Η.-Μ. Chang and Ρ_ Ρ ·- But · But et al. "Pharmacology and Applications of Chinese material Medica", 'p. 1041-1046, title "Huang Qi", World Scientific Publishing Co., Singapo re, 1987]. It is known that Huangmin decoction, purified low-molecular weight part (ie, the part with molecular weight between 25,000-35,000), and decoction containing astragalus medicinal mixture can also restore the immunity of the local immune response system due to foreign limb transplant Reactivity (D._T.chu et al., "Immunotherapy with Chinese medicinal herbs. I ...", J. Clin. Lab. Immunol., 25, 1 19-123 (1988)), and reverse the cause loop Immunosuppressive response induced by phosphatidamine (D.-T. Chu et al., "Immunotherapy with Chinese medicinal herbs II ..", J. Clin. Lab. Immunol., 25, 125-129 (1988) ) To promote cytotoxicity caused by rIL-2 in LAK cells (D.-T. Chu et al., "Fractionated extract of dWraga / w membranaceus' \ J. Clin. Lab. Immunol., 25, 183-187 (1988)), Promoting the immune response in immunosuppressive mice [KS Zhao et al., `` Enhancement of the immune response in mice by Astragalus membranaceus extracts '', Immunopharmacology, 20, 225-234 (1990)], Stimulation list Nuclear cell response [Y. sun et al ·, "preliminary observation on the effects of the Chinese medicinal herbs ... J. Biol. Response Modifiers, 2, 227-237 (1983)], and stimulate the bone marrow hematopoietic ability of mice [M. Rou et al., "The effect of Radix astragali on mouse marrow hemopoiesis" , J. Trad. Chin. Med ·, 3 (3), 199-204 (1983); S.-I. Miura et al ·, "Effect of a traditional Chinese herbal medicine… Int. J. This paper standard applies to China National Standard (CNS) A4 Specification (210 X 297 mm). ----- (Please read the precautions on the back before filling out this page) Order ----- 1240629 A7 B7 Employees ’Cooperatives, Intellectual Property Bureau, Ministry of Economic Affairs Printed 5. Description of Invention (;)
Immunopharmacol·,7(11),771-780 (1989); and Y. Ohnishi et al·,“Effects of Juzen-taiho-toh (TJ-48)···”,Exp. Hematol·, 18, 18-22 (1990)]。Immunopharmacol ·, 7 (11), 771-780 (1989); and Y. Ohnishi et al ·, "Effects of Juzen-taiho-toh (TJ-48) ···", Exp. Hematol ·, 18, 18- 22 (1990)].
Liu之美國專利案第4,843,067號中揭露了一種藥學組 成物’其係由內含黃蔑多醣(可由 或dWraga/似 中萃取而得)及當歸 多醣所組成。該黃芪多醣可以水由黃芪根部粉末中萃得或 以酒精沉澱而得。Verbiscar之美國專利案第5,2868,467號 中揭露了一種來自西黃蓍膠 iragMani/zaWragac植物之,免疫調節性多醣,其係 在低溫下製備而成,以”保持多醣結構的完整性,不致有化 學或組態變化”。Josephson等人之美國專利案第5,336,506 號中揭露了以諸如***糖半乳聚糖(分離自落葉松(lar/x 及甘露聚糖這類植物性多醣所形成的複合物 ,與其他藥劑一起作爲能傳遞藥劑至可進行胞飮作用之標 的細胞受體的藥劑來使用。Adams等人之美國專利案第 5,1 16,969號則揭露了一種適合用於密度梯度分離過程之特 別細的***糖半乳聚糖產物。June等人之美國專利案第 5,478,576號則揭露了一種純化的***糖半乳聚糖(同樣 來自Lar/x 、其裂解產物、及其之修飾物等, 同樣可用來傳遞藥劑至可進行胞飮作用之標的細胞受體。 Lewis等人之美國專利案第5,589,591號則揭露了一種不含 內毒素之多醣,例如***糖半乳聚糖、糊精、甘露聚糖 、及***膠;其係藉由超過濾法由一種不純的多醣中製 6 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) " ---------^--------- (請先閱讀背面之注意事項再填寫本頁) 1240629 A7 B7 五、發明說明(Φ ) 備而成,首先通過一種可去掉低分子量部分的薄膜,留下 濾液,之後再通過另一種可去除較高分子量部分之薄膜, 同樣仍留下濾液部分。 但上述這些參考文獻,均著重在產物中的多醣部分(即 ,***糖半乳聚糖),並可能使用可排除***糖半乳聚 糖蛋白質之技術。 經濟部智慧財產局員工消費合作社印製 ***糖半乳聚糖蛋白質,亦存在於開花植物中,並 廣泛存在於高等植物中。***糖半乳聚糖蛋白質(AGPs) ’有時又稱***糖半乳聚糖胜肽,係爲內含高比例碳水 化合物及低量蛋白質(通常低於10%)之醣蛋白,雖然已知 亦有含高量蛋白質之AGPs。在由植物所分離出之富含羥 基脯氨酸之醣蛋白中,AGPs的特點是低蛋白質含量及其 能與β-葡萄糖基亞里夫(Yariv)試劑,即1,3,5-三-(4-β-葡萄 糖基呋喃氧苯基偶氮)-2,4,6-三羥基苯,結合的能力[J.H. yariv et al.,Biochem. J·,85,383-388 (1962); R.L. Anderson et al·,Aust· J· plant physiol·,4,143-158 (1977)]。AGPs 係 爲***膠(即一種***膠樹senega/)之膠狀滲出 物)之一部分,常被當作乳化劑、防止結晶劑、及風味劑被 添力口到食品中。自 Nicotiana alata 、NicotianaU.S. Patent No. 4,843,067 to Liu discloses a pharmaceutical composition 'which is composed of a polysaccharide containing xanthophyll (which can be obtained by extracting from or dWraga) and angelica polysaccharide. The astragalus polysaccharide can be obtained by extracting water from astragalus root powder or precipitating with alcohol. Verbiscar U.S. Patent No. 5,2868,467 discloses an immunomodulating polysaccharide from the iragMani / zaWragac plant of tragacanth, which is prepared at low temperature to "maintain the integrity of the polysaccharide structure, No chemical or configuration changes. " U.S. Patent No. 5,336,506 to Josephson et al. Discloses a complex formed by botanical polysaccharides such as arabinose galactan (isolated from larch (lar / x and mannan), etc. Agents that deliver agents to cellular receptors that are capable of carrying out the cell-killing action. U.S. Patent No. 5,1,969, Adams et al. Discloses a particularly fine arabinose galactose suitable for use in density gradient separation processes. Glycan products. June et al. US Patent No. 5,478,576 discloses a purified arabinose galactan (also from Lar / x, its lysate, and its modifications, etc.), which can also be used to deliver pharmaceuticals to Target cell receptor capable of carrying out cell cyst effect. Lewis et al. US Patent No. 5,589,591 discloses an endotoxin-free polysaccharide, such as arabinogalactan, dextrin, mannan, and acacia ; It is made from an impure polysaccharide by ultrafiltration. 6 The paper size is applicable to China National Standard (CNS) A4 (210 X 297 mm) " --------- ^ --- - ----- (Please read the notes on the back before filling this page) 1240629 A7 B7 V. Description of the invention (Φ) Prepared by first passing a film that can remove the low molecular weight part, leaving the filtrate, and then passing Another thin film that removes higher molecular weight fractions still leaves a filtrate fraction. However, these references above all focus on the polysaccharide fraction (ie, arabinogalactan) in the product, and may use arabinose that can be excluded Technology of galactoprotein. Arabinogalactan protein printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs is also found in flowering plants and widely in higher plants. Arabinogalactan proteins (AGPs ) 'Sometimes also known as arabinogalactan peptides, are glycoproteins that contain a high proportion of carbohydrates and low amounts of protein (usually less than 10%), although AGPs containing high amounts of protein are also known. Among the hydroxyproline-rich glycoproteins isolated from plants, AGPs are characterized by a low protein content and their ability to interact with β-glucosyl Yarif reagents, namely 1,3,5- -(4-β-glucosylfuranoxyphenylazo) -2,4,6-trihydroxybenzene, binding ability [JH yariv et al., Biochem. J ·, 85, 383-388 (1962); RL Anderson et al ·, Aust · J · plant physiol ·, 4,143-158 (1977)]. AGPs are part of gum arabic (ie, a gum exudate of gum arabica senega /) and are often used as Used as emulsifier, prevent crystallizer, and flavor from being added to food. From Nicotiana alata, Nicotiana
及 comm以mi 等植物中分離出 AGP 基因的方法揭露於Chen等人所提之美國專利案第 5,646,〇29號中。至於有關AGP的詳細討論,請參考E.A. Nothnagel 之”Proteglycans and related compounds in plant cells”,Int. Rev. cytology,174, 195-291 (1997)。 7 本紙張尺度適用中國國家標準(CNS)A4規格(210 x 297公釐) 經濟部智慧財產局員工消費合作社印製 1240629 a7 B7 五、發明說明() 上述所提的這些文獻,在此均以參考文獻方式倂入本 發明中。 發明槪述 本發明之第一態樣係提供一種分離自黃民 根部之純化的***糖半乳聚糖組成物。 本發明之第二態樣係提供一種***糖半乳聚糖蛋白 質組成物,其係具有平均分子量至少爲100 KD且係分離 自上述本發明第一態樣之純化的***糖半乳聚糖組成物 中。 本發明之第三態樣係提供一種可供靜脈注射之*** 糖半乳聚糖配方,其係包含一種藥學有效量之本發明第一 態樣之純化的***糖半乳聚糖組成物,或本發明第二態 樣之***糖半乳聚糖蛋白質組成物,及一種可供靜脈注 射的輔藥溶液。 本發明之第三態樣係提供一種藉由施用本發明第一態 樣之純化的***糖半乳聚糖組成物,或本發明第二態樣 之***糖半乳聚糖蛋白質組成物來治療哺乳動物疾病的 方法(例如刺激造血,引發成熟巨核細胞增生,刺激IL-ip 、:[L-6、TNF-α、IFN-γ、GM-CSF、或 G-CSF 之生成,刺 激嗜中性白血球細胞的生成或其作用,治療嗜中性白血球 細胞過少,貧血,或血小板過少,加速因曝露(例如因意外 或非治療性的曝露,以及藥學治療下的曝露)於細胞毒性物 質處理或輻射照射後的復原情形,治療惡病質,嘔吐,或 藥物脫癮症狀,或改善生物反應或保護B型肝炎患者之肝 8 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐Γ " ----U------------- (請先閱讀背面之注意事項再填寫本頁) 1240629 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明(fc) 臟細胞),包括在該接受治療的哺乳動物身上注射一藥學有 效量之本發明第一態樣之純化的***糖半乳聚糖組成物 ,或本發明第二態樣之***糖半乳聚糖蛋白質組成物, 特別是本發明第三態樣之一種可供靜脈注射之***糖半 乳聚糖配方;或者與至少一種其他藥劑(例如能刺激造血之 藥劑)合用。 本發明之第五態樣係提供一種製備本發明第一態樣之 純化的***糖半乳聚糖組成物,及本發明第二態樣之阿 拉伯糖半乳聚糖蛋白質組成物的方法。 附圖簡單說明 圖1示出人類癌症患者接受化療後,其無進一步治療 者及以本發明純化之***糖半乳聚糖組成物、或以G-CSF治療時,其治療後白血球平均値隨天數變化之情形。 圖2示出人類癌症患者接受化療後,其無進一步治療 者及以本發明純化之***糖半乳聚糖組成物、或以G-CSF治療時,其治療後總症狀値隨天數變化之情形。 圖3不出卡g右夫斯基表現指標(Karnofsky performance Index)上的數値。 發明詳述 定義 “***糖半乳聚糖蛋白質”或”AGP”通常可爲β-葡萄 糖基亞里夫(Yariv)試劑所沉澱,且屬高度醣化之蛋白質, 其碳水化合物約佔分子量之至少50%,且主要之碳水化合 物組成爲***糖及半乳糖,且該***糖殘基多半位於 9 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ----U----^--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1240629 A7 B7 五、發明說明(β) 末端。其可與AGPs之單株抗體MAC207進行專一性的反 應。 “***糖半乳聚糖蛋白質組成物”係指一種組成物, 其至少包含如上述之至少70%,特別是至少80%,更特別 是至少90%之組成物重量之***糖半乳聚糖蛋白質,及 相關之***糖半乳聚糖及其他多醣。 “純化之***糖半乳聚糖組成物”乃是一種包含阿拉 伯糖半乳聚糖蛋白質(如上述)及相關之***糖半乳聚糖 與其他多醣之組成物。 “哺乳動物”包含人類及非-人類之哺乳動物,例如寵物 (貓、狗等)及家畜(牛、馬 '綿羊、山羊、豬等)。 “疾病”包含一種動物之任一種不健康的情況,包括因 醫療(如,副作用)所致之不健康的情況,例如一種疾病狀 態,其補血效果係具療效的,特別是包括本發明之”藥理及 用途”部分所記載的那些疾病狀態。 “藥學上可接受的輔藥”係指一種對製備藥學組成物有 用之輔藥,一般係安全、無毒、且喜好的,並包括可用於 獸醫用藥及人類用藥之輔藥。這類輔藥可以是固體、液體 、半固體、或氣霧組成物形式、及氣體形式。 “藥學可接受量”係指一種藥量,當被施用至動物體身 上以治療疾病時,該藥量足以有效地治療該疾病。 “治療”一種疾病包括預防已感染但尙未出現病徵之動 物不致發病(預防治療),抑制疾病(減緩或抑制疾病的發展) ,提供該疾病之症狀或副作用緩解(包括妤減療法),及緩 10 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ----------^---------^^^1 (請先閱讀背面之注意事項再填寫本頁) 1240629 B7 濟 部 智 慧 財 產 局 員 工 消 費 合 社 印 製 五、發明說明( 解該疾病(使疾病退化)。 純化之***糖半乳聚糖組成物 本發明純化之***糖半乳聚糖組成物之定義如上述 ,其係分離自黃其(jWMga/似 根部,且較好 是分离隹自 Astragalus membranaceus Bge. var. mongholicus Hsiao 或 (Fisch) Bge.之根部;且較 好是生長在內蒙古或中國山西省之黃芪(儿 植物之根部,特別是前者;且該植物根部較好是來自兩年 生之黃茛。其具有一典型的糖組成物(由該組成物之甲醇萃 取物之三甲基矽基衍生物的GLC分析中判定),內含約5-15%(莫耳百分比),特別是1〇%(莫耳百分比)之***糖 (Ara) ; 1.5°/。(莫耳百分比)以下,特別是1%(莫耳百分比)以 下之Rha ;高達約4%(莫耳百分比)之GalA ;約3%至7%( 莫耳百分比)間之Gal;及約70%至90%(莫耳百分比)間之 Glc ;其中Ara : Gal之比例不低於1.5 : 1 ;特別是不低於 1.75 : 1 ;且特別是不低於3 : 1 ;其灰份不高於2%(重量百 分比);重金屬含量不高於lOppm ;且羥基脯氨酸含量不高 於〇·1%,特別是不高於0.05%。其幾乎不含任何內毒素(即 ’依製造商手冊說明所進行之內毒素測試 (endospecy)[Seikagaku corporation,Tokyo,Japan])後,其含 量低於1.0 EU/毫克,特別是低於〇·8 EU/毫克,更特別是 低於0.5 EU/毫克,最好是低於0.3 EU/毫克);溶於水可達 至少20毫克/毫升,且水溶液pH値介於約4.5至6.5間; 且平均分子量介於20至60 kD間,特別是介於25至40 ----K----^--------- (請先閱讀背面之注意事項再填寫本頁) 11 經濟部智慧財產局員工消費合作钍印製 1240629 a7 B7 五、發明說明() kD間,且最好是介於27至35 kD間。爲方便起見,以卞 均以” PAGC”來代表本物質。 製備純化之***糖半乳聚糖組成物 純化之***糖半乳聚糖組成物係在有或無鹼金屬鹽( 特別是磷酸二氫鉀或磷酸二氫鈉)這類共萃物(co-extract)# 在下,以熱水(溫度一般不低於80°C,特別是不低於9〇t; ,特別是約100 °C ),在該溫度下萃取黃茛(△的ga/如 曬乾後之根部(一般係於無菌下將乾燥的***以超過濾水搓洗,再以諸如70%酒精之無菌溶液沖洗後 ,將根部切成薄片,並於無菌下風乾,以下稱此製備爲”碎 片(drink chips)”)一段時間,並視需要重複進行許多次萃取 步驟,讓根部欲求之***糖半乳聚糖蛋白質及相關之多 醣可儘量溶出(一般係在約100°C,萃取三次,每次3小時) 。所有在製備根部碎片後進行的步驟均係使用無菌設備及 試劑在無菌環境下操作。將熱水萃出物濃縮(於60-70°C下 真空濃縮至約1公升/公斤之乾燥根部),之後以低碳數之 醇類(例如酒精,室溫下終濃度爲70%)沉澱。再以更低碳 數之醇類來淸洗這些低碳數醇類的沉澱物(一般是以95%酒 精淸洗三次),並以適當濃度懸浮於水中等待進一步處理( 一般係18-20%重量/體積)。之後將懸浮液中不溶於水的 物質去除,例如藉由低碳數烷醇(約35%之乙醇)加以沉澱 去除。之後,低烷醇沉澱物(例如,35%之酒精沉澱物)之上 淸液再以高濃度之低碳數烷醇作進一步沉澱’例如 80%之酒精,特別是60-70%之酒精,將內含***糖半乳 12 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ---------^--------- Γ铸先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1240629 A7 B7 五、發明說明(/0) 'Λ 聚糖蛋白質及相關多醣之***糖半乳聚糖組成物之粗產 物沉澱出來。將沉澱再度溶於水中,並乾燥(噴霧乾燥’避 免過度加熱),以分離出***糖半乳聚糖組成物之粗產物 。該組成物粗產物一般係爲淡黃色粉末’可溶於水’濃度 至少爲100毫克/毫升,特別是至少200毫克/毫升,因乾 燥所損失的乾重約低於15%,且內毒素含量低於〇·5 ’特 別是低於0.3 EU/毫克。 之後再以離子交換色層層析法進一步純化***糖半 乳聚糖組成物之粗產物。將其溶於水,或是將沉澱溶解後 之溶液調整至適當濃度(一般約2%),之後進行超過濾,去 除掉低分子量部分,以減少溶液體積(例如,以可排除分子 量在5 kD下之超過濾系統(5K MWCO UF系統)進行過濾) 。再將超過濾後之上淸液流經陽離子交換管柱(例如,SP Sepharose陽離子交換管柱,以pH 5·20之20 mM NaOAc 緩衝液平衡),再將餾出液裝塡至陰離子交換管柱中(例如 ,Q Sepharose陰離子交換管柱,以同樣的NaOAc緩衝液 平衡)。從陰離子交換管柱中流出的液體可直接拿來製備本 發明第二態樣之***糖半乳聚糖蛋白質組成物,並可濃 縮乾燥形成一種適合用來製備***糖半乳聚糖蛋白質組 成物之中間產物,或直接用來製備一種純化的***糖半 乳聚糖組成物。在製備純化之***糖半乳聚糖組成物時 ,可用適當能過濾細菌之微過濾膜(例如Ο.ΐμπι之濾膜), 進行超過濾,去除鹽類並減少溶液體積(例如以8Κ MWCO UF系統)。將超過濾後之上淸液濃縮(50-60°C下,20-26%) 13 本紙張尺度綱中闘家鮮(CNS)A4娜(21() x 297公爱) ' - --------^---------' (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印制衣 1240629 A? _ B7 五、發明說明(ί丨) ,之後以低碳數烷醇(約80-90%之乙醇)加以沉澱。進一步 淸洗沉澱(例如以無水酒精,淸洗三次),並乾燥(60-70°C之 真空烤箱),可獲得純化之***糖半乳聚糖組成物。 ***糖半乳聚糖蛋白質組成物 本發明之***糖半乳聚糖蛋白質組成物之定義乃是 如上述自黃民似membra而ce⑽)根部分離出來的阿 拉伯糖半乳聚糖蛋白質組成物;且較好是自 membranaceus Bge. Var. mongholicus (Bge) Hsiao ^ A. (Fisch) Bge.之根部分離出來的;且較好是生 長在內冡古或中國山西省之黃民(i 根部, 特別是前者;且該植物根部較好是來自兩年生之黃芪。其 具有一典型約45°/。至75%(莫耳百分比)之糖組成物,特別 是約50%至70%(莫耳百分比)間之***糖(Ara) ; 2%至 4%(莫耳百分比)間之Rha;約4%至6%(莫耳百分比)間之 GalA ;約8%至25%(莫耳百分比)間、特別是1〇%至20%間 之Gal ;及約5%至25%(莫耳百分比)間之Glc ;其中Ara : Gal之比例不低於2 : 1 ;特別是不低於3 : 1 ;且特別是不 低於4 ·· 1 ;其灰份不高於2%(重量百分比);重金屬含量不 高於10 ppm ;且羥基脯氨酸含量不高於〇·2%,特別是不 局於0.3%。其幾乎不含任何內毒素(同上述定義);溶於水 後濃度至少可達20毫克/毫升,且水溶液pH値介於約4.5 至6.5間;且平均分子量不低於1〇〇 kD間,特別是介於 150至350 kD間。其內含約95%碳水化合物(包括可將阿 拉伯糖半乳聚糖蛋白質核心醣化之碳水化合物在內),及約 14 本紙張尺度適用中國國家標準(CNS)A4規格mo X 297公釐) ---------^-------- (請先閱讀背面之注意事項再填寫本頁) 1240629 A7 一 B7 五、發明說明(l2〇 5%蛋白質。約佔其氨基酸總量20%之羥基脯氨酸則是阿拉 伯糖半乳聚糖蛋白質之特點。爲方便起見,以下均以 ” AGPC”來代表本物質。 製備***糖半乳聚糖蛋白質組成物 本發明之***糖半乳聚糖蛋白質組成物係藉由100 K MWCO超過濾系統,將上述***糖半乳聚糖組成物或 離子交換管柱純化步驟所得之餾出物(或將餾出物濃縮乾燥 後之固態中間物)進一步純化而製得。上述”製備***糖 半乳聚糖蛋白質組成物”中陰離子交換管柱中所得的餾出物 ,係直接加到100 K MWCO超過濾系統中。將此100 K MWCO超過濾系統中留存的濾液進一步濃縮,並以低碳數 的烷醇加以沉澱,或者進一步淸洗、乾燥,均與製備純化 的***糖半乳聚糖組成物類似,可得***糖半乳聚糖 蛋白質組成物。 藥理及用途And comm's method of isolating the AGP gene from mi and other plants is disclosed in US Patent No. 5,646, 〇29 filed by Chen et al. For a detailed discussion of AGP, please refer to "Proteglycans and related compounds in plant cells" by E.A. Nothnagel, Int. Rev. cytology, 174, 195-291 (1997). 7 This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1240629 a7 B7 V. Description of the invention () These documents mentioned above are based on References are incorporated into the present invention. Summary of the Invention A first aspect of the present invention provides a purified arabinose galactan composition isolated from Huang Min's root. A second aspect of the present invention provides an arabinose galactan protein composition having a mean molecular weight of at least 100 KD and a purified arabinose galactan composition isolated from the first aspect of the present invention. In. A third aspect of the present invention provides an arabinose galactan formulation for intravenous injection, which comprises a pharmaceutically effective amount of the purified arabinose galactan composition of the first aspect of the present invention, or The arabinogalactan protein composition of the second aspect of the present invention, and an adjuvant solution for intravenous injection. A third aspect of the present invention provides a treatment by administering the purified arabinose galactan composition of the first aspect of the present invention, or the arabinose galactan protein composition of the second aspect of the present invention for treatment. Methods for mammalian diseases (such as stimulating hematopoiesis, triggering proliferation of mature megakaryocytes, stimulating IL-ip, [L-6, TNF-α, IFN-γ, GM-CSF, or G-CSF production, stimulating neutrophil The formation or effect of white blood cells, the treatment of too few neutrophils, anemia, or too few platelets, accelerates the exposure (such as accidental or non-therapeutic exposure, and exposure under pharmaceutical treatment) to cytotoxic substance treatment or radiation Recovery after irradiation, treatment of cachexia, vomiting, or drug withdrawal symptoms, or to improve the biological response or protect the liver of patients with hepatitis B. 8 This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm Γ " ---- U ------------- (Please read the notes on the back before filling out this page) 1240629 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs fc) dirty cells) The method includes injecting a pharmaceutically effective amount of the purified arabinose galactan composition of the first aspect of the present invention or the arabinose galactan protein composition of the second aspect of the present invention into the mammal being treated. In particular, a third aspect of the present invention is an arabinose galactan formulation that can be injected intravenously; or combined with at least one other agent (such as a agent that can stimulate hematopoietic). A fifth aspect of the present invention provides a Method for preparing the purified arabinose galactan composition of the first aspect of the present invention and the arabinose galactan protein composition of the second aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a human cancer patient After receiving chemotherapy, if there is no further treatment, and the arabinose galactan composition purified according to the present invention, or when treated with G-CSF, the average leukocyte count after treatment varies with the number of days. Figure 2 shows human cancer After the patient receives chemotherapy, if there is no further treatment, and the arabinose galactan composition purified according to the present invention, or when treated with G-CSF, the total symptoms after treatment will not change. Fig. 3 does not show the number on the Karnofsky performance index. Detailed description of the invention Definition "Arabinose galactan protein" or "AGP" can usually be β-glucose Precipitated by Yariv reagent and a highly saccharified protein, the carbohydrates account for at least 50% of the molecular weight, and the main carbohydrate composition is arabinose and galactose, and the arabinose residue is mostly located 9 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) ---- U ---- ^ --------- (Please read the precautions on the back before filling in this Page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1240629 A7 B7 V. The invention description (β) end. It can specifically react with AGPs monoclonal antibody MAC207. "Arabinose galactan protein composition" means a composition comprising at least 70%, in particular at least 80%, and more particularly at least 90% by weight of the composition of arabinose galactan as described above Protein, and related arabinose galactans and other polysaccharides. The "purified arabinose galactan composition" is a composition comprising arabinose galactan protein (as described above) and related arabinose galactan and other polysaccharides. "Mammal" includes human and non-human mammals, such as pets (cats, dogs, etc.) and domestic animals (cows, horses, sheep, goats, pigs, etc.). "Disease" includes any unhealthy condition of an animal, including unhealthy conditions caused by medical treatment (eg, side effects), such as a disease state, and its blood-reinforcing effect is curative, especially including the "pharmacological and Disease states described in the "Usage" section. "Pharmaceutically acceptable adjuvant" means an adjuvant that is useful for the preparation of a pharmaceutical composition, is generally safe, non-toxic, and preferred, and includes adjuvants that can be used in veterinary medicine and human medicine. Such adjuvants can be in the form of a solid, liquid, semi-solid, or aerosol composition, and a gaseous form. "Pharmaceutically acceptable amount" means a drug amount which, when administered to the body of an animal to treat a disease, is sufficient to effectively treat the disease. "Treatment" of a disease includes preventing the infection of an animal that has no signs of disease (preventive treatment), suppressing the disease (slowing or inhibiting the development of the disease), providing relief from the symptoms or side effects of the disease (including mitigation therapy), and 10 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) ---------- ^ --------- ^^^ 1 (Please read the back first Please pay attention to this page and fill in this page again) 1240629 B7 Printed by the Consumers ’Cooperative of the Ministry of Intellectual Property Bureau of the Ministry of Education 5. Description of the invention (Solving the disease (deteriorating the disease). The glycogalactan composition is defined as described above, which is isolated from Huang Qi (jWMga / like root, and preferably isolated from the root of Astragalus membranaceus Bge. Var. Mongholicus Hsiao or (Fisch) Bge .; and more Fortunately, the roots of astragalus (children's plant, especially the former) growing in Inner Mongolia or Shanxi Province of China; and the roots of the plant are preferably biennial yellow buttercups. It has a typical sugar composition (the methanol of the composition Extracts The trimethylsilyl derivative was judged in the GLC analysis), which contained about 5-15% (molar percentage), especially 10% (molar percentage) of arabinose (Ara); 1.5 ° /. Ear percentage), especially Rha below 1% (mole percentage); GalA up to about 4% (mole percentage); Gal between about 3% and 7% (mole percentage); and about 70% to Glc between 90% (percent of mole); where the ratio of Ara: Gal is not less than 1.5: 1; especially not less than 1.75: 1; and especially not less than 3: 1; its ash content is not more than 2 % (Weight percentage); heavy metal content is not higher than 10ppm; and hydroxyproline content is not higher than 0.1%, especially not higher than 0.05%. It contains almost no endotoxin (ie, according to the manufacturer's manual After the endospecy test [Seikagaku corporation, Tokyo, Japan]), its content is less than 1.0 EU / mg, especially less than 0.8 EU / mg, more specifically less than 0.5 EU / mg, Preferably less than 0.3 EU / mg); soluble in water up to at least 20 mg / ml, and the pH of the aqueous solution is between about 4.5 to 6.5; and the average molecular weight is between 20 and 60 kD, especially It is between 25 and 40 ---- K ---- ^ --------- (Please read the precautions on the back before filling out this page) 1240629 a7 B7 V. Description of the invention () kD, and preferably between 27 and 35 kD. For convenience, "PAGC" is used to represent this substance. Preparation of purified arabinose galactan composition The purified arabinose galactan composition is co-extracted (co- with or without alkali metal salts, especially potassium dihydrogen phosphate or sodium dihydrogen phosphate). extract) # Under the conditions, hot water (temperature is generally not lower than 80 ° C, especially not lower than 90 °; especially about 100 ° C), at this temperature, yellow buttercup (△ ga / such as sun Dry roots (generally, the dried roots are washed with ultra-filtered water under aseptic conditions, and then rinsed with a sterile solution such as 70% alcohol, then the roots are cut into thin slices and air-dried under aseptic conditions, hereinafter referred to as "" "Drink chips") for a period of time, and repeat the extraction steps as many times as necessary, so that the arabinogalactan protein and related polysaccharides that the root desires can be dissolved as much as possible (generally at about 100 ° C, extracted three times, 3 hours each time). All steps performed after the preparation of root fragments are performed in a sterile environment using sterile equipment and reagents. The hot water extract is concentrated (concentrated under vacuum at 60-70 ° C to about 1 liter / Kilograms of dried roots), then low-carbon Alcohol (such as alcohol, the final concentration of 70% at room temperature) precipitation. The lower carbon number alcohols are used to wash these low carbon number alcohol precipitates (usually three times with 95% alcohol) , And suspended in water at an appropriate concentration for further processing (generally 18-20% weight / volume). After that, the water-insoluble substances in the suspension are removed, for example, by low-carbon alkanol (about 35% ethanol) After the precipitation is removed, the lower alkanol precipitate (for example, 35% alcohol precipitate) is used for further precipitation with a high concentration of low-carbon alkanol, such as 80% alcohol, especially 60-70 % Of alcohol, will contain arabinose galacto 12 This paper size applies to Chinese National Standard (CNS) A4 specifications (210 X 297 mm) --------- ^ --------- Γ Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1240629 A7 B7 V. Description of the invention (/ 0) 'Λ glycan protein and related polysaccharides arabinose galactan composition The crude product precipitated out. The precipitate was dissolved in water again and dried (spray-dried 'to avoid excessive heating) to isolate the crude product of the arabinose galactan composition. The crude product of this composition is generally a light yellow powder 'soluble in water' at a concentration of at least 100 mg / ml, especially at least 200 mg / ml, the dry weight lost by drying is less than about 15%, and the endotoxin content Below 0.5 ', especially below 0.3 EU / mg. The crude product of the arabinogalactan composition was further purified by ion exchange chromatography. Dissolve it in water, or adjust the solution after the precipitate is dissolved to an appropriate concentration (usually about 2%), and then perform ultrafiltration to remove the low molecular weight part to reduce the solution volume (for example, to exclude the molecular weight at 5 kD The following ultrafiltration system (5K MWCO UF system) is used for filtration). After ultrafiltration, the supernatant liquid is passed through a cation exchange column (for example, SP Sepharose cation exchange column, equilibrated with 20 mM NaOAc buffer, pH 5.20), and the distillate is packed into the anion exchange tube. In a column (for example, a Q Sepharose anion exchange column, equilibrated with the same NaOAc buffer). The liquid flowing out from the anion exchange column can be directly used to prepare the arabinogalactan protein composition of the second aspect of the present invention, and can be concentrated and dried to form a suitable arabinogalactan protein composition. The intermediate product is used directly to prepare a purified arabinose galactan composition. When preparing the purified arabinose galactan composition, a microfiltration membrane (for example, a filter membrane of 0.ΐμπι) capable of filtering bacteria can be used for ultrafiltration to remove salts and reduce the solution volume (for example, 8K MWCO UF system). Concentrate the ultrafiltration solution after ultrafiltration (50-60 ° C, 20-26%) 13 In the standard paper of this paper, 闘 家 鲜 (CNS) A4 娜 (21 () x 297 公 爱) '----- ---- ^ --------- '(Please read the notes on the back before filling out this page) Printed clothing 1240629 A? _ B7 by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs丨), followed by precipitation with a low-carbon alkanol (about 80-90% ethanol). Further washing the precipitate (for example, three times with absolute alcohol) and drying (60-70 ° C vacuum oven) to obtain a purified arabinose galactan composition. Arabinose galactan protein composition The definition of arabinose galactan protein composition of the present invention is the arabinose galactan protein composition isolated from the root of a membra-like membra as described above; and Var. Mongholicus (Bge) Hsiao ^ A. (Fisch) Bge. Is preferably isolated from the root of membranaceus Bge. Var. Mongholicus (Bge) Hsiao ^ A. (Fisch) Bge .; The former; and the root of the plant is preferably from biennial astragalus. It has a sugar composition that is typically about 45 ° to 75% (mole percent), especially about 50% to 70% (mole percent) Arabinose (Ara); Rha between 2% and 4% (percent of moles); GalA between about 4% and 6% (percent of moles); between about 8% and 25% (percent of moles); In particular, Gal between 10% and 20%; and Glc between approximately 5% and 25% (molar percentage); wherein the ratio of Ara: Gal is not less than 2: 1; especially not less than 3: 1; And especially not less than 4 ·· 1; its ash content is not higher than 2% (weight percent); heavy metal content is not higher than 10 ppm; and hydroxyproline content is not higher than 0.2%, especially not less than 0.3%. It contains almost no endotoxin (as defined above); it can be dissolved in water at a concentration of at least 20 mg / ml, and the pH of the aqueous solution is between about 4.5 to 6.5 ; And the average molecular weight is not less than 100 kD, especially between 150 and 350 kD. It contains about 95% carbohydrates (including carbohydrates that can saccharify arabinose galactan protein core) , And about 14 paper sizes are applicable to Chinese National Standard (CNS) A4 specification mo X 297 mm) --------- ^ -------- (Please read the notes on the back before filling This page) 1240629 A7-B7 V. Description of the invention (1205% protein. Hydroxyproline, which accounts for about 20% of its total amino acids, is a characteristic feature of arabinogalactan protein. For convenience, the following are all "AGPC" is used to represent this substance. Preparation of arabinogalactan protein composition The arabinogalactan protein composition of the present invention is composed of the aforementioned arabinogalactan by a 100 K MWCO ultrafiltration system Or the distillate obtained from the purification step of the ion exchange column (or the distillate The solid intermediate after shrinking and drying) was further purified. The distillate obtained from the anion exchange column in the above "Preparation of arabinogalactan protein composition" was directly added to a 100 K MWCO ultrafiltration system The filtrate remaining in this 100 K MWCO ultrafiltration system is further concentrated and precipitated with a low carbon number alkanol, or further washed and dried, which are similar to the preparation of purified arabinose galactan composition. The arabinose galactan protein composition was obtained. Pharmacology and use
本發明第一態樣之純化的***糖半乳聚糖組成物或 本發明第二態樣之***糖半乳聚糖蛋白質組成物之有利 的活性,已在數種測試中獲得證實,並可知有下列用途。 治療化療患者嗜中性白血球細胞過少症 1·從活化的人類周邊血液單核細胞(PBMC)中製造細胞素 免疫及造血系統需藉助數種細胞素的交互作用才能被 激活,實施例3中檢驗了 PAGC在體外誘發生成細胞素的 能力。PAGC能啓動經PHA活化後之人類PBMC明顯、且 與劑量相關地釋放 IL-Ιβ、IL-6、TNF-α、IFN-γ、GM-CSF 15 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) (請先閱讀背面之注意事項再填寫本頁) tr--------- 經濟部智慧財產局員工消費合作社印製 經濟部智慧財產局員工消費合作社印製 1240629 A7 -------------B7 五、發明說明(、')) 及G-CSF。已知IL-6、GM-CSF及G-CSF這三種細胞素可 在體外及體內影響嗜中性白血球細胞的製造和/或活化。已 知IL-6本身或與其他細胞素一起,可刺激骨髓中的巨核細 胞成熟,並可恢復老鼠及非人類靈長類動物周邊血液中血 小板的數目。這些數據顯示PAGC可藉由製造與造血功能 相關之細胞素來刺激骨髓抑制患者自行製造嗜中性白血球 細胞並恢復其血小板數値。 2.復原經氟化尿嘧啶處理之老鼠體內GM-CFC代細胞 (GM_CFC progenitor cells)數目 實施例5是PAGC之短期、老鼠、體外造血代細胞的 測試模型。氟化尿嘧啶被廣泛用來去除老鼠骨髓中的代細 胞及細胞培育中會產生細胞群落的細胞單元(colony forming units in culture, CFU-C)。由於未被激活的幹細胞 不會受氟化尿嘧啶的影響,因此經過此種處理後的老鼠, 其會沿著一已知、可預期的時間曲線復原[A. M. Yeager et al·,uThe effects of 5-fluorouracil on hematopoiesis: studies of murine megakaryocyte-CFC, granulocyte-macrophage-CFC,and peripheral blood cell levels’’,Exp. Hematol.,11, 944-952 (1983)]。由實施例5中的表格可知在氟化尿嘧啶 處理後第4天,隨著PAGC劑量的增加,每隻老鼠體內 GM-CFC的數量也跟著上升。當所施用PAGC劑量爲200 毫克/公斤時,每隻老鼠平均所增加的GM-CFC爲控制組老 鼠的3.3倍(ρ<0·01);若是以100毫克/公斤之PAGC來處 理,則每隻老鼠平均所增加的GM-CFC爲控制組老鼠的 16 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ---------訂---------. (請先閱讀背面之注意事項再填寫本頁) 1240629 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明(外) 1.8倍(p<0.01);若是以50毫克/公斤之PAGC來處理,則 每隻老鼠平均所增加的GM-CFC與控制組相去不遠。這些 數據顯示PAGC可促進氟化尿嘧啶所誘發之骨髓抑制老鼠 體內骨髓代細胞(GM-CFC)的復原,其係藉由PAGC可促進 骨髓抑制老鼠體內周邊白血球細胞數値的增加所致。 3.回復接受亞致死劑量之輻射照射的老鼠體內周邊白血球 細胞數値 以亞致死劑量之輻射量來照射BALB/c老鼠’之後再 依據實施例6之步驟由皮下注射生理食鹽水或各種劑量之 PAGC到老鼠體內。相較於正常老鼠,接受輻射照射之老 鼠在輻射照射後第Η天,其WBC的數目會下降到正常老 鼠値之12%。相較於僅注射生理食鹽水的老鼠而言,以 PAGC治療可增加老鼠體內WBC的總數。以1〇〇或300毫 克/公斤之PAGC治療的老鼠,其體內WBC總數可比僅注 射生理食鹽水的老鼠早7-9天,回復至正常鼠數値的80%( 正常値:6000-10000 WBC/mm3血液)。在PAGC處理的老 鼠中,約22-23天後,其WBC數値即可回復正常;而僅注 射生理食鹽水的老鼠此時的數値則僅爲正常値的46%。此 外,也可由周邊血液抹片染色來得知不同的WBC値。從 這些資訊中可計算出嗜中性白血球細胞及淋巴細胞的絕對 値。相較於僅注射生理食鹽水的老鼠,以PAGC治療可增 加嗜中性白血球細胞數目的絕對値。同時也能增加淋巴細 胞數目的絕對値。 4·回復化療患者體內白血球細胞數値 17 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公® ) ----. (請先閱讀背面之注意事項再填寫本頁) ----訂---- 1240629 A7 ___ B7 五、發明說明(X、) 所有第二期臨床試驗均係在中華人民共和國內完成。 試驗中分析了一小群參與實驗時其WBC數値即低於3 X 109 WBC/L之患者的數據:其中168位患者係接受pAGC 治療,59位患者係接受G-CSF治療,另23位患者則未接 受任何治療。治療流程詳述於實施例9中。如圖1所示’ PAGC可穩定地增加接受化療後患者體內的WBC數値’且 此增加的情形可持續到第14天。雖然施用G-CSF ’可使 WBC增加的速度較快,但卻無法持續,到第7天後’患者 體內的WBC數値即開始下降。相較於未接受任何治療的 控制組而言,這兩組治療組都可使WBC數値回復到正常 。相較於未接受任何治療的控制組而言,PAGC治療組之 WBC數値可於第10天及第14天達到明顯差異;但PAGC 組與G-CSF組在第14天時,兩組數値並無明顯差異。上 述結果顯示對接受化療治療之肺癌、腸胃癌及乳癌患者使 用PAGC作爲治療佐劑,不僅安全且是患者可以容忍的。 PAGC治療可回復化療患者體內的WBC數値。且臨床上以 PAGC進行治療時,也未發現任何嚴重的副作用。 治療化療患者血小板細胞過少症 1.回復接受亞致死劑量之輻射照射的老鼠體內周邊血小板 細胞數値 在第〇天時,以亞致死劑量之輻射量來照射BALB/c 老鼠,之後再依據實施例6之步驟以生理食鹽水或各種劑 量之PAGC來進行治療。相較於正常老鼠,接受幅射照射 之老鼠在輻射照射後第1〇天,其WBC的數目會下降到正 18 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) (請先閱讀背面之注意事項再填寫本頁) II--------- 經濟部智慧財產局員工消費合作社印製 經濟部智慧財產局員工消費合作社印製 1240629 A7 B7 五、發明說明(4) 常老鼠値之7%。以皮下注射100或300毫克/公斤之 PAGC治療的老鼠,可明顯提局老體內周邊血小板細胞數 値。相較於僅接受生理食鹽水注射的老鼠而言’ PAGC可 比控制組早5-6天,誘發血小板細胞數目回復至正常老鼠 的80%(正常値:8-12 X 1〇6血小板/mm3血液)。^PAGC 處理的老鼠,在輻射照射後約24-25天’其血小板數値即 可回復正常;而僅注射生理食鹽水的老鼠此時的數値則僅 爲正常値的72%。這些結果顯示PAGC是一種相當有效能 促進血小板發展的藥劑,因此對治療血小板數目過少相當 有用。以同樣方式在爲期20天的療程中,以1〇〇及250毫 克/公斤之AGPC進行治療的老鼠,相較於控制組’在所有 時間點進行測試時,均可改善其紅血球及血小板數目;顯 示PAGC在此模型系統中也有相同的療效。 2.骨髓巨核細胞的增生及成熟 PAGC .可加速恢復輻射照射動物模型中所誘發之骨髓 抑制動物其體內周邊血小板數値,如前段文字所述;此顯 示PAGC可刺激骨髓巨核細胞的增生和/或成熟。關於這個 可能性,係以實施例4所述之體外液態培育系統 liqid culture system)進行硏究。由劑量滴定曲線可知,此模 型中最佳的PAGC劑量爲100-200微毫克/毫升,且ED50 是30-40微毫克/毫升。此硏究顯示單獨使用PAGC即可促 進體外骨髓巨核細胞的增生和/或成熟。此外,PAGC亦可 和次佳劑量之IL-3(50 pg/ml)—起協同作用,以劑量相關的 方式增加乙醯膽鹼酯酶(AchE)的含量。由經200微毫克/毫 19 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公爱) ---------^--------- (請先閱讀背面之注意事項再填寫本頁) 1240629 A/ B7 五、發明說明() 升之PAGC及50 pg/ml之IL-3處理過之細胞培育物中所 獲得的AchE含量,相當於以最佳劑量之IL-3處理過後之 培育物中的AchE含量。适些數據顯不PAGC可提尚骨髓 巨核細胞對較低劑量之1L_3的反應性,或是IL_3可增加 骨髓巨核細胞對PAGC的反應性。因化療或輻射照射會破 壞包括可製造細胞素之細胞在內的細胞及組織’因此接受 此種治療的患者其體內內生性細胞素的含量會較低。這麼 低的細胞素含量將無法支持造血系統的正常功能’因而無 法產生足夠數目的造血細胞以恢復受抑制的骨髓功能。因 此,PAGC乃是能修復此種情況的最佳物質。此硏究結果 顯示PAGC可在內生性細胞素不足的情況下,促進骨髓巨 核細胞的增生和/或成熟。如上所示,PAGC可刺激周邊血 小板數目恢復正常,且此種作用似乎是藉由誘發骨髓巨核 細胞的增生和/或成熟而達成的。此數據顯示PAGC對治療 因骨髓抑制所致之血小板數目過少症可能很有用。 3.增加化療後血小板細胞的數値 第二期臨床試驗係如實施例9所述。試驗中分析了一 小群參與實驗時其WBC數値即低於4 X 109 WBC/L、且血 小板數値低於90 X l〇9/L之患者的數據:其中54位患者係 接受PAGC治療。這些患者的血小板數値在第7天增加到 高於100 X l〇9/L,並持續增加至第14天,如表9所示。 這些數據顯示PAGC可增加這些化療患者體內的血小板數 値。 改善癌症患者的生活品質 20 尺度適用中國國家標準(cnS)A4規格(210 X 297公釐) " ^ (請先閱讀背面之注意事項再填寫本頁) — 訂 · 經濟部智慧財產局員工消費合作社印製 經濟部智慧財產局員工消費合作社印副衣 1240629 A7 __B7 五、發明說明(d) 第一期臨床試驗係如實施例9所述。如實施例9所述 ’以改善化療症狀及卡諾夫斯基表現指標(Karnofsky performance Index)之數値作爲評估所有參與試驗之患者生 活品質的依據。記錄並計算治療期間由化療所引起的症狀 ,包括疲憊及倦怠、不適、盜汗、呼吸短促及沒有食慾在 內。如圖2所示,隨著治療進展,顯示PagC組的療效最 快,此可由症狀指數下跌及其較G-CSF組更快恢復至正常 數値(數値=〇)這點上看出。此外,相較於G-CSF組, PAGC組在桌10天至弟14天期間,也表現出比統計數字 更明顯的改善(Ρ<〇·〇1)。然而,但G-CSF組與控制組(未接 受任何治療)則在整個14天的監測期間,都無任何明顯統 計上的差異。這些數據顯示PAGC可改善化療患者的症狀 ,而關於這點,G-CSF則似乎無任何效用。分析治療前卡 諾夫斯基表現指標(Karnofsky performance Index)數値低於 70之一小群患者之症狀改善指數。相較於G-CSF組及未接 受任何治療之控制組而言,PAGC組可顯著改善症狀,其 統計上的差異分別爲ρ<〇·〇1及p<0.0001,如圖3所示。大 部分PAGC組中的患者的指數都比其他兩組爲高。 預防接受化療之癌症患者體內嗜中性白血球細胞過少 在前面段落中,已知PAGC可有效地治療化療所引發 的嗜中性白血球細胞過少症。這些數據顯示PAGC對預防 及治療嗜中性白血球細胞過少一樣有效,目前也預計要在 中華人民共和國內進行臨床試驗。 治療接受輻射照射之癌症患者體內嗜中性白血球細胞過少 21 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) " (請先閱讀背面之注意事項再填寫本頁) tr' A7 B7 經濟部智慧財產局員工消費合作社印製 1240629 五、發明說明(q) 前面的藥理數據也顯示PAGC可有效地恢復輻射照射 所引發的嗜中性白血球細胞過少症。目前已開始在中華人 民共和國內進行臨床試驗,以了解PAGC治療非-哈金氏淋 巴瘤癌症患者在接受輻射照射後所引起的嗜中性白血球細 胞過少症的情形。 治療接受化療之癌症患者之貧血現象 以亞致死劑量(4.25 Gy)之輻射量來照射BALB/c老鼠 ,之後再依據實施例6之步驟以生理食鹽水或各種劑量之 PAGC來進行治療。相較於正常老鼠,接受輻射照射之老 鼠在輻射照射後第Π天,其RBC的數目會下降到正常老 鼠値之55%。經PAGC處理的老鼠在第17天時可得相當 高的RBC數値,而僅注射生理食鹽水的老鼠此時的數値則 仍舊很低。以皮下注射或3〇〇毫克/公斤之PAGC治 療的老鼠,可明顯提高老體內周邊RBC細胞數値。相較於 僅接受生理食鹽水注射的老鼠而言,PAGC可比控制組早 4-6天,誘發RBC數目回復至正常老鼠的80%(正常値: 8·5-11 X 106 RBC /mm3血液)。經PAGC處理的老鼠,約在 20-22天其RBC數値即可回復正常;而僅注射生理食鹽水 的老鼠此時的數値則僅爲正常値的65%。這些結果顯示 PAGC是一種相當有效能促進紅血球代細胞發育和/或移動 的藥劑,因此對治療輻射照射或化療所引起的貧血相當有 用。以同樣方式在爲期20天的療程中,以100及250毫克 /公斤之AGPC進行治療的老鼠’相較於控制組’在所有時 間點進行測試時,均可改善其紅血球及血小板數目;顯示 22 1本纸張尺度適用中國國家標準(CNS)A4規格(210 X 297公爱) " (請先閱讀背面之注意事項再填寫本頁)The advantageous activity of the purified arabinose galactan composition of the first aspect of the present invention or the arabinose galactan protein composition of the second aspect of the present invention has been confirmed in several tests, and it is known that It has the following uses. Treatment of neutropenia in chemotherapy patients 1. To produce cytokine immunity and hematopoietic systems from activated human peripheral blood mononuclear cells (PBMCs) requires the interaction of several cytokines to be activated, which is tested in Example 3. The ability of PAGC to induce cytokine production in vitro was demonstrated. PAGC can activate human PBMC after PHA activation, and release IL-Ιβ, IL-6, TNF-α, IFN-γ, GM-CSF obviously and dose-dependently. 15 This paper size applies to Chinese National Standard (CNS) A4 specifications. (210 X 297 mm) (Please read the notes on the back before filling out this page) tr --------- Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1240629 A7 ------------- B7 V. Description of the Invention (, ')) and G-CSF. It is known that three cytokines, IL-6, GM-CSF, and G-CSF, can affect the production and / or activation of neutrophils in vitro and in vivo. It is known that IL-6 itself or together with other cytokines can stimulate megakaryocyte maturation in the bone marrow and restore the number of platelets in the peripheral blood of mice and non-human primates. These data show that PAGC can stimulate patients with bone marrow suppression to make neutrophils and restore their platelet count by making cytokines related to hematopoietic function. 2. Recovery of GM-CFC progenitor cells in mice treated with fluorinated uracil. Example 5 is a test model of PAGC's short-term, mouse, and in vitro hematopoietic cells. Fluorinated uracil is widely used to remove progenitor cells in mouse bone marrow and colony forming units in culture (CFU-C) in cell culture. Since unactivated stem cells are not affected by fluorinated uracil, the treated mice will recover along a known and predictable time curve [AM Yeager et al., UThe effects of 5 -fluorouracil on hematopoiesis: studies of murine megakaryocyte-CFC, granulocyte-macrophage-CFC, and peripheral blood cell levels '', Exp. Hematol., 11, 944-952 (1983)]. From the table in Example 5, it can be seen that the amount of GM-CFC in each mouse also increased with the increase of the dose of PAGC on the fourth day after the uracil fluoride treatment. When the dose of PAGC administered was 200 mg / kg, the average increase in GM-CFC per mouse was 3.3 times that of the control group (ρ <0.01); if it was treated with 100 mg / kg PAGC, each The average increase of GM-CFC per mouse is 16 paper sizes of the control group of rats, which is applicable to China National Standard (CNS) A4 (210 X 297 mm) --------- Order ------ ---. (Please read the notes on the back before filling out this page) 1240629 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (outside) 1.8 times (p <0.01); if it is 50 mg / For the treatment of kg of PAGC, the average increase of GM-CFC per mouse is not far from that of the control group. These data show that PAGC can promote the recovery of bone marrow generation cells (GM-CFC) in bone marrow-suppressed mice induced by fluorinated uracil. PAGC can increase the number of peripheral white blood cells in bone marrow-suppressed mice. 3. Resume the number of peripheral white blood cells in the mice that received sublethal doses of radiation 値 irradiate BALB / c mice with sublethal doses of radiation and then inject subcutaneous saline or various doses according to the procedure of Example 6 PAGC into mice. Compared with normal rats, the number of WBCs in the rats exposed to radiation decreased to 12% of that in the normal rats on the day after radiation exposure. Compared to mice injected with saline alone, treatment with PAGC increased the total number of WBCs in the mice. In mice treated with 100 or 300 mg / kg PAGC, the total number of WBCs in the body was 7-9 days earlier than that of rats injected with normal saline alone, and returned to 80% of normal rats (normal: 6000-10000 WBC). / mm3 blood). In the old rats treated with PAGC, the WBC count returned to normal after about 22-23 days, while the rats injected with saline only had 46% of the normal count at this time. In addition, peripheral blood smears can be used to learn about different WBCs. From this information, the absolute 値 of neutrophils and lymphocytes can be calculated. Compared with mice injected with saline alone, PAGC treatment increased the absolute number of neutrophils. It can also increase the absolute number of lymphocytes. 4 · Number of white blood cells in patients receiving chemotherapy 値 17 This paper size is applicable to Chinese National Standard (CNS) A4 (210 X 297 males) ----. (Please read the precautions on the back before filling this page)- --Order ---- 1240629 A7 ___ B7 V. Description of Invention (X,) All Phase II clinical trials were completed in the People's Republic of China. The trial analyzed the data of a small group of patients whose WBC number was less than 3 X 109 WBC / L when participating in the experiment: 168 patients were treated with pAGC, 59 patients were treated with G-CSF, and another 23 The patient was not receiving any treatment. The treatment process is detailed in Example 9. As shown in Fig. 1, 'PAGC can stably increase the number of WBCs in patients after receiving chemotherapy', and this increase can continue to the 14th day. Although the administration of G-CSF 'can increase the WBC faster, it cannot be sustained, and the WBC number in the patient's body will begin to decrease after the 7th day. Compared with the control group who did not receive any treatment, both groups of treatment groups returned WBC count to normal. Compared with the control group that did not receive any treatment, the number of WBCs in the PAGC treatment group could reach a significant difference on the 10th and 14th days; but on the 14th day, the PAGC group and the G-CSF group had two groups.値 There is no significant difference. The above results show that the use of PAGC as an adjuvant for lung cancer, gastrointestinal cancer and breast cancer patients receiving chemotherapy is not only safe but also tolerable by patients. PAGC treatment can restore the number of WBCs in chemotherapy patients. In addition, no serious side effects were found in the clinical treatment with PAGC. Treatment of Chemotherapy Patients with Low Platelet Cells 1. Resume the number of peripheral platelet cells in rats that received sublethal doses of radiation. On day 0, irradiate BALB / c mice with sublethal doses of radiation, and then follow the examples Step 6 is treated with physiological saline or various doses of PAGC. Compared with normal mice, the number of WBCs of rats exposed to radiation will decrease to exactly 18 days after radiation exposure. The paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ( Please read the notes on the back before filling out this page) II --------- Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1240629 A7 B7 4) 7% of Chang rats. Mice treated with subcutaneous injection of 100 or 300 mg / kg PAGC can significantly increase the number of peripheral platelet cells in the old body. Compared with mice that received saline injection only, PAGC could induce platelet cell numbers to return to 80% of normal mice 5-6 days earlier than the control group (normal: 8-12 X 106 platelets / mm3 blood). ). ^ PAGC-treated mice returned to normal about 24-25 days after radiation exposure, while rats injected with saline only returned 72% of normal numbers. These results show that PAGC is a potent agent that promotes platelet development and is therefore quite useful for treating too few platelets. In the same way, rats treated with 100 and 250 mg / kg of AGPC during a 20-day course of treatment improved their red blood cells and platelet numbers compared to the control group when tested at all time points. ; Shows that PAGC has the same effect in this model system. 2. Proliferation of bone marrow megakaryocytes and mature PAGC. It can accelerate the recovery of bone marrow suppression in the animal model of bone marrow suppression induced by radiation irradiation, as described in the previous paragraph; this shows that PAGC can stimulate the proliferation of bone marrow megakaryocytes and / Or mature. This possibility was investigated using the in vitro liquid culture system (Liqid Culture System) described in Example 4. From the dose titration curve, we know that the optimal PAGC dose in this model is 100-200 micromg / ml, and the ED50 is 30-40 micromg / ml. This study shows that PAGC alone can promote the proliferation and / or maturation of bone marrow megakaryocytes in vitro. In addition, PAGC also works synergistically with a suboptimal dose of IL-3 (50 pg / ml) to increase acetylcholinesterase (AchE) content in a dose-dependent manner. Made by 200 micrograms / milligrams 19 This paper size is applicable to China National Standard (CNS) A4 specifications (210 X 297 public love) --------- ^ --------- (Please read first Note on the back, please fill out this page again) 1240629 A / B7 V. Description of the invention () The AchE content obtained in the cell culture treated with 1 liter of PAGC and 50 pg / ml of IL-3 is equivalent to the optimal dose AchE content in IL-3 treated cultures. Appropriate data shows that PAGC can improve the response of bone marrow megakaryocytes to lower doses of 1L_3, or that IL_3 can increase the response of bone marrow megakaryocytes to PAGC. Because chemotherapy or radiation can damage cells and tissues, including cells that can produce cytokines, patients who receive this treatment will have lower levels of endogenous cytokines. Such a low cytokine content will not be able to support the normal function of the hematopoietic system 'and therefore cannot produce a sufficient number of hematopoietic cells to restore the suppressed bone marrow function. Therefore, PAGC is the best substance to repair this situation. The results of this study show that PAGC can promote the proliferation and / or maturation of bone marrow megakaryocytes in the absence of endogenous cytokines. As shown above, PAGC can stimulate the number of peripheral blood platelets to return to normal, and this effect seems to be achieved by inducing the proliferation and / or maturation of bone marrow megakaryocytes. This data suggests that PAGC may be useful in treating hypoplasia due to bone marrow suppression. 3. Increasing the number of platelet cells after chemotherapy. The second phase clinical trial is as described in Example 9. The data of a small group of patients participating in the experiment when their WBC number was less than 4 X 109 WBC / L and platelet number was less than 90 X 10 / L were analyzed in the trial: 54 patients received PAGC treatment . The platelet count of these patients increased to more than 100 × 109 / L on day 7, and continued to increase to day 14, as shown in Table 9. These data show that PAGC can increase platelet counts in these chemotherapy patients. Improving the quality of life of cancer patients 20 scales applicable to China National Standard (cnS) A4 specifications (210 X 297 mm) " ^ (Please read the precautions on the back before filling out this page) — Order · Consumption by the Intellectual Property Bureau of the Ministry of Economic Affairs Cooperative printed by the Intellectual Property Bureau of the Ministry of Economic Affairs. Consumption printed by the cooperative of the employees. Cooperative printed 1240629 A7 __B7 V. Description of the invention (d) The first phase clinical trial is as described in Example 9. As described in Example 9, the number of improving chemotherapy symptoms and Karnofsky performance index (Karnofsky performance index) was used as the basis for evaluating the quality of life of all patients participating in the trial. Record and calculate symptoms caused by chemotherapy during treatment, including fatigue and burnout, discomfort, night sweats, shortness of breath, and lack of appetite. As shown in Figure 2, as the treatment progressed, the PagC group showed the fastest curative effect, which can be seen from the decrease in the symptom index and its faster return to normal number (number = 値) than the G-CSF group. In addition, compared with the G-CSF group, the PAGC group also showed a more significant improvement than the statistics during the period of 10 days to 14 days (P < 0.001). However, there were no significant statistical differences between the G-CSF group and the control group (without any treatment) throughout the 14-day monitoring period. These data show that PAGC improves symptoms in chemotherapy patients, and in this regard, G-CSF seems to have no effect. Analyze the symptom improvement index of a small group of patients with a Karnofsky performance index below 70 before treatment. Compared with the G-CSF group and the control group not receiving any treatment, the PAGC group can significantly improve symptoms, and the statistical differences are ρ < 0.001 and p < 0.0001, respectively, as shown in FIG. 3. Patients in most PAGC groups had higher indices than the other two groups. Preventing too few neutrophils in cancer patients undergoing chemotherapy In the previous paragraph, PAGC is known to be effective in treating neutropenia caused by chemotherapy. These data show that PAGC is equally effective in preventing and treating too few neutrophils, and clinical trials are currently expected in the People's Republic of China. Too little neutrophil cells in the treatment of cancer patients receiving radiation exposure 21 This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) " (Please read the precautions on the back before filling this page) tr 'A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1240629 V. Description of the Invention (q) The preceding pharmacological data also show that PAGC can effectively recover the neutrophilic leukocytosis caused by radiation exposure. Clinical trials in the People's Republic of China have begun to understand PAGC in treating patients with non-Harkin's lymphoma cancer who have received neutropenia after radiation exposure. Anemia in cancer patients treated with chemotherapy. BALB / c mice were irradiated with a sub-lethal dose (4.25 Gy) of radiation, and then treated with physiological saline or various doses of PAGC according to the procedure of Example 6. Compared with normal rats, the number of RBCs in the rats exposed to radiation will drop to 55% of that in normal rats on day Π after irradiation. On the 17th day, the rats treated with PAGC could obtain a relatively high RBC count, while the rats injected with saline only still had a low count at this time. Mice treated with subcutaneous injection or 300 mg / kg PAGC can significantly increase the number of peripheral RBC cells in the old body. Compared with mice receiving saline injection only, PAGC can induce RBC to return to 80% of normal mice 4-6 days earlier than the control group (normal 値: 8 · 5-11 X 106 RBC / mm3 blood) . In rats treated with PAGC, their RBC counts returned to normal in about 20-22 days, while rats injected with physiological saline only had 65% of normal counts at this time. These results show that PAGC is a very effective agent that promotes the development and / or migration of red blood cell cells, and is therefore very useful for treating anemia caused by radiation exposure or chemotherapy. In the same manner, rats treated with AGPC at 100 and 250 mg / kg during the 20-day course of treatment "improved their red blood cell and platelet numbers when tested at all time points compared to the control group"; 22 1 This paper size applies to China National Standard (CNS) A4 (210 X 297 public love) " (Please read the precautions on the back before filling this page)
經濟部智慧財產局員工消費合作社印製 1240629 A/ B7 五、發明說明(/ ) PAGC在此模型系統中也有相同的療效。 實施例9中的第二期臨床試驗旨在硏究化療患者其 WBC的恢復情形,因此其條件是要依據WBC數値來進行 分析試驗。目前以規劃要在PRC(中華人民共和國)內硏究 以PAGC治療貧血、及化療所引起的貧血之臨床試驗。 單獨移動周邊血液代細胞或與G-CSF —起來治療接受周 邊血液代細胞移植之患者 造血幹細胞及包括諸如BFU-E(bUrst-f〇rming unit -erythroid)這類會產生細胞群落、GM-CFC(生成粒細胞巨嗜 細胞群落)、及CFU-Mix(會產生細胞群落之混合物)在內之 代細胞上均含有CD4抗原。CD34*細胞之流動細胞儀分析 結果提供了 一種計算移植組成物(graft composition)最好的 方式。早在1975年,即已提出人類周邊血液系統中存在有 造血代細胞(PBPC)這樣的描述。這些周邊血液代細胞僅佔 總細胞數的一小部分,因大部分的代細胞均位於骨髓中。 1976年時,首先有人提出化療後的恢復期間,人類周邊血 液系統中的造血代細胞數目會上升。最近,更有人報導說 細胞群落刺激因子(colony stimulating factor)G-CSF 及 GM-CSF,可直接提高PBPC的數目。相較於單獨使用,化療 時合倂使用細胞群落刺激因子,可大幅增加PBPC的數目 。目前,G-CSF被用來在人類自體及異體移植中,移動 PBPC。相較於傳統的骨髓移植,移植PBPC可使移植進行 得更快速。如果與化療或其他細胞素合倂使用的話,可更 大幅提高G-CSF治療所產生的PBPC數目。單獨使用 23 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ' ---------^--------- (請先閱讀背面之注意事項再填寫本頁) A7 1240629 ____B7___ 五、發明說明(、.丨) PAGC或與G-CSF協同使用,來誘發PBPC移動的能力詳 述於實施例7。如實施例7中的表格結果所示,PAGC可提 高循環之GM-CFC量約6倍之多。此外,PAGC與G-CSF 協同使用時,可提高循環之GM-CFC量約83倍之多。此 增加量遠較單獨使用G-CSF時所增加的39倍量還要高。 結果顯示PAGC可提高循環之GM-CFC的量,或與G-CSF 協同增加正常老鼠體內循環之GM-CFC的量。PAGC也可 增加循環之BFU-E的量約3倍,如表格所示,並與G-CSF 協同增加循環之BFU-E的量約9.5倍。此增加量遠較單獨 使用G-CSF時所增加的4.5倍量還要高。在以環磷醯胺處 理之老鼠的類似實驗中,相較於未接受治療的老鼠’其 CD34+Lin細胞的數會增加。諸如PAGC這類,可與G-CSF 協同作用以增加PBPC數目的藥劑也很有效。這類協同作 可降低需從捐贈者體內獲取PBPC細胞所需花費的費用。 此外,這類的合倂治療,對於當接受移植之患者對單獨使 用G-CSF反應不佳時也有幫助,或是無法使用化撩之患者 也有幫助。同樣的,AGPC對移植也很有效。 治療惡病質 化療及輻射照射最常見的一種副作用就是造成患者食 慾降低及體重減輕。實施例8顯示PAGC可使經化療藥劑 環磷醯胺及氟化尿嘧啶處理後之老鼠的體重增加。相較於 單獨以環磷醯胺處理後之老鼠,經PAGC處理過之老鼠’ 其體重減輕較少,並可更快恢復其原來體重。但這些差異 在統計上,還未達明顯差異。 24 本紙張尺度適^Ϊ國家鮮(CNS)A4祕(210 X 297公餐1 ' '一* U--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1240629 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明(π” 如上述,PAGC明顯可改善第二期臨床試驗患者的生 活品質。其中所測量的一項參數爲食慾。此與老鼠模型硏 究結果,顯示PAGC可幫助改善患者之惡病質’其係一種 由諸如癌症這種慢性病或癌症治療所造成的一種身爲上的 浪費及營養不良現象。 與G-CSF合倂治療接受抑制骨髓治療之癌症患者以促進 嗜中性白血球細胞恢復並降低G_CSF用量 前面的討論顯示PAGC可刺激細胞素的生成’特別是 人類周邊血液單核細胞所產生的G_CSF ;並可促進因輻射 照射所致之骨髓抑制動物模型中GM_CFC及周邊WBC數 目的恢復,及使第二期臨床試驗之癌症患者接受化療後體 內WBC値回復至正常。這些結果顯示PAGC可藉由製造 多種內生性細胞素來間接作用於造血系統上,這些細胞素 則可彼此協同作用來恢復嗜中性白血球細胞數目。同時, PAGC亦可和IL-3協同作用來促進骨髓巨核細胞的增生/成 熟。 此外,施用PAGC不僅安全,且可爲臨床試驗之患者 所容忍。而使用G-CSF時倂發之嚴重副作用例如骨頭痛、 肌肉痛、頭痛、疲憊、噁心、嘔吐、下痢等,在施用 PAGC時並未發現。此與上述之協同作用,顯示PagC可 與G-CSF合倂使用來降低外因性G-CSF的用量,並促進接 受骨髓抑治療之癌症患者,其體內嗜中性白血球細胞數目 的復原。 在高劑量細胞毒性治療及自體或異體血液代細胞移植後, 25 本纸張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 裝---------tr--------- (請先閱讀背面之注意事項再填寫本頁) 1240629 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明(4) 合倂使用G-CSF治療以促進嗜中性白血球細胞復原 目前以高劑量化療和/或輻射照射及支持性的血液代細 胞(BPC)移植來治療許多癌症患者,例如乳癌、淋巴癌及多 重骨髓癌患者。移植BPC可加速恢復造血功能。通常在移 植BPC後會配合使用G-CSF,以加速恢復嗜中性白血球細 胞的數目,以預防因感染所致之嗜中性白血球細胞過少所 引起的發燒,並縮短住院期,及降低抗生素用量。如實施 例9第二期臨床試驗所示,PAGC可恢復化療癌症患者的 WBC數値;此與前段文字總結之藥理效用,顯示合倂使用 PAGC與G-CSF,對移植BPC後加速恢復嗜中性白血球細 胞的數目非常有用。 改善及修飾B型肝炎帶原者之生物反應性及保護肝臟細胞 細胞素在對抗病毒感染上扮演了相當重要的角色。細 胞素係由與免疫系統相關的細胞所製造,例如巨嗜細胞; 而CD4+及CD8+T淋巴細胞則對個體由諸如B型肝炎病毒 (HBV)感染中恢復有較直接的影響。從動物模型到人體硏 究,可淸楚地知道細胞免疫反應可能對解決HBV感染及其 疾病病理上扮演了一些角色。在急性HBV感染中,強烈的 多株細胞免疫反應乃是必需的。第1型細胞素的釋出特徵 是會由CD4+ T淋巴細胞製造生成IL-2及INF-γ ;並藉由 IL-2及INF-γ來誘發並維持細胞免疫性,這點對於對抗病 毒感染乃是相當重要的[c· A. Biron,“cytokines in the generation of immune response to, and regulation of, virus infection”,Cur. 〇pin. Immunol·,6,530-538 (1994)]。CD4+ 26 本紙張尺度適用中國國家標準(CNS)A4規格(210 x 297公釐) ---------^---------^^^1 (請先閱讀背面之注意事項再填寫本頁) 1240629 A7 B7 五、發明說明(A) 及CD8+細胞所釋放的細胞素對於向下調節 (downregulation)HBV之複製上也扮演了相當重要的角色。 如果急性反應上出現了缺陷,就會變成慢性HBV感染。這 些結果顯示強化第1型反應或強化在肝臟局部地區製造適 當的細胞素可能對治療慢性HBV感染非常有用[M.J. Koziel,Sem. Liver Disease,19(2),157-161 (1999)]。PAGC 係由傳統中藥植物(心似cews Far· (AM))所製得,並早已被用來刺激免疫及造血 系統。其被廣泛用來治療各種貧血患者,這類患者的病均 很類似化療或輻射照射所誘發的骨髓抑制情形(除嗜中性白 血球細胞過少外,還有血小板過少及貧血)。此外,已知 AM可於體外刺激老鼠胰臟細胞增生,且此細胞增生現象 係與劑量呈正相關;並可提高已接種了 S-180肉瘤細胞之 動物體內胰臟細胞中的天然殺手細胞(Natural killer,NK)活 性;並提高具細胞毒性之T-淋巴細胞(cytotoxic T lymphocytes,CTL)活性。CTL所製造之細胞素可控制體內 病毒感染,而病毒專一性之CTL所製造的INF-γ及INF-a ,可增加CTL淸除感染病毒的能力[L. G. Guidotti et al·, “Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice’’, Proc. Natl· Acad. Sci. USA,91,764-3768 (1994)]。此外, 如上述,PAGC可於以PHA活化後,刺激人類PBMC製造 生成 IL-Ιβ、IL-6、TNF-α、TNF-γ、GM-CSF、及 G-CSF。 這些結果顯示PAGC可藉由調控細胞素的生成來間接影響 2Ί 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ----------- (請先閱讀背面之注意事項再填寫本頁) --訂---- Φ. 經濟部智慧財產局員工消費合作社印製 1240629 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明() 免疫系統。由AM製得的粗萃取物已被用來治療慢性肝炎 患者,以降低其體內過高的IgG量,並降低ALT値,改善 患者的免疫及肝臟功能[Y· Liu,“therapeutic effect of oral solution from Astragalus in the treatment of 70 chronic hepatitis B patients”,Jiang Su Chung Yao,15(12),38 (1994)]。此外,已知AM的分餾物具有如前述能增加免疫 功能的能力。這些結果顯示PAGC可用來改善及修飾B型 肝炎帶原者之生物反應性及保護肝臟細胞。 B型肝炎患者之E疫苗佐劑 由所製備而成的多醣,已被用作 B型肝炎疫苗的佐劑來治療慢性B型肝炎。已知其對於轉 換B型肝炎e抗原(HBeAg)爲血淸陰性及消除B型肝炎病 毒DNA(HBV-DNA)具有令人滿意的效果[S.M· Wu et al., “The therapeutic observation on the combined Polyporus polysaccharide with hepatitis B vaccine in the treatment of chronic hepatitis J. Chin. Infectious Disease, 13(3), 187-189 (1995); Η· Z· Shu et al·,“The therapeutic observation on the Polyporus polysaccharide with large dose of hepatitis B vaccine in the treatment of 64 cases of chronic hepatitis B patients”,Med. J. NDFSC,6(4),21 1-212 (1996)]。已知由 AM製得的萃取物可將B型肝炎e抗原(HBeAg)及抗B型 肝炎病毒核心抗原(anti HBc)轉換爲血淸陰性,並去除B型 肝炎患者之B型肝炎病毒DNA(HBV-DNA)[C. K. Liu et al., “Clinical and experimental studies on effects of chronic 28 本紙張尺度適用中國國家標準(CNS)A4規格(210 x 297公釐) ^--------- (請先閱讀背面之注意事項再填寫本頁) 1240629 A7 B7 五、發明說明(A) hepatitis B treated with Astragli composita”,Chung Kuo Chung His I Chieh Ho Tsa Chin,16(7),394-397 (1996); P. L· Chen et al·,“Polysaccharide from Astragalus in treating 33 cases of chronic active hepatitis B patients’’,New Drugs Clin. Remedies,11 (2),75-76 (1991)]。上述這些結果顯示 PAGC可能可以作爲B型肝炎患者之疫苗佐劑。 治療***勒戒之脫癱症狀 患者開始戒除使用***時,一般會出現脫癮症狀。 就傳統中醫(Tranditional Chinese Medicine, TCM)觀點而言 ,所謂的脫癮症狀就是氣(能量)不足。就中醫師的觀察而 言,如果能強化氣,就能加速藥物戒除過程。實施例9關 於PAGC臨床試驗的結果之一,就是可改善癌症化療患者 的生活品質。藉由評估與化療相關之病徵的改善與否,來 評定生活品質是否獲得改善;這些包括疲倦及疲憊、不適 、冒冷汗、呼吸急促及缺乏食慾。這些症狀都是典型”氣” 不足的一種表現,也是傳統中醫所認爲之脫癮症狀的典型 表現。因此,PAGC除了能強化患者的”氣”外,還可治療 欲戒除使用***物患者的脫癮症狀。 預防及治療接受化療或輻射照射之癌症患者的嘔吐症狀 化療和/或輻射照射最常出現的副作用就是噁心及嘔吐 。雖然目前化療或輻射照射技術已有大幅改良,但仍有相 當高比例的患者會出現噁心的症狀,因此業者也在持續找 寺目§降低追類副作用的方法。雖然市面上有許多這類能抗 嚼心的藥(例如5-羥色胺受體頡抗劑、類皮質激素及多巴胺 29 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公爱) 丨——丨----- (請先閱讀背面之注意事項再填寫本頁) 訂--- 經濟部智慧財產局員工消費合作社印製 1240629 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明(J) 受體頡抗劑),但是,這類藥物也有其本身的其他副作用。 和這些藥物相關的症狀爲輕微頭痛、便秘、無法入睡、睡 不著、肌肉及舌頭不自主地顫動及鎭靜。雖然關於噁心的 神經藥理目前尙不淸楚,但與完全控制噁心這項目標相關 的作法,包括儘量讓患者感到方便舒適,及提供患者能減 少住院及臥床時間的治療,進而促進患者生活品質。在中 國境內進行的臨床試驗顯示PAGC對化療患者有益,特別 是可改善其生活品質。從這些硏究所得的結果以及參與硏 究的患者,都支持PAGC可改善患者生活品質的結論,此 顯示PAGC可能在預防及治療化療所引起的噁心症狀上扮 演了一些角色。 降低或取代洗腎患者所用的血紅素生成素(erythropoietin) 之用量 EPOGEN(Epoetin alfa,erythropoietin)係在 1989 年被 核准用來治療洗腎患者其與貧血相關的慢性腎衰竭。此藥 品讓患者能過一個較正常、活躍的生活。在EPOGEN問世 前,90%的洗腎患者都有貧血,造成患者經常感到疲倦及 全身無力,並因而影響其工作能力。今天,大部分的洗腎 患者都會同時接受EPOGEN治療來提高或維持其體內紅血 球量。在臨床試驗期間,最常看到與EP0GEN相關的嚴重 副作用爲高血壓、頭痛、中風、噁心/嘔吐,及肌肉血栓發 作;一般如果出現這些副作用時,醫師會建議患者降低 EPOGEN用量。PAGC硏究顯示,其可促進接受亞致死劑 量之輻射照射的老鼠體內周邊紅血球數目恢復正常,如實 30 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ----------^---------^^^1 (請先閱讀背面之注意事項再填寫本頁) 1240629 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明(J) 施例6所示。以同樣方式在爲期20天的療程中’以100及 250毫克/公斤之AGPC進行治療的老鼠’相較於控制組’ 在所有時間點進行測試時,均可改善其紅血球及血小板數 目;顯示PAGC在此模型系統中也有相同的療效。此外’ 如實施例7所示,PAGC也可增加循環之BFU_E的量,並 與G-CSF協同增加循環之正常老鼠體內循環之BFU_E的 量。此外,經PAGC處理後的正常鼠及經環磷醯胺處理之 老鼠,其周邊吸血液中的TER-119+細胞數目都明顯增高許 多。TER-119抗原係從初期的紅血球母細胞期間一直到成 熟紅血球階段,都會表現在紅血球細胞表面。表現了 TER_ 119+細胞的數目增加,即表示PAGC可刺激骨髓中的紅血 球代細胞之分化、增生及成熟,並促使這些細胞移動到周 邊血液中。所有這些結果都顯示在以老鼠進行的硏究中, PAGC可促進紅血球的製造及成熟。由於在臨床試驗中使 用PAGC不僅安全,且從未發現任何嚴重的副作用,因此 PAGC可能可以降低或甚至取代EPOGEN,來治療洗腎患 者的貧血現象。 從上述這些結果可知,當動物或人類患者被刻意曝露 於輻射線及細胞毒性劑下,相較於未經處理的動物或人類 患者而言,這種處理可加速該動物或人類患者復原,而同 時PAGC亦會加速動物或人類患者由因曝露(即,意外曝露 或不具療效性地曝露)於輻射線及細胞毒性劑下所引起的傷 害中復原。 依據實施例6之程序,以同樣方式在爲期20天的療程 31 本紙張尺度適用中國國家標準(CNS)A4規袼(21〇 x 297公爱) ----U----^--------- (請先閱讀背面之注意事項再填寫本頁) 1240629 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明) 中,以100及250毫克/公斤之AGPC進行治療的老鼠,相 較於輻射照射控制組,在所有時間點進行測試時’均可3夂 善其紅血球及血小板數目;顯示PAGC在此模型系統中也 有相同的療效。如果與本篇硏究的其他PAGC數據一起討 論的話,AGPC與PAGC的同樣都具有療效,因此適合用 於所討論過的PAGC之各種藥學應用。 藥學配方及施用 一般來說,本發明第一態樣之純化之***糖半乳聚 糖組成物或本發明第二態樣之***糖半乳聚糖蛋白質組 成物之藥學有效量,將會以靜脈注射方式,單獨地或與至 少一種其他藥劑,特別是能刺激造血的藥劑,一起施用至 個體身上。藥學有效量將視疾病、其嚴重性、施用個體之 年齡及健康情形,與其他因素而有所不同。對刺激造血而 言,包括巨核細胞增生或成熟,刺激生成IL-Ιβ、IL-6、 TNF-α、INF-γ、GM-CSF或G-CSF,刺激嗜中性白血球細 胞的生成或作用,治療嗜中性白血球細胞過少,貧血或血 小板細胞數目過少症;對一般體形及體重的人類來說,本 發明第一態樣之純化之***糖半乳聚糖組成物的藥學有 效星約介於50至100毫克/天,特別是約介於100至5〇〇 毫克/天,特別是約250毫克/天。對治療惡病質、噁心、 或脫癮症狀、或改善生物反應性或保護B型肝炎患者之肝 臟細胞而言,類似上述的劑量也屬藥學有效量。因本發明 ***糖半乳聚糖蛋白質組成物本身所含的***糖半乳 聚糖蛋白質量較高,因此預計其藥性應較強,因此其藥學 32 本紙張尺度適用中關家標準(CNS)A4規格⑽x 297公爱)------- ----------^ --------- (請先閱讀背面之注意事項再填寫本頁) 1240629 Α7 Β7 五、發明說明u。) 有效量將較低。,例如介於純化之***糖半乳聚糖組成 物之藥學有效量的10%至50%之間。習知技藝者應能在無 需更進一步實驗下,依據本說明書揭露的事實及其本身技 藝,來判斷對一特定疾病應給予多少藥學有效量之本發明 組成物。 一般來說,本發明第一態樣之純化之***糖半乳聚 糖組成物或本發明第二態樣之***糖半乳聚糖蛋白質組 成物將以藥學配方方式由靜脈注射施用。配方將包含本發 明第一態樣之純化之***糖半乳聚糖組成物或本發明第 二態樣之***糖半乳聚糖蛋白質組成物,及一適合供靜 脈注射的佐劑溶液。可供靜脈注射之適當的佐劑溶液乃是 習知技藝者熟知的,這些及製作配方的方法均可參考 Alfonso AR: Remington’s Pharmaceutical Science,17th ed., Mack Publishing Company,Easton,PA,1985。供靜脈注射 之適當的佐劑溶液包括水、生理食鹽水、葡萄糖水溶液等 類似物。 一般來說,若當作造血劑來施用,本發明第一態樣之 純化之***糖半乳聚糖組成物或本發明第二態樣之阿拉 伯糖半乳聚糖蛋白質組成物將以靜脈注射方式來施用,特 別是可連續注射數分鐘或一或數小時之連續性注射,例如 連續注射15分鐘。組成物中本發明化合物的含量將視組成 物的種類、單位劑量大小、佐劑種類及其他習知因素而有 所不同。一般來說,組成物最終可包含0.001%至10%(重 量百分比)之本發明化合物,較好是介於0.01%至1%間, Λ勹 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) (請先閱讀背面之注意事項再填寫本頁) 裝 訂--- 經濟部智慧財產局員工消費合作社印製 1240629 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明(y ) 其餘則爲佐劑。 本發明第一態樣之純化之***糖半乳聚糖組成物或 本發明第二態樣之***糖半乳聚糖蛋白質組成物也可選 擇性地與至少一種其他可治療疾病的藥劑合倂施用,特別 是能刺激造血之其他藥劑,例如血紅素生成素、血小板生 成素、粒細胞群落刺激因子(G-CSF)等類似物。 實施例 本發明可由下列非限制性實施例做進一步說明。所有 純化的***糖半乳聚糖組成物及***糖半乳聚糖蛋白 質組成物均經過體積篩選色層層析法(size exclusion chromatography)分析。 體積篩選色層層析法(size exclusion chromatography)的 程序如下:Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1240629 A / B7 V. Description of Invention (/) PAGC has the same effect in this model system. The second phase of the clinical trial in Example 9 was designed to investigate the recovery of WBC in chemotherapy patients. Therefore, the condition is to perform an analysis test based on the number of WBCs. At present, it is planned to investigate clinical trials for treating anemia with PAGC and anemia caused by chemotherapy in PRC (People's Republic of China). Moving peripheral blood cells alone or together with G-CSF to treat patients receiving peripheral blood cell transplants and including hematopoietic stem cells and cells such as BFU-E (bUrst-fomming unit -erythroid) that produce cell populations, GM-CFC (Generating granulocyte macrophage colonies), and CFU-Mix (a mixture that produces cell colonies) all contain CD4 antigen on the progeny cells. The analysis of CD34 * cells by flow cytometry provides the best way to calculate the graft composition. As early as 1975, the description of the existence of hematopoietic cells (PBPCs) in the peripheral blood system of humans has been proposed. These peripheral blood progenitor cells account for only a small fraction of the total number of cells because most of the progenitor cells are located in the bone marrow. In 1976, it was first suggested that during the recovery period after chemotherapy, the number of hematopoietic cells in the human peripheral blood system would increase. Recently, it has been reported that colony stimulating factor G-CSF and GM-CSF can directly increase the number of PBPCs. Compared with single use, the combined use of cell population stimulating factors during chemotherapy can significantly increase the number of PBPCs. G-CSF is currently used to move PBPCs in human autologous and allogeneic transplants. Compared with traditional bone marrow transplantation, transplantation of PBPC can make transplantation faster. If used in combination with chemotherapy or other cytokines, the number of PBPCs produced by G-CSF treatment can be significantly increased. Use 23 paper sizes separately. Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) '--------- ^ --------- (Please read the notes on the back first (Fill in this page again) A7 1240629 ____B7___ 5. Description of the Invention (,. 丨) The ability of PAGC or G-CSF to cooperate to induce the movement of PBPC is detailed in Example 7. As shown in the table results in Example 7, PAGC can increase the amount of GM-CFC in the cycle by about 6 times. In addition, when PAGC and G-CSF are used together, the amount of GM-CFC can be increased by about 83 times. This increase is much higher than the 39-fold increase when G-CSF is used alone. The results showed that PAGC could increase the amount of circulating GM-CFC, or synergize with G-CSF to increase the amount of circulating GM-CFC in normal mice. PAGC can also increase the amount of circulating BFU-E by about 3 times, as shown in the table, and cooperate with G-CSF to increase the amount of circulating BFU-E by about 9.5 times. This increase is much higher than the 4.5-fold increase when G-CSF is used alone. In a similar experiment in mice treated with cyclophosphamide, the number of CD34 + Lin cells increased compared to untreated mice '. Agents such as PAGC that work synergistically with G-CSF to increase the number of PBPCs are also effective. Such synergy can reduce the cost of obtaining PBPC cells from a donor. In addition, this type of combined treatment is also helpful when patients receiving transplantation do not respond well to G-CSF alone, or patients who cannot use chemical conversion. Similarly, AGPC is also effective for transplantation. Treatment of cachexia One of the most common side effects of chemotherapy and radiation exposure is the loss of appetite and weight loss. Example 8 shows that PAGC can increase the weight of mice treated with the chemotherapeutic agents cyclophosphamide and fluorinated uracil. Compared to mice treated with cyclophosphamide alone, PAGC-treated mice ' have less weight loss and can recover their original weight faster. However, these differences are not statistically significant. 24 This paper is suitable for the size of the national fresh (CNS) A4 secret (210 X 297 meals 1 '' a * U --------- (Please read the precautions on the back before filling out this page) Ministry of Economy Printed by the Consumer Property Cooperative of the Intellectual Property Bureau 1240629 A7 B7 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the Invention (π) As mentioned above, PAGC can obviously improve the quality of life of patients in Phase II clinical trials. The parameter is appetite. The results of this study with a mouse model show that PAGC can help improve cachexia in patients. It is a kind of physical waste and malnutrition caused by chronic diseases such as cancer or cancer treatment. And G -CSF combined treatment of cancer patients receiving bone marrow suppression therapy to promote the recovery of neutrophil cells and reduce the amount of G_CSF. The previous discussion showed that PAGC can stimulate the production of cytokines, especially G_CSF produced by human peripheral blood mononuclear cells; and Can promote the recovery of GM_CFC and surrounding WBC numbers in bone marrow suppression animal models caused by radiation exposure, and enable WB in vivo in patients with cancer in the second phase of clinical trials after chemotherapy C 値 returned to normal. These results show that PAGC can indirectly act on the hematopoietic system by manufacturing a variety of endogenous cytokines, and these cytokines can cooperate with each other to restore the number of neutrophils. At the same time, PAGC and IL can also -3 works synergistically to promote the proliferation / maturation of bone marrow megakaryocytes. In addition, PAGC administration is not only safe, but can be tolerated by patients in clinical trials. Serious side effects such as bone headache, muscle pain, headache when using G-CSF , Fatigue, nausea, vomiting, diarrhea, etc. were not found during the administration of PAGC. This synergy with the above shows that PagC can be used in combination with G-CSF to reduce the amount of exogenous G-CSF and promote the acceptance of bone marrow suppression Recovery of the number of neutrophils in the treated cancer patients. After high-dose cytotoxic treatment and autologous or allogeneic hematopoietic cell transplantation, the paper size of this paper applies Chinese National Standard (CNS) A4 (210 X 297 mm) installed --------- tr --------- (Please read the precautions on the back before filling out this page) 1240629 Employees' cooperation on intellectual property of the Ministry of Economic Affairs Print A7 B7 V. Description of the invention (4) Combination of G-CSF treatment to promote the recovery of neutrophil leukocytes At present, many high-dose chemotherapy and / or radiation exposure and supportive blood cell (BPC) transplantation are used to treat many Cancer patients, such as breast cancer, lymphoma, and multiple bone marrow cancer. Transplantation of BPC can accelerate the recovery of hematopoietic function. G-CSF is usually used after transplantation of BPC to accelerate the recovery of the number of neutrophils to prevent infection due to infection. Fever caused by too few neutrophils, shortened the length of hospital stay, and reduced the amount of antibiotics. As shown in the second phase clinical trial of Example 9, PAGC can restore the WBC number of chemotherapy cancer patients; this and the pharmacological effects summarized in the previous paragraph show that the combined use of PAGC and G-CSF can accelerate the recovery of neutropenia after transplantation of BPC The number of sexual white blood cells is very useful. Improve and modify the bioreactivity of hepatitis B carriers and protect liver cells. Cytokines play a very important role in fighting viral infections. Cytokines are produced by cells associated with the immune system, such as macrophages; CD4 + and CD8 + T lymphocytes have a more direct effect on individuals' recovery from infections such as hepatitis B virus (HBV). From animal models to human studies, it is clear that cellular immune responses may play a role in addressing HBV infection and its pathology. In acute HBV infection, a strong multi-cell immune response is required. The release characteristics of type 1 cytokines are that IL-2 and INF-γ are produced by CD4 + T lymphocytes; and IL-2 and INF-γ are used to induce and maintain cellular immunity, which is important for combating viral infections. It is quite important [c. A. Biron, "cytokines in the generation of immune response to, and regulation of, virus infection", Cur. Opin. Immunol., 6, 530-538 (1994)]. CD4 + 26 This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) --------- ^ --------- ^^^ 1 (Please read the Note: Please fill in this page again) 1240629 A7 B7 V. Description of the invention (A) and cytokines released by CD8 + cells also play a very important role in downregulation of HBV replication. If there is a defect in the acute response, it becomes a chronic HBV infection. These results suggest that enhancing type 1 responses or enhancing the production of appropriate cytokines in localized areas of the liver may be very useful in treating chronic HBV infection [M.J. Koziel, Sem. Liver Disease, 19 (2), 157-161 (1999)]. PAGC is made from traditional Chinese medicine plants (like cews Far · (AM)) and has long been used to stimulate the immune and hematopoietic system. It is widely used to treat patients with various types of anemia, whose disease is very similar to that of bone marrow suppression induced by chemotherapy or radiation (in addition to too few neutrophils, too few platelets and anemia). In addition, AM is known to stimulate rat pancreatic cell proliferation in vitro, and this cell proliferation phenomenon is positively correlated with dose; it can also increase natural killer cells in pancreatic cells of animals inoculated with S-180 sarcoma cells (Natural killer (NK) activity; and increase cytotoxic T lymphocytes (CTL) activity. Cytokines produced by CTL can control viral infections in the body, and INF-γ and INF-a produced by virus-specific CTL can increase the ability of CTL to eliminate virus infection [LG Guidotti et al., "Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice '', Proc. Natl · Acad. Sci. USA, 91, 764-3768 (1994)]. In addition, as described above, PAGC can stimulate humans after being activated with PHA. PBMC manufactures IL-1β, IL-6, TNF-α, TNF-γ, GM-CSF, and G-CSF. These results show that PAGC can indirectly affect the production of cytokines by regulating cytokine production 2Ί This paper is applicable to China Standard (CNS) A4 specification (210 X 297 mm) ----------- (Please read the precautions on the back before filling out this page)-Order ---- Φ. Intellectual Property of the Ministry of Economic Affairs Printed by the Bureau ’s Consumer Cooperatives 1240629 A7 B7 Printed by the Intellectual Property Bureau of the Ministry of Economy ’s Consumer Cooperatives 5. Printed on the invention () Immune system. Crude extracts made by AM have been used to treat patients with chronic hepatitis to reduce their internal health High IgG levels and lower ALT 値, improve Patients' immune and liver function [Y · Liu, "therapeutic effect of oral solution from Astragalus in the treatment of 70 chronic hepatitis B patients", Jiang Su Chung Yao, 15 (12), 38 (1994)]. In addition, it is known The fraction of AM has the ability to increase immune function as described above. These results show that PAGC can be used to improve and modify the bioreactivity of hepatitis B carriers and protect liver cells. E vaccine adjuvants for patients with hepatitis B were prepared The resulting polysaccharide has been used as an adjuvant for hepatitis B vaccine to treat chronic hepatitis B. It is known to convert hepatitis B e-antigen (HBeAg) to be blood-negative and eliminate hepatitis B virus DNA (HBV- DNA) has a satisfactory effect [SM · Wu et al., "The therapeutic observation on the combined Polyporus polysaccharide with hepatitis B vaccine in the treatment of chronic hepatitis J. Chin. Infectious Disease, 13 (3), 187-189 (1995); Η · Z · Shu et al ·, "The therapeutic observation on the Polyporus polysaccharide with large dose of hepatitis B vaccine in the treatment of 64 cases of chronic hepatitis B patients ", Med. J. NDFSC, 6 (4), 21 1-212 (1996)]. It is known that the extract made by AM can convert hepatitis B e antigen (HBeAg) and anti-HBc core antigen (anti HBc) to blood sac negativity, and remove hepatitis B virus DNA from patients with hepatitis B ( HBV-DNA) [CK Liu et al., "Clinical and experimental studies on effects of chronic 28 This paper is sized to the Chinese National Standard (CNS) A4 (210 x 297 mm) ^ --------- (Please read the precautions on the back before filling this page) 1240629 A7 B7 V. Description of Invention (A) Hepatitis B treated with Astragli composita ", Chung Kuo Chung His I Chieh Ho Tsa Chin, 16 (7), 394-397 ( 1996); P. L. Chen et al., "Polysaccharide from Astragalus in treating 33 cases of chronic active hepatitis B patients", New Drugs Clin. Remedies, 11 (2), 75-76 (1991)]. The results show that PAGC may be used as a vaccine adjuvant for patients with hepatitis B. The symptoms of withdrawal are generally seen when patients with narcotic withdrawal and palsy symptoms begin to withdraw from the use of narcotics. From the perspective of Traditional Chinese Medicine (TCM) In terms of The symptom of withdrawal is Qi (energy) deficiency. According to the observation of Chinese medicine practitioners, if Qi can be strengthened, the drug withdrawal process can be accelerated. One of the results of the clinical trial of PAGC in Example 9 is that it can improve cancer chemotherapy patients. Quality of life. The quality of life is assessed by assessing the improvement of chemotherapy-related symptoms; these include fatigue and exhaustion, discomfort, cold sweats, shortness of breath, and lack of appetite. These symptoms are typically "qi" deficiency A manifestation is also a typical manifestation of the symptoms of withdrawal as considered by traditional Chinese medicine. Therefore, in addition to strengthening the "qi" of patients, PAGC can also treat the symptoms of withdrawal of patients who want to quit using anesthetic drugs. Prevention and treatment of chemotherapy Nausea and vomiting are the most common side effects of chemotherapy and / or radiation exposure in cancer patients with radiation or radiation. Although the current chemotherapy or radiation technology has been greatly improved, a significant proportion of patients will experience nausea. Therefore, the industry is also continuously looking for ways to reduce the side effects of chasing. Although the market Many such chewing-resistant drugs (such as serotonin receptor antagonists, corticosteroids, and dopamine 29) This paper size applies to China National Standard (CNS) A4 (210 X 297 public love) 丨 —— 丨- ---- (Please read the precautions on the back before filling this page) Order --- Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 1240629 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (J ) Receptor antagonists), however, these drugs also have other side effects of their own. Symptoms associated with these drugs are mild headaches, constipation, inability to fall asleep, sleeplessness, involuntary vibrations and quiescence of muscles and tongue. Although the neuropharmacology of nausea is currently unclear, approaches related to the goal of complete control of nausea include making patients as comfortable and comfortable as possible, and providing patients with treatments that reduce hospitalization and bed time, thereby promoting patients' quality of life. Clinical trials conducted in China have shown that PAGC is beneficial to chemotherapy patients, especially to improve their quality of life. The results from these studies and the patients participating in the study support the conclusion that PAGC can improve the quality of life of patients, which shows that PAGC may play some roles in preventing and treating the nausea caused by chemotherapy. Reduce or replace the amount of erythropoietin used in dialysis patients. EPOGEN (Epoetin alfa, erythropoietin) was approved in 1989 for the treatment of chronic kidney failure associated with anemia in patients with dialysis. This medicine allows patients to live a more normal and active life. Prior to the advent of EPOGEN, 90% of dialysis patients had anemia, which often caused patients to feel tired and weak, and thus affected their ability to work. Today, most dialysis patients receive EPOGEN at the same time to increase or maintain their red blood cells. During clinical trials, severe side effects associated with EP0GEN are most commonly seen as hypertension, headache, stroke, nausea / vomiting, and muscle thrombosis; in general, physicians will advise patients to reduce the amount of EPOGEN if these side effects occur. PAGC research has shown that it can promote the recovery of the number of red blood cells around the body of rats exposed to sub-lethal doses of radiation. It is true that this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ----- ----- ^ --------- ^^^ 1 (Please read the notes on the back before filling in this page) 1240629 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs J) As shown in Example 6. In the same way, mice 'treated with 100 and 250 mg / kg AGPC during a 20-day course of treatment' improved their red blood cell and platelet numbers when tested at all time points compared to the control group; show PAGC has the same effect in this model system. In addition, as shown in Example 7, PAGC can also increase the amount of circulating BFU_E, and cooperate with G-CSF to increase the amount of circulating BFU_E in normal mice. In addition, the number of TER-119 + cells in the peripheral blood drawn by PAGC-treated normal mice and cyclophosphamide-treated mice increased significantly. The TER-119 antigen system appears on the surface of red blood cells from the early period of red blood cells to mature red blood cells. It shows that the increase in the number of TER_119 + cells means that PAGC can stimulate the differentiation, proliferation and maturation of red blood cell cells in the bone marrow, and promote the migration of these cells to the surrounding blood. All these results show that PAGC promotes the production and maturation of red blood cells in a mouse-based study. Because the use of PAGC in clinical trials is not only safe, and has never seen any serious side effects, PAGC may reduce or even replace EPOGEN to treat anemia in patients with dialysis. From these results, it can be seen that when an animal or human patient is deliberately exposed to radiation and cytotoxic agents, this treatment can accelerate the recovery of the animal or human patient compared to an untreated animal or human patient, and At the same time, PAGC will also accelerate the recovery of animal or human patients from injuries caused by exposure (ie, accidental or ineffective exposure) to radiation and cytotoxic agents. In accordance with the procedure of Example 6, in the same way during a 20-day course of treatment, 31 paper sizes apply the Chinese National Standard (CNS) A4 Regulations (21 × x 297 public love) ---- U ---- ^- -------- (Please read the notes on the back before filling out this page) 1240629 Printed by A7 B7, Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, V. Invention Description), 100 and 250 mg / kg of AGPC Compared with the radiation control group, the treated mice were able to improve their number of red blood cells and platelets when tested at all time points; it was shown that PAGC had the same effect in this model system. If discussed in conjunction with other PAGC data studied in this article, AGPC and PAGC are equally effective and therefore suitable for the various pharmaceutical applications of PAGC discussed. Pharmaceutical Formulation and Administration Generally speaking, the pharmaceutically effective amount of the purified arabinose galactan composition of the first aspect of the present invention or the arabinose galactan protein composition of the second aspect of the present invention will be Intravenous injections are administered to an individual, either alone or together with at least one other agent, particularly one that stimulates hematopoiesis. A pharmaceutically effective amount will vary from other factors depending on the disease, its severity, the age and health of the individual being administered. For stimulation of hematopoiesis, including megakaryocyte proliferation or maturation, stimulation of IL-Ιβ, IL-6, TNF-α, INF-γ, GM-CSF or G-CSF, stimulation of neutrophil production or action, For the treatment of too few neutrophilic leukocytes, anemia or too few platelet cells; for humans of general body shape and body weight, the pharmaceutically effective star of the purified arabinose galactan composition of the first aspect of the present invention is between 50 to 100 mg / day, especially about 100 to 500 mg / day, especially about 250 mg / day. For the treatment of cachexia, nausea, or withdrawal symptoms, or to improve bioreactivity or protect liver cells in patients with hepatitis B, a dose similar to the above is also a pharmaceutically effective amount. Because the arabinose galactan protein composition of the present invention itself contains higher quality arabinose galactan protein, it is expected that its medicinal properties should be stronger, so its pharmacy is 32. The paper standard is applicable to the CNS A4 specifications ⑽ x 297 public love) ------- ---------- ^ --------- (Please read the precautions on the back before filling this page) 1240629 Α7 Β7 Fifth, the invention description u. ) The effective amount will be lower. For example, between 10% and 50% of the pharmaceutically effective amount of the purified arabinose galactan composition. A skilled artisan should be able to determine, without further experimentation, how much of a composition of the invention should be administered to a particular disease based on the facts disclosed in this specification and its own technology. Generally, the purified arabinose galactosaccharide composition of the first aspect of the present invention or the arabinose galactan protein composition of the second aspect of the present invention will be administered by intravenous injection in a pharmaceutical formulation. The formulation will include the purified arabinose galactan composition of the first aspect of the present invention or the arabinose galactan protein composition of the second aspect of the present invention, and an adjuvant solution suitable for intravenous injection. Suitable adjuvant solutions for intravenous injection are well known to those skilled in the art. For these and methods of formulating, refer to Alfonso AR: Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, PA, 1985. Suitable adjuvant solutions for intravenous injection include water, physiological saline, aqueous glucose and the like. Generally, if administered as a hematopoietic agent, the purified arabinose galactan composition of the first aspect of the present invention or the arabinose galactan protein composition of the second aspect of the present invention will be injected intravenously. It can be administered by way of injection, especially by continuous injection for several minutes or continuous injection for one or several hours, such as continuous injection for 15 minutes. The content of the compound of the present invention in the composition will vary depending on the type of composition, the size of the unit dose, the type of adjuvant, and other conventional factors. Generally speaking, the composition may finally contain the compound of the present invention from 0.001% to 10% by weight, preferably between 0.01% and 1%. The paper size is applicable to the Chinese National Standard (CNS) A4 specification ( 210 X 297 mm) (Please read the notes on the back before filling out this page) Binding --- Printed by the Employees 'Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1240629 Printed by the Employees' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 (Y) The rest are adjuvants. The purified arabinose galactan composition of the first aspect of the present invention or the arabinose galactan protein composition of the second aspect of the present invention can also be selectively combined with at least one other agent capable of treating diseases Administration, especially other agents that stimulate hematopoiesis, such as heme-producing hormone, thrombopoietin, granulocyte community stimulating factor (G-CSF) and the like. Examples The invention is further illustrated by the following non-limiting examples. All purified arabinogalactan compositions and arabinogalactan protein compositions were analyzed by size exclusion chromatography. The procedure for size exclusion chromatography is as follows:
Shimadzu HPLC系統,其上配備了 SCL-10A系統控制 子、LC-10AD幫浦、DGU-4A排氣子、RID-6A折射率偵測 器、及SPD-10AV UV偵測器,並使用以0.2N氯化鈉平衡 之 GS-701 及 GS-620 管柱(shodex Ashipak,7.6 X 500 mm) 進行分析。裝載之樣量爲80 μ§(濃度爲2 mg/ml之4〇 μΐ樣 品溶液),並以1毫升/分鐘的速率將樣品洗出。並以不同 濃度的標準物來製備校準曲線;再由此校準曲線來決定分 子量。樣品平均分子量(Μπ^Σ^ΜΟ/ΣΑί)係以第2·4版的 Shimadzu SEC軟體由校準曲線上定出。 糖含量及本發明PAGC與AGPC組成物係由三甲基砂 甲基醣苷衍生物之GLC分析來決定。在此方法中,先將多 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ---------^ --------- (請先閱讀背面之注意事項再填寫本頁) 1240629 經濟部智慧財產局員工消費合作社印製 A7 Β7___ 五、發明說明(ρ) 醣於氫氯酸化之甲醇中甲醇化,接著以三甲基矽衍生物 (TMS)來產生揮發性的單醣衍生物。淸除後,以氣液色層 層析法(GLC)來分析衍生物,其係使用配備了火焰式離子 偵測器(FID)之DB-1管柱來做分析。並以myo-insitol爲內 標,與組成物樣品一起作分析來定量含糖量及組成物。 以色度分析儀來分析羥基輔胺酸含量。先以氫氯酸將 樣品水解,之後再以過硼酸鈉(溶於氫氧化鈉之硼溶液)、 氫氯酸及二甲基氨基苯甲醛來處理。於色度儀上分析最終 溶液之光學密度,至於羥基輔胺酸的含量則係由以同樣方 式製備而成之各種羥基輔胺酸濃度的校準曲線中來決定。 實施例1 製備***糖半乳聚糖組成物 步驟A· ”碎片(drink chips)”程序 將300公斤曬乾的黃民⑽acws)***製成碎片,並去掉其中任何污染部分,在無菌下淸洗並 以超過濾水搓洗,然後浸泡在70%酒精中隔夜,切成厚約 3-5毫米的細片,在60-70°C的無菌烘箱中烘乾。這些烘乾 後的”碎片”因烘烤所失去的水重約<15%。 步驟B.***糖半乳聚糖組成物之粗萃取液 將200公斤,以步驟A製成的”碎片”,用UF水(以 10K MWCO UF系統進行超過濾之去離子水)於100°C下萃 取3次,每次3小時。於60-70°C下,將萃取液減壓濃縮至 約200公升。在濃縮液中加入95%的酒精,使酒精終濃度 爲70%,並於室溫下攪拌15分鐘,以便將多醣沉澱。傾出 上淸液,以95%酒精淸洗沉澱3次。將沉澱懸浮於UF水 35 I紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) " " _ ---------^--------* (請先閱讀背面之注意事項再填寫本頁) 1240629 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明(A ) 中,濃度約18-20%(由折射率偵知),再加入95%酒精’使 酒精終濃度爲35%。將酒精懸浮液離心’丟棄沉澱部分’ 再度加入95%酒精,並攪拌使酒精終濃度爲70%。收集沉 澱部分,再以UF水將其溶解,噴霧乾燥後可得***糖 半乳聚糖組成物之粗產物。其因乾燥所失去的水重約<15% 。內毒素含量約<〇·3 EU/毫克。 步驟C.***糖半乳聚糖組成物之純化 將3.5公斤,依步驟B製成的***糖半乳聚糖組成 物之粗產物,用UF水溶解成2%(175公升)。以5K MWCO UF系統將溶液過濾至終體積爲35-40公升。藉由添加1.0 M NaOAc緩衝液pH5.2的緩衝溶液,將濃縮液濃度調整 爲20 mM NaOAc之緩衝液,ρΗ5·2。將溶液載入Q Sepharose陽離子樹脂交換管柱中,然後收集3-3.5倍體積 的餾出液。將所收集由Q Sepharose陽離子樹脂交換管柱 中流出的液體以0.1 μιη濾膜過濾後,再用8K MWCO UF 系統進行超過濾。將留存的液體以濃縮系統於50-6(TC下濃 縮至20-26%,再添加無水酒精進行沉澱,酒精終濃度爲 80-90%。以無水酒精淸洗沉澱3次,然後在60-70°C下烘 乾可得純化之***糖半乳聚糖組成物。 純化之***糖半乳聚糖組成物爲一種白色粉末,溶 於水、生理食鹽水及5%的葡萄糖水中,濃度爲20毫克/毫 升,且其含水量£6.0%。組成物所含灰份4.0%,重金屬 SlO ppm,內毒素iO.l EU/mg。組成物水溶液之pH値介於 4.5及6.5間。組成物之含糖量^85%(以葡萄糖爲標準物, 36 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ------------------ (請先閱讀背面之注意事項再填寫本頁) 1240629 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明(w) 由甲酚-硫酸法偵知),且Ai*a : Gal比例11.5 : 1(由組成物 之TMS-甲基糖苷衍生物之GLC分析中得知)。 實施例2 製備***糖半乳聚糖蛋白質組成物 將實施例1、步驟C,由Q Sepharose陽離子樹脂交換 管柱中所收集的液體以100K MWCO UF系統進行超過濾。 將超過濾後留存的液體進一步濃縮,並再添加無水酒精進 行沉澱,酒精終濃度爲80-90%。以無水酒精淸洗沉澱3次 ,然後在60-70°C下烘乾可得純化之***糖半乳聚糖蛋白 質組成物。 純化之***糖半乳聚糖蛋白質組成物爲一種白色粉 末,溶於水、生理食鹽水及5%的葡萄糖水中,濃度爲20 毫克/毫升,且其水溶液之pH値介於4.5及6.5間。組成 物含重金屬ppm,內毒素£ 0.5 EU/mg,且Ara : Gal 比例22 ·· 1,並含S 0.2%之羥基脯氨酸。組成物之平均分子 量爲之100kD〇 實施例3 由活化之人類周邊血液單核細胞中製備細胞素 依Boyum所述之方法製備人類周邊血液單核細胞 (PBMC)[A. Boyum,“Isolation of mononuclear cells and granulocytes from human blood …“,Scan. J. Lab· Invest., 97,77-89 (1968)]。約25毫升/捐贈人之人類血液血塊黃層 (buffy coat)樣品,係由史丹佛大學醫學中心血庫取得。將 每一血塊黃層(buffy coat)樣品,於室溫下輕輕地重新懸浮 於總體積1〇〇毫升、無鈣-、無鎂-之Hank平衡鹽溶液 (HBSS,Gibco)。將25毫升的細胞懸浮液放入內含15毫升 η 表紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公爱1 " ----- I I I ^ · I------I (請先閱讀背面之注意事項再填寫本頁) 1240629 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明(p)Shimadzu HPLC system, equipped with SCL-10A system controller, LC-10AD pump, DGU-4A exhaust, RID-6A refractive index detector, and SPD-10AV UV detector, and uses 0.2 N-NaCl equilibrated GS-701 and GS-620 columns (shodex Ashipak, 7.6 X 500 mm) were analyzed. The sample load was 80 μ§ (40 μΐ sample solution at a concentration of 2 mg / ml), and the sample was washed out at a rate of 1 ml / min. A calibration curve was prepared with standards of different concentrations; the calibration curve was then used to determine the molecular weight. The average molecular weight of the sample (Mπ ^ Σ ^ ΜΟ / ΣΑί) was determined from the calibration curve using Shimadzu SEC software version 2.4. The sugar content and the composition of the PAGC and AGPC of the present invention were determined by GLC analysis of trimethyl sand methyl glycoside derivatives. In this method, first apply multiple paper sizes to Chinese National Standard (CNS) A4 (210 X 297 mm) --------- ^ --------- (Please read first Note on the back, please fill out this page again) 1240629 Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7___ V. Description of the invention (ρ) Methanolation of sugar in hydrochloric acid-methanol, followed by trimethyl silicon derivative (TMS ) To produce volatile monosaccharide derivatives. After elimination, the derivatives were analyzed by gas-liquid chromatography (GLC), which was performed using a DB-1 column equipped with a flame ion detector (FID). The myo-insitol was used as an internal standard to analyze sugar composition and composition together with the composition sample. A chroma analyzer was used to analyze the content of hydroxy couric acid. The sample was hydrolyzed with hydrochloric acid and then treated with sodium perborate (a boron solution in sodium hydroxide), hydrochloric acid and dimethylaminobenzaldehyde. The optical density of the final solution was analyzed on a colorimeter. As for the content of hydroxycoamino acid, it was determined from the calibration curve of various hydroxycoamino acid concentrations prepared in the same way. Example 1 Preparation of arabinogalactan composition Step A. "Drink chips" procedure 300 kg of dried Huang Mincao aws) roots were made into chips, and any contaminated parts were removed, and rinsed under aseptic conditions. It was scrubbed with ultra-filtered water, then immersed in 70% alcohol overnight, cut into thin pieces about 3-5 mm thick, and dried in a sterile oven at 60-70 ° C. These dried "chips" lose about 15% of the water lost from baking. Step B. The crude extract of the arabinose galactan composition is 200 kg of "fragments" made in step A, with UF water (deionized water ultrafiltered with a 10K MWCO UF system) at 100 ° C Extract 3 times for 3 hours each. The extract was concentrated under reduced pressure to about 200 liters at 60-70 ° C. Add 95% alcohol to the concentrate to make the final alcohol concentration 70%, and stir at room temperature for 15 minutes to precipitate the polysaccharide. Decant the supernatant and rinse the pellet 3 times with 95% alcohol. Suspend the precipitate in UF water 35 I paper size applicable to Chinese National Standard (CNS) A4 specification (210 X 297 mm) " " _ --------- ^ -------- * (Please read the precautions on the back before filling out this page) 1240629 Printed by the Consumer Property Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 5. In the description of the invention (A), the concentration is about 18-20% (detected by the refractive index), and then Add 95% alcohol 'to bring the final alcohol concentration to 35%. Centrifuge the alcohol suspension 'discard the precipitate' and add 95% alcohol again and stir to a final alcohol concentration of 70%. The precipitate was collected and dissolved in UF water. After spray drying, the crude product of the arabinose galactan composition was obtained. The water lost due to drying weighs about < 15%. The endotoxin content is about < 0.3 EU / mg. Step C. Purification of the arabinose galactan composition 3.5 kg of the crude product of the arabinose galactan composition prepared in step B was dissolved in UF water to 2% (175 liters). The solution was filtered with a 5K MWCO UF system to a final volume of 35-40 liters. The concentration of the concentrate was adjusted to a buffer solution of 20 mM NaOAc by adding a 1.0 M NaOAc buffer pH 5.2 buffer solution, ρΗ5.2. The solution was loaded into a Q Sepharose cationic resin exchange column, and a 3-3.5-fold volume of distillate was collected. The collected liquid flowing from the Q Sepharose cation resin exchange column was filtered through a 0.1 μm filter membrane, and then ultra-filtered with an 8K MWCO UF system. The remaining liquid was concentrated in a concentration system at 50-6 ° C to 20-26%, and then anhydrous alcohol was added for precipitation. The final alcohol concentration was 80-90%. The precipitate was rinsed with anhydrous alcohol 3 times, and then at 60- The purified arabinose galactan composition can be obtained by drying at 70 ° C. The purified arabinose galactan composition is a white powder, which is soluble in water, physiological saline and 5% glucose water at a concentration of 20 mg / ml, and its water content is £ 6.0%. The composition contains 4.0% ash, S10 ppm of heavy metal, iO.l EU / mg of endotoxin. The pH of the composition aqueous solution is between 4.5 and 6.5. Composition Sugar content ^ 85% (with glucose as standard, 36 paper sizes are applicable to China National Standard (CNS) A4 specifications (210 X 297 mm) ---------------- -(Please read the notes on the back before filling out this page) 1240629 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (w) Detected by the cresol-sulfuric acid method), and Ai * a: Gal ratio 11.5: 1 (obtained from GLC analysis of the TMS-methyl glycoside derivative of the composition). Example 2 Preparation of arabinose galactan protein The liquid collected in the Q Sepharose cationic resin exchange column in Example 1 and Step C was ultra-filtered using a 100K MWCO UF system. The liquid remaining after the ultra-filtration was further concentrated, and then anhydrous alcohol was added for precipitation. The final concentration of alcohol is 80-90%. The precipitate is washed three times with anhydrous alcohol, and then dried at 60-70 ° C to obtain a purified arabinose galactan protein composition. Purified arabinose galactopolymer The glycoprotein composition is a white powder that is soluble in water, physiological saline and 5% glucose water at a concentration of 20 mg / ml, and the pH of its aqueous solution is between 4.5 and 6.5. The composition contains heavy metals in ppm, within Toxin £ 0.5 EU / mg, Ara: Gal ratio 22 ·· 1, and containing 0.2% hydroxyproline. The average molecular weight of the composition is 100kD. Example 3 From activated human peripheral blood mononuclear cells Preparation of Cytokines Human peripheral blood mononuclear cells (PBMCs) were prepared according to the method described by Boyum [A. Boyum, "Isolation of mononuclear cells and granulocytes from human blood ...", Scan. J. Lab. Invest., 97, 77 -89 (1968)]. About 25ml / donor of human blood buffy coat sample was obtained from the blood bank of Stanford University Medical Center. Each buffy coat sample was taken at room temperature. Resuspend gently in a total volume of 100 ml, calcium-free, magnesium-free Hank's balanced salt solution (HBSS, Gibco). Put 25 ml of cell suspension into 15 ml η table paper size applicable to China National Standard (CNS) A4 specifications (210 X 297 Public Love 1 " ----- III ^ · I ------ I (Please read the precautions on the back before filling this page) 1240629 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of Invention (p)
Ficoll-Paque (Pharmacia LKB biotechnlogy,Inc·)的 50 毫升 離心管內,以Beckman GPR桌上型離心機(GH-3.7型轉子) 在15 °C以4 0 0 X g的速度離心3 0分鐘。離心後’將界面間 的PBMC懸浮液轉移至另一 50毫升的離心管中,重新懸 浮於45毫升HBSS中,並在15°C以354 X g的速度離心10 分鐘。將上淸液丟棄,並將細胞沉澱重新懸浮於45毫升 HBSS中,並在15°C以265 X g的速度離心10分鐘。將細 胞沉澱重新懸浮於10毫升X-vivo組織培養基質中(Bio Whittaker,MD)並計算細胞數目。在下列實驗中使用聚苯乙 烯管(Falcon #2057,Becton Dickson)及來自兩位不同人之 PBMC。將PBMC稀釋成4 X 106/毫升,在終濃度爲0.3 pg/ml 之 0.5 毫升的植物凝血素 P(PHA-P,Pharmacia 27-3707-01)存在下,將1毫升的細胞懸浮液與0.5毫升各種濃 度之本發明組成物溶液之一,一同培育。每根管子內的總 體積爲2毫升。以另一單獨用PHA處理的細胞溶液當作控 制組。在37°C、7%C02下培育24小時,將這些離心管以 Beckman GPR桌上型離心機(GH-3.7型轉子)在15°C以1600 X g的速度離心10分鐘,收集上淸液,在分析前先儲存於-70°C。以一般常用的ELISA試劑套組來測量細胞素含量, 如人類細胞素 IL-Ιβ、IL-6、TNF-α、GM-CSF、及 G-CSF(R&D Systems, MN)與人類 INF-γ (Endogen,MA)。以可 讀取微量盤光度的儀器(Thermo max, Molecular Devices, CA)來讀取各培養孔之光學密度。經軟體計算後,以上淸 液中所含細胞素含量(pg/ml)來表示。所有結果都以樣品對 38 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) 裝—U—訂--------- (請先閱讀背面之注意事項再填寫本頁) 1240629 PAGC ELISA (S/C) pg/ml IL-1 11-6 TNF IFN GM-CSF G-CSF 25 9.1 11.8 4.9 3.1 6.5 49.9 10 4.3 5.9 2.3 1.2 2.1 16.5 A7 B7 五、發明說明(祚) 控制組比例(S/C)來表示,其中S表示以PHA及測試樣品 刺激PBMC後所產生的細胞素量,且C是單獨以PHA刺 激PBMC後所產生的細胞素量。下表總結了 PAGC可增加 活化之人類PBMC所產生的細胞素量。 實施例4 骨髓巨核細胞之增生/成熟 以9-14週大的G3H/HeJ雄鼠來進行此實驗。用來分 析巨核細胞成熟與否的液態培育物分析,乃是一種可硏究 骨髓巨核細胞(周邊血小板代細胞)增生/成熟之體外試驗。 詳細方法參見 S.A. Burstein,“Interleukin 3 promotes maturation of murine megakaryocytes in vitro’’,Blood Cells, 11,469-479 (1986)。首先分離出老鼠正常骨髓單核細胞, 並於室溫下將其懸浮於內含0.5 mM二異丙基氟化磷酸鹽 (DFP,sigma,St· Louis, MO)之基質中20分鐘,以將內生之 乙醯膽鹼酯酶(AchE)去活化。淸洗細胞、重新懸浮於5% FCS-IMDM (Gibco BRL,Gaithersburg,MD)並以 4 X 106/毫 升之濃度放置於組織培養塑膠瓶中,基質細胞及巨核細胞 會黏附於瓶底,藉此方式將其分離出來。將內含重新懸浮 之骨髓細胞的組織培養瓶中於37°C、5%C02下培育1.5小 時。收集那些未黏附於組織瓶中的細胞,並將其以1 X 1〇6/ 毫升之濃度懸浮於 1% Nutridoma SP (Boehringer mannheim, 39 本紙張尺度適用中國國家標準(CNS)A4規格(210 χ 297公釐) K^--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1240629 A7 B7 五、發明說明(V])Ficoll-Paque (Pharmacia LKB biotechnlogy, Inc.) was centrifuged in a 50 ml centrifuge tube with a Beckman GPR desktop centrifuge (GH-3.7 rotor) at 15 ° C for 30 minutes at 400 x g. After centrifugation, the PBMC suspension between the interfaces was transferred to another 50 ml centrifuge tube, resuspended in 45 ml HBSS, and centrifuged at 15 ° C for 10 minutes at 354 X g. The supernatant was discarded, and the cell pellet was resuspended in 45 ml of HBSS, and centrifuged at 265 X g for 10 minutes at 15 ° C. The cell pellet was resuspended in 10 ml of X-vivo tissue culture substrate (Bio Whittaker, MD) and the number of cells was counted. Polystyrene tubes (Falcon # 2057, Becton Dickson) and PBMC from two different people were used in the following experiments. Dilute PBMC to 4 X 106 / ml in the presence of 0.5 ml of phytohexin P (PHA-P, Pharmacia 27-3707-01) at a final concentration of 0.3 pg / ml. Mix 1 ml of cell suspension with 0.5 One milliliter of one of the solutions of the composition of the present invention was cultivated together. The total volume in each tube is 2 ml. Another cell solution treated with PHA alone was used as the control group. Incubate at 37 ° C and 7% C02 for 24 hours. Centrifuge these tubes in a Beckman GPR tabletop centrifuge (GH-3.7 rotor) at 15 ° C for 10 minutes at 1600 X g to collect the supernatant. , Store at -70 ° C before analysis. Cytokines are measured using commonly used ELISA reagent kits, such as human cytokines IL-Ιβ, IL-6, TNF-α, GM-CSF, and G-CSF (R & D Systems, MN) and human INF- γ (Endogen, MA). The optical density of each well was read with a microplate-ready instrument (Thermo max, Molecular Devices, CA). After calculation by software, the cytokine content (pg / ml) contained in the above mash was expressed. All results are based on samples on 38 paper sizes. Applicable to China National Standard (CNS) A4 (210 X 297 mm). Packing—U—Order —-------- (Please read the notes on the back before filling This page) 1240629 PAGC ELISA (S / C) pg / ml IL-1 11-6 TNF IFN GM-CSF G-CSF 25 9.1 11.8 4.9 3.1 6.5 49.9 10 4.3 5.9 2.3 1.2 2.1 16.5 A7 B7 V. Description of the invention (祚) Control group ratio (S / C), where S is the amount of cytokines produced by stimulating PBMC with PHA and test samples, and C is the amount of cytokines produced by stimulating PBMC with PHA alone. The following table summarizes that PAGC can increase the amount of cytokines produced by activated human PBMCs. Example 4 Proliferation / maturity of bone marrow megakaryocytes This experiment was performed with G3H / HeJ male mice 9-14 weeks old. The liquid culture analysis used to analyze the maturation of megakaryocytes is an in vitro test that can investigate the proliferation / maturity of bone marrow megakaryocytes (peripheral platelet generation cells). For detailed methods, see SA Burstein, "Interleukin 3 promotes maturation of murine megakaryocytes in vitro", Blood Cells, 11,469-479 (1986). First, normal mouse bone marrow mononuclear cells were isolated and suspended at room temperature. Deactivate the endogenous acetylcholinesterase (AchE) in a matrix containing 0.5 mM diisopropyl fluoride phosphate (DFP, sigma, St. Louis, MO) for 20 minutes. Wash the cells, Resuspend in 5% FCS-IMDM (Gibco BRL, Gaithersburg, MD) and place it in a tissue culture plastic bottle at a concentration of 4 X 106 / ml. The stromal cells and megakaryocytes will adhere to the bottom of the bottle to isolate them. Come out. Incubate the tissue culture flask containing resuspended bone marrow cells at 37 ° C, 5% CO2 for 1.5 hours. Collect those cells that are not adhered to the tissue flask, and place them at 1 x 106 / ml. The concentration is suspended in 1% Nutridoma SP (Boehringer mannheim, 39. This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) K ^ --------- (Please read the note on the back first (Please fill in this page for matters) Member of Intellectual Property Bureau, Ministry of Economic Affairs Co-op printed 1240629 A7 B7 V. invention is described in (V])
Indianapolis, IN)-IMDM中以便進行分析。將細胞加到96 孔洞,U-型組織培養盤中,以每個培養孔1〇5個細胞’總 體積0.2毫升,內含各種PAGC濃度,及有或無50 pg/ml 之老鼠重組 IL-3(R&D Systems,Minneapolis,MN)。於 37 C 、5%C02下培育這些培育物7天。PAGC對巨核細胞的活 性係藉由分析AchE活性的上升與否來測定,其係爲囑齒 類動物巨核細胞相當專一的細胞標誌物。7天後’將培養 盤離心並將上淸液丟棄。在每個培養孔中加入第I溶液 (0.2% Triton X-100, ImM EDTA, 0.12 mM NaCl, 50 mM HEPES,pH 7.5),及 0.2 毫升、20 μΐ 之 6.27 mM 之碘化硫 代乙醯膽驗(sigma,St. Louis,MO)(碘化硫代乙醯膽驗之終 濃度爲0.57 mM)。將組織培養盤在37°C下培育4小時,之 後由每個孔洞取出10 μΐ之反應混合物放到96孔洞的黑色 盤中(black plate)(Labsystems,Helsinki,Finland),之後加入 10μ1、0.4 mM溶於DMSO之香豆素苯順-丁烯二醯亞胺 (coumarinphenylmaleimide, CMP, Molecular probes Inc., Eugene,OR),及0·2毫升之第II溶液(5 mM乙酸鈉, ImM EDTA,0.2% Triton X-100, pH 5.0)。輕輕搖動培養盤 使液體混合均勻。以390 nm之激發光濾片及460 nm之發 射光濾片於螢光計中測量所釋出的螢光強度(Fliioroskan II, Labsystems,Helsinki,finland)。該螢光強度即與所培育巨 核細胞製造的AchE量成正比。PAGC濃度介於12.5 pg/ml 及400 pg/ml(波峰出現在濃度爲200 |Lig/ml時)時,可增加 所培育巨核細胞數目,且PAGC濃度介於12.5 pg/ml及 40 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) -----------螓& (請先閱讀背面之注意事項再填寫本頁) 訂--- SI. 經濟部智慧財產局員工消費合作社印製 經濟部智慧財產局員工消費合作社印製 1240629 A7 ^__B7 五、發明說明(0) 400 pg/ml(波峰出現在濃度爲200 pg/ml時)時,可與50 Pg/ml之IL-3 —起協同增加所培育巨核細胞數目(遠超過50 至200 pg/ml時所增加之巨核細胞數目)。 實施例5 復原經氟化尿嘧啶處理之老鼠體內GM-CFC 代細胞數目 於第〇天,將150毫克/公斤之氟化尿[I密症(sigma)注 射到8-10週大的BALB/c雌鼠腹膜內;並於第1-3天,分 別由皮下注入生理食鹽水、各種濃度之PAGC或100 pg/公 斤之重組人類G-CSF(Neupogen,Amgen)。並於第4天,將 實驗老鼠犧牲。收集雌鼠骨髓,計算後,將5 X 104個白血 球細胞培養在35毫米的培養盤中,每個培養盤中放了 1毫 升之甲基纖維素完全培養基(complete methylcellulose medium)及波草促細胞***素之胰臟細胞培養基(pokeweed mitogen spleen cell conditioned medium)作爲可提供群落刺 激因子的來源物(StemCell Technologies, Inc)。於 37°C下培 育6-7天後,以Nikon Diaphot顯微鏡(30-60倍)評估細胞 數超過50個的細胞群落。下表顯示PAGC可促進經氟化脲 嘧啶處理後之老鼠GM-CFC代細胞的復原。 處理 生理 PAGC PAGC PAGC G-CSF 食鹽水 50 mg/Kg 100 mg/Kg 200 mg/Kg 100 pg/Kg GM-CFC 601 608 1075 2020 1196 /femur 實施例6 復原接受亞致死劑量之輻射照射的老鼠體內周 邊白血球細胞數値 41 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) ^--------- (請先閱讀背面之注意事項再填寫本頁) 1240629 A7 B7 五、發明說明(0) 以平均體重爲20克的BALB/c雌鼠來進行此實驗。每 一次實驗前5天,在老鼠未酸化的飮水中加入40毫克/升 之新黴素(neomycin,Sigma,St. Louis, MO)。隨機挑選控制 組或實驗組之老鼠,每組各6隻,並於第0天以4.25 Gy 之 X 射線(250 KVP,0.35 mm Cu filter,Philips,Germany)來 進行輻射照射。輻射照射後,老鼠體內周邊白血球及血小 板的數目都比正常鼠來得低,至於紅血球細胞數目則略低 於正常値。由皮下注射1〇〇及300 mg/Kg之PAGC。最初 5天(從第〇天至第4天),每天注射一次,之後則是每週3 次,連續注射3週;其中第一劑係在輻射照射後4-5小時 內施打。總計施用14劑量之PAGC。控制組則是經皮下注 射0.1毫升的生理食鹽水。實驗期間,每週由老鼠尾部靜 脈抽血兩次,並將血液樣品存放在塗覆了 EDTA的管子中 (Sarstedt,Germany),並以 Serono 9010+細胞計數器(Serono Baker Diagnostics Inc.,Allentown,PA)來計算分析周邊白血 球、血小板及紅血球的數目,以及血紅素含量。相較於控 制組,1〇〇及300 mg/Kg之PAGC可明顯(ρ<0·01)促進 輻射照射後第1 7至3 0天(實驗後)之老鼠白血球細胞 的復原;並可明顯(ρ<0·05,一般係ρ<〇.〇1)促進輻射 照射後第14至25天之老鼠血小板細胞的復原;還 可明顯(ρ<〇.〇5,一般係ρ<〇.〇1)促進輻射照射後第 14至25天之老鼠紅血球細胞的復原。在20天硏究 期間,以相同方式施用100及300 mg/Kg之AGPC,無 論在哪一個時間點測試,都可改善輻射照射後之老 42 $紙張尺度適用中國國家標準(CNS)A4規格(210 x 297公釐) " (請先閱讀背面之注意事項再填寫本頁) f裝----W----訂---- 經濟部智慧財產局員工消費合作社印製 1240629 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明說明(w) 體內紅血球及血小板的復原,顯示其與PAGC有相 同的效益。 實施例7 單獨以PAGC或與粒細胞群落刺激因子(G-CSF)—起來移動周邊血液代細胞 讓8-10週大的BALB/c老鼠自由取用酸化水及食物。 正常老鼠則依下列方式處理7天,每天一次:生理食鹽水 (200 μ卜皮下注射),PAGC(100 mg/Kg,皮下注射),G-CSF(100 pg/Kg,皮下注射)或 PAGC+G_CSF(100 mg/Kg 之 PAGC+100 pg/Kg之G-CSF,皮下注射)。每隻老鼠的注射 量約200μ1。每一組有五隻老鼠。經過七天上述處理後, 第8天時在老鼠腹膜內注射20單位的肝素(Elkins-Sinn, Inc·,Cherry Hill,NJ),然後以吸入30分鐘C02的方式將老 鼠犧牲,並以心臟穿刺方式收集周邊血液〇UFicoll-Paque(pharmacia biotech AP,Uppsala,Sweden)密度離心方 式分離出周邊血液單核細胞,以磷酸緩衝液淸洗兩次,重 新懸浮於培養基中並以細胞計數器計算細胞數目。將介於 2·5 X 104及1 X 105間的周邊血液單核細胞培養在35毫米 的培養盤中,每個培養盤中放了 1毫升之甲基纖維素完全 培養基(complete methylcellulose medium)及重組之 IL-3+IL-6 幹細胞因子(Stem cell Technologies Inc.,Vancouver, B.C_)。於37°C下培育7-14天後,以Nikon Diaphot顯微 鏡(30-150倍)評估細胞數超過50個的細胞群落。評估可形 成粒細巨嗜細胞群落(GM-CFC)及 BFU-E(burst-forming unit -erythroid)。下表顯示PAGC可與G-CSF協同增加正常鼠 43 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) """ .—U— ^--------1 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1240629 A7 _______ B7 五、發明說明(d) 體盘fj環之GM-CFC與BFU-E 數曰。 生理食鹽水 PAGC 100 mg/Ka G-CSF 100 μβ/Kg PAGC, 100 mg/Kg G-CSF,100 pg/Kg GM-CFC, 100/ml 0.8 4.6 30 64 BFU-E, 100/ml 1.0 3.1 4.5 9.5 在另一類似實驗中,以200毫克/公斤的環磷醯胺來 處理老鼠,之後再以生理貫鹽水或100或300毫克/公斤 的PAGC處理11天,收集PBMC後重新懸浮成2 X 107細 胞/毫升,以螢光素異硫氰酸-抗-CD34及PE-細胞系標誌物 (CD3、CD4、CD6、CD19、CDllb、GR-1、CD41 及丁61:-119)(PhannaCia,San Diego, CA)進行染色,再以流體細胞計 (FACSCalibur,Becton Dickson,San Jose,CA)分析,可看到 PAGC會加速CD34*Lin_細胞移動進入周邊血液中。 實施例8 PAGC可恢復氟化脲嘧啶或環磷醯胺處理過之 老鼠體重 以>10週大的BALB/C雌鼠來進行此實驗。將10隻 老鼠隨機分成5組··分別是正常控制組、環磷醯胺(CY)-處 理組、氟化胧嘧啶(FU)-處理組、CY+PAGC-處理組、或 FU+PAGC-處理組。對正常控制組而言,第〇天時由腹膜 內注射生理食鹽水,然後由第1天至第12天,則是由皮下 注射生理食鹽水。對環磷醯胺(CY)-處理組或CY+PAGC-處 理組’則是在第0天時由腹膜內注射200毫克/公斤的環磷 醯胺’然後由第1天至第12天,則是由皮下注射生理食鹽 水或200毫克/公斤的PAGC。對氟化脲嘧啶(FU)-處理組或 44 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) -n hi n n n 一:口,a n I n (請先閱讀背面之注意事項再填寫本頁) 1240629 A7 五、發明說明u>) FU+PAGC-處理組,則是在第〇天時由腹膜內注射150毫 克/公斤的FU,然後由第1天至第12天,則是由皮下注射 生理食鹽水或200毫克/公斤的PAGC。老鼠在第0天時稱 一次體重,之後每隔一天再稱一次體重,直到第12天。結 果顯示經CY+PAGC-處理及FU+PAGC-處理過的老鼠,其 體重減輕最少,且比單獨以CY或FU處理之老鼠更快恢復 其原體重。 實施例9 在中國所進行的PAGC人體臨床試驗 在中國所進行的PAGC人體臨床試驗係依據中國衛生 部之規定來計進行。 第一期臨床試驗,PAGC係以正常生理食鹽水稀釋並 以靜脈注射的方式施用到32位正常的志願者體內,並連續 灌注7天。施用3次臨床劑量(250毫克/天)後未發現任何 嚴重的臨床副作用。 第二期臨床試驗則是評估PAGC是否對減輕肺癌、腸 胃癌或乳癌患者在接受化療後所引起的白血球過少(<4 X 1〇9 WBC/L)現象有所助益。總計六個醫學中心、487位患 者參與了這次臨床試驗,其中328位患者屬於PAGC治療 組,84位患者屬於G-CSF治療組,及75位控制組患者。 在整個14天的化療期間’若任何時候患者得WBC値低於 4 X 109 WBC/L,即將該患者納入任三組之一。對第一組患 者,將250毫克PAGC溶於500毫升生理食鹽水中後,再 每天一次靜脈注射到患者體內,連續注射7天。除了觀察 施用PAGC那7天外,再持續觀察患者7天。G-CSF治療 45 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) (請先閱讀背面之注意事項再填寫本頁) ----訂---------線 經濟部智慧財產局員工消費合作社印製 經濟部智慧財產局員工消費合作社印製 1240629 A7 B7 五、發明說明(吶) 組的患者,則每天由皮下注射75吨的G_CSF 一次,連續 注射5天’除注射的5天外’再繼續觀察患者9天。至於 控制組,則在化療後不再給予其他可強化血液系統的藥物 ,並觀察患者Η天。如此觀察所有3組患者其治療後η 天的WBC、RBC及血小板數値。同時也藉由藥物改善患 者化療症狀及卡諾夫斯基表現指標(Karn〇fsky performance Index)兩者,來評論其改善患者生活品質的效果。與化療 相關的症狀包括疲憊及倦怠、不適、盜汗、呼吸短促及沒 有食慾在內,並由合格的中醫師進行判定。每一項之分數 如下:〇分-沒有症狀,1分-症狀輕微,2分-中等 症狀及3分-症狀嚴重。因此,最嚴重的例子將會獲得 15分。卡諾夫斯基表現指標(Karnofsky performance Index) 則是WHO用來評估一般健康狀態的方法。圖1顯示化療 後,白血球細胞數目每天的變化情形;圖2則是化療後, 總症狀分數每天的變化情形;圖3顯示以卡諾夫斯基表現 指標(Karnofsky performance Index)表示之分數(PAGC 與 G-CSF比較:U=18.36,Ρ<0·01 ; PAGC與控制組比較: U=15.62,ρ<〇.〇〇〇1)。下表顯示化療後平均血小板數値與 天數的變化情形;所有結果都顯示以250毫克PAGC治療 7天’確實可促進WBC及血小板細胞數目恢復,改善化療 所引起的症狀,並改善化療患者的卡諾夫斯基表現指標, 整體來說’在統計上比控制組明顯來得好;且一般也比以 75 pg的G-CSF治療5天的效果來得好。 46 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐) " ----------訂-------— (請先閱讀背面之注意事項再填寫本頁) 1240629 A7 __ B7 、發明說明(〇>) 血小板,109/L ,PAGC-治療組患者(η=54) 第〇天 第4天 第7天 第10天 第14天 65 ±21 87土61* 118土94** 149土107** 178土 111** 氺ρ<0·05,**ρ<0·001 本發明已藉由實施例加以說明,但習知技藝者在看了 本說明書後,將可了解有許多等同的技術可應用於本發明 中,但這些等同的應用應仍屬於本發明範疇中。 (請先閱讀背面之注意事項再填寫本頁) 訂--- 經濟部智慧財產局員工消費合作社印製 47 本紙張尺度適用中國國家標準(CNS)A4規格(210 X 297公釐)Indianapolis, IN) -IMDM for analysis. Add cells to a 96-well, U-shaped tissue culture dish, with 105 cells per well, with a total volume of 0.2 ml, containing various PAGC concentrations, and with or without 50 pg / ml mouse recombinant IL- 3 (R & D Systems, Minneapolis, MN). These cultures were incubated at 37 C, 5% C02 for 7 days. PAGC activity on megakaryocytes is determined by analyzing the increase in AchE activity, and it is a very specific cell marker for megakaryocytes in dental animals. After 7 days' the culture plate was centrifuged and the supernatant was discarded. Add the first solution (0.2% Triton X-100, ImM EDTA, 0.12 mM NaCl, 50 mM HEPES, pH 7.5) to each culture well, and 0.2 ml, 20 μΐ of 6.27 mM thiothioacetate iodide Test (sigma, St. Louis, MO) (final thioacetate iodide test concentration is 0.57 mM). Incubate the tissue culture plate at 37 ° C for 4 hours, and then take out 10 μΐ of the reaction mixture from each well and place it into a 96-well black plate (Labsystems, Helsinki, Finland), and then add 10 μ1, 0.4 mM Coumarinphenylmaleimide, CMP, Molecular probes Inc., Eugene, OR in DMSO and 0.2 ml of a second solution (5 mM sodium acetate, ImM EDTA, 0.2 % Triton X-100, pH 5.0). Gently shake the culture plate to mix the liquid evenly. The emitted fluorescence intensity was measured in a fluorometer using an excitation light filter of 390 nm and an emission filter of 460 nm (Fliioroskan II, Labsystems, Helsinki, Finland). This fluorescence intensity is directly proportional to the amount of AchE produced by the cultured megakaryocytes. PAGC concentrations between 12.5 pg / ml and 400 pg / ml (peaks at a concentration of 200 | Lig / ml) can increase the number of megakaryocytes cultured, and PAGC concentrations between 12.5 pg / ml and 40 paper sizes Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) ----------- 螓 & (Please read the notes on the back before filling this page) Order --- SI. Economy Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and printed by 1240629 A7 ^ __ B7 V. Description of the invention (0) 400 pg / ml (when the peak appears at a concentration of 200 pg / ml) IL-3 at 50 Pg / ml works together to increase the number of megakaryocytes cultivated (far more than 50 to 200 pg / ml). Example 5 Reconstitution of GM-CFC generation cells in mice treated with fluorinated uracil. On day 0, 150 mg / kg of fluorinated urine [I sigma] was injected into BALB / c. Female mice were intraperitoneally; and on day 1-3, physiological saline, various concentrations of PAGC or 100 pg / kg of recombinant human G-CSF (Neupogen, Amgen) were injected subcutaneously. And on the 4th day, the experimental mice were sacrificed. Bone marrow was collected from female mice. After calculation, 5 X 104 white blood cells were cultured in 35 mm culture plates. Each culture plate was filled with 1 ml of complete methylcellulose medium and prokaryotic cells. Pokeweed mitogen spleen cell conditioned medium is a source of community stimulating factors (StemCell Technologies, Inc). After 6-7 days of incubation at 37 ° C, cell populations with more than 50 cells were evaluated with a Nikon Diaphot microscope (30-60x). The following table shows that PAGC can promote the recovery of GM-CFC generation cells in mice treated with uracil fluoride. Treatment of physiological PAGC PAGC PAGC G-CSF saline 50 mg / Kg 100 mg / Kg 200 mg / Kg 100 pg / Kg GM-CFC 601 608 1075 2020 1196 / femur Example 6 Restoration of rats exposed to sublethal doses of radiation Peripheral white blood cell count: 41 This paper is sized for Chinese National Standard (CNS) A4 (210 X 297 mm) ^ --------- (Please read the precautions on the back before filling this page) 1240629 A7 B7 V. Description of the invention (0) This experiment was performed on female BALB / c mice with an average weight of 20 grams. Five days before each experiment, 40 mg / L of neomycin (neomycin, Sigma, St. Louis, MO) was added to the unacidified mash of rats. Six mice were randomly selected from the control group or the experimental group, and irradiated with 4.25 Gy X-rays (250 KVP, 0.35 mm Cu filter, Philips, Germany) on day 0. After radiation exposure, the number of white blood cells and blood platelets around the mice was lower than that of normal mice, while the number of red blood cells was slightly lower than normal. PAGC was injected subcutaneously at 100 and 300 mg / Kg. The first 5 days (from day 0 to day 4) were given once a day, and then three times a week for three consecutive weeks; the first dose was administered within 4-5 hours after radiation exposure. A total of 14 doses of PAGC were administered. The control group was subcutaneously injected with 0.1 ml of physiological saline. During the experiment, blood was drawn from the tail vein of the mice twice a week, and blood samples were stored in EDTA-coated tubes (Sarstedt, Germany) and counted with a Serono 9010+ cell counter (Serono Baker Diagnostics Inc., Allentown, PA). ) To calculate and analyze the number of peripheral white blood cells, platelets and red blood cells, and the hemoglobin content. Compared with the control group, 100 and 300 mg / Kg PAGC significantly (ρ < 0.01) promoted the recovery of white blood cells in mice from 17 to 30 days (after the experiment) after irradiation; (Ρ < 0.05, general ρ < 0.01) promotes the recovery of rat platelet cells from 14 to 25 days after radiation exposure; it is also obvious (ρ < 0.05, generally ρ < 〇.〇 1) Promote the recovery of red blood cells in mice from 14 to 25 days after irradiation. During the 20-day study period, applying AGPC at 100 and 300 mg / Kg in the same manner, no matter at which time point the test was performed, the old $ 42 paper after radiation exposure could be improved to the Chinese National Standard (CNS) A4 specification ( 210 x 297 mm) " (Please read the notes on the back before filling out this page) f -------------------- Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs Consumer Cooperatives 1240629 A7 B7 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. 5. Description of the invention (w) The recovery of red blood cells and platelets in the body shows that it has the same benefits as PAGC. Example 7 PAGC alone or with granulocyte colony stimulating factor (G-CSF) —moving peripheral blood generation cells to allow 8-10 week old BALB / c mice to freely access acidified water and food. Normal mice were treated for 7 days, once a day as follows: physiological saline (200 μ subcutaneous injection), PAGC (100 mg / Kg, subcutaneous injection), G-CSF (100 pg / Kg, subcutaneous injection) or PAGC + G_CSF (100 mg / Kg PAGC + 100 pg / Kg G-CSF, subcutaneous injection). The injection volume of each mouse was about 200 μ1. There are five mice in each group. After seven days of the above treatment, mice were injected intraperitoneally with 20 units of heparin (Elkins-Sinn, Inc., Cherry Hill, NJ) on the eighth day, and then the rats were sacrificed by inhaling C02 for 30 minutes, and cardiac puncture was performed. Peripheral blood was collected. UFicoll-Paque (pharmacia biotech AP, Uppsala, Sweden) density centrifugation was used to isolate peripheral blood mononuclear cells, rinsed twice with phosphate buffered saline, resuspended in culture medium, and calculated the number of cells with a cytometer. Peripheral blood mononuclear cells between 2.5 x 104 and 1 x 105 were cultured in 35 mm culture plates, and 1 ml of complete methylcellulose medium and Recombinant IL-3 + IL-6 stem cell factor (Stem cell Technologies Inc., Vancouver, B.C.). After 7-14 days of incubation at 37 ° C, cell populations with more than 50 cells were evaluated with a Nikon Diaphot microscope (30-150 times). The formation of GM-CFC and BFU-E (burst-forming unit-erythroid) was evaluated. The following table shows that PAGC and G-CSF can increase normal rats. The paper size is applicable to Chinese National Standard (CNS) A4 (210 X 297 mm). &Quot; " " .—U— ^ ------ --1 (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economy 1240629 A7 _______ B7 V. Description of the invention (d) GM-CFC and BFU-E numbers of the body disk fj ring Said. Physiological saline PAGC 100 mg / Ka G-CSF 100 μβ / Kg PAGC, 100 mg / Kg G-CSF, 100 pg / Kg GM-CFC, 100 / ml 0.8 4.6 30 64 BFU-E, 100 / ml 1.0 3.1 4.5 9.5 In another similar experiment, rats were treated with 200 mg / kg of cyclophosphamide, and then treated with physiological saline or 100 or 300 mg / kg of PAGC for 11 days. PBMCs were collected and resuspended into 2 X 107 Cells / ml, with fluorescein isothiocyanate-anti-CD34 and PE-cell line markers (CD3, CD4, CD6, CD19, CDllb, GR-1, CD41 and Ding 61: -119) (PhannaCia, San Diego, CA) for staining and analysis with a fluid cytometer (FACSCalibur, Becton Dickson, San Jose, CA). It can be seen that PAGC accelerates the movement of CD34 * Lin_ cells into the surrounding blood. Example 8 PAGC can restore the weight of mice treated with fluorinated uracil or cyclophosphamide. This experiment was performed with female BALB / C rats> 10 weeks old. Ten mice were randomly divided into 5 groups: normal control group, cyclophosphamide (CY) -treated group, fluorpyrimidine (FU) -treated group, CY + PAGC-treated group, or FU + PAGC- Processing group. In the normal control group, physiological saline was injected intraperitoneally on day 0, and then physiological saline was injected subcutaneously from day 1 to day 12. For the cyclophosphamide (CY) -treated group or CY + PAGC-treated group, the intraperitoneal injection of 200 mg / kg of cyclophosphamide was performed on day 0, and then from day 1 to day 12, It is a subcutaneous injection of saline or 200 mg / kg of PAGC. For the fluorinated uracil (FU) -treatment group or 44 paper sizes, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) applies -n hi nnn One: mouth, an I n (Please read the note on the back first Please fill in this page again) 1240629 A7 V. Description of invention u &); FU + PAGC-treatment group, 150 mg / kg of FU was injected intraperitoneally on day 0, and then from day 1 to day 12, It is a subcutaneous injection of saline or 200 mg / kg of PAGC. The rats were weighed once on day 0 and then again every other day until day 12. The results showed that CY + PAGC-treated and FU + PAGC-treated mice had the least weight loss and recovered their original weight faster than mice treated with CY or FU alone. Example 9 PAGC human clinical trial conducted in China PAGC human clinical trial conducted in China was conducted in accordance with the regulations of the Ministry of Health of China. In the first phase of clinical trials, PAGC was diluted with normal saline and administered intravenously to 32 normal volunteers, and was continuously perfused for 7 days. No serious clinical side effects were found after three clinical doses (250 mg / day). The second phase of the clinical trial was to evaluate whether PAGC was helpful in reducing the incidence of low white blood cells (< 4 X 109 WBC / L) caused by chemotherapy in patients with lung, gastrointestinal or breast cancer. A total of 487 patients from six medical centers participated in this clinical trial, of which 328 patients belonged to the PAGC treatment group, 84 patients belonged to the G-CSF treatment group, and 75 patients in the control group. During the entire 14-day chemotherapy period, if the patient has a WBC below 4 X 109 WBC / L at any time, the patient will be included in any of the three groups. For the first group of patients, 250 mg of PAGC was dissolved in 500 ml of physiological saline, and then intravenously injected into the patient once a day for 7 consecutive days. Except for the 7 days that PAGC was administered, the patients were continuously observed for another 7 days. G-CSF treatment 45 This paper size is applicable to Chinese National Standard (CNS) A4 specification (210 X 297 mm) (Please read the precautions on the back before filling this page) ---- Order -------- -Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs and printed by 1240629 A7 B7 On day 'except 5 days of injection' continue to observe the patient for 9 days. As for the control group, other drugs that strengthen the blood system will not be given after chemotherapy, and the patients will be observed the next day. In this way, the WBC, RBC, and platelet counts of all three groups of patients were observed n days after treatment. At the same time, the effect of improving the quality of life of patients was also reviewed by using drugs to improve the patients' chemotherapy symptoms and Karnofsky performance index. Symptoms related to chemotherapy include fatigue and burnout, discomfort, night sweats, shortness of breath, and lack of appetite, and are determined by a qualified Chinese medicine practitioner. The score for each item is as follows: 0-no symptoms, 1-mild symptoms, 2-moderate symptoms and 3-severe symptoms. Therefore, the worst case will score 15 points. The Karnofsky performance index is the method WHO uses to assess general health. Figure 1 shows the daily changes in the number of white blood cells after chemotherapy; Figure 2 shows the daily changes in total symptom scores after chemotherapy; Figure 3 shows the score (PAGC) expressed by Karnofsky performance index Compared with G-CSF: U = 18.36, P <0.01; PAGC compared with control group: U = 15.62, ρ < 0.0.00). The table below shows the changes in mean platelet counts and days after chemotherapy. All results show that treatment with 250 mg of PAGC for 7 days does indeed promote the recovery of WBC and platelet cell numbers, improve the symptoms caused by chemotherapy, and improve the cardiology of chemotherapy patients. The overall performance index of Novsky was statistically significantly better than that of the control group; and it was generally better than the effect of 5 days of treatment with 75 pg of G-CSF. 46 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) " ---------- Order ----------- (Please read the precautions on the back before (Fill in this page) 1240629 A7 __ B7 、 Explanation of the invention (〇 >) Platelet, 109 / L, PAGC-treatment group patients (η = 54) Day 0 Day 4 Day 7 Day 10 Day 14 65 ± 21 87 soil 61 * 118 soil 94 ** 149 soil 107 ** 178 soil 111 ** 氺 ρ < 0,05, ** ρ < 0 · 001 The present invention has been described by way of examples, but those skilled in the art are watching After completing this description, it will be understood that there are many equivalent technologies that can be applied to the present invention, but these equivalent applications should still belong to the scope of the present invention. (Please read the precautions on the back before filling out this page) Order --- Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 47 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)
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CN (1) | CN1179979C (en) |
AU (1) | AU6203600A (en) |
HK (1) | HK1046290B (en) |
MY (1) | MY131426A (en) |
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CN100509843C (en) * | 2000-06-29 | 2009-07-08 | 法蒙金希公司 | Acid-modified arabinogalactan protein composition |
US7601368B2 (en) | 2004-09-01 | 2009-10-13 | Lupin Limited | Purified Arabinogalactan-Protein (AGP) composition useful in the treatment psoriasis and other disorders |
CN1854158B (en) * | 2005-04-25 | 2011-09-07 | 蒋来高 | Extraction of astragalus mongholicus polysaccharose by super-critical fluid technology |
SG172029A1 (en) * | 2008-12-15 | 2011-07-28 | Ecopharm Llc | Treating idiopathic thrombocytopenic purpura with compositions comprising extracts of astragalus membranaceus |
WO2016028714A1 (en) * | 2014-08-18 | 2016-02-25 | Pharmagenesis, Inc. | Polygalacturonan rhamnogalacturonan1 (pgrg1) composition |
CN104147254A (en) * | 2014-08-21 | 2014-11-19 | 陈勇 | Traditional Chinese medicine preparation for treating leucocytopenia after chemotherapy |
CN109432242A (en) * | 2018-11-07 | 2019-03-08 | 郑毅男 | A kind of larch arabinogalactan preparation method and its application in terms of medical treatment |
CN110551230B (en) * | 2019-09-21 | 2022-02-15 | 天津赛诺制药有限公司 | Preparation method of astragalus polysaccharide |
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US5589591A (en) * | 1986-07-03 | 1996-12-31 | Advanced Magnetics, Inc. | Endotoxin-free polysaccharides |
US4944946A (en) * | 1987-01-27 | 1990-07-31 | Yaguang Liu | Pharmaceutical composition for inhibiting viruses and increasing immune function (PII) |
CN1027168C (en) * | 1989-05-13 | 1994-12-28 | 储大同 | Bioregulator made from Inter leukin-2 |
IT1238685B (en) * | 1990-02-09 | 1993-09-01 | Indena Spa | IMMUNOMODULATING ACTION POLYSACCHARIDES FROM ASTRAGALUS MEMBRANACEOUS AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM |
US5116969B1 (en) * | 1990-04-26 | 1997-04-01 | Larex International Inc | Ultrarefined arabinogalactan product |
ES2049561B1 (en) * | 1991-04-27 | 1994-12-16 | Andromaco Lab | PROCEDURE FOR OBTAINING POLYMERS WITH ACTIVITY ON THE HEMATOPOYETIC SYSTEM. |
US5770217A (en) * | 1997-07-02 | 1998-06-23 | Atlatl, Inc. | Dietary supplement for hematological, immune and appetite enhancement |
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HK1046290A1 (en) | 2003-01-03 |
WO2001000682A1 (en) | 2001-01-04 |
EP1200482A1 (en) | 2002-05-02 |
CN1368979A (en) | 2002-09-11 |
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