TWI308068B

TWI308068B -

Info

Publication number: TWI308068B
Application number: TW91113159A
Authority: TW
Inventors: Gaylen M Zentner; Miroslav Baudys; Maria Jurek; Wolfram Samlowski
Original assignee: Macromed Inc
Priority date: 2002-06-13
Filing date: 2002-06-17
Publication date: 2009-04-01

Description

1308068 A7 _B7 五、發明説明（1 ) 本案請求2001年6月14日申請之美國臨時申請案案號60/298,594的權利。發明之枝術領域 .本發明隸屬於淋巴激素局部緩釋至溫血動物之技術領域。更明確的說，本發明係關於用於局部性控制或局部並全身性控制細胞增殖疾病的治療組合物及方法。發明背景在探究免疫系統之生物學上，最近的硏究成果使得免疫反應之重要調節子得以鑑定出來，這些免疫調節子通常稱之爲細胞激素或者淋巴激素（若其由淋巴細胞生產）。 Rosenberg, Steven A. "The immunotherapy and Gene Therapy.” J. Clin. Oncology 10:180-199(1992)。細胞激素是小蛋白質，其主要地由免疫系統中可增進其他細胞增殖與分化的細胞分泌，但並非僅能由這種細胞分泌。細胞激素的範例包括間白素、干擾素、血球生成性集落刺激因子（CSF)、及前發炎因子，例如腫瘤壞死因子（TNF)。施用細胞激素及相關免疫調節子會導致患有多種類型腫瘤的病患產生目標腫瘤反應。Sarna, Gregory 等人，“A Pilot Study of Intralymphatic Interleukin-2. II. Clinical and Biological Effects", J. Biol. Response Modifiers, 9:81-86(1990).但是目前的細胞激素用藥方式經常伴隨著毒性，如此限制了這些藥劑的治療價値。本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）先閱背之注項再 % % 本頁經濟部智慧財產局員工消費合作社印製 -4 - 1308068 A7 B7 五、發明説明（2 經濟部智慧財產局員工消費合作社印製間白素-2(IL-2)是一種由正常週邊血液淋巴細胞所生產的淋巴激素，這些淋巴細胞一旦暴露於植物外源凝集素、抗原、或其他刺激物之後，會誘使經過抗原或促細胞分裂劑刺激的T細胞增生，此發現係由Morgan, D.A.等人首先記載於 Science (1976)，193:1007-1008。IL-2 作用在三種主要類型的淋巴細胞：T細胞、B細胞、及NK細胞，IL-2刺激這些細胞增生且增強其分化功能。IL-2可藉由刺激NK 細胞而達到固有的或天生的宿主抵抗力、且可藉由剌激T 細胞及B細胞而達到對抗原專一的後天免疫反應力。最初是藉由培養人類週邊血液淋巴細胞（PBL)或其他IL-2生產細胞株來生產IL-2,例如記載於美國專利案第4,401，756號。重組DNA技術提供PBL及其他IL-2生產細胞株之外的另一種選擇。Taniguchi，T.等人，Nature( 1983), 302:305-310 及 Devos, R·，Nucleic Acids Research (1983)，1 1:4307-4323 業已記載選殖人類IL-2基因且於微生物中表現該基因。美國專利案第4,5 1 8,584號揭示並請求IL-2之突變蛋白質，其中正常出現在野生型或天然分子中第125位置的半洸胺酸被中性胺基酸（例如絲胺酸或丙胺酸）取代。美國專利案第4,5 30,787號及第4,569,790號揭示並請求純化重組型天然IL-2及其突變蛋白質的方法，以及IL-2的純化形式。美國專利案第4,604,377號揭示適用於重建作用 (reconstitution)的IL-2組成物，其係由經氧化、自微生物生產的重組型IL-2於藥學上可接受的水性載劑中所組成者。對付腫瘤抗原時，命名爲「幫助者T細胞」的一亞群請先閲讀— 背之注1308068 A7 _B7 V. INSTRUCTIONS (1) This application claims the right of U.S. Provisional Application No. 60/298,594, filed on June 14, 2001. FIELD OF THE INVENTION The present invention is directed to the technical field of localized release of lymphokines to warm-blooded animals. More specifically, the present invention relates to therapeutic compositions and methods for the localized control or topical and systemic control of cell proliferative disorders. BACKGROUND OF THE INVENTION In exploring the biology of the immune system, recent research has led to the identification of important regulators of immune responses, often referred to as cytokines or lymphoid hormones (if they are produced by lymphocytes). Rosenberg, Steven A. "The immunotherapy and Gene Therapy.” J. Clin. Oncology 10:180-199 (1992). Cytokines are small proteins that are primarily cells that promote proliferation and differentiation of other cells in the immune system. Secretion, but not exclusively, can be secreted by such cells. Examples of cytokines include interleukins, interferons, hematopoietic colony-stimulating factor (CSF), and pro-inflammatory factors such as tumor necrosis factor (TNF). And related immunomodulators can cause target tumor responses in patients with multiple types of tumors. Sarna, Gregory et al., "A Pilot Study of Intralymphatic Interleukin-2. II. Clinical and Biological Effects", J. Biol. Response Modifiers , 9:81-86 (1990). However, current cytokine regimens are often accompanied by toxicity, thus limiting the therapeutic cost of these agents. This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm). Read the back of the item and then save %%. Page on the page, Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative Printed -4 - 1308068 A7 B7 V. Invention Description (2 Department of Economics, Intellectual Property Office, Staff Consumer Cooperative, Printed Interleukin-2 (IL-2) is a lymphohormone produced by normal peripheral blood lymphocytes. Once exposed to plant lectins, antigens, or other After the stimuli, it induces proliferation of T cells stimulated by antigen or mitogens. This finding was first described by Morgan, DA et al. in Science (1976), 193: 1007-1008. IL-2 acts on three main types. Types of lymphocytes: T cells, B cells, and NK cells, IL-2 stimulates these cells to proliferate and enhance their differentiation. IL-2 can achieve innate or innate host resistance by stimulating NK cells, and Acquires acquired antigenic response to antigen by stimulating T cells and B cells. Initially, IL-2 is produced by culturing human peripheral blood lymphocytes (PBL) or other IL-2 producing cell lines. For example, it is described in U.S. Patent No. 4,401,756. Recombinant DNA technology provides an alternative to PBL and other IL-2 producing cell lines. Taniguchi, T. et al., Nature (1983), 302: 305-310 and Devos, R., Nucleic Acids Research (1983), 1 1:4307-4323 has documented the selection of the human IL-2 gene and its expression in microorganisms. U.S. Patent No. 4,5,8,584 discloses and requests IL- a mutant protein of 2, wherein the semi-proline which is normally present at position 125 in the wild-type or natural molecule is substituted with a neutral amino acid such as serine or alanine. U.S. Patent No. 4,5,30,787 and A method for purifying recombinant native IL-2 and its mutant protein, and a purified form of IL-2, is disclosed in U.S. Patent No. 4,569,790, the disclosure of which is incorporated herein by reference. It consists of oxidized, microbial-produced recombinant IL-2 in a pharmaceutically acceptable aqueous carrier. When dealing with tumor antigens, a subgroup named "Helper T cells" should be read first - back Note

I 妾訂辦本纸張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -5- 1308068 A7 B7 五、發明説明（3 ) (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Η消費合作社印製淋巴細胞會分泌少量IL-2。此IL-2局部作用在腫瘤抗原刺激之位置，藉此活化了調控全身性腫瘤細胞破壞作用的胞毒性T細胞及天然殺手細胞。經靜脈內、經淋巴管內、及經病變處內部施用IL-2 ,已使得一些腫瘤病患在臨床上有顯著反應。已證實全身性施用高劑量的重組型IL-2會導致小鼠之經確定轉移的惡性腫瘤消退，且刺激人類之經淋巴激素活化的殺手細胞，與滲透腫瘤之淋巴細胞。Rosenberg 等人，J· Exp. Med. (1985)1 61:1 1 69- 1 1 88 ; Rosenberg, S.等人，New Eng. J. Med.(1985)31 3:1485- 1492 ; Rosenberg 等人， Science( 1 986)233:1318- 1321)。但是嚴重的毒性（低血壓及水腫）限制了經靜脈及經淋巴管施用IL-2的劑量與效力。 Hoover, H.C.Jr.等人，Cancer Res. (1984)，44(4):1671-6。全身性施用淋巴激素的毒性作用並不令人意外，因爲這些淋巴激素係調節局部細胞交互作用且一般狀況下只分泌極少量。再者，諸如間白素-4(IL-4)、阿法干擾素（α -INF)、及加瑪干擾素（T -INF)之類的其他淋巴激素曾經用於刺激對抗腫瘤細胞之免疫反應。如同IL-2，目前用藥方式具有不利之副作用。爲了要預防發生全身性IL-2用藥時的毒性，有幾位硏究員曾經檢測在病變處內部注射IL-2。此方法排除了全身性施用IL-2所伴隨的毒性。但是需要多次經病變處內部注射才可以達到最佳治療功效。Bubenik，等人，immunology Letters. 23:287-292(1989/1990) Borden, Ernest C.及 Sondel, Paul M，Cancer. 1990 年 2 月 1 日；65(3 附刊）··8〇〇- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X297公釐） -6- 1308068 A7 B7 五、發明説明（4 ) 14 ； Rosenberg, Steven A.等人，108:853-864( 1988)。因此，此注射法對許多病人而言不實際，尤其是沒有潛在病變而無可供注射之腫瘤位置時。美國專利案第6,045,788號揭示，爲了要活化及/或刺激病人的免疫系統但不造成實質毒性，低劑量的天然與重組型IL-2可長時間（例如，三個月以上）連續地施用至病人身上。但是，此方法需要長時間的頻繁用藥，另一種選擇是，冀求一種該專利案中並未揭示之最理想的緩釋組合物。除了潛在的毒性，全身施用IL-2之另一個困擾是快速的腎排出。當經靜脈內供給IL-2時，11^2被快速排出，其最初排出半衰期及終止排出半衰期分別是6至12分鐘、及 40至80分鐘。因此，冀求一種IL-2組合物，其局部施用至溫血動物之後可形成包含IL-2之積貯處。此種含有IL-2的積貯處提供持續的、緩釋的IL-2，藉此足以刺激胞毒性T淋巴細胞 (CTTL’s)的增生但不引起不欲的副作用，CTTL’s可局部性作用、或局部並全身性作用。作爲本發明組合物中可注射用的或可植入的聚合性藥物載劑的最理想材料應該是生物可降解的、可與藥物相容的、且可使用簡單的、安全的溶劑（例如水）製備的、及施用後不需要額外的聚合反應或其他形成共價鍵的反應。發明槪述本發明提供一種治療性組合物，其包括具有熱可逆之本纸張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (讀先閲讀背面之注意事項再填寫本頁) 4 訂經濟部智慧財產局員工消費合作社印製 1308068 A7 B7 五、發明説明（5 ) (請先閲讀背面之注意事項再填寫本頁) 凝膠化性質之生物可降解的嵌段（block)共聚體藥物載劑及有效量之淋巴激素，該組合物可經腫瘤內部或經腫瘤週邊施用並形成包含淋巴激素之積貯處（depot)。該包含淋巴激素之積貯處提供持續釋出的、延緩釋出的淋巴激素（例如 IL- 2 )，其足以刺激胞毒性T淋巴細胞之生成，該τ淋巴細胞可局部性作用、或局部並全身性作用，但是不會造成任何明顯不欲之全身性副作用。“淋巴激素”乙詞表示具有 IL-2或其他間白素活性之任何藥劑，包括天然的、重組的及突變的IL-2或其他間白素，及其等同物或衍生物。較佳的淋巴激素可選自由間白素-2(IL-2)、間白素-4、間白素-12 及其衍生物所組成之類群。經濟部智慧財產局員工消費合作社印製本發明也提供一種將高毒性藥物，即，IL-2或其他間白素，轉化爲具有最低之不欲副作用且充分有效之藥物的方法；此方法包括將IL-2或其他間白素倂入一種生物可降解的藥物載劑中，該載劑能夠在局部用藥之後形成包含藥物之積貯處。較好，此生物可降解的藥物載劑具有反向的熱凝膠化性質，易言之，這類藥物載劑在低於體溫時呈液體狀，加溫之，則轉化爲凝膠或固體狀，且等於體溫或接近體溫時以凝膠狀存在。本發明於下文說明中將普遍提及 IL-2,除非特別指明，此IL-2乙詞之教示擴及上文定義之其餘藥劑。IL-2較好是重組型人類IL-2。本發明另提供一種治療性或預防性治療溫血動物宿主之腫瘤負擔的方法，包括對該宿主局部性施用一組合物，該組合物包括有效量之IL-2、或其他淋巴激素，其係與具本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐） -8- 1308068 A7 B7 _ 五、發明説明（7 ) 第1B圖說明IL-2自實例1之IL-2組合物活體外釋出的結果，其係利用細胞增殖分析法分析並檢視° 第2圖說明實例1之IL-2組合物之細胞毒性分析結果，其係測定專一性細胞溶解百分比。第3圖說明施用實例1之IL-2組合物對纖維肉瘤腫瘤生長的影響，其係測定經歷一段時間之腫瘤大小的變化。第4圖說明IL-2自實例4之IL-2組合物活體外釋出的結果，其係利用ELISA檢視。第5圖說明施用實例4之IL-2組合物對腫瘤生長之劑量漸增的影響，其係測定經歷一段時間之腫瘤大小的變化〇第6圖說明重複施用實例4之IL-2組合物對腫瘤生長之影響，其係測定經歷一段時間之腫瘤大小的變化。第7圖說明重複施用實例4之IL-2組合物對於MetA 纖維肉瘤腫瘤模型中之小鼠生存率的影響。第8圖說明重複施用實例4之IL-2組合物對於B16黑色素瘤腫瘤模型中之小鼠生存率的影響。經濟部智慧財產局員工消費合作社印製發明詳述在揭露與說明本發明之治療組合物及利用該組合物於藥物輸送之方法之前，需了解本發明並不限於本文中揭示的特定配製、方法步驟、及材料。這些特定的配製、方法步驟、.及材料在屬於本發明範疇之內可有一些變化。也應了解的是，本文所採用的用詞僅僅是用來說明特定實施體本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -10- 1308068 A7 ___ B7 五、發明説明（8 ) 系而非用於限制，因爲本發明之範圍將只受本文附加的申請專利範圍及其均等物之限制。除非本文內容淸楚指明，一定要注意的是，本說明書及附加的申請專利範圍中所使用的定冠詞V’、 ‘‘an”、及 “ the ”包括複數的指定對象。舉例而言，當提及用於輸送“ 一藥物”之組合物時，包括一種、兩種、或多種藥物之涵義。下列用詞將用來說明及請求本發明，其係根據下文列示的定義。 "非經腸的”乙詞表示經腫瘤內部、經腫瘤週邊、經病變處週邊、經病變處區內部、經鞘膜內、經腹膜內、及經腹腔內。 "細胞增殖疾病”乙詞包括細胞增殖及分化之疾病，通常思及贅生的或惡性的，例如腫瘤。腫瘤諸如癌瘤、肉瘤、黑色素瘤、骨髓瘤 '肉瘤等。腫瘤乙詞也包括腫瘤前組織及細胞，咸知該等組織或細胞若不加以治療將會發展爲真正的腫瘤。細胞增殖疾病之其他範例爲疣。 “治療性”治療乙詞表示當經過該技術領域之任何方法確認病人已經發展成腫瘤之後（即，已經確定有腫瘤負擔之後），將一藥物施用至該病人以達到減輕或消除已經存在的腫瘤負擔之目的的方法。 "預防性”治療乙詞表示在已經提供治療性治療之後，爲了預防腫瘤復發的用藥方法。 "治療有效量”乙詞表示本發明之方法或組成物中每個活性組份的量足以顯現對病患有意義的益處，即，生命延長本紙張尺度適用中.國國家標準（CNS ) A4規格.（210X297公釐〉 (請先閲讀背面之注意事項再填寫本頁) 4 訂經濟部智慧財產局員工消費合作社印製 -11 - 1308068 A7 B7 五、發明説明（11) 合成器內合成兩種或更多種的共聚體物系。由上述兩種製法製備的混合物可與水混合，以形成具有相同或不同凝膠化性質及凝膠品質之聚合物溶液。 ‘‘溶液”經溶解的”' “溶解的”及所有其他類似用詞表示一溶液或溶解狀態，包括均質溶液、膠質溶液、或任何明顯地單一膠態，例如乳液或懸浮液。 "生物可降解的”乙詞表示嵌段共聚體可在體內被化學性斷裂或降解以形成無毒性的組份。降解速率可與藥物釋出速率相同或不同。 "藥物”乙詞表示用於治療性目的之具有生物活性或藥物活性的任何有機或無機化合物或物質。 “肽”、“多肚”、“寡肽”及“蛋白質”等詞用於表示肽藥物或蛋白質藥物時，彼此可交互使用；除非特別指明，不應限定彼等在任何特定分子量、肽序列或長度、生物活性之領域或治療用途。經濟部智慧財產局員工消費合作社印製 “間白素”乙詞表示具有間白素活性（尤其是IL-2及IL-1 2活性）之任何蛋白質/多肽藥劑、或其衍生物、類似物或模擬物；包括天然及重組型IL-2及IL-12，天然與重組型間白素構成之藥學上可接受的融合蛋白質、其衍生物及混合物。 “hIL-2”乙詞表示呈現以人類間白素_2爲特徵之活性範圍的蛋白質。詳言之，此蛋白質一定能夠刺激依賴ML — 2之細胞溶解T細胞與幫助者（helper)T細胞株的增殖，其標準分析法可參見 S.Gillis 等人，J. Immunol.( 1978) 120:2027- 本紙張尺度適用中國國家揉準（CNS ) A4規格（21〇χ：297公釐） -14 - 1308068 A7 B7 五、發明説明（12) 2032 及 J· Watson，J. Exp. Med.(1979) 150:15 10-15 19。經修飾之IL-2及IL-12也涵括在此定義內，只要其生物活性未被修飾破壞。 “PLGA”乙詞表示自乳酸與乙醇酸之縮合共聚反應、或藉由乳酸與乙醇酸之開環共聚反應所衍生的共聚體或共聚體基團。乳酸與乳酸鹽兩詞可交互使用，乙醇酸及乙醇酸鹽兩詞也可交互使用。 “PLAM乙詞表示自乳酸縮合反應、或藉由丙交酯之開環聚合反應所衍生之聚合物。 "PLGA”乙詞表示自乙醇酸縮合反應、或藉由乙交酯之開環聚合反應所衍生之聚合物。 “生物可降解之聚酯或聚（原酸酯）”乙詞表示任何生物可降解之聚酯或聚（原酸酯），該等聚酯以選自D，L-丙交酯、D-丙交酯、L-丙交酯' D，L-乳酸、D-乳酸、L-乳酸、乙交酯、乙醇酸、ε -己內酯、ε -羥基己酸' r -丁內酯、r ·羥基丁酸、6 -戊內酯、5 -羥基戊酸、羥基丁酸、羥基丁二酸、及其共聚體所組成之類群之單體所合成者爲佳。 “ReGel®”乙詞表示MacroMed公司之商標名，其用於 —類低分子量、生物可降解之具有反向熱凝膠化性質的嵌段共聚體，其揭示於美國專利案第6,004,573、6，117,949、 6,201，072、及6,287,588號，這些專利案皆倂入本發明作爲參考。此商標名也包括揭示於懸而未決之美國專利申請案編號09/906,041及09/559,799中的組成物，這些專利申請案亦倂入本發明作爲參考。生物可降解之藥物載劑包括本紙張尺度適用中國國家標準（CNS ) A4规格（2丨〇><297公釐） (請先閲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 -15- 1308068 A7 B7 五、發明説明（13) ΑΒΑ型或BAB型之三嵌段共聚體或其混合物，其中A嵌段是相當疏水性的且含有生物可降解的聚酯或聚（原酸酯）；而 B嵌段是相當親水性的且包含聚乙二醇（PEG);該共聚體具有5 0.1至83重量％之疏水性含量、17至49.9重量％之親水性$量、且嵌段共聚體之總分子量介於2000至8000之間。此藥物載劑在溫度低於哺乳動物之體溫時呈現水溶解性；且在溫度等於生理學上之哺乳動物體溫時，進行可逆性熱凝膠化之後變成凝膠。生物可降解的疏水性A嵌段包含一聚酯或聚（原酸酯），其中該聚酯係由選自D，L-丙交酯、D-丙交酯、L-丙交酯、D，L-乳酸、D-乳酸、L-乳酸、乙交酯、乙醇酸' ε -己內酯、ε -羥基己酸、7 -丁內酯、τ -羥基丁酸、戊內酯、羥基戊酸、羥基丁酸、羥基丁二酸、及其共聚體所組成之類群之單體所合成者，且具有平均分子量爲約600至3000。親水性Β嵌段部分較好是具有平均分子量爲約500至2200之聚乙二醇（PEG)。當嵌段共聚體爲可溶性時（於溫度低於凝膠化溫度诗）的濃度，可視爲官能性濃度。一般而言，可使用低至3重量％及高達約50重量％之嵌段共聚體濃度，這些濃度下的嵌段共聚體仍有官能性。但以濃度介於約5至40重量％之間爲較佳，且濃度介於約10至30重量％之間爲最佳。在較低官能性濃度範圔時，相轉移可導致稀薄凝膠或黏性液體的形成，然而這些物系仍可適用於本發明之目的。I 妾Order the paper size for the Chinese National Standard (CNS) A4 specification (210X297 mm) -5- 1308068 A7 B7 V. Invention description (3) (Please read the note on the back and fill in this page) The intellectual property bureau, the consumer cooperative, printed lymphocytes secreted a small amount of IL-2. This IL-2 acts locally at the site of tumor antigen stimuli, thereby activating cytotoxic T cells and natural killer cells that regulate the destruction of systemic tumor cells. Intravenous, intralymphatic, and intralesional administration of IL-2 has led to a significant clinical response in some cancer patients. Systemic administration of high doses of recombinant IL-2 has been shown to result in regression of malignant tumors in mice that have been determined to metastasize, and to stimulate lymphocyte-activated killer cells in humans, as well as lymphocytes that infiltrate tumors. Rosenberg et al., J. Exp. Med. (1985) 1 61:1 1 69- 1 1 88; Rosenberg, S. et al., New Eng. J. Med. (1985) 31 3:1485-1492; Rosenberg et al. People, Science (1 986) 233: 1318 - 1321). However, severe toxicity (hypotension and edema) limits the dose and efficacy of IL-2 administered intravenously and via lymphatics. Hoover, H. C. Jr. et al., Cancer Res. (1984), 44(4): 1671-6. The toxic effects of systemic administration of lymphokines are not surprising, as these lymphatic hormones regulate local cellular interactions and in general only secrete very small amounts. Furthermore, other lymphoid hormones such as interleukin-4 (IL-4), alpha interferon (α-INF), and gamma interferon (T-INF) have been used to stimulate immunity against tumor cells. reaction. Like IL-2, current regimens have unfavorable side effects. In order to prevent the toxicity of systemic IL-2, several investigators have tested the injection of IL-2 into the lesion. This method precludes the toxicity associated with systemic administration of IL-2. However, multiple injections through the lesion are required to achieve optimal therapeutic efficacy. Bubenik, et al., immunology Letters. 23:287-292 (1989/1990) Borden, Ernest C. and Sondel, Paul M, Cancer. February 1, 1990; 65 (3 Supplement)··8〇〇- This paper scale applies to the Chinese National Standard (CNS) Α4 specification (210X297 mm) -6- 1308068 A7 B7 V. Inventive Note (4) 14; Rosenberg, Steven A. et al., 108: 853-864 (1988). Therefore, this injection method is not practical for many patients, especially when there are no potential lesions and there are no tumor sites available for injection. U.S. Patent No. 6,045,788 discloses that in order to activate and/or stimulate a patient's immune system without causing substantial toxicity, low doses of natural and recombinant IL-2 can be administered continuously (for example, more than three months) continuously to On the patient. However, this method requires frequent administration over a long period of time, and another option is to request an optimal sustained release composition not disclosed in the patent. In addition to the potential toxicity, another problem with systemic administration of IL-2 is rapid renal discharge. When IL-2 was intravenously administered, 11^2 was rapidly discharged, and its initial half-life and half-life of termination were 6 to 12 minutes, and 40 to 80 minutes, respectively. Therefore, an IL-2 composition which is topically applied to a warm-blooded animal can form a reservoir containing IL-2. This IL-2 containing reservoir provides sustained, sustained release of IL-2, which is sufficient to stimulate the proliferation of cytotoxic T lymphocytes (CTTL's) without causing undesirable side effects, CTTL's can be localized, or Local and systemic effects. The most desirable material for the injectable or implantable polymeric drug carrier in the compositions of the present invention should be biodegradable, pharmaceutically compatible, and simple, safe solvent (e.g., water) There is no need for additional polymerization or other reactions to form covalent bonds after preparation and after application. BRIEF SUMMARY OF THE INVENTION The present invention provides a therapeutic composition comprising a thermally reversible paper scale applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) (Read the first note on the back side and fill out this page) 4 Ordered Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1308068 A7 B7 V. Description of Invention (5) (Please read the note on the back and fill out this page) Gel-degradable biodegradable block copolymer A pharmaceutical carrier and an effective amount of a lymphokine that can be administered intratumorally or via the periphery of the tumor and form a depot comprising lymphokines. The reservoir containing lymphatic hormone provides a sustained release, delayed release of a lymphokine (eg, IL-2) sufficient to stimulate the production of cytotoxic T lymphocytes that may be localized, or localized. Systemic, but does not cause any apparent systemic side effects. The term "lymphocytic hormone" means any agent having IL-2 or other interferon activity, including natural, recombinant and mutated IL-2 or other interleukins, and equivalents or derivatives thereof. The preferred lymphoid hormone may be selected from the group consisting of interleukin-2 (IL-2), interleukin-4, interleukin-12 and its derivatives. Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, The present invention also provides a method for converting a highly toxic drug, i.e., IL-2 or other interleukin, into a drug having a minimum of undesirable side effects and being sufficiently effective; IL-2 or other interferon is incorporated into a biodegradable drug carrier that is capable of forming a drug-containing reservoir after topical administration. Preferably, the biodegradable drug carrier has reverse thermal gelation properties. In other words, such drug carriers are liquid at a temperature below body temperature, and if heated, they are converted to a gel or solid. It is in the form of a gel when it is at or near body temperature. The present invention will be generally referred to in the following description of IL-2, and unless otherwise specified, the teachings of this IL-2 term extend to the remaining agents as defined above. IL-2 is preferably recombinant human IL-2. The invention further provides a method of therapeutically or prophylactically treating a tumor burden in a host of a warm-blooded animal comprising topically administering to the host a composition comprising an effective amount of IL-2, or another lymphosteroid, Applicable to the Chinese National Standard (CNS) A4 Specification (210 X 297 mm) -8- 1308068 A7 B7 _ V. Invention Description (7) Figure 1B illustrates the IL-2 combination of IL-2 from Example 1. The results of in vitro release of the material were analyzed and examined by cell proliferation assay. Fig. 2 is a graph showing the results of cytotoxicity analysis of the IL-2 composition of Example 1, which is a measure of the percentage of specific cell lysis. Fig. 3 is a graph showing the effect of administration of the IL-2 composition of Example 1 on the growth of fibrosarcoma tumors, which was measured for changes in tumor size over a period of time. Figure 4 illustrates the in vitro release of IL-2 from the IL-2 composition of Example 4, which was examined by ELISA. Figure 5 is a graph showing the effect of administration of the IL-2 composition of Example 4 on the dose escalation of tumor growth, which is a measure of the change in tumor size over a period of time. Figure 6 illustrates the repeated application of the IL-2 composition pair of Example 4. The effect of tumor growth, which is a measure of the change in tumor size over a period of time. Figure 7 illustrates the effect of repeated administration of the IL-2 composition of Example 4 on the survival rate of mice in the MetA fibrosarcoma tumor model. Figure 8 illustrates the effect of repeated administration of the IL-2 composition of Example 4 on the survival rate of mice in the B16 melanoma tumor model. Detailed Description of the Invention Before exposing and explaining the therapeutic composition of the present invention and the method of using the same for drug delivery, it is to be understood that the present invention is not limited to the particular formulation or method disclosed herein. Steps, and materials. These particular formulations, method steps, and materials may vary within the scope of the invention. It should also be understood that the terms used in this article are only used to describe the specific Chinese version of the paper. The Chinese National Standard (CNS) A4 specification (210X297 mm) -10- 1308068 A7 ___ B7 V. Invention Description (8 Is not intended to be limiting, as the scope of the invention is limited only by the scope of the appended claims and their equivalents. Unless otherwise stated in this document, it must be noted that the definite titles V', ''an', and 'the' used in this specification and the appended claims are intended to include the plural. And the meaning of one, two, or more drugs when used to deliver a "one drug" composition. The following terms are used to describe and claim the invention, which is defined according to the following list. The word "intestinal" means the inside of the tumor, the periphery of the tumor, the periphery of the lesion, the inside of the lesion, the intrathecal, the intraperitoneal, and the peritoneal cavity. "cell proliferative diseases" includes diseases of cell proliferation and differentiation, usually thinking of axillary or malignant, such as tumors. Tumors such as carcinoma, sarcoma, melanoma, myeloma, sarcoma, etc. Pre-neoplastic tissues and cells, which are known to be true tumors if left untreated. Other examples of cell proliferative diseases are 疣. "Therapeutic" treatment refers to any method that passes through the field of technology. After confirming that the patient has developed a tumor (ie, after a tumor burden has been determined), a drug is administered to the patient to achieve a goal of reducing or eliminating the burden of the existing tumor. "Preventive" A method of administration for preventing tumor recurrence after therapeutic treatment has been provided. "Therapeutically effective amount" means that the amount of each active ingredient in the method or composition of the present invention is sufficient to exhibit a benefit to the meaning of the disease, i.e., the life extension of the paper scale is applicable to the National Standard (CNS) A4. Specifications. (210X297 mm) (Please read the notes on the back and fill out this page) 4 Customs Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed -11 - 1308068 A7 B7 V. Invention Description (11) Synthesizer Or more than one type of interpolymer system. The mixture prepared by the above two methods can be mixed with water to form a polymer solution having the same or different gelation properties and gel quality. ''Solution' is dissolved "'Soluble' and all other similar terms mean a solution or dissolved state, including a homogeneous solution, a glial solution, or any apparently single colloid, such as an emulsion or suspension. "Biodegradable" The block interpolymers can be chemically cleaved or degraded in vivo to form non-toxic components. The rate of degradation can be the same or different from the drug release rate. Any organic or inorganic compound or substance having biological activity or pharmaceutically active activity for therapeutic purposes. The terms "peptide", "poly", "oligopeptide" and "protein" are used to mean peptide drugs or protein drugs when They can be used interchangeably; unless otherwise specified, they should not be limited to any specific molecular weight, peptide sequence or length, biological activity, or therapeutic use. The Ministry of Economic Affairs, Intellectual Property Office, and the Consumer Cooperative, printed the word "Interleukin". Any protein/polypeptide agent, or a derivative, analog or mimetic thereof, of interleukin activity (especially IL-2 and IL-1 2 activity); including natural and recombinant IL-2 and IL-12, natural and A pharmaceutically acceptable fusion protein, a derivative thereof, and a mixture of recombinant interleukins. The word "hIL-2" means a protein exhibiting an activity range characterized by human interleukin-2. In detail, this Proteins must be able to stimulate the proliferation of ML-2-dependent cytolytic T cells and helper T cell lines. For standard assays, see S. Gillis et al., J. Immunol. (1978) 120 :2027- This paper size is applicable to China National Standard (CNS) A4 specification (21〇χ: 297 mm) -14 - 1308068 A7 B7 V. Invention description (12) 2032 and J. Watson, J. Exp. Med. (1979) 150:15 10-15 19. Modified IL-2 and IL-12 are also included in this definition as long as their biological activity is not modified by modification. "PLGA" is a term derived from lactic acid and glycolic acid. Condensation copolymerization, or interpolymer or interpolymer groups derived by ring-opening copolymerization of lactic acid with glycolic acid. The words lactic acid and lactate can be used interchangeably, and the terms glycolic acid and glycolate can also be used interchangeably. "PLAM" means a polymer derived from a lactic acid condensation reaction or a ring-opening polymerization reaction of lactide. "PLGA" means a condensation reaction from glycolic acid or ring-opening polymerization by glycolide. The polymer derived from the reaction. "Biodegradable polyester or poly(orthoester)" means any biodegradable polyester or poly(orthoester) selected from D, L-lactide, D- Lactide, L-lactide 'D, L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, ε-caprolactone, ε-hydroxycaproic acid 'r-butyrolactone, r Preferably, a monomer such as hydroxybutyric acid, 6-valerolactone, 5-hydroxyvaleric acid, hydroxybutyric acid, hydroxysuccinic acid, or a copolymer thereof is synthesized. "ReGel®" is a trademark of MacroMed Corporation, which is used in the class of low molecular weight, biodegradable block copolymers having reverse thermal gelation properties as disclosed in U.S. Patent No. 6,004,573,6. 117, 949, 6, 201, 072, and 6, 287, 588, each of which is incorporated herein by reference. This brand name also includes the compositions disclosed in the laid-open U.S. Patent Application Serial Nos. 09/906,041, the entire disclosure of which is incorporated herein by reference. Biodegradable drug carriers include the Chinese National Standard (CNS) A4 specification (2丨〇><297 mm) for this paper size (please read the notes on the back and fill out this page) Bureau employee consumption cooperative printing -15- 1308068 A7 B7 V. Description of invention (13) Triblock copolymers of the ΑΒΑ or BAB type or mixtures thereof, wherein the A block is quite hydrophobic and contains biodegradable poly Ester or poly(orthoester); and B block is quite hydrophilic and comprises polyethylene glycol (PEG); the interpolymer has a hydrophobic content of 5 0.1 to 83% by weight and a hydrophilicity of 17 to 49.9% by weight The amount of the material and the total molecular weight of the block copolymer are between 2,000 and 8,000. The drug carrier exhibits water solubility when the temperature is lower than the body temperature of the mammal; and becomes a gel after reversible thermogelation at a temperature equal to the physiological body temperature of the mammal. The biodegradable hydrophobic A block comprises a polyester or a poly(orthoester), wherein the polyester is selected from the group consisting of D, L-lactide, D-lactide, L-lactide, D , L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid 'ε-caprolactone, ε-hydroxycaproic acid, 7-butyrolactone, tau-hydroxybutyric acid, valerolactone, hydroxypenta A monomer of a group consisting of acid, hydroxybutyric acid, hydroxysuccinic acid, and a copolymer thereof is synthesized and has an average molecular weight of about 600 to 3,000. The hydrophilic hydrazine block moiety is preferably polyethylene glycol (PEG) having an average molecular weight of from about 500 to 2,200. The concentration of the block interpolymer when it is soluble (at a temperature below the gelation temperature) can be considered as the functional concentration. In general, block copolymer concentrations as low as 3% by weight and up to about 50% by weight can be used, and the block interpolymers at these concentrations are still functional. However, it is preferred to have a concentration of between about 5 and 40% by weight, and preferably between about 10 and 30% by weight. At lower functional concentrations, phase transfer can result in the formation of thin gels or viscous liquids, however these lines are still suitable for the purposes of the present invention.

本發明之組合物也可含有增進且促成重建作用 (reconstitution)的藥劑，此藥劑其含有聚乙二醇（PEG)、PEG 本紙張尺度適用中國國家標準（CNS ) A4規格( 210X297公釐） (請先閲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 -16- 1308068 A7 B7 五、發明説明（14) 衍生物、或PEG與PEG衍生物之混合物，該PEG或PEG衍生物之分子量爲150至1100道爾吞。該PEG衍生物包括事先與下列化合物衍生而得的PEG ,該化合物係選自由D,L-丙交酯、D-丙交酯' L-丙交酯、D，L-乳酸、D-乳酸、L-乳酸、乙交酯、乙醇酸、及其共聚體所組成之類群。PEG衍生物也可以是以 R'-CO-CMPEGhCO-R2 或 V-CMPEGhCO-R2 表示的一員，其中R1及R2分別選自由11及C,至》：,。烷基所組成之類群。在低於凝膠化溫度下，可將生物可降解之共聚體與藥物（例如IL-2)所組成的混合物製備成該共聚體之水溶液或單一膠態，藉以形成IL-2輸液，其中IL-2可部份或完全溶解。然後將此IL-2輸液經由局部輸藥途徑，例如經腫瘤內部或經腫瘤週邊，施用至病患，施用部位會進行可逆性熱凝膠化作用，因爲體溫將高於凝膠化溫度。此用藥方法實質上減輕、或在許多實例中排除了 IL-2的全身性毒性，並將免疫系統對腫瘤細胞之反應加強至最大。本發明之IL-2組合物之其他潛在應用包括在手術介入/腫瘤切除的位置予以局部性用藥，藉以殺死手術後仍殘留的腫瘤細胞。治療惡性腫瘤之其他可能的局部性輸藥途徑包括經腹膜內用藥，此適用於位在腹膜內的腫瘤/轉移物；及經頭顱內用藥，此適用於位在頭骨內的腫瘤。本發明也提供一種通用方法，可將高毒性之細胞激素及相關藥物，例如淋巴激素，包括11^-2、11^-4、11^12及彼等之衍生物及模擬物，轉化爲有效的抗腫瘤藥劑，同時將本紙張尺度適用中國國家標準（CNS ) Α4規格（2丨0X297公釐） (請先閱请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 -17- 1308068 A7 B7 五、發明説明（15) 不利的副作用減至最低。本發明之組合物提供自局部積貯處持續輸送藥物，該積貯處係由位在單一個或數個局部化的腫瘤位置之腫瘤內部/腫瘤週邊形成者，其誘發局部性或局部並全身性免疫反應。經由形成腫瘤內部/腫瘤週邊之局部持續性輸藥積貯處，可達到在腫瘤位置有相當高的局部細胞激素穩定態濃度。IL-2的局部濃度高，會刺激對腫瘤專一之CTTL的活化與生成，該CTTL可局部性並全身性攻擊腫瘤組織，此時全身的IL-2總量仍夠低而足以避免明顯的不欲副作用。經濟部智慧財產局員工消費合作社印製本發明之組合物及方法適用於治療可由任何惡性組織或腫瘤前組織所形成之表現出腫瘤疾病的任何腫瘤，包括癌瘤、肉瘤、黑色素瘤、骨髓瘤、肉瘤等。本發明之組合物及方法提供一種方式可將高毒性藥物，特別是細胞激素（例如IL-2、IL-4、IL-12、及其衍生物與模擬物），轉化爲沒有明顯不欲之副作用的有效治療性組合物。當與全身治療所需之IL-2及其他淋巴激素的劑量相比較下，儘管所施用之淋巴激素總劑量被減少了，本發明之組合物仍提供較佳或可供比較的藥學功效。於本發明實施體系之一，IL-2倂入具有反向熱凝膠化性質之生物可降解的嵌段共聚體藥物載劑（ReGel®)，該載劑呈可溶解形式、或爲懸浮液或其他膠態形式，且在低於該載劑之凝膠化溫度下保存。經腫瘤內部或經腫瘤週邊將本發明組合物施用至病人或其體溫尚於ReGel®凝.膠化溫度的患病動物（溫血哺乳動物宿主）之後，該組合物形成局部積貯本紙張尺度適用中國國家標準（CNS ) A4規格（210X：297公釐） -18- 1308068 A7 B7 五、發明説明（16) 處，其提烘連續釋出、持續釋出之IL-2。此IL-2/ ReGel®組合物也可含有多種添加劑，例如聚醇類（包括糖類）' 界面活性劑、胺基酸、其他蛋白質及緩衝鹽類。這些添加劑可作爲一特定淋巴激素之官能性及/或物理性安定劑，該淋巴激素包括IL-2、IL-4、IL-12及其衍生物與模擬物，其於注射之前呈液體狀態、或注射之後在局部IL-2積貯處位置的凝膠中。本發明之組合物可一次施用，但是較好在每日至每月的基礎上重複施用以提供改良的治療效果，例如縮小腫瘤體積，如此可長期管理腫瘤疾病、或導致腫瘤完全消失或不再復發。由於活體內會自動地化學性降解此生物可降解之嵌段共聚體，歷時數天至四週（由藥物載劑種類決定），故在相同注射位置重複用藥是可以達成的。一般而言，本發明之組合物可經腫瘤週邊或經腫瘤內部施用至經過公信之診斷用顯像技術確認後的腫瘤位置。於治療需要精確的腫瘤定位之惡性腫瘤時，有一種施用本發明之IL-2組合物的特殊途徑爲經頭顱內用藥以治療頭骨內部的腫瘤。另一種本發明之IL-2組合物的特殊局部用藥途徑係用於治療不需要精確腫瘤定位之惡性腫瘤，其爲經腹膜內施用IL-2、以治療位在腹膜內的原位腫瘤或腫瘤轉移物。另一種不需要任何精密顯像技術的特殊用藥途徑爲，經皮下注射本發明之IL-2組合物至位於皮膚內之腫瘤（例如，原位性及轉移性的黑色素瘤腫瘤）旁邊。另一種特殊用藥途徑是將IL-2組合物局部施用在手術介入/腫瘤切除的位本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）諳先閲讀背面之注意事項頁經濟部智慧財產局員工消費合作社印製 -19- 1308068 A7 B7 經濟部智慧財產局員工消費合作社印製五、發明説明（17) 置，藉以殺死手術後殘留的腫瘤細胞。施用之最佳劑量取決於腫瘤種類、宿主類型、腫瘤位置、用藥途徑、規劃及順序、現存的腫瘤負擔（病況）、細胞激素之種類，特別是使用IL-2、IL-4、IL-12及其衍生物與模擬物、或其他淋巴激素。本發明之IL-2組合物所用的一般劑量範圍在1000 I.U.至1χ108Ι.υ.。本發明之方法包括對溫血哺乳動物宿主施用一藥學上有效量的藥物，特別是IL-2,該溫血哺乳動物宿主包括小鼠、大鼠、兔子、靈長動物、豬或人類宿主，較好是人類病患。由於原料的生物相容性及凝膠之塑性，此種系統不會對周圍組織造成不欲之毒性或機械性傷害，且於一指定的時間間隔內，其可被生物完全降解爲乳酸、乙醇酸、或其他對應單體、及聚乙二醇。可藉由適當設計及製備多種藥物載劑，來控制IL-2釋出速率、凝膠強度、凝膠化溫度及降解率。例如，可調整A嵌段B嵌段之重量百分比、含有A嵌段之單體的莫爾百分比、及ΑΒΑ三嵌段共聚體或 ΒΑΒ三嵌段共聚體之分子量及聚合度分佈性。IL-2的釋出也可藉由校正聚合物於藥物輸液中的濃度予以控制。將一種含有IL-2之聚合物溶液（即，藥物輸液）的劑型施用至動物體。由於嵌段共聚體之反向熱凝膠化性質，當組合物之溫度上升至體溫時、此組合物隨後自動變成凝膠以形成藥物積貯處。於許多情況下，IL-2將被調製成每毫升藥物輸液含有約1000 I.U.至lxl08I,U.的IL-2。在有些情況下，可藉由添加多種添加劑至此水溶液或懸浮液，來本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁)The compositions of the present invention may also contain an agent which enhances and promotes reconstitution. The agent contains polyethylene glycol (PEG), PEG. The paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) ( Please read the notes on the back and fill out this page. Ordering the Ministry of Economic Affairs, Intellectual Property Office, Staff and Consumer Cooperatives Printed-16-1308068 A7 B7 V. INSTRUCTIONS (14) Derivatives, or a mixture of PEG and PEG derivatives, the PEG Or the molecular weight of the PEG derivative is from 150 to 1100 Dow. The PEG derivative includes PEG derived in advance from the following compounds selected from the group consisting of D, L-lactide, D-lactide 'L-lactide, D, L-lactic acid, D-lactic acid, A group consisting of L-lactic acid, glycolide, glycolic acid, and copolymers thereof. The PEG derivative may also be a member represented by R'-CO-CMPEGhCO-R2 or V-CMPEGhCO-R2, wherein R1 and R2 are respectively selected from 11 and C, to ":,". A group consisting of alkyl groups. At a temperature below the gelation temperature, a mixture of a biodegradable interpolymer and a drug (eg, IL-2) can be prepared as an aqueous solution or a single colloidal state of the interpolymer to form an IL-2 infusion, wherein IL -2 can be partially or completely dissolved. This IL-2 infusion is then administered to the patient via a local drug delivery route, for example, via the tumor or via the periphery of the tumor, and the site of administration undergoes reversible thermogelation because the body temperature will be above the gelation temperature. This method of administration substantially alleviates, or in many cases excludes, the systemic toxicity of IL-2 and maximizes the response of the immune system to tumor cells. Other potential applications of the IL-2 compositions of the present invention include topical administration at the site of surgical intervention/tumor resection to kill tumor cells that remain after surgery. Other possible localized routes of delivery for the treatment of malignant tumors include intraperitoneal administration, which is suitable for intratumoral tumors/metastasis; and intracranial administration, which applies to tumors located within the skull. The present invention also provides a general method for converting highly toxic cytokines and related drugs, such as lymphokines, including 11^-2, 11^-4, 11^12, and their derivatives and mimetics into effective ones. The anti-tumor agent, at the same time, the paper size is applicable to the Chinese National Standard (CNS) Α4 specification (2丨0X297 mm) (please read the note on the back and fill in the page first) The Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative -17-1308068 A7 B7 V. INSTRUCTIONS (15) Unfavorable side effects are minimized. The compositions of the present invention provide for the continuous delivery of a drug from a localized reservoir that is formed by a tumor/intratumoral periphery of a single or several localized tumor sites, which induces local or local and systemic Sexual immune response. A fairly high local cytokine steady state concentration at the tumor site can be achieved by the formation of a local persistent drug reservoir at the periphery of the tumor/tumor. The high local concentration of IL-2 stimulates the activation and production of tumor-specific CTTL, which can localize and systemically attack tumor tissue. At this time, the total amount of IL-2 in the whole body is still low enough to avoid obvious Want side effects. The Ministry of Economic Affairs Intellectual Property Office employee consumption cooperative printed the composition and method of the present invention for treating any tumor which can be formed by any malignant tissue or pre-neoplastic tissue and exhibits a tumor disease, including cancer, sarcoma, melanoma, myeloma , sarcoma, etc. The compositions and methods of the present invention provide a means by which highly toxic drugs, particularly cytokines such as IL-2, IL-4, IL-12, and derivatives and mimetics thereof, can be converted to no apparently undesirable An effective therapeutic composition for side effects. When compared to the dose of IL-2 and other lymphokines required for systemic treatment, the compositions of the present invention provide better or comparable pharmaceutical efficacy, although the total dose of lymphokine administered is reduced. In one of the embodiments of the present invention, IL-2 incorporates a biodegradable block interpolymer drug carrier (ReGel®) having reverse thermal gelation properties in a soluble form or as a suspension. Or other colloidal form and stored below the gelation temperature of the carrier. The composition forms a local storage paper size after administration of the composition of the invention to the patient or to a diseased animal (warm-blooded mammalian host) whose body temperature is still at ReGel® gelatinization temperature, either inside the tumor or via the periphery of the tumor. Applicable to China National Standard (CNS) A4 specification (210X: 297 mm) -18- 1308068 A7 B7 V. Inventive Note (16), which is continuously released and continuously released IL-2. The IL-2/ReGel® composition may also contain various additives such as polyalcohols (including sugars), interfacial activators, amino acids, other proteins, and buffer salts. These additives may act as a functional and/or physical stabilizer for a particular lymphokine, including IL-2, IL-4, IL-12 and its derivatives and mimetics, which are in a liquid state prior to injection, Or in the gel at the location of the local IL-2 reservoir after injection. The composition of the present invention can be administered at a time, but is preferably administered repeatedly on a daily to monthly basis to provide an improved therapeutic effect, such as reducing the tumor volume, so that the tumor disease can be managed for a long period of time, or the tumor completely disappears or no longer occurs. relapse. Since the biodegradable block interpolymer is automatically chemically degraded in vivo for several days to four weeks (depending on the type of drug carrier), repeated administration at the same injection site is achievable. In general, the compositions of the present invention can be administered to the periphery of the tumor or via the interior of the tumor to the location of the tumor after confirmation by a diagnostic imaging technique. In the treatment of malignant tumors requiring precise tumor localization, a particular route of administration of the IL-2 compositions of the present invention is intratracheal administration to treat tumors within the skull. Another special topical route of the IL-2 composition of the invention is for the treatment of malignant tumors that do not require precise tumor localization, which is administered intraperitoneally with IL-2 to treat an orthotopic tumor or tumor located in the peritoneum. Transfer. Another special route of administration that does not require any precision imaging technique is to subcutaneously inject the IL-2 composition of the present invention to a tumor located in the skin (e.g., an in situ and metastatic melanoma tumor). Another special route of administration is to apply the IL-2 composition locally in the surgical intervention/tumor resection. The paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm). Read the back of the note on the back page. Property Bureau Staff Consumer Cooperatives Printed -19- 1308068 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Printed five, invention instructions (17) to kill tumor cells remaining after surgery. The optimal dose to be administered depends on the type of tumor, the type of host, the location of the tumor, the route of administration, the planning and sequence, the existing tumor burden (condition), the type of cytokines, especially the use of IL-2, IL-4, IL-12. And its derivatives and mimics, or other lymphoid hormones. The IL-2 composition of the present invention is generally used in a dosage range of from 1000 I.U. to 1χ108Ι.υ. The method of the invention comprises administering to a warm-blooded mammalian host a pharmaceutically effective amount of a drug, particularly IL-2, comprising a mouse, rat, rabbit, primate, porcine or human host. It is better for human patients. Due to the biocompatibility of the raw materials and the plasticity of the gel, such systems do not cause undesirable toxicity or mechanical damage to the surrounding tissue, and can be completely biodegraded into lactic acid and ethanol at a specified time interval. Acid, or other corresponding monomer, and polyethylene glycol. The release rate, gel strength, gelation temperature and degradation rate of IL-2 can be controlled by appropriately designing and preparing a plurality of drug carriers. For example, the weight percentage of the A block B block, the Mohr percentage of the monomer containing the A block, and the molecular weight and degree of polymerization distribution of the ruthenium triblock interpolymer or the ruthenium triblock interpolymer can be adjusted. The release of IL-2 can also be controlled by correcting the concentration of the polymer in the drug infusion. A dosage form containing a polymer solution of IL-2 (i.e., a drug infusion) is administered to the animal. Due to the reverse thermal gelation properties of the block interpolymer, when the temperature of the composition rises to body temperature, the composition then automatically turns into a gel to form a drug reservoir. In many cases, IL-2 will be formulated to contain about 1000 I.U. to lxl08I, IL-2 per milliliter of drug infusion. In some cases, the Chinese National Standard (CNS) A4 specification (210X297 mm) can be applied to this paper scale by adding various additives to this aqueous solution or suspension (please read the back note before filling this page)

-20- 1308068 A7 B7 五、發明説明（18) 增加間白素的官能安定性或生理安定性。可使用諸如聚醇類（包括糖類）、胺基酸、界面活性劑、防腐劑、抗氧化劑、安定劑、張力校正劑、其他蛋白質及某些鹽類等添加劑。這些添加劑可輕易倂入該藥物輸液中。顯然爲最有效之劑量是導致腫瘤體積縮小 '腫瘤完全消失或不再復發的劑量，且此劑量對宿主沒有毒性或爲可接受的毒性。此最佳劑量由許多因素決定，例如，根據宿主種類及腫瘤種類、用藥途徑、規劃及順序、現存的腫瘤負擔、IL-2之種類、及毒性的定義。於本發明之實施體系之一，IL-2(20xl06I.U.)與 ReGel®混合而形成一液體組合物，然後將其注射至腫瘤週邊。當溫度升高至體溫時，IL-2/ReGel®於注射位置形成一凝膠，藉此延緩IL-2自積貯處釋出，該積貯處中的IL-2緩慢且持續地釋出歷數天之久。自凝膠釋出的IL-2具有活性且可局部性並全身性刺激CTTL。相反的，單次注射習知組合物（即，不含有ReGel®載劑者）中所含的IL-2，已證實對控制腫瘤生長無效。因此，經由局部注射一劑量的IL-2(若全身性施用此劑量，將未能達到治療功效），本發明之IL- 2/ReGeI®組合物提供局部性、或局部並全身性抗腫瘤治療〇茲提供下列實施例以說明製備本發明組成物之方法、及使用本發明組成物之方法。實例1 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公瘦） (請先閲讀背面之注意事項再填寫本頁) -訂-20- 1308068 A7 B7 V. INSTRUCTIONS (18) Increase the functional stability or physiological stability of interleukin. Additives such as polyalcohols (including sugars), amino acids, surfactants, preservatives, antioxidants, stabilizers, tonicity correctors, other proteins, and certain salts can be used. These additives can be easily incorporated into the drug infusion. Obviously the most effective dose is the dose that causes the tumor to shrink in size 'the tumor completely disappears or no longer relapses, and this dose is not toxic or acceptable to the host. This optimal dose is determined by a number of factors, for example, based on the type of host and the type of tumor, route of administration, planning and sequence, existing tumor burden, type of IL-2, and toxicity. In one of the embodiments of the present invention, IL-2 (20xl06I.U.) is mixed with ReGel® to form a liquid composition which is then injected into the periphery of the tumor. When the temperature rises to body temperature, IL-2/ReGel® forms a gel at the injection site, thereby delaying the release of IL-2 from the reservoir, where IL-2 is slowly and continuously released. It lasted for a few days. IL-2 released from the gel is active and can locally and systemically stimulate CTTL. In contrast, IL-2 contained in a single injection of a conventional composition (i.e., without ReGel® carrier) has been shown to be ineffective in controlling tumor growth. Thus, the IL-2/ReGeI® composition of the present invention provides local, or local, and systemic anti-tumor therapy via local injection of a dose of IL-2 (which would not achieve therapeutic efficacy if administered systemically). The following examples are provided to illustrate the preparation of the compositions of the present invention, and methods of using the compositions of the present invention. Example 1 This paper scale applies to China National Standard (CNS) A4 specification (210X297) (Please read the note on the back and fill in this page) -

經濟部智慧財產局員工消費合作社印製 -21 - 1308068 Δ7 Α7 Β7 五、發明説明（19) 此實例說明IL-2自本發明之IL-2組合物活體外釋出的情形。此實例中的生物可降解之嵌段共聚體載劑爲23重量％之嵌段共聚體溶液，該嵌段共聚體之PLG/PEG-1000重量比爲.2.4 ' L/G莫爾比爲75/25，分子量爲4,000道爾吞，且凝膠化溫度爲14t 。將含有 50,000 I.U.(3 微克）之 IL-2(Proleukin®，可購自 Chiron公司）之固定體積（1毫升）的滅菌IL-2組合物放入50 毫升的組織培養燒瓶的底部，重複做兩套實驗。在25毫升之組織培養基（RPMI1640，Bio Whittaker公司）存在下，經濟部智慧財產局員工消費合作社印製於37°C 培養該燒瓶。在預先選定的時間點，抽出含有釋出物之培養基等份（0.5毫升），予以適當稀釋之後 '分析 IL-2含量。IL-2含量之分析係利用專一酵素鍵結免疫分析法（OptEIA人類IL-2套組，Pharmingen廠），並於3H-胸腺核苷被攝入細胞之後（於37°C 培養24小時）、利用CTTL-20指標細胞進行量化之細胞增殖分析法。針對後述方法，藉由連續稀釋IL-2標準物，以製備劑量反應曲線。結果係根據逐漸累積之IL-2釋出量及進行的時間作圖（第1A圖爲利用ELISA測定的IL-2 ;第1B圖爲利用細胞增殖分析法測定的IL-2)。兩種方法皆顯示IL-2釋出量可被定量，持續3 至4天，且釋出的IL-2完全具有生物活性。實例2. 此實例說明自本發明之IL-2組合物釋出之IL-2誘生胞本紙張尺度適用中國國家標準（CNS ) A4規格（21〇x297公釐） -22- 1308068 A7 B7 五、發明説明（20) 毒性淋巴細胞的能力，其係利用實例1所述之相同IL-2組合物。經濟部智慧財產局員工消費合作社印製將固定體積（0.5毫升）之如實例1所述的滅菌IL-2組合物，其含有逐漸增加劑量的IL-2(Proleukin® ; 12,500 I.U.; 25,000 I.U_ ; 50,000 I.U.)，放入25毫升的組織培養燒瓶的底部，重複做兩套實驗。在含有小鼠脾臟細胞之25毫升組織培養基（RPMI1640, Bio Whittaker公司）的存在下，於 37°C 培養該燒瓶歷三天。回收經活化的淋巴細胞，於”Cr 釋出分析法中分析該淋巴細胞殺死RD-995纖維肉瘤腫瘤細胞的能力，此能力係藉由測定發生溶解之腫瘤細胞百分比 (%)。如第2圖所示，與釋出期剛開始時添加至釋出培養基的游離IL-2相比較，由ReGel®釋出的IL-2完全具有生物活性。第2圖中的E:T細胞比，表示作用細胞（經栝化之淋巴細胞）相對於標的細胞（RD-995腫瘤細胞）的比例，每份樣品中的標的細胞數目維持固定（104)。總而言之，由本發明組合物釋出之IL-2完全具有生物活性，且此組合物可用來作爲一種有效的輸藥系統、以利於活體內持續地以局部性經腫瘤週邊方式輸送IL-2。實例3 此實例說明於小鼠經腫瘤週邊單次注射本發明之IL-2 組合物之腫瘤縮小功效。將RD-995纖維肉瘤腫瘤細胞經皮下植入小鼠 (C3H/HEN)。當此實體腫瘤爲4-5毫米大小時，將實例1所本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） -23- 1308068 A7 B7 五、發明説明（21) 述之0_2毫升的IL-2組合物注射至小鼠，即，在腫瘤周圍的相對位置注射2x0.1毫升。此IL-2組合物含有漸增之1乙_ 2 劑量，10,000 I.U. ; 25,000 I.U.或 50,000 I.U.。對照組僅僅注射藥物載劑。每隔一天，歷3週，測量腫瘤大小（第 3圖）。與對照組比較，每個實驗組的腫瘤生長都受到限制〇實例4 此實例說明活體外IL-2自藥物載劑釋出的情形，該載劑具有大於2(TC 之凝膠化溫度。此實例中的生物可降解之聚合物載劑爲13重量％之嵌段共聚體溶液，該嵌段共聚體之PEG(1000)/PEG(1450)重量比爲20/80、PLG/PEG重量比爲2.06、 L/G莫甯比爲 85/15、分子量爲4,800道爾吞、且凝膠化溫度爲 26°C 。將固定體積（1毫升）之滅菌IL-2組合物，其含有50,〇〇〇 I.U.(3微克）的IL-2(Proleukin®),放入 50毫升的組織培養燒瓶的底部，重複做兩套實驗。在25毫升組織培養基 (RPMI1640，Bio Whittaker 公司）的存在下，於 37°C 培養該燒瓶。在預先選定的時間點，抽出含有釋出物之培養基等份（0.5毫升），予以適當稀釋之後、利用專一酵素鍵結免疫分析法（OptEIA人類IL-2套組，Pharmingen廠）分析IL-2含量。結果係根據逐漸累積之IL-2釋出量相對於時間作圖（第4圖）。於活體外，ReGel®中攜帶的IL-2以生物活性形式自凝膠積貯處定量地釋出，歷時4天。本紙張尺度適用中國國家標準（CNS ) A4规格（210 X 297公釐） f靖先閲讀背面之注意事項再填寫本頁〕訂Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives -21 - 1308068 Δ7 Α7 Β7 V. INSTRUCTION DESCRIPTION (19) This example illustrates the release of IL-2 from the IL-2 composition of the present invention in vitro. The biodegradable block interpolymer carrier in this example is a 23% by weight block copolymer solution having a PLG/PEG-1000 weight ratio of .2.4 'L/G molar ratio of 75. /25, molecular weight 4,000 doxane, and gelation temperature of 14t. Place a fixed volume (1 ml) of sterile IL-2 containing 50,000 IU (3 μg) of IL-2 (Proleukin®, available from Chiron) into the bottom of a 50 ml tissue culture flask and repeat the two Set of experiments. The flask was cultured at 37 ° C in the presence of 25 ml of tissue culture medium (RPMI 1640, Bio Whittaker). At a pre-selected time point, an aliquot of the medium containing the liberated material (0.5 ml) was withdrawn and appropriately diluted to 'analyze the IL-2 content. The analysis of IL-2 content was performed using a specific enzyme-binding immunoassay (OptEIA Human IL-2 kit, Pharmingen Plant), and after the 3H-thymidine was taken into the cells (cultured at 37 ° C for 24 hours), Cell proliferation assays quantified using CTTL-20 indicator cells. A dose response curve was prepared by serial dilution of the IL-2 standard for the method described below. The results were based on the cumulative amount of IL-2 released and the time of administration (Fig. 1A shows IL-2 by ELISA; Figure 1B shows IL-2 measured by cell proliferation assay). Both methods show that IL-2 release can be quantified for 3 to 4 days, and the released IL-2 is fully biologically active. Example 2. This example illustrates the IL-2 induced cell paper release from the IL-2 composition of the present invention. The Chinese National Standard (CNS) A4 specification (21〇x297 mm) -22- 1308068 A7 B7 BRIEF DESCRIPTION OF THE INVENTION (20) The ability of toxic lymphocytes using the same IL-2 composition as described in Example 1. The Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, printed a fixed volume (0.5 ml) of the sterile IL-2 composition as described in Example 1, which contained a gradually increasing dose of IL-2 (Proleukin®; 12,500 IU; 25,000 I. U_; 50,000 IU), placed in the bottom of a 25 ml tissue culture flask, and repeated two sets of experiments. The flask was incubated at 37 ° C for three days in the presence of 25 ml of tissue culture medium (RPMI 1640, Bio Whittaker) containing mouse spleen cells. The activated lymphocytes were recovered and analyzed for their ability to kill RD-995 fibrosarcoma tumor cells in a Cr release assay by measuring the percentage (%) of tumor cells that were lysed. As shown, the IL-2 released by ReGel® is completely biologically active compared to the free IL-2 added to the release medium at the beginning of the release period. The E:T cell ratio in Figure 2 indicates The ratio of the target cells (deuterated lymphocytes) to the target cells (RD-995 tumor cells), the number of target cells in each sample remains fixed (104). In summary, IL-2 released from the composition of the invention It is completely biologically active, and this composition can be used as an effective drug delivery system to facilitate continuous localized delivery of IL-2 in a peri-tumor manner in vivo. Example 3 This example illustrates a single pass through the tumor periphery of a mouse. Injection of the IL-2 composition of the present invention for tumor shrinking efficacy. RD-995 fibrosarcoma tumor cells were subcutaneously implanted into mice (C3H/HEN). When the solid tumor was 4-5 mm in size, Example 1 was The paper size is suitable China National Standard (CNS) A4 Specification (210X297 mm) -23- 1308068 A7 B7 V. INSTRUCTIONS (21) The 0-2 ml IL-2 composition is injected into mice, ie, injected at a relative position around the tumor. 2 x 0.1 ml. This IL-2 composition contains an increasing dose of 1 B _ 2, 10,000 IU; 25,000 IU or 50,000 IU. The control group only injected the drug carrier. Every other day, for 3 weeks, the tumor size was measured ( Figure 3). Tumor growth was limited in each experimental group compared to the control group. Example 4 This example illustrates the release of IL-2 from a drug carrier in vitro. The carrier has a concentration greater than 2 (TC Gelation temperature. The biodegradable polymer carrier in this example is a 13% by weight block copolymer solution having a weight ratio of PEG(1000)/PEG(1450) of 20/80, PLG. The weight ratio of /PEG is 2.06, the L/G molar ratio is 85/15, the molecular weight is 4,800 dox, and the gelation temperature is 26 ° C. A fixed volume (1 ml) of the sterilized IL-2 composition, It contains 50, 〇〇〇IU (3 μg) of IL-2 (Proleukin®) and is placed in the bottom of a 50 ml tissue culture flask. Two sets of experiments were repeated. The flask was incubated at 37 ° C in the presence of 25 ml of tissue culture medium (RPMI 1640, Bio Whittaker). At a pre-selected time point, an aliquot of the medium containing the effluent was withdrawn (0.5 ML), after appropriate dilution, was analyzed for IL-2 content using a specific enzyme-binding immunoassay (OptEIA Human IL-2 kit, Pharmingen Plant). The results are plotted against time based on the cumulative amount of IL-2 released (Fig. 4). In vitro, IL-2 carried in ReGel® was quantitatively released from the gel reservoir in a biologically active form for 4 days. This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) f Jing Xian read the back of the note and fill out this page]

經濟部智慧財產局員工消費合作社印製 -24- 1308068 A7 _B7 五、發明説明（22) 實例5 此實例說明於小鼠經腫瘤週邊單次注射IL-2組合物的腫瘤縮小功效。此IL-2組合物與實例4所述者相同。 .將RD-995纖維肉瘤腫瘤細胞經皮下植入小鼠 (C3H/HEN)。當此實體腫瘤爲4-5毫米大小時，將含有漸增之 IL-2 劑量（500,000 I.U. ; 2,000,000 I.U.或 4,000,000 I.U.)之0.2毫升IL-2組合物注射至小鼠。對照組中，經腫瘤週邊注射僅僅含有ReGel®之組合物（無藥物）、或習知之 IL-2組合物（500,000 I.U.)。每隔一天，歷26天，測量腫瘤大小（第5圖）。與對照組比較，每個ReGel®/IL-2實驗組中的腫瘤生長都受到限制，約歷3週。於用藥之後的第3 天與第5天測量每組動物的血壓。血壓値皆保持在生理學上的正常範圍內，未測得不利的血壓減少，顯示本發明之 IL-2組合物不會導致不利的副作用。實例6 此實例說明於小鼠經腫瘤週邊單次注射IL-2組合物的腫瘤縮小功效。此IL-2組合物與實例4所述者相同。將RD-995纖維肉瘤腫瘤細胞經皮下植入小鼠 (C3H/HEN)。當此實體腫瘤爲4-5毫米大小時，將含有 2,000,000 I.U.之IL-2之0.2毫升IL-2組合物注射至小鼠（在腫瘤周圍的相對位置注射2x0.1毫升）。對照組中，經腫瘤週邊注射僅僅含有藥物載劑之組合物（無藥物）、或習知之本紙張尺度適用中固國家標準（CNS ) A4規格（210X297公釐） (請先閱讀背面之注意事項再填寫本頁) 訂Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives -24- 1308068 A7 _B7 V. INSTRUCTIONS (22) EXAMPLE 5 This example illustrates the tumor shrinking effect of a single injection of IL-2 composition in mice around the tumor. This IL-2 composition was identical to that described in Example 4. RD-995 fibrosarcoma tumor cells were subcutaneously implanted into mice (C3H/HEN). When the solid tumor was 4-5 mm in size, 0.2 ml of the IL-2 composition containing the increasing IL-2 dose (500,000 I.U.; 2,000,000 I.U. or 4,000,000 I.U.) was injected into the mouse. In the control group, a composition containing only ReGel® (no drug) or a conventional IL-2 composition (500,000 I.U.) was injected around the tumor. Tumor size was measured every other day for 26 days (Fig. 5). Tumor growth was limited in each ReGel®/IL-2 experimental group compared to the control group for approximately 3 weeks. The blood pressure of each group of animals was measured on days 3 and 5 after administration. The blood pressure 値 was kept within the physiologically normal range, and no unfavorable blood pressure reduction was measured, indicating that the IL-2 composition of the present invention does not cause adverse side effects. Example 6 This example illustrates the tumor shrinking efficacy of a single injection of an IL-2 composition in a mouse surrounding a tumor. This IL-2 composition was identical to that described in Example 4. RD-995 fibrosarcoma tumor cells were subcutaneously implanted into mice (C3H/HEN). When the solid tumor was 4-5 mm in size, 0.2 ml of IL-2 composition containing 2,000,000 I.U. of IL-2 was injected into the mouse (2 x 0.1 ml was injected at a relative position around the tumor). In the control group, the composition containing only the drug carrier (no drug) was injected through the periphery of the tumor, or the conventional paper size was applicable to the China National Standard (CNS) A4 specification (210×297 mm) (please read the notes on the back first) Fill in this page again)

經濟部智慧財產局員工消費合作社印製 -25- 1308068 A7 B7 五、發明説明（23) IL-2組合物（500,000 I.U.)。另一對照組之處理爲使用習知之IL-2組合物（180,000 I.U·)，全身性給藥（經皮下）、每日兩次（B.I.D.)，連續5天（此爲小鼠全身性施用IL-2之最大可耐受劑量）。除了習知之全身性B.I.D.組之外，在第7、14 、.及21天重複用藥。每隔一天測量腫瘤大小（第6圖）。與對照組比較，IL-2組合物組中的腫瘤生長都受到限制，約歷41/2週。每組動物於第一次用藥之後的第8天測量血壓。血壓値皆保持在生理學上之正常範圍內，未測得不利的血壓降低。實例7 此實例說明於MethA腹膜內腫瘤之小鼠模型中每週經腹膜內注射IL-2組合物的結果。此IL-2組合物與實例4所述者相同。將106個MethA纖維肉瘤腫瘤細胞經腹膜內注射至小鼠（BalbC)〇3天後（第0天，第7圖），將含有漸增劑量之 IL-2(100,000 I.U. ; 500,000 I.U.或 2,000,000 I.U·)之 0.2 毫升IL-2組合物經腹膜內注射至小鼠（分組爲每組各10隻鼠）。對照組由如下小鼠組成：承受腫瘤但無治療的小鼠；經注射無藥物之組合物的小鼠；經習知全身性施用IL-2治療之小鼠（ 1 80,000 I.U.;經皮下；每日兩次；歷五天）；及經腹膜內注射500,000 I.U.之習知IL-2的小鼠。除了習知之全身性B.I.D.組之外，在第7、14、21、28、及35天重複用藥。追蹤小鼠的存活率（第7圖）。與使用習知IL-2組合物本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事項再填寫本頁) 訂Printed by the Consumer Intellectual Property Office of the Ministry of Economic Affairs -25- 1308068 A7 B7 V. INSTRUCTIONS (23) IL-2 composition (500,000 I.U.). The other control group was treated with a conventional IL-2 composition (180,000 IU·), systemically administered (subcutaneously), twice daily (BID) for 5 consecutive days (this is a systemic administration of IL to mice). -2 the maximum tolerable dose). The drugs were repeated on days 7, 14, and 21, except for the conventional systemic B.I.D. group. Tumor size was measured every other day (Fig. 6). Tumor growth in the IL-2 composition group was limited compared to the control group for about 41/2 weeks. Blood pressure was measured on the 8th day after the first dose of each group of animals. The blood pressure sputum was kept within the physiologically normal range, and unfavorable blood pressure was not measured. Example 7 This example illustrates the results of weekly intraperitoneal injection of an IL-2 composition in a mouse model of MethA intraperitoneal tumor. This IL-2 composition was the same as described in Example 4. 106 MethA fibrosarcoma tumor cells were injected intraperitoneally into mice (BalbC) for 3 days (Day 0, Figure 7) and will contain increasing doses of IL-2 (100,000 IU; 500,000 IU or 2,000,000 IU) The 0.2 ml IL-2 composition was injected intraperitoneally into mice (grouped into 10 mice each). The control group consisted of mice that were tumor-bearing but not treated; mice injected with drug-free compositions; mice that were routinely administered with IL-2 (1 80,000 IU; subcutaneous; each Twice a day; five days); and 500,000 IU of conventional IL-2 mice were injected intraperitoneally. The drugs were repeated on days 7, 14, 21, 28, and 35, except for the conventional systemic B.I.D. group. The survival rate of the mice was followed (Fig. 7). Use of the traditional IL-2 composition This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) (please read the notes on the back and fill out this page)

經濟部智慧財產局員工消費合作社印製 -26- 1308068 A7 B7 五、發明説明（24) 之陽性對照組比較，證實本發明之IL-2組合物提供了更好的存活數據。 (請先閲讀背面之注意事項再填寫本頁) 實例8 .此實例說明於承受黑色素瘤實體腫瘤之小鼠、每週經腫瘤週邊注射本發明之IL-2組合物的存活率。此il-2組合物與實例4所述者相同。將B16黑色素瘤腫瘤細胞經皮下植入小鼠（C57/B16)。當此實體腫瘤爲4-5毫米大小時，將含有2,000,000 I.U.之 IL-2之0.2毫升之本發明IL-2組合物注射至小鼠（在腫瘤周圍的相對位置注射2x0.1毫升），小鼠分組爲每組各8隻鼠。對照組中，經腫瘤週邊注射僅僅含有藥物載劑之組合物（無藥物）' 或習知之IL-2組合物（2,000,000 I.U.)。在第7、經濟部智慧財產局員工消費合作社印製 14、21 ' 28、及35天重複用藥。追蹤小鼠之腫瘤生長情形及存活率。每隔一天測量腫瘤大小（第6圖）。與對照組比較，IL-2組合物組中的腫瘤生長都受到限制，約歷41 /2週。每組動物於第1、2、3、4、及5天（第一次用藥之後）測量血壓，且在第14、15、16、17、及18天（第三次用藥之後）重複測量血壓。血壓値皆保持在生理學上之正常範圍內，未測得不利的血壓降低。與對照組比較，施用本發明之 IL-2組合物的腫瘤生長明顯變慢了。與使用習知IL-2組合物之陽性對照組比較，本發明之IL-2組合物提供了更好的存活數據（第8圖）。本紙張尺度適用中國國家標準（CNS ) A4規格（2丨0X297公釐） -27- 1308068 A7 B7 五、發明説明（25) 實例9 (請先閲讀背面之注意事項再填寫本頁) 此實例說明於小鼠經腫瘤週邊每週注射IL-2組合物' 對全身性免疫力之活化作用。藥物載劑與實例1所述者相同。經濟部智慧財產局員工消費合作社印製將RD-995纖維肉瘤腫瘤細胞經皮下植入小鼠 (C3H/HEN)，形成兩個腫瘤，兩側各一個腫瘤。當此實體腫瘤爲4-5毫米大小時，將含有2,000,000 I.U.之IL-2之實例 1所述的0.2毫升IL-2組合物注射至小鼠（分組爲每組各1〇隻鼠），於腫瘤周圍的相對位置注射2x0.1毫升，注射位置全部在小鼠右側腫瘤（5隻鼠）、或全部在左側腫瘤（5隻鼠）。對照組由如下小鼠組成：承受兩個腫瘤但無治療的小鼠 ;僅僅注射藥物載劑的小鼠（5隻鼠注射於左側腫瘤處、且5 隻鼠注射於右側腫瘤處）；及經腫瘤週邊注射習知IL-2組合物（2,000,000 I.U.)之小鼠（5隻鼠注射於左側腫瘤處、且5隻鼠注射於右側腫瘤處）。各組中，每週重複用藥於相同之預先選定的腫瘤位置，連續8週。使用IL-2組合物之組別中 ,經治療與未經治療之腫瘤生長皆受到限制，而對照組兩側之腫瘤皆有成長。因此，施用IL-2組合物之組別的存活數據比對照組（包括經腫瘤週邊施用習知IL-2之組Sil)更好，證明全身性免疫反應只能經由本發明之IL-2組合物才可達成。實例10 此實例說明於小鼠經腫瘤週邊每週注射IL-2組合物、本紙張尺度適用中國國家楼準（CNS ) A4規格.（210X 297公釐） -28- 1308068 A7 B7 五、發明説明（26) 對全身性免疫力之活化作用。藥物載劑與實例1所述者相同。將RD-995纖維肉瘤腫瘤細胞經皮下植入小鼠 (C3H/HEN)，形成兩個腫瘤，兩側各一個腫瘤。當此實體腫瘤爲4-5毫米大小時，將含有2,000,000 I.U.之IL-2之實例 4所述的0.2毫升IL-2組合物注射至小鼠（分組爲每組各10 隻鼠），於腫瘤周圍的相對位置注射2x0.1毫升，注射位置全部在小鼠右側腫瘤（5隻鼠）、或全部在左側腫瘤（5隻鼠）。對照組由如下小鼠組成：承受兩個腫瘤但無治療的小鼠 ;僅僅注射藥物載劑的小鼠（5隻鼠注射於左側腫瘤處、且5 隻鼠注射於右側腫瘤處）；及經腫瘤週邊注射習知IL-2組合物（2,000,000 I.U.)之小鼠（5隻鼠注射於左側腫瘤處、且5隻鼠注射於右側腫瘤處）。各組中，每週重複用藥於相同之預先選定的腫瘤位置，連續8週。使用IL-2組合物之組別中 ,經治療與未經治療之腫瘤生長皆受到限制，而對照組兩側之腫瘤皆有成長。因此，施用IL-2組合物之組別的存活數據比對照組（包括經腫瘤週邊施用習知IL-2之組別）更好，證明全身性免疫反應只能經由本發明之IL-2組合物才可達成。提供上述實施例僅僅爲了說明之目的，不意圖、且絕不應該以任何方式來限制本發明。在不背離本發明之精神與範疇之前提下，可對本發明之化合物及方法進行多種修飾。熟悉該項技術者將立即了解何種修飾可進行、卻不背離本發明之精神與範疇；本發明之精神與範疇只受限於下 ^紙張尺度適用中關家標準（CNS )八4祕（21GX297公釐) (請先閲讀背面之注意事項再填寫本頁)Printed by the Intellectual Property Office of the Ministry of Economic Affairs, Employees' Consortium -26- 1308068 A7 B7 5. Comparison of the positive control group of the invention (24), confirming that the IL-2 composition of the present invention provides better survival data. (Please read the precautions on the back and then fill out this page.) Example 8. This example illustrates the survival rate of the IL-2 composition of the present invention injected per week around the tumor in mice bearing melanoma solid tumors. This il-2 composition was the same as described in Example 4. B16 melanoma tumor cells were subcutaneously implanted into mice (C57/B16). When the solid tumor is 4-5 mm in size, 0.2 ml of the IL-2 composition of the invention containing 2,000,000 IU of IL-2 is injected into the mouse (injection of 2 x 0.1 ml at a relative position around the tumor), small Rats were grouped into 8 rats each. In the control group, a composition containing only a drug carrier (no drug) or a conventional IL-2 composition (2,000,000 I.U.) was injected via the periphery of the tumor. In the seventh, the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives, printed 14, 21 '28, and 35 days of repeated medication. The tumor growth and survival rate of the mice were followed. Tumor size was measured every other day (Fig. 6). Compared to the control group, tumor growth was limited in the IL-2 composition group, approximately 41 /2 weeks. Blood pressure was measured on days 1, 2, 3, 4, and 5 (after the first dose), and blood pressure was measured repeatedly on days 14, 15, 16, 17, and 18 (after the third dose). . The blood pressure sputum was kept within the physiologically normal range, and unfavorable blood pressure was not measured. Tumor growth with the administration of the IL-2 composition of the present invention was significantly slower compared to the control group. The IL-2 composition of the present invention provided better survival data (Fig. 8) as compared to the positive control group using the conventional IL-2 composition. This paper scale applies to Chinese National Standard (CNS) A4 specification (2丨0X297 mm) -27- 1308068 A7 B7 V. Invention description (25) Example 9 (Please read the note on the back and fill out this page) Activation of systemic immunity in mice by weekly injection of IL-2 composition around the tumor. The drug carrier was the same as described in Example 1. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives. RD-995 fibrosarcoma tumor cells were implanted subcutaneously into mice (C3H/HEN) to form two tumors with one tumor on each side. When the solid tumor was 4-5 mm in size, 0.2 ml of IL-2 composition described in Example 1 containing 2,000,000 IU of IL-2 was injected into mice (grouped into 1 each group). The relative position around the tumor was injected 2 x 0.1 ml, and the injection site was all in the right tumor of the mouse (5 mice), or all of the tumors on the left side (5 mice). The control group consisted of mice that received two tumors but were untreated; mice injected with drug vehicle alone (5 rats injected into the left tumor and 5 rats injected into the right tumor); Mice of the conventional IL-2 composition (2,000,000 IU) were injected around the tumor (5 mice were injected at the left tumor and 5 rats were injected at the right tumor). In each group, the same pre-selected tumor location was repeated weekly for 8 weeks. In the group using the IL-2 composition, both the treated and untreated tumors were limited in growth, while the tumors on both sides of the control group grew. Therefore, the survival data of the group administered with the IL-2 composition was better than that of the control group (including the group administered with the conventional IL-2 via the tumor periphery), demonstrating that the systemic immune response can only be via the IL-2 combination of the present invention. Things can be achieved. Example 10 This example illustrates the weekly injection of IL-2 composition in mice via the periphery of the tumor. This paper scale applies to the Chinese National Standard (CNS) A4 specification. (210X 297 mm) -28- 1308068 A7 B7 V. Description of the invention (26) Activation of systemic immunity. The drug carrier was the same as described in Example 1. RD-995 fibrosarcoma tumor cells were subcutaneously implanted into mice (C3H/HEN) to form two tumors, one on each side of the tumor. When the solid tumor was 4-5 mm in size, 0.2 ml of IL-2 composition described in Example 4 containing 2,000,000 IU of IL-2 was injected into mice (grouped into 10 mice each) for tumor The surrounding relative position was injected 2 x 0.1 ml, and the injection site was all in the right tumor of the mouse (5 mice), or all of the tumors on the left side (5 rats). The control group consisted of mice that received two tumors but were untreated; mice injected with drug vehicle alone (5 rats injected into the left tumor and 5 rats injected into the right tumor); Mice of the conventional IL-2 composition (2,000,000 IU) were injected around the tumor (5 mice were injected at the left tumor and 5 rats were injected at the right tumor). In each group, the same pre-selected tumor location was repeated weekly for 8 weeks. In the group using the IL-2 composition, both the treated and untreated tumors were limited in growth, while the tumors on both sides of the control group grew. Therefore, the survival data of the group administered with the IL-2 composition is better than that of the control group (including the group of conventional IL-2 administered by the tumor), demonstrating that the systemic immune response can only be via the IL-2 combination of the present invention. Things can be achieved. The above-described embodiments are provided for the purpose of illustration only, and are not intended to be in any way limiting. Various modifications of the compounds and methods of the present invention can be made without departing from the spirit and scope of the invention. Those skilled in the art will immediately understand which modifications can be made without departing from the spirit and scope of the present invention; the spirit and scope of the present invention is limited only by the application of the Chinese National Standards (CNS) 8 (4) 21GX297mm) (Please read the notes on the back and fill out this page)

.•IT.•IT

經濟部智慧財產局員工消費合作社印製 -29- 1308068 A7 B7 經濟部智慧財產局員工消費合作社印製五、發明説明（27)文之申請專利範圍及其功能均等物。 (請先閲讀背面之注意事項再填寫本頁) 訂本紙張尺度適用中國國家標準（CNS ) A4規格<2丨〇><297公釐） -30-Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing -29- 1308068 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (27) The patent application scope and functional equivalents. (Please read the notes on the back and fill out this page.) The paper size is applicable to the Chinese National Standard (CNS) A4 specification <2丨〇><297 mm) -30-

Claims

Attachment 7A; 1308068 (Please read the note on the back and then fill out this page) Patent application No. 91113159 is replaced by the Chinese patent application scope. May 2, 1993, May 2, 1993. 1. A local or partial and systemic A pharmaceutical composition for controlling a cell proliferative disorder, comprising: a) as an active ingredient, 〇〇〇 to 1 x 108 I_U./ml of a lymphokine selected from interleukin-2 (IL-2) 'IL- 2 derivatives and IL-2 mimetic, and b) 3 to 50% by weight of a biodegradable quinone or quinone type triblock interpolymer, wherein the triblock interpolymer comprises i) 51 to 83% by weight a biodegradable hydrophobic hydrazine block comprising a biodegradable polyester or poly(orthoester), and ii) 17 to 49% by weight of a hydrophilic B block comprising polyethylene glycol (PEG) Wherein the triblock interpolymer has a weight average molecular weight of from 2,000 to 4,990 and has a thermoreversible gelling property; the Ministry of Economic Affairs, Intellectual Property Office, employee consumption cooperative, and wherein the pharmaceutical composition is formed after application a reservoir of lymphatic hormones and provides continuity Out, delaying the release of the lymphatic hormone. 2. The composition of claim 1, further comprising an agent that enhances and promotes reconstitution, comprising polyethylene glycol (PEG), a PEG derivative, or a mixture of PEG and a PEG derivative The weight average molecular weight of the PEG or PEG derivative is 150 to 1100. The size of the paper is applicable to the Chinese National Standard (CNS) A4 specification (2丨0X 297 mm) _ 1 · 1308068 A8 B8 C8 D8 6. Patent application Scope 2 3. The composition of claim i, wherein the composition is an injectable liquid prior to administration. 4_ The composition of claim 1, further comprising a bio-reactable additive selected from the group consisting of polyalcohols (including sugars), surfactants, amino acids, proteins, preservatives, and anti-corrosion agents. A group of oxidants, stabilizers, tonicity correctors, buffer salts, and equivalents thereof. 5. The composition of claim 2, wherein the pEG derivative comprises PE previously derived from the following compound (3, the compound is selected from the group consisting of D, L-lactide, D-lactide a group consisting of _ L-lactide, D, L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, and an interpolymer thereof, as in the composition of claim 2 Wherein the peg derivative is represented by R^CO-CMPEGhCO-R2 or R--0-(PEG)-C0-R2, wherein R1 and R2 are each selected from the group consisting of hydrazine and C! to Ci. The composition of any one of claims 1 to 6 which can be used for local control or for local and systemic control of cell proliferative diseases in warm-blooded animals. 8. Combination of claim 7 The cell proliferative disease is a tumor or a sputum. 9. The composition of claim 7 which can be administered parenterally. The parenteral route is selected from the inside of the tumor, through the periphery of the tumor, Composed of the periphery of the lesion, the interior of the lesion, the intrathecal, the intraperitoneal, and the transabdominal 10. The composition of claim 7 of the patent scope, wherein the composition is (please read the note on the back and then fill out this page). Installed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed Paper Scale for China National Exercise $ ( CNS ) A4 Specification (210X297 mm) -2 - ABCD 1308068 VI. The scope of patent application can be applied from once a day to once a month. Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Consumer Cooperatives (please apply) Read the notes on the back and fill out this page.) This paper size applies to China National Ladder (CNS) A4 specification (21〇X297 mm).