201106947 六、發明說明： 【發明所屬之技術領域】 本發明係有關一種醫藥配方，其含有正亞丁基苯酞及 多元酸酐。本發明也揭示此配方用於治療腫瘤的用途。 【先前技術】 正亞丁基苯敵（B dp h)為從當歸（Angelica sinensis)分離 出來的化學化合物。其可用於治療各種不同腫瘤，例如多 鲁形神經膠胚細胞瘤（glioblastoma multiforme)及乳腺癌。參 見’例如 ’ Tsai 等人，Clin. Cancer Res. 2005，11(9): 3475-3484 及 Tsai 等人，J Neurochem. 2006，99(4): 1251-62。然而’以選擇性及持續的方式將正亞丁基苯酞遞 送至癌症部位對於其於有效癌症治療方面的用途係重要 的。對於治療腦癌尤其重要，其中該藥物由於血腦障壁 (blood brain barrier)而難以達到疾病區域。所以需要研發用 φ 於遞送該藥物的有效方式》 【發明内容】 本發明係以一種醫藥配方的發現為基礎，正亞丁基苯 酞可從該醫藥配方歷經長時間，例如，多於3〇天，逐^釋 放。 在一方面中，本發明描述一種醫藥配方的特徵，該醫 藥配方含有⑴正亞丁基苯敵及（ii)_聚合物，彼等m在 一起。 201106947 該聚合物可為聚（乳酸_共聚合乙醇酸）、聚葡萄胺糖 (chitosan)、膠原、水凝膠或多元酸酐，該多元酸酐係由雙（對 叛基笨氧基）丙燒、雙（賴基笨氧基）丁烧、雙（對叛基苯氧 基）戊烷、雙（對羧基苯氧基）庚烷雙（對羧基苯氧基）己烷、 雙(對羧基苯氧基)辛烷、間苯二甲酸、M苯二丙酸、十二 烷一酸、草酸、丙二酸、丁二酸、戊二酸、己二酸、庚二 酸、辛一酸、壬二酸、癸二酸、鄰苯二甲酸、間苯二甲酸、 對苯二曱酸或其混合物製備而成。 該配方之一實例為正亞丁基苯酞與多元酸酐 p(CPP-SA)的混合物’該多元酸酐p(cpp_SA)係由雙(對羧基 苯氧基）丙烧（cpp)與該癸二酸（SA)製備而成。在該多元酸 野中’該雙（賴基苯氧基）丙燒與該癸二酸之間的比例較 佳為1 . 2至1 · 1〇 (例如，i : 4)。該正亞丁基苯醜的重量 百分比為該配方的3%至2〇%(例如，1〇%^該配方可呈粉 末、膜；Uwa㈣、薄片、棒、微球、奈韓、糊或膠 態。 在另-方面中，本發明描述上述用於治療腫瘤的醫藥 配方的特徵。能治療的腫瘤之實例包括，但不限於，多形 神經膠胚細胞瘤、肺癌、肝“胞癌、結腸癌、黑色素瘤： 乳腺癌、神經胚細胞瘤、畸胎瘤或人類白血病。 而且上述配方在製造有用於治療腫瘤的藥劑時的用 在本發明的範圍以内。 節 在下文說明中說明本發明的一或更多具體實施例 。從此說明及申請專利ϋ圍將使本發明的其他特徵 的細、目 201106947 的及優點顯而易見。 【實施方式】 用於實現本發明的正亞丁基苯酞在商業上可，例如， 從Lancaster Synthesis有限公司（υκ)取得。其也可從當歸 的氣仿萃取物分離出來。參見，例如，Tsai等人，CUn以以以201106947 VI. Description of the Invention: [Technical Field to Which the Invention Is Applicable] The present invention relates to a pharmaceutical formulation comprising n-butylene benzoquinone and a polybasic acid anhydride. The invention also discloses the use of this formulation for the treatment of tumors. [Prior Art] Orthobutylidene (B dp h) is a chemical compound isolated from Angelica sinensis. It can be used to treat a variety of different tumors, such as glioblastoma multiforme and breast cancer. See, for example, 'Tsai et al., Clin. Cancer Res. 2005, 11(9): 3475-3484 and Tsai et al, J Neurochem. 2006, 99(4): 1251-62. However, the delivery of n-butylidenephthalide to cancer sites in a selective and sustained manner is important for its use in effective cancer treatment. It is especially important for the treatment of brain cancer, where the drug is difficult to reach the disease area due to the blood brain barrier. Therefore, there is a need to develop an effective means for delivering the drug with φ. [SUMMARY OF THE INVENTION The present invention is based on the discovery of a pharmaceutical formulation from which the n-butyl phenyl hydrazine can be subjected to a long period of time, for example, more than 3 days. , release by ^. In one aspect, the invention features a pharmaceutical formulation comprising (1) n-butylene benzoate and (ii) polymer, all of which are together. 201106947 The polymer may be poly(lactic acid-co-glycolic acid), polyglycosic sugar (chitosan), collagen, hydrogel or polybasic acid anhydride, and the polybasic acid anhydride is made of bis(p-stupyloxy)propene. Bis(Lydyloxy)butane, bis(p-phenoxyphenoxy)pentane, bis(p-carboxyphenoxy)heptane bis(p-carboxyphenoxy)hexane, bis(p-carboxyphenoxy) Base) octane, isophthalic acid, M phenyldipropionic acid, dodecane monoacid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, octanoic acid, bismuth It is prepared from acid, azelaic acid, phthalic acid, isophthalic acid, terephthalic acid or a mixture thereof. An example of such a formulation is a mixture of n-butylene benzoquinone and polybasic anhydride p(CPP-SA). The polybasic anhydride p(cpp_SA) is derived from bis(p-carboxyphenoxy)propane (cpp) and the sebacic acid. (SA) prepared. The ratio between the bis(Leptylphenoxy)propane and the sebacic acid in the polybasic acid field is preferably from 1.2 to 1 · 1 Torr (e.g., i: 4). The weight percentage of the n-butylene benzene is 3% to 2% of the formulation (for example, 1%%), the formulation may be in the form of a powder or a film; Uwa (four), flakes, rods, microspheres, naihan, paste or colloidal state. In another aspect, the invention features the above-described pharmaceutical formulations for treating tumors. Examples of treatable tumors include, but are not limited to, polymorphic glioma, lung cancer, liver "cell carcinoma, colon cancer" Melanoma: breast cancer, neuroblastoma, teratoma or human leukemia. Moreover, the above formulation is used within the scope of the present invention in the manufacture of a medicament for treating a tumor. Section 1 of the present invention is explained in the following description. Or more specific embodiments. The details of the other features of the present invention will be apparent from the description and the claims. The embodiments of the present invention are obvious. For example, obtained from Lancaster Synthesis Co., Ltd. (υκ). It can also be isolated from the airborne extract of Angelica. See, for example, Tsai et al., CUn
Res. 2005，1 1(9): 3475-3484。 該正亞丁基苯酞化合物，無論用購買或分離的方式， 可進一步經由驟彿塔層析法（fUsh e〇iumn chromatography)、高效液相層析法、結晶法或任何其他適 合方法加以純化。 用於實現本發明的聚合物可任意從商業上取得或可經 由此技藝中習知的方法製備而成。舉例來說，可使二元酸 化合物在醋酸酐中迴流而獲得一多元酸奸。Res. 2005, 1 1(9): 3475-3484. The n-butylene benzoquinone compound, whether purchased or isolated, may be further purified by flash chromatography, high performance liquid chromatography, crystallization or any other suitable method. The polymers used to practice the invention are commercially available commercially or can be prepared by methods known in the art. For example, a dibasic acid compound can be refluxed in acetic anhydride to obtain a polyacid.
該聚合物可為一共聚物。 由二不同聚多元酸酐部分利用 見，例如，Domb等人，journai 3373-3386 〇 有關一實例，此一共聚物可 溶融聚縮合法製備而成。參 〇f 聚合物 science，1987, 25: 所獲得的聚合物可藉由任何適合的方法予以純化而且 藉由NMR、MS或FT-IR來界定其特徵。 為了製備本發明的配方，可於想要的比例(例如，ι〇 重量份的正亞丁基苯耿及9G重量份的多元酸肝）下混合該 亞丁基苯酞與該聚合物，例如，多元酸酐。有關另一實例， 可將該亞T基苯醜與多元酸㈣於—溶劑（例如，二氣甲烧) 201106947 内而且接著移除該溶劑以提供一乾粉》 由此所獲得的混合物可進一步加工成各種不同的形 態’例如膜片、薄片、棒、微球、奈米球、糊或膠。舉例 來說，可使用一模子把該混合物壓縮成膜片。 在此使用的措辭“醫藥配方”係用於意指一組成，其⑴ 係適用於投藥給人類或其他哺乳動物或其可經處理，例 如，殺菌，使其適用於此投藥，及（ϋ)包含至少一孳物（例 如，正亞丁基苯酞）及上述聚合物之至少其一。此配方可為 任何可遞送一藥物的裝置的部分或全部，該裝置包括丸 劑、膠囊、凝膠、針劑（depot)、醫藥可植入的裝置（例如， 支架（其包括自膨式支架’球囊擴張式支架、塗藥血管支架 (drug-eluting stent)及覆膜支架（stent_graft))' 移植（例如， 主動脈移植）、人造心臟瓣膜、腦脊趙液分流器 (cerebrospinal fluid shunt)、起搏點電極（pacemaker electrode)、心律調節器導線及生物吸收性植入體 (bioerodable impiant)等），及外部操縱裝置（例如藥物裝置 及導管，其包括可釋放藥物的導管，例如，由於加熱該導 管尖端的結果）。該醫藥配方也可包括一或更多其他添加 物，舉例來說醫藥上可接受的賦形劑、載劑、穿透增強劑、 安定劑、緩衝劑或其他以物理方式聯合該藥物的材料，及/ 或用於增進該給藥形態的遞送性及/或該藥物的功效的聚 合物。該配方可為，舉例來說，液體、懸浮液、如錠劑、 丸劑、膠囊（包括微膠囊）的固體、乳液、微胞、藥膏、凝 膠、乳液、針劑（其包括皮下植人針劑）或在植人裝置，例 201106947 如支架等，上的塗層。該配方可舉例來說被施於外部，例 如以貼片的形態，或部分施於外部且部分植入的裝置，或 完全植入或經皮下注入。 該措辭藥物”意指在人類或其他哺乳動物體内局部及 /或全身具有生物活性的材料。藥物的實例係揭示於MerckThe polymer can be a copolymer. Partial use of two different polyanhydrides is described, for example, in Domb et al., Journai 3373-3386. For an example, the copolymer can be prepared by melt-condensation condensation.参 f Polymer science, 1987, 25: The obtained polymer can be purified by any suitable method and characterized by NMR, MS or FT-IR. To prepare the formulation of the present invention, the butylene benzoquinone can be mixed with the polymer in a desired ratio (for example, ι by weight of n-butylene benzoquinone and 9 g parts by weight of polyacid liver), for example, Anhydride. In another example, the sub-T-phenyl benzene and the polybasic acid (tetra) may be in a solvent (eg, a gas-fired) 201106947 and then the solvent is removed to provide a dry powder. The mixture thus obtained may be further processed. In a variety of different forms 'such as diaphragms, sheets, rods, microspheres, nanospheres, pastes or glues. For example, the mold can be compressed into a membrane using a mold. The phrase "pharmaceutical formulation" as used herein is used to mean a composition (1) which is suitable for administration to a human or other mammal or which can be treated, for example, sterilized, to render it suitable for administration, and (ϋ) At least one of the imides (eg, n-butylene benzoquinone) and at least one of the above polymers is included. The formulation may be part or all of any device that delivers a drug, including pills, capsules, gels, depots, medical implantable devices (eg, stents (including self-expanding stents) Balloon-expandable stents, drug-eluting stents, and stent grafts (stent_graft)' transplantation (eg, aortic graft), artificial heart valves, cerebrospinal fluid shunt, a pacemaker electrode, a heart rhythm regulator lead, and a bioerodable implant, etc., and an external manipulation device (eg, a drug device and a catheter including a drug-releasing catheter, for example, due to heating The result of the catheter tip). The pharmaceutical formulation may also include one or more other additives, such as pharmaceutically acceptable excipients, carriers, penetration enhancers, stabilizers, buffers, or other materials that physically associate the drug, And/or a polymer for enhancing the delivery of the administered form and/or the efficacy of the drug. The formulation may be, for example, a liquid, a suspension, a solid such as a lozenge, a pill, a capsule (including microcapsules), an emulsion, a microcapsule, an ointment, a gel, an emulsion, an injection (which includes a subcutaneous implant). Or in the implanting device, for example 201106947 such as a bracket, etc., on the coating. The formulation can be applied, for example, to the exterior, such as in the form of a patch, or partially applied externally and partially implanted, or fully or subcutaneously. The phrase "drug" means a material that is biologically active locally and/or systemically in a human or other mammal. Examples of drugs are disclosed in Merck
Index、the Physicians Desk Reference 及美國專利案第 6,297,337號第11攔第16行至第12攔第58行，及US φ 2003/0224974的第0045段中，在此為了所有目的將其全文 併入本文。藥物可舉例來說為用於治療、預防、診斷、治 癒或減輕症狀或患病，其包括維他命及礦物質補充劑；影 響哺乳動物的構造或機能的物質；前藥，在其被置於生理 環境中之後變為生物活性或更具活性的物質；及藥物的代 謝物。診斷劑的實例為含有放射線元素的造影劑、含有舉 例來說碘、酶及螢光物質等的對比劑。 本發明的配方也可含有適合的添加物。這些添加物可 .# 於此配方的任何製備階段時包括於此配方内。為了賦予預 期的效果該等添加物在此配方内的想要濃度，如熟於此藝 之士所習知，可利用習用方法來分析》 本發明的配方’藉由與流體接觸，釋放出正亞丁基苯 酞一抗腫藥劑。因此，本發明亦係關於藉由投予有效量的 此配方給有需要的病患而治療腫瘤的方法。此配方中的亞 丁基苯酞係以特定時期，例如20、30、35、40、50、60天， 緩慢且持續釋放至鄰近的組織内。 如此處所用的，該措辭“治療”係定義為投予有效量的 201106947 此配方給病患，該病患有腫瘤、腫瘤徵兆、腫瘤衍生的疾 病或症狀或易患腫瘤的體質，以達到治癒、緩和、減輕、 醫治或改善腫瘤、腫瘤徵兆、腫瘤衍生的疾病或症狀或易 患腫瘤的體質的目的。 “病患’’指的是人或非人動物。非人動物的實例包括所 有脊椎動物’例如，哺乳物，諸如非人靈長類動物（特別是 高級靈長類動物）、狗、齧齒類動物（例如，小鼠或大鼠）、 天竺鼠、描及非哺乳動物類，諸如鳥類、兩棲動物、攸行 動物等等。在較佳的具體實施例中，該病患為人類。在另 一具體實施例中，該病患為實驗動物或適合當作疾病模型 的動物。待治療腫瘤、癌或其他細胞增殖症狀的病患可經 由此症狀的標準診斷技術加以辨別。 腫瘤為經由細胞的不正常生長所形成的瘤或病害。其Index, the Physicians Desk Reference and U.S. Patent No. 6,297,337, No. 11 to Line 16, line 12, line 58, and US φ 2003/0224974, paragraph 0045, hereby incorporated herein in its entirety for all purposes. . The medicament may, for example, be used to treat, prevent, diagnose, cure or alleviate symptoms or illnesses, including vitamins and mineral supplements; substances that affect the structure or function of the mammal; prodrugs, which are placed in the physiology A substance that becomes biologically active or more active in the environment; and a metabolite of the drug. Examples of the diagnostic agent are a contrast agent containing a radioactive element, and a contrast agent containing, for example, iodine, an enzyme, a fluorescent substance, and the like. The formulations of the invention may also contain suitable additives. These additives can be included in this formulation at any stage of preparation of this formulation. In order to impart the desired effect, the desired concentration of such additives in the formulation, as is known to those skilled in the art, can be analyzed by conventional methods. The formulation of the present invention is released by contact with a fluid. Butylene benzoquinone anti-tumor agent. Accordingly, the present invention is also directed to a method of treating a tumor by administering an effective amount of the formulation to a patient in need thereof. The butyl benzoquinone in this formulation is slowly and continuously released into adjacent tissues for a specific period of time, for example 20, 30, 35, 40, 50, 60 days. As used herein, the phrase "treatment" is defined as the administration of an effective amount of 201106947 to a patient suffering from a tumor, a tumor sign, a tumor-derived disease or condition, or a susceptibility to a tumor to achieve a cure. , mitigating, mitigating, treating or ameliorating the purpose of tumors, tumor signs, tumor-derived diseases or symptoms, or susceptibility to tumors. "Patient" refers to a human or non-human animal. Examples of non-human animals include all vertebrates 'eg, mammals, such as non-human primates (especially high-grade primates), dogs, rodents Animals (eg, mice or rats), guinea pigs, non-mammals, such as birds, amphibians, cockroaches, etc. In a preferred embodiment, the patient is a human. In a specific embodiment, the patient is an experimental animal or an animal suitable for use as a disease model. Patients suffering from tumor, cancer or other cell proliferation symptoms can be identified by standard diagnostic techniques for this condition. a tumor or disease formed by normal growth.
病患治療效果的 由靜脈注射投予，或經皮下 癌）。癌指的是一疾病的種 制的生長、侵害及有時候轉 但不限於，多形神經膠胚細 、黑色素瘤、乳腺癌、神 治療方法可於體内或體外進行，單獨 聯合。在體内方法中，將一化合物 般而言，該化合物或配方係製備於 J如’ _生理食鹽水）中而且經口或經 皮下、肌肉内、鞘内、腹膜内、直 201106947 腸内、葉鞘内、鼻内、胃内、氣管内或肺内注射或植入。 較佳地，該配方可經皮下、肌肉内、靜脈内、組織間 或顱内植入癌症患者體内。在一具體實施例中，該配方， 以各種不同形態，可被植入該癌症部分或其近處而不管該 腫瘤有沒有被移除。 所需的劑量取決於投予途徑的選擇；該配方的本質； 該患者患病的特質；該病患的體型、重量、表面積、年齡 ^ 及性別；其他被投予的藥物；及主治醫師的意見。適合的 劑量為在0.01至100 mg/kg的範圍内。所需劑量的變化能 綜觀各式各樣可取得的化合物及各種不同投予途徑的效率 而預期。 該配方可配合手術或放射線治療一起使用。也可與一 或更多其他化學治療藥劑合併》該等化學治療藥劑可為， 舉例來說，如拓撲替康（topotecan)的喜樹驗類 (camptothecins)、如都沙辛（doxorubicin)的蒽環類抗生素 # (anthracycline antibiotics)、如環磷醯胺的烷化劑類或如紫 杉醇（paclitaxel)、帝摩多（temozolomide)或卡氣芥 (carmustin)的抗微管藥劑（antimicrotubule agent) 〇 不用進一步闡明，咸相信上述說明已經能適當完成本 發明。因此，將下列實施例解釋為僅為例示，而且不會以 任何方式限制揭示内容的其餘部分。在此所引用的所有刊 物在此以引用方式將其全文併入本文。 實施例1:聚合物的合成 201106947 SA預聚物 SA單體由醇再結晶兩次。使2 7 g SA單體在6〇如醋 酸酐中於135至14〇〇C時在真空（1〇-4托耳）之下迴流3〇分 鐘》把未反應的醋酸酐移除 <>使SA預聚物在真空之下於 60。(：時乾燥而且接著溶於乾燥甲苯中。將此溶液加至於體 積比1 · 10的乾燥乙醚與石油醚的i: i v/v混合物而且使其 靜置過夜而使該SA預聚物（10: 1 v/v)沈派出來。等該乙喊 及石油醚被移除之後，使該SA預聚物在真空之下乾燥。 CPP預聚物 使3 g CPP單體以5〇 mi醋酸酐於mvc時在真空（1〇·4 托耳）之下迴流30分鐘。冷卻之後，過濾該反應混合物。 藉由移除一些未反應的醋酸酐而濃縮濾液而且使該CPp預 聚物於o°c下結晶化。移除剩餘的未反應的醋酸酐。以醚 清洗該CPP預聚物而且在真空之下乾燥。將DMF及乾燥 醚（DMF :乾燥醚=1 : 9)依序加至該CPP預聚物。經過12 小時之後，移除DMF及謎而且再度使該cpp預聚物晶體 在真空之下乾燥。 P(CPP-SA)共聚物 把比例20 : 80的CPP預聚物及δΑ預聚物填入一玻璃 試管（2x20 cm)内而且於180。(：下在一油浴内加熱i分鐘。 把壓力降至10-4mmHg。在整個聚合過程中每15分鐘消除 真空。以二氮甲烷清洗該試管而且接著添加石油醚以使 201106947 P(CPP-S A)共聚物沈澱出來，以無水醚清洗該共聚物而且在 真空之下乾燥。 藉由IR及1H NMR來定義該P(CPP_SA)共聚物的特 徵。參見圖1及2。在此iR光譜研究中，於1^12 % 下觀察到酸酐鍵的特徵信號。在此iH NMR光譜研究中， 於6.9至8.2 ppm下觀察到CPP的芳香族部分的特徵信號 而且於1.3 ppm下測得SA的亞甲基部分的特徵信號。再 者’根據該1H NMR光譜研究中的CPP及sA的特徵峰強 度鑑別出該共聚物内的CPP及S Α的比例為1 : 4至1 : 5。The therapeutic effect of the patient is administered intravenously or subcutaneously). Cancer refers to the growth, aggression and sometimes the conversion of a disease. However, it is not limited to polymorphism, melanoma, breast cancer, and God's treatment. It can be combined in vivo or in vitro. In an in vivo method, a compound or a formulation is prepared in a J, such as 'physiological saline, and is orally, subcutaneously, intramuscularly, intrathecally, intraperitoneally, straight, 201106947, intestine, Intrathecal, intranasal, intragastric, intratracheal or intrapulmonary injection or implantation. Preferably, the formulation can be implanted into a cancer patient subcutaneously, intramuscularly, intravenously, interstitially or intracranically. In a specific embodiment, the formulation, in a variety of different forms, can be implanted into or near the cancer portion regardless of whether the tumor has been removed. The dosage required will depend on the choice of route of administration; the nature of the formulation; the trait of the patient's illness; the size, weight, surface area, age and sex of the patient; other administered drugs; and the attending physician's opinion. A suitable dose is in the range of 0.01 to 100 mg/kg. Changes in the required dose can be expected from a wide range of available compounds and the efficiency of the various routes of administration. This formula can be used in conjunction with surgery or radiation therapy. It may also be combined with one or more other chemotherapeutic agents, such as camptothecins of topotecan, such as doxorubicin Anthracycline antibiotics, alkylating agents such as cyclophosphamide or antimicrotubule agents such as paclitaxel, temozolomide or carmustin It is further clarified that the above description has been able to properly complete the present invention. Therefore, the following examples are to be construed as illustrative only and not limiting the remainder of the disclosure in any manner. All publications cited herein are hereby incorporated by reference in their entirety. Example 1: Synthesis of polymer 201106947 SA prepolymer The SA monomer was recrystallized twice from an alcohol. 2 7 g of SA monomer is refluxed under vacuum (1〇-4Torr) for 3 〇 at 135 to 14 〇〇C in acetic anhydride, and the unreacted acetic anhydride is removed <> The SA prepolymer was allowed to stand at 60 under vacuum. (: Dry at room temperature and then dissolved in dry toluene. This solution was added to a mixture of dry ether and petroleum ether in an i: iv/v ratio of 1 · 10 and allowed to stand overnight to make the SA prepolymer (10) : 1 v/v) Sink out. After the B is shouted and the petroleum ether is removed, the SA prepolymer is dried under vacuum. The CPP prepolymer makes 3 g of CPP monomer as 5 〇mi acetic anhydride. It was refluxed under vacuum (1 〇·4 Torr) for 30 minutes at mvc. After cooling, the reaction mixture was filtered. The filtrate was concentrated by removing some unreacted acetic anhydride and the CPp prepolymer was allowed to o°. Crystallization under c. The remaining unreacted acetic anhydride was removed. The CPP prepolymer was washed with ether and dried under vacuum. DMF and dry ether (DMF: dry ether = 1:9) were added sequentially to the CPP prepolymer. After 12 hours, DMF and mystery were removed and the cpp prepolymer crystals were again dried under vacuum. P(CPP-SA) copolymer gave a ratio of 20:80 CPP prepolymer and δΑ The prepolymer was filled in a glass test tube (2 x 20 cm) and at 180. (: heated in an oil bath for 1 minute. The pressure was lowered to 10-4 mmHg. The vacuum was removed every 15 minutes throughout the polymerization. The tube was rinsed with diazomethane and then petroleum ether was added to precipitate the 201106947 P(CPP-S A) copolymer, which was washed with anhydrous ether and dried under vacuum. The characteristics of the P(CPP_SA) copolymer were defined by IR and 1H NMR. See Figures 1 and 2. In this iR spectroscopy study, the characteristic signal of the acid anhydride bond was observed at 1 ^ 12 %. Here iH NMR In the spectroscopy study, the characteristic signal of the aromatic portion of CPP was observed at 6.9 to 8.2 ppm and the characteristic signal of the methylene portion of SA was measured at 1.3 ppm. Further, 'based on the CPP in the 1H NMR spectroscopy study The characteristic peak intensity of sA identifies the ratio of CPP and S 内 in the copolymer from 1:4 to 1:5.
Bdph-p(CPP-SA)配方 使p(CPP-S A)聚合物與Bdph混合以提供含有以重量計 3%或10%的Bdph的混合物。也製備成含有97%p(CPP-SA) 及3% 1，3-雙（2-氯乙基）-1_亞硝基脲（BCNU)的混合物。使該 混合物以10% (w/v)的濃度溶於二氣曱烷中。使該溶液在真 鲁空之下乾燥72小時。如Walter等人，Cancer Res. 1994，54(8): 2207-12，Leong 等人 ’ j Biomed Mater Res· 1985, 19(8): 941-55 ;及 Storm 等人 ’ j Neurooncol. 2002, 56(3): 209-17 中所說明的’利用不銹鋼模（内徑，13 mm)在 Carver Press 的輕微壓力之下於200 psi下壓縮由此所獲得的乾燥粉末 以形成 Bdph p(CPP-SA)圓盤（100 mg/圓盤）。 實施例2:釋放動力學 把Bdph-p(CPP-SA)圓盤置於具有1.0 ml的0.1 Μ磷 11 201106947 酸鹽緩衝食鹽水（pH 7·4)及1.0 ml正辛醇的閃爍瓶内而且 於37。(：下培養。藉由新鮮緩衝液於各不同時間點時替換該 溶液。如 Weingart 等人，Int. J. Cancer. 1995, 62(5): 605-9 中說明的，利用一光譜儀測量於3丨〇 nm的波長下的吸收度 以測量Bdph於該緩衝液中的濃度。獲致Bdph持續釋放。 參見圖2。 實施例3 ·體内控制釋放及細胞毒作用（Cytotoxic effect) 進行分析以檢查p(CPP-SA)-10% Bdph對於惡性神經 膠質瘤RG2的生長抑制作用。據發現該p(CPP_SA)_l〇% Bdph的生長抑制作用與控制組相比為5〇%。此外，在 P(CPP-SA)-10% Bdph的治療之後觀察經逐漸改變及卸下的 培養平板底部的GBM腫瘤細胞之形態學（圖4)。比起未反 應的細胞，大部分卸下的GBM細胞在經過p(CPP-SA)-10% Bdph治療之後凋亡（圖4B & c)。 注意數據係表示成平均值士 SD或SE (分別為標準偏差 及標準誤差）。經由學生氏t_檢定（Student，s卜如〇及 Mantel-Cox檢定來分析統計顯著性。利用Kaplan-Meier方 法進行存活分析。將<〇.〇5的p值視為顯著的。 實施例4:凋亡途徑及由p(cpp_SA)_Bdph所誘發的Nurr 移位 為了確認以寡聚合去氧核苷酸為基礎的微陣列分析的 結果’藉由RT-PCR檢查經Bdpll治療的RG2細胞内的孤兒 12 201106947 受體（orphan receptors) NOR-l、Nurrl 及 Nur77 的表現。利 用Bdph治療特定時期之後，於二GBM細胞株内以時間相 依的方式誘發該Nurr77的Mrna表現。從Bdph治療之後半 小時至該治療之後6小時明顯誘發出Nurr77 mRNA表現（圖 5)。Nur77，其在Bdph治療之後被高度誘發出來，據發現 與生長抑制及細胞凋亡有牵連，暗示Nur77誘發可能是 GBM細胞内Bdph-誘發凋亡的初期現象。 為了檢查是否回應Bdph而發生Nur77的移位，利用對 應的Nur77特有抗體進行該Nur77的免疫螢光檢查定位而 且利用螢光顯微鏡來測量。結果顯示Nur77在細胞核内比 在細胞液内更多許多。經過Bdph治療24小時之後，把 Nur77從該細胞核移位至該細胞質（圖6)。為了進一步確 認，藉由西方氏墨潰分析法（Western blot analysis)檢查細胞 液及細胞核部分。免疫轉潰法（Immunoblotting)顯示Nur77 主要位於欠缺Bdph治療的細胞核内（圖7)。 最後，利用西方氏墨潰分析法測定Bdph-誘發Nur77 的基因表現所涉及的傳信途徑。如圖8所示，經過Bdph 治療1小時之後JNK、AKT、ERK顯然均被磷酸化。再者， MTT分析結果顯示在細胞利用5至20 nM的pJNK抑制劑 預處理而且於RG2細胞内利用Bdph治療之後細胞成活力 提高了。 實施例5 :動物研究 從國家實驗室動物中心（台北，台灣）獲得雄性F344大 13 201106947 鼠（230至260g)及雄性Foxnl nu/nu小鼠（10至12週）。所 有程序遵守國立東華大學（花蓮，台灣）的實驗室動物中心 及中國醫藥大學附設醫院（台中，台灣）的標準作業程序。 於動物實驗中使用RG2細胞（大鼠GBM)及DBTRG-05MG 細胞（人類GBM)以監視p(CPP-SA)-3%或10% Bdph配方及 p(CPP-SA)-3% BCNU的抗腫瘤活性。 同基因F344大鼠（6隻/組）接受1χ1〇6個RG2細胞的皮 下背部植入體（subcutaneous back implants)。藉由皮下植入 體利用 p(CPP-SA)-3%、p(CPP-SA)-10% Bdph 配方、 p(CPP-SA)-3% BCNU或在該腫瘤細胞植入之後從原始注入 部位移除的至少1.5 cm之單獨聚合物的治療各組内的動 物。 此外，Foxnl nu/nu小鼠（5至6隻/組）接受lx 1〇6個 DBTRG-05MG 細胞的皮下植入，p(CPP-SA)-3%、 p(CPP-SA)-10°/〇 Bdph 配方、p(CPP-SA)-3% BCNU 或在該腫 瘤細胞植入之後從原始注入部位移除的至少1.5 cm之單獨 聚合物的皮下植入。 利用測徑器測量腫瘤大小而且將體積計算為L X Η X W X 0.5236。當腫瘤的體積在大鼠體内超過25 cm3而且在 小鼠體内超過1 〇〇〇 mm3時則將動物放棄。利用該日期來計 算大鼠及小鼠的最終存活日期。 實施例6 : p(CPP-SA)-Bdph在動物模型内的治療效果 F344大鼠及免疫缺陷裸小鼠接受5χ1〇4個RG2細胞而 201106947 且利用 p(CPP-SA)-3% Bdph、p(CPP-SA)-10% Bdph' 單獨 p(CPP-SA)或 p(CPP-SA)-3% BCNU 做皮下治療。與該 p(CPP-SA)-3% Bdph 治療組、p(CPP-SA)-3% BCNU 治療組 及p(CPP-SA)治療組（圖9A，p < 0.005)相比時，該 p(CPP-SA)-10% Bdph治療組觀察到顯著的抑制效果。 該RG2組第30曰時的平均腫瘤大小為2070.79 土 784.90 mm3 ’ 該 p(CPP-SA)治療組為 1586.30 ± 243.69 鲁 mm3，該 p(CPP-SA)-30/〇 Bdph 治療組為 346.71 ± 521.68 mm3，該 p(CPP-SA)-10% Bdph 治療組為 87.89 ± 167.44 mm3’ 以及該 p(CPP-SA)-3% BCNU 治療組為 357.48 ± 27.30 mm3 〇 ki-67的免疫組織化學染色（immunoshitochemical stain)，其指示細胞增殖，顯示該p(CPP-S A)-10% Bdph治 療組明顯減少。此外，該硫胱氨酸蛋白酶（caspase)的免疫 組織化學染色，其指示凋亡，顯示該p(CPP-SA)-10% Bdph • 治療組明顯減少。 最後，在該p(CPP-SA)-10% Bdph治療組的動物體内， 藉由體重及各個不同器官的組織分析來評估時，沒有觀察 到與藥物有關的毒性，但是在該p(CPP-SA)-3% BCNU治療 組中觀察到顯著體重喪失（圖9B)。 實施例7 : p(CPP-SA)-Bdph在異種皮移植腫瘤生長方面的 治療效果 給裸小鼠注入人類DBTRG-05MG細胞而且在第5日植 15 201106947 入 p(CPP-SA)-Bdph(0%'3%及 10%)。在該 3%及 10%Bdph-膜片治療組中觀察到腫瘤生長的明顯抑制（圖10)。控制組 在第39曰時的腫瘤大小平均值為1098.46 ± 170.11 mm3， 該 p(CPP-SA)-3% Bdph 治療組為 605.8 ± 98.8 mm3 而且該 p(CPP-SA)-10% Bdph 治療組為 504.4 ± 38.9 mm3 (圖 10C ; p < 0.05)。 實施例8 : Bdph抑制人類多形神經膠胚細胞瘤的遷移及侵 襲 利用 BioCoat細胞基質侵襲艙（matrigel invasion chamber)系統（BD Bioscience， Bedford, ΜΑ)檢查 DBTRG-05MG細胞株的侵襲力。BD細胞基質本體係由在 含有8 μιη細孔的聚對苯二甲酸乙二酯（PET)膜上層黏蛋白 (laminin)、膝原 IV、巢蛋白 / 内肌動蛋白（ni do gen/ent action) 及蛋白多醣（proteoglycan)組成。參見圖12及16。在體内 遷移分析中’在 Falcon培養杯（culture insert) (BD Bioscience)上的應用低細孔密度 PET徑跡蝕刻膜 (track-etched membranes)。將該膜置於細胞基質艙（Matrigel chamber)或Falcon培養杯的上方孔與下方孔之間。首先使 該等細胞再懸浮於含有10%胎牛血清的PRMI 1640内而且 播種於該艙的上方孔内（每孔50,000個細胞）。於37。(：下培 養24小時之後’利用Liu著色劑（Handsel科技股份有限公 司，台北，台灣）將透過該膜侵入或遷移的細胞染色而且在 顯微鏡之下計數。各實驗重覆進行三次。 201106947 用上述系統檢查Bdph對於人類多形神經膠胚細胞瘤 的遷移及侵襲的影響。據發現Bdph以劑量相依的方式抑制 人類多形神經膠胚細胞瘤的遷移及侵襲。參見圖12至14。 實施例9 : Bdph經由壓抑Axl而抑制腫瘤遷移及侵襲 利用反轉錄酶（reverse transcriptase)-聚合酶連鎖反應 (RT-PCR)檢查Bdph對於GBM細胞株内的基因表現圖譜的 影響以闡釋Bdph對惡性腦瘤起作用的可能機制。據發現該 Axl受體酪胺酸激酶（rtk)的mRNA表現在dbph存在的情 況之下調降。再者，經由將pCDNA3.0-Axl質粒轉染至該 GBM細胞株内所引起的Axl過度表現可能逆轉Bdph對於 Axl媒介的增殖、遷移及侵襲的抑制效果。 另外進行西方氏墨潰分析法以檢查在Bdph存在的情 況之下該等GBM細胞的Axl蛋白含量。結果（圖11)顯示該 Axl蛋白含量降低了。另外參見圖15及17。 現在已能接受蛋白酪胺酸激酶在細胞增殖及分化的調 節中以及在包括人類神經膠質瘤的許多瘤形成的發端中扮 演重要的角色。除此之外，據記載腫瘤遷移及侵襲中涉及 Axh上述結果暗示Bdph抑制Αχ1受體酪胺酸激酶的蛋白 表現而且藉以抑制該GBM細胞株的遷移及侵襲能力。 其他具體實施例 5本說明中所揭示的所有特徵可以任何組合合併。用於 相同、等效或相似目的的選擇性特徵均可替代此說明書中 17 201106947 ) 所揭不的各特徵。因此，除非另行明碟說明，否則所揭示 的各特徵僅為等效或相似特徵的基因系列的實例。 從上述說明，熟於此藝之士可輕易確定本發明的基本 特徵而且不會恃離其精神及範圍，可進行本發明的各種 不同改變及修飾使其適於各種不同用法及條件。因此，其 他具體實施例也在下列申請專利範圍的範圍以内。 【圖式簡單說明】 圖1為顯示P(CPP-SA)共聚物的1H NMR光譜的圖形。參 CPP的芳香族部分的特徵信號於6 9至8 2 ppm下觀察到。 另一 SA的亞甲基部分的特徵信號於13 ppm下觀察到。 圖2為顯示p(CPP_SA)共聚物的FTIr光譜的圖形eCpp 與SA之間的酸酐鍵的特徵信號於i812 76 cin-i下觀察到。 圖 3 為顯示 p(cpp_SA)_2〇/〇 BCNU 膜片（♦)、 p(CPP-SA)-30/〇 Bdph 膜片⑷及 p(CPP-SA)_10〇/〇 Bdph 膜片 (▲)的釋放動力學的圖形。在所有配方中均觀察到Bdph或 鲁 BCNU的持續釋放。 圖4A至4D為顯示p(cpp-SA)-10% Bdph-誘發大鼠惡 性神經膠質瘤細胞的生長抑制的圖形及照片，其中（A)以 10% Bdph-膜片治療大鼠惡性神經膠質瘤細胞株R(} 24小 時。藉由MTT分析測定細胞成活力。此數據表示3個獨立 實驗的具有標準偏差的平均值。*p< 〇〇5。（B)利用膜片治 療的RG2惡性神經膠質瘤細胞的細胞形態學^ (c)利用 p(CPP-SA)-3% Bdph治療24小時的RG2惡性神經膠質瘤細 18 201106947 胞的細胞形態學。（D)利用P(CPP-SA)-10% Bdph治療24小 時的RG2惡性神經膠質瘤細胞的細胞形態學。 圖5為顯示Bdph-誘發Nur77轉錄的照片。以IC50濃 度的Bdph治療大鼠GBM細胞（RG2)達到指定時間（〇、〇.5、 1、3及6小時）。以藥物塔養之後，收集細胞而且分離出全 部RNA。用GADPH當作内標準物。 圖6為顯示Bdph-誘發Nur77從細胞核遷移至細胞質 的一組照片，其中以Bdph (100 pg/mL)治療DBTRG-05NG 細胞24小時，接著以抗-Nur77抗體然後對應的Rhodamine-共軛抗-IgG二級抗體進行免疫細胞化學染色以顯示Nur77 蛋白。同時，以DAPI把細胞染色以顯示出細胞核。以螢 光顯微鏡把螢光影像視覺化。 圖7為顯示Bdph-誘發RG2細胞内的Nur77核質移位 的一組照片。把RG2細胞植入10cm碟子内而且培養至90% 聚滿（confluence)。以Bdph (100 pg/ml)治療細胞達到不同 φ 時期（〇、6、12、24及48小時）。獲取細胞而且分離出細胞 核及細胞質部分。用西方氏墨潰分析法監視測定細胞核及 細胞質的Nur77的量。 圖8為顯示該Bdph-誘發生長抑制中的發信途徑的影 響的一組照片。依指示以Bdph (100 pg/ml)治療RG2細胞 達到0至180分鐘的各種不同時期。為pJNK、pPKC、ρΑΚΤ、 ΑΚΤ、pERK、ERK、JNK、ρρ38及ρ38抗體進行西方氏墨 潰分析。用/3 -肌動蛋白的表現當作内控制組。 圖9Α至9C為顯示經由同基因大鼠GBM模型中的 19 201106947 p(CPP-SA)-Bdph抑制腫瘤生長的圖形及一組照片。同基因 F344大鼠（6隻/組）接受lxl06個rG2細胞的皮下背部植入 體。把RG2細胞（5 X 1〇6)皮下植入F344大鼠的後側腹内。 在RG2細胞移植5日之後，僅植入R(32細胞的7大鼠為控 制組（·）而且其他大鼠分別以膜片（□)、3〇/〇Bdpli-膜片（▲)、 10%Bdph-膜片（_)及3% BCNU-膜片（X)治療。圖9A把該腫 瘤大小表示成具有標準偏差的平均值。圖9B中的體重也表 示成具有標準偏差的平均值。圖9C於GBM組織内進行免 疫組織化學染色的分析（於該RG2細胞植入之後第30曰）。 該膜片組、3% Bdph-膜片組、i〇〇/0 Bdph-膜片組及3% BCNU-膜片組的片段的代表性照片。GBM腫瘤（對於ki-67的免疫 組織化學染色）、分裂的硫胱氨酸蛋白酶3及Nur77。把該 Ki-67-、分裂的硫胱氨酸蛋白酶3-或Nur77-正性細胞染成 褐色（400x)。 圖10A至10D為顯示p(CPP-SA)-Bdph抑制異種皮移 植人類GBM裸小鼠模型中的腫瘤生長的圖形及一組照 片。把 DBTRG-05MG 細胞（2 X 1〇6)皮下植入裸 F〇xnl nu/nu 小鼠的後側腹區域内。在DBTRG-05MG細胞移植5日之 後，分別以膜片（♦)、3%Bdph-膜片⑷及1()〇/oBdph膜片（▲) 治療小鼠。（A)把該腫瘤大小表示成具有標準偏差的平均 值。（B)體重也表示成具有標準偏差的平均值。（c)及（D)照 片顯示以該膜片、3% Bdph-膜片或10% Bdph-膜片治療裸 小鼠體内的腫瘤。P<〇.01. 圖11為顯示Bdph調降Axl蛋白表現的西方氏墨潰法 20 201106947 結果的一組照片。 圖12顯示評估腫瘤細胞遷移及侵襲分析的孔杯系統。 圖1 3為顯示Bdph以劑量相依的方式抑制腫瘤細胞遷 移的一組照片及圖形。 圖14為顯示Bdph以劑量相依的方式抑制腫瘤細胞侵 襲的一組照片及圖形。 圖15為顯示藉由DBTRG腦瘤細胞内的Axl表現逆轉 ^ Bdph抑制的腫瘤細胞增殖的圖形。 圖16為顯示用於偵測細胞遷移及侵襲的OrisTM系統 的一組照片及圖形。 圖17為顯示Bdph抑制的細胞遷移可能以劑量相依的 方式被DBTRG腦瘤細胞内的Axl表現逆轉的一組照片及圖 形。 圖1 8為顯示BP抑制的細胞侵襲可能被DBTRG腦瘤 細胞内的Axl表現逆轉的一組照片及圖形。 21Bdph-p (CPP-SA) formulation The p(CPP-S A) polymer was mixed with Bdph to provide a mixture containing 3% or 10% by weight of Bdph. A mixture containing 97% p(CPP-SA) and 3% 1,3-bis(2-chloroethyl)-1_nitrosourea (BCNU) was also prepared. The mixture was dissolved in dioxane at a concentration of 10% (w/v). The solution was allowed to dry under vacuum for 72 hours. For example, Walter et al., Cancer Res. 1994, 54(8): 2207-12, Leong et al.' j Biomed Mater Res. 1985, 19(8): 941-55; and Storm et al.' j Neurooncol. 2002, 56 (3): The dried powder obtained by compressing the thus obtained dry powder at 200 psi under a slight pressure of Carver Press using a stainless steel mold (inner diameter, 13 mm) as described in 209-17 to form Bdph p (CPP-SA) ) Disc (100 mg / disc). Example 2: Release kinetics Bdph-p (CPP-SA) discs were placed in scintillation vials with 1.0 ml of 0.1 Μphosphorus 11 201106947 acid buffered saline (pH 7.4) and 1.0 ml of n-octanol. And at 37. (: Lower culture. Replace the solution at various time points with fresh buffer. As described in Weingart et al., Int. J. Cancer. 1995, 62(5): 605-9, measured with a spectrometer Absorbance at a wavelength of 3 丨〇 nm to measure the concentration of Bdph in the buffer. A sustained release of Bdph is obtained. See Figure 2. Example 3 • In vivo controlled release and Cytotoxic effect Analysis is performed to check The growth inhibitory effect of p(CPP-SA)-10% Bdph on malignant glioma RG2. It was found that the growth inhibition of p(CPP_SA)_l〇% Bdph was 〇% compared with the control group. (CPP-SA) After treatment with -10% Bdph, the morphology of GBM tumor cells at the bottom of the gradually altered and unloaded culture plates was observed (Fig. 4). Most of the unloaded GBM cells were compared to unreacted cells. Apoptosis after treatment with p(CPP-SA)-10% Bdph (Fig. 4B & c). Note that the data are expressed as mean ± SD or SE (standard deviation and standard error, respectively). (Student, S. and Mantel-Cox assays to analyze statistical significance. Using Kaplan-Me The ier method was used for survival analysis. The p value of <〇.〇5 was considered to be significant. Example 4: Apoptotic pathway and Nurr shift induced by p(cpp_SA)_Bdph in order to confirm the oligomerization of deoxynucleoside Results of acid-based microarray analysis 'Performance of NOR-l, Nurrl, and Nur77 by orphan receptors in the BD2 cells treated with Bdpll by RT-PCR. After treatment with Bdph for a specific period of time, The Mrna expression of Nurr77 was induced in a time-dependent manner in two GBM cell lines. Nurr77 mRNA expression was significantly induced from half an hour after Bdph treatment to 6 hours after the treatment (Fig. 5). Nur77, which was highly heightened after Bdph treatment It was found to be implicated in growth inhibition and apoptosis, suggesting that Nur77 induction may be an initial phenomenon of Bdph-induced apoptosis in GBM cells. In order to check whether Nur77 is translocated in response to Bdph, the corresponding Nur77-specific antibody is used. The immunofluorescence location of the Nur77 was performed and measured using a fluorescence microscope. The results showed that Nur77 was much more in the nucleus than in the cytosol. 24 times after treatment with Bdph After that, Nur77 was translocated from the nucleus to the cytoplasm (Fig. 6). For further confirmation, the cell fluid and nuclear fraction were examined by Western blot analysis. Immunoblotting showed that Nur77 was mainly located in the nucleus of Bdph-deficient cells (Fig. 7). Finally, the Western blot analysis method was used to determine the signaling pathway involved in Bdph-induced Nur77 gene expression. As shown in Figure 8, JNK, AKT, and ERK were apparently phosphorylated after 1 hour of Bdph treatment. Furthermore, MTT analysis showed an increase in cell viability after pretreatment with cells with 5 to 20 nM of pJNK inhibitor and treatment with Bdph in RG2 cells. Example 5: Animal Studies Male F344 large 13 201106947 rats (230 to 260 g) and male Foxnl nu/nu mice (10 to 12 weeks) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). All procedures are in compliance with the standard operating procedures of the Laboratory Animal Center of the National Dong Hwa University (Hualien, Taiwan) and the Hospital of China Medical University (Taichung, Taiwan). RG2 cells (rat GBM) and DBTRG-05MG cells (human GBM) were used in animal experiments to monitor p(CPP-SA)-3% or 10% Bdph formulation and p(CPP-SA)-3% BCNU resistance Tumor activity. Isogenic F344 rats (6/group) received subcutaneous back implants of 1χ1〇6 RG2 cells. By subcutaneous implant using p(CPP-SA)-3%, p(CPP-SA)-10% Bdph formula, p(CPP-SA)-3% BCNU or from the original injection after implantation of the tumor cells Animals within each group were treated with a separate polymer of at least 1.5 cm removed. In addition, Foxnl nu/nu mice (5 to 6/group) received subcutaneous implantation of lx 1〇6 DBTRG-05MG cells, p(CPP-SA)-3%, p(CPP-SA)-10° /〇Bdph formulation, p(CPP-SA)-3% BCNU or subcutaneous implantation of at least 1.5 cm of individual polymer removed from the original injection site after implantation of the tumor cells. Tumor size was measured using a caliper and the volume was calculated as L X Η X W X 0.5236. The animals were abandoned when the volume of the tumor exceeded 25 cm3 in the rat and exceeded 1 〇〇〇 mm3 in the mouse. This date was used to calculate the final survival dates of rats and mice. Example 6: Therapeutic effect of p(CPP-SA)-Bdph in an animal model F344 rats and immunodeficient nude mice received 5χ1〇4 RG2 cells and 201106947 and utilized p(CPP-SA)-3% Bdph, p(CPP-SA)-10% Bdph' alone p(CPP-SA) or p(CPP-SA)-3% BCNU for subcutaneous treatment. When compared with the p(CPP-SA)-3% Bdph treatment group, p(CPP-SA)-3% BCNU treatment group, and p(CPP-SA) treatment group (Fig. 9A, p < 0.005), A significant inhibitory effect was observed in the p(CPP-SA)-10% Bdph treated group. The mean tumor size at 30th RG of the RG2 group was 2070.79 784.90 mm3'. The p(CPP-SA) treatment group was 1586.30 ± 243.69 hmmm3, and the p(CPP-SA)-30/〇Bdph treatment group was 346.71 ± 521.68 mm3, the p(CPP-SA)-10% Bdph treatment group was 87.89 ± 167.44 mm3' and the p(CPP-SA)-3% BCNU treatment group was 357.48 ± 27.30 mm3 〇ki-67 immunohistochemical staining (immunoshitochemical stain), which indicates cell proliferation, showing a marked decrease in the p(CPP-S A)-10% Bdph treatment group. Furthermore, immunohistochemical staining of the cysteine protease (caspase), which indicates apoptosis, showed a significant reduction in the p(CPP-SA)-10% Bdph• treatment group. Finally, in the p(CPP-SA)-10% Bdph-treated group, no drug-related toxicity was observed when evaluated by body weight and tissue analysis of various organs, but in the p (CPP) Significant weight loss was observed in the -SA)-3% BCNU treatment group (Fig. 9B). Example 7: Therapeutic effect of p(CPP-SA)-Bdph on growth of xenograft skin tumors Nude mice were injected with human DBTRG-05MG cells and implanted on day 5 15 201106947 into p(CPP-SA)-Bdph ( 0% '3% and 10%). Significant inhibition of tumor growth was observed in the 3% and 10% Bdph-membrane treated groups (Figure 10). The mean tumor size at the 39th week of the control group was 1098.46 ± 170.11 mm3, and the p(CPP-SA)-3% Bdph treatment group was 605.8 ± 98.8 mm3 and the p(CPP-SA)-10% Bdph treatment group It was 504.4 ± 38.9 mm3 (Fig. 10C; p < 0.05). Example 8: Bdph inhibits migration and invasion of human polymorphic neutrophil blastoma The invasiveness of DBTRG-05MG cell line was examined using the BioCoat cell matelel invasion chamber system (BD Bioscience, Bedford, ΜΑ). BD Cell Matrix This system consists of a poly(ethylene terephthalate) (PET) membrane containing 8 μιηη pores, laminin, knee progenitor IV, nestin/inner actin (ni do gen/ent action) And proteoglycan composition. See Figures 12 and 16. Low pore density PET track-etched membranes were applied to Falcon culture inserts (BD Bioscience) in in vivo migration assays. The membrane was placed between the upper and lower wells of a Matrigel chamber or Falcon culture cup. The cells were first resuspended in PRMI 1640 containing 10% fetal bovine serum and seeded in the upper well of the chamber (50,000 cells per well). At 37. (After 24 hours of culture), cells invaded or migrated through the membrane were stained with a Liu coloring agent (Handsel Technology Co., Ltd., Taipei, Taiwan) and counted under a microscope. Each experiment was repeated three times. Systematic examination of the effects of Bdph on migration and invasion of human polymorphonuclear blastoma. It was found that Bdph inhibited migration and invasion of human polymorphonan blastoma in a dose-dependent manner. See Figures 12 to 14. : Bdph inhibits tumor migration and invasion via repression of Axl. Reverse transcriptase-polymerase chain reaction (RT-PCR) is used to examine the effect of Bdph on gene expression profiles in GBM cell lines to elucidate Bdph's role in malignant brain tumors. Possible mechanism of action. The mRNA expression of the Axl receptor tyrosine kinase (rtk) was found to be downregulated in the presence of dbph. Furthermore, the pCDNA3.0-Axl plasmid was transfected into the GBM cell line. The overexpression of Axl may reverse the inhibitory effect of Bdph on the proliferation, migration and invasion of Axl media. The Axl protein content of these GBM cells in the presence of Bdph. The results (Figure 11) show a decrease in the Axl protein content. See also Figures 15 and 17. Protein tyrosine kinase is now accepted for cell proliferation and differentiation. In addition, it plays an important role in the regulation of many neoplasias including human gliomas. In addition, it is documented that Axh is involved in tumor migration and invasion. The above results suggest that Bdph inhibits the protein of Αχ1 receptor tyrosine kinase. Performance and thereby inhibiting the migration and invasion abilities of the GBM cell line.Other Specific Embodiments 5 All features disclosed in this specification can be combined in any combination. Selective features for the same, equivalent or similar purposes can be substituted for this specification.中17 201106947 ) Various features not revealed. Thus, unless expressly stated otherwise, the features disclosed are merely examples of the gene series of equivalent or similar features. From the above description, those skilled in the art can readily determine the basic characteristics of the invention and do not depart from the spirit and scope of the invention. Accordingly, other specific embodiments are also within the scope of the following claims. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the 1H NMR spectrum of a P(CPP-SA) copolymer. The characteristic signal of the aromatic portion of the reference CPP was observed at 6 9 to 8 2 ppm. The characteristic signal of the methylene moiety of the other SA was observed at 13 ppm. Figure 2 is a graph showing the characteristic signal of the anhydride bond between the graph eCpp and SA of the FTIr spectrum of the p(CPP_SA) copolymer observed at i812 76 cin-i. Figure 3 shows p(cpp_SA)_2〇/〇BCNU diaphragm (♦), p(CPP-SA)-30/〇Bdph diaphragm (4) and p(CPP-SA)_10〇/〇Bdph diaphragm (▲) The release dynamics of the graph. Sustained release of Bdph or Lu BCNU was observed in all formulations. Figures 4A to 4D are graphs and photographs showing growth inhibition of p(cpp-SA)-10% Bdph-induced rat malignant glioma cells, wherein (A) treatment of rat malignant glia with 10% Bdph-membrane Tumor cell line R (} 24 hours. Cell viability was determined by MTT assay. This data represents the mean of standard deviations for 3 independent experiments. *p< 〇〇 5. (B) RG2 malignancy treated with a patch Cell morphology of glioma cells^ (c) Cell morphology of RG2 malignant glioma cells treated with p(CPP-SA)-3% Bdph for 24 hours. (D) Using P(CPP-SA) ) Cell morphology of RG2 malignant glioma cells treated with -10% Bdph for 24 hours. Figure 5 is a photograph showing Bdph-induced Nur77 transcription. Rat GBM cells (RG2) were treated with IC50 concentration of Bdph for a specified time (〇 , 〇.5, 1, 3, and 6 hours). After raising the drug tower, the cells were collected and all RNA was isolated. GADPH was used as an internal standard. Figure 6 shows a Bdph-induced Nur77 migration from the nucleus to the cytoplasm. Group photo, in which DBTRG-05NG cells were treated with Bdph (100 pg/mL) for 24 hours. Immunocytochemical staining with the anti-Nur77 antibody followed by the corresponding Rhodamine-conjugated anti-IgG secondary antibody to reveal the Nur77 protein. At the same time, the cells were stained with DAPI to reveal the nucleus. The fluorescence image was visualized by a fluorescence microscope. Figure 7 is a set of photographs showing the shift of Nur77 nucleoplasm in Bdph-induced RG2 cells. RG2 cells were seeded in 10 cm dishes and grown to 90% confluence. Treated with Bdph (100 pg/ml). The cells reached different φ periods (〇, 6, 12, 24, and 48 hours). The cells were harvested and the nucleus and cytoplasm fraction were isolated. The amount of Nur77 in the nucleus and cytoplasm was monitored by Western blot analysis. A set of photographs of the effects of Bdph-induced signaling pathways in growth inhibition. RG2 cells were treated with Bdph (100 pg/ml) for various periods ranging from 0 to 180 minutes. pJNK, pPKC, ρΑΚΤ, ΑΚΤ, pERK , ERK, JNK, ρρ38, and ρ38 antibodies were analyzed by Western blotting. The expression of /3 -actin was used as the internal control group. Figures 9A to 9C are shown in the GBM model via syngeneic rat 19 201106947 p (CPP-SA)-Bdph inhibition of tumor growth and a set of photographs. Isogenic F344 rats (6/group) received subcutaneous dorsal implants of lxl06 rG2 cells. RG2 cells (5 X 1〇6) were subcutaneously implanted into the posterior flank of F344 rats. After 5 days of RG2 cell transplantation, only R was implanted (the 7 rats of 32 cells were the control group (·) and the other rats were respectively with the diaphragm (□), 3〇/〇 Bdpli-membrane (▲), 10 %Bdph-diaphragm (_) and 3% BCNU-diaphragm (X) treatment. Figure 9A shows the tumor size as an average with standard deviation. The body weight in Figure 9B is also expressed as an average with standard deviation. Figure 9C is an analysis of immunohistochemical staining in GBM tissue (30th after RG2 cell implantation). The membrane group, 3% Bdph-membrane group, i〇〇/0 Bdph-membrane group and Representative photographs of fragments of the 3% BCNU-membrane group. GBM tumors (immunohistochemical staining for ki-67), cleaved thiocyl protease 3 and Nur77. The Ki-67-, split thios Lysinase 3- or Nur77-positive cells stained brown (400x). Figures 10A to 10D are graphs showing the growth of tumor in p(CPP-SA)-Bdph inhibiting xenografted human GBM nude mouse model and Group photo. DBTRG-05MG cells (2 X 1〇6) were subcutaneously implanted into the posterior ventral region of nude F〇xnl nu/nu mice. After 5 days of DBTRG-05MG cell transplantation Mice were treated with patch (♦), 3% Bdph-membrane (4), and 1 () 〇/oBdph patch (▲). (A) The tumor size was expressed as the mean with standard deviation. (B) Body weight is also expressed as an average with standard deviation. (c) and (D) photographs show treatment of tumors in nude mice with this membrane, 3% Bdph-membrane or 10% Bdph-membrane. P< Fig. 11 is a set of photographs showing the results of Bdph downregulating Axl protein expression by Western blotting method 201104469. Figure 12 shows the well cup system for assessing tumor cell migration and invasion analysis. Figure 1 3 shows the dose of Bdph A set of photographs and graphs that inhibit tumor cell migration in a dependent manner. Figure 14 is a set of photographs and graphs showing that Bdph inhibits tumor cell invasion in a dose-dependent manner. Figure 15 shows reversal of Axl expression in DBTRG brain tumor cells. ^Bdph-inhibited tumor cell proliferation pattern. Figure 16 is a set of photographs and graphs showing the OrisTM system for detecting cell migration and invasion. Figure 17 is a graph showing that Bdph-inhibited cell migration may be DBTRG brain in a dose-dependent manner. Reversal of Axl expression in tumor cells Group photographs and graphs Figure 18 is a set of photographs and graphs showing that BP-inhibited cell invasion may be reversed by Axl expression in DBTRG brain tumor cells.