TW200906386A - Compositions with anti-angiogenic activity - Google Patents

Abstract

The present invention relates to a composition for treating disorders or diseases associated with angiogenesis.

Description

200906386 ψ 九、發明說明： 【發明所屬之技術領域】 本發明是相關用於治療與血管過度增生有關病況或疾病之組成 物。 【先前技術】 血管新生是癌細胞轉移的主要途徑之一（Zetter，B.R.，Αηη.200906386 九 IX, invention description: [Technical field to which the invention pertains] The present invention relates to a composition for treating a condition or disease associated with hypervascular hyperplasia. [Prior Art] Angiogenesis is one of the main pathways for cancer cell metastasis (Zetter, B.R., Αηη.

Med. 49: .424, 1998)。持續性且無法受調控的血管新生發生 於疾病的許多階段上，也發生於癌細胞轉移過程，以及不正常血 管内皮細胞增生的病變上。新生的血f讓癌細胞由原發處轉移至 1環系統中；因而許多癌症可以血㈣度做為歸上的預後指 ‘ p血g d度越间’其轉移潛能越高。軸内的血管新生由血 管新生誘導因子所調控，這些誘導因子的種類包含纖維母細胞生 長因子’以及血管时細胞生長因子。此相_也會活化控制 原發位及轉移位血管新生的血管新生抑制因子，例如細⑽咖 。目驗輪抑悔，以输種類之天然 血管新生抑_子制於抗觸潛力，正在被積極地研 發虽中。這種藥劑可能毒性較低，且較不易5丨起抗藥性。 血管新生也絲㈣慢性㈣ 為血管新生在㈣病變情白血癌«。一般認 常的血管新生也與其他疾除此之外’過多不正 病的主要特徵為新生血管侵 =血官新生疾病，該疾 兄、，同膜或角膜。再者，年齡相關之 200906386 黃斑視覺退制是目為血管自Bmeh，s membrane麵巾入侵導 致視網膜下血管纖化组織增生所致。新生血管娜也與糖尿^視 網膜病變、早產兒視_病變、角膜移殖排斥、新生血管性青光 眼’以及眼睛水純後纖維增生症有關。其他與血管新生有關的 疾病，包括，但並不限於，風祕關節炎、紅雜狼瘡、多發性 動脈炎、鐮刀型細胞貧血、退化性關節炎、靜脈阻塞、動脈阻塞、 頸部阻塞，及動脈硬化。針對㈣血管新生的練方法可以消除 或減緩這些疾病。目前’ _發展_血科生藥雛佳的療法 之臨床試驗正在進行當中（US專利第6518298號）。 【發明内容】 ST104P係由一個4, 5-二羥基萘-2, 7_二磺酸以亞曱基橋聯所構成 四個次體（tetrameric)的環狀化合物（PohB. L. et al· Tetra.Med. 49: .424, 1998). Persistent and unregulated angiogenesis occurs at many stages of the disease, as well as in cancer cell metastasis and in abnormal vascular endothelial cell hyperplasia. The newly born blood f causes the cancer cells to be transferred from the primary to the 1-ring system; thus, many cancers can have a blood (four) degree as the prognosis of the ‘p blood gd degree', and the higher the metastatic potential. Intraaxial angiogenesis is regulated by angiogenesis-inducing factors, and the types of these inducing factors include fibroblast growth factor' and vascular cell growth factor. This phase also activates angiogenesis inhibitors that control angiogenesis in the primary and metastatic sites, such as fine (10) coffee. The examination of the round of repentance, in order to lose the type of natural angiogenesis _ sub-system in the anti-touch potential, is being actively researched and developed. This agent may be less toxic and less susceptible to drug resistance. Angiogenesis is also silky (four) chronic (four) for angiogenesis in (four) disease white blood cancer. Commonly recognized angiogenesis is also associated with other diseases. The main feature of too many unhealthy diseases is neovascular invasion = blood stasis, the disease, the same membrane or the cornea. Furthermore, the age-related 200906386 macular vision retreat is caused by the invasion of the subretinal vascular fibrotic tissue caused by the invasion of Bmeh, s membrane face towel. Neovascularization is also associated with urinary urethral retinopathy, premature infant vision, lesions, corneal transplant rejection, neovascular glaucoma, and post-pion hyperplasia. Other diseases associated with angiogenesis, including, but not limited to, wind-induced arthritis, red lupus, polyarteritis, sickle cell anemia, degenerative arthritis, venous obstruction, arterial occlusion, neck obstruction, and arteriosclerosis. The method of (IV) angiogenesis can eliminate or slow down these diseases. A clinical trial of the current ' _ development _ blood medicinal remedy therapy is underway (US Patent No. 6518298). SUMMARY OF THE INVENTION ST104P is a tetrameric cyclic compound composed of a 4,5-dihydroxynaphthalene-2,7-disulfonic acid bridged by a fluorenylene group (PohB. L. et al· Tetra.

Letters 30 (8) : 1005-1008, 1989; Poh B L and Lim E S, Tetrahedron 46 (10) ： 3651-3658, 1990a; Poh B. L. et.al., Tetrahedron 46 (12) : 4679-4386, 1990b)。它是一個多硫酸化、 環狀、由四萘次體合成的大環狀化合物，且亦是水溶性且細胞毒 性很低的化合物。ST104P曾被指出具有抗病毒及抗血栓形成的作 用（US 專利第 5166173 號，第 5196452 號，第 5312837 號，第 5441983 號’第5409959號），但是ST104P不曾被指出具有抗血管新生的 活性而可以用來治療與血管新生有關的病變，包括，但不限於， 癌症。理論上有抗病毒及抗血栓活性的化合物也可能有其他的活 200906386 性，本發明敘述-個化合組成物ST腑具抗血管新生的活性，可 以用來治療癌症及其他與血管新生有關的病變。 本發明的另-個目的是提供一種包含ST綱p的治療方法來治療 •癌症及其他與血管新生有關的病變，包括，但不限於，糖尿病眼 病變、年齡相關黃斑視覺退化、風濕性關節炎、退化性關節炎、 動脈硬化、眼晶體血管新生、視網膜血管新生及全身性狼療、紅 斑性狼瘡、多發性動脈炎、鐮刀型細胞貧血症、靜脈阻塞、動脈 阻塞、頸部阻塞。 本發明是關於-個在體外和體内皆具有抑制血管新生活性的低毒 性多硫化環狀化合物，ST104P。ST104P有極佳的水溶性，低細胞 毒性。ST104P抑制血管内皮細胞分泌基質金屬蛋白酶(mai：rix 1 metalloproteinase，MMP) JT104P也阻擾血管内皮細胞的移動及 # 管狀形成。STi〇4p抑制雞胚***血管新生，且此作用呈劑量關 係性。再者，重複對具有路易士肺癌的小鼠施予STio^p，可以延 緩其腫瘤的生長並延長罹癌小鼠之壽命。STi〇4P以埋殖片或眼藥 水方式投予也可以抑制bFGF引起的大鼠眼角膜血管新生。這些結 果顯示ST104P可以抑制血管新生並用來治療癌症及其他與血管新 生有關的疾病和症狀。 本發明的其中一個方面是提供一種抗癌藥物，經由抑制腫瘤内血 7 200906386 9 管新生來治療人或騎的癌症。此無於醫藥上可以接受的载體 中包含以亞甲基橋連接的4個4, 5-二絲萘-2, 7-二顧次體的 環狀化合物’這個載體可以適用於口、舌下、直腸、鼻腔、***、 腹腔、經皮、表皮，關節内’癌内、眼球内、眼球表面或注射方 式投藥。 廷個載體可以適用於製成賴、膠囊、顆粒、溶液、乳劑、塞劑、 貼布、眼藥水、埋殖片或粉劑。 本發明另-方面是提供抑制血管新生之組合物絲抑制人或動物 癌症的血管新生，它包含由4個4, 5-二經基萘-2, 7-二續酸次體以 亞甲基橋連接起來的環狀化合物在醫藥可以接受誠射，這個 載體可以適用於口、舌下、直腸、鼻腔、***、腹腔、經皮、表 皮’關即内，癌内、眼球内、眼球表面或注射方式投藥。這個載 體可以適用於製成旋劑、膠囊、顆粒、溶液、乳劑、塞劑、貼布、 眼藥水、埋殖片或粉劑。 本發明另—方面是提供—個血管新生抑綱來治療人或動物其他 ^血苢新生有關的非癌症病況或疾病。它包含由4個4, 5-二羥基 萘-2’7-二磺酸次體以亞甲基橋連接起來的環狀化合物在醫藥可 以接文的载體中’這個載體可以適用於口、舌下、直腸、鼻腔、 丢L腹腔、經皮、表皮，關節内，癌内、眼球内、眼球表面或 200906386 ，賴刊翻峨_、職、顆粒、溶 液礼劑、塞劑、貼布、眼藥水、埋殖片或粉劑。 疾贼錄包括，但视於，糖尿病眼病變、 ㈣叙Γ喊覺舰、眼晶體血管触、視_血管新生、多 脈炎、鐮刀型細胞貧血症、靜脈/1 且塞、動脈阻塞、頸部阻 ㈣=硬化、顺性闕節炎、全身性狼瘡、紅斑性狼瘡，及退 ，即炎。醫藥可接受的載體可以適用於口服、舌下、直腸、 鼻腔、***、腹腔、癌内、關節内、眼球内、眼球表面、表皮、 經皮’及其他注射方法之投付式。醫葯可接受的倾可以適用 於錠劑、膠囊、顆粒、溶液、乳化液'塞劑、貼布、眼藥水、埋 殖片或粉末劑型。 醫药可接受的輪可以義於口服、舌下、直腸、鼻腔、***、 腹腔、癌内、關節内、眼球内（玻璃狀體内、眼臉内）、眼球表面、 表皮、、工皮&其他注射方法之投予方劑型可以是單位劑型。 可以以傳統製峨術製備，這些技術包括將有效成分與醫藥載體 或輔型劑結合。 【實施方式】 實施例1 材料及方法 200906386 試劑 重組鹼性纖維母細胞生長因子(bFGF)由R仙系統(Minneapolis， MN)購得。 M^trigel 由 BD PharMingen (La Jolla, CA)購得。 , ST104P原液由ST104P粉末溶於食鹽水或PBS製備（lmg/ml 或其他所需濃度）。 ( 細胞培養 人類臍帶靜脈内皮細胞(HUVEC，3-6世代）由臍帶靜脈分離培養 於RPMI-1640 培養基(Life Technologies, Gaithersburg, MD) + 15% FCS (PAA，Austria) + 20#g/ml /ml 豬的肝素（Sigma • Chemical Co.)及100 # g/ml内皮細胞生長添加物 (Calbiochem，La Jolla，CA)。牛動脈内皮細胞（BAEC), 3TC 細 l' . v 胞，LL2 路易士肺癌細胞培養於 DMEM (Gibco BRL，Rochvi 1 le，MD) + 10% FCS + 2 mM 麩醯胺 + lOO/zg/ml 盤尼西林 + lOOyg/ml 鏈 黴素（Gibco BRL，Rochville，MD)於 37°C，5% C〇2 培養箱中。 人血管内皮細胞(EA.hy926)培養於DMEM(GibcoBRL，Rochville， MD) + 10% FCS + 100"M sodium hypoxanthine + 0. 4//M 胺喋呤， 16/zM 胸苷（HAT, Gibco BRL，Rochville，MD) + 2mM 麵醯胺 + 盤尼西林 + 100"g/ml 鍵徽素(Gibco BRL，Rochville 10 200906386 MD)於37°C，5% C〇2培養箱中。 細胞***試驗 ST104P對不同細胞的毒性以結晶紫染色方法分析。2_4 χ ι〇3細胞 /孔培養於96孔培養皿中。以不同濃度ST104P處理48小時，然 後用2. 5%戊二酸·在室溫固定15分鐘’以0.1%結晶紫（溶解於2〇% 甲醇）染色’以50%乙醇+ 〇. 1%乙酸溶解，然後以59〇nm測量 吸光值(ELISA讀取機，Dynatech Lab. Chantilly, VA) 細胞移動試驗 丘管内皮細胞以不同濃度ST104P處理6-12小時，然後以胰蛋白 酶取下’離心’加上DMEM + 0.1% BSA，將之種植於Boyden chamber 上腔中（1.2 x 105 /400# 1)。下腔加入 2〇〇# 1 DMEM，内含 100 //g/ml bFGF作為化學吸引劑。不含bFGF的做為負對照（任意移 Γ 動）。上下腔之間以聚碳酸酯膜分開（8/ζιη孔徑；Nucleopore, , Costar，Chambridge，ΜΑ)。膜塗上 〇· 005% 白明膠，在 5% c〇2， 37 C培養箱中培養2-4小時後，在膜上的細胞拿出來以乙醇固 定，10% Giemsa (Merck, Germany)染色，移動至反面的細胞在 顯微鏡下5個不同地方計數平均值土標準差。 管狀形成試驗 200906386Letters 30 (8): 1005-1008, 1989; Poh B L and Lim E S, Tetrahedron 46 (10): 3651-3658, 1990a; Poh B. L. et.al., Tetrahedron 46 (12): 4679-4386, 1990b). It is a polysulfated, cyclic, macrocyclic compound synthesized from tetrasene, and is also a water-soluble and cytotoxic compound. ST104P has been pointed out to have antiviral and antithrombotic effects (US Patent No. 5,166,173, No. 5,194,452, No. 5,312,837, No. 544,1983, No. 5,409,959), but ST104P has not been shown to have anti-angiogenic activity. Used to treat diseases associated with angiogenesis, including, but not limited to, cancer. In principle, compounds with antiviral and antithrombotic activity may also have other activities of 200906386. The present invention describes a compound composition ST anti-angiogenesis activity, which can be used to treat cancer and other angiogenesis-related diseases. . Another object of the present invention is to provide a method of treatment comprising ST class p to treat cancer and other angiogenesis-related diseases including, but not limited to, diabetic eye lesions, age-related macular degeneration, rheumatoid arthritis , degenerative arthritis, arteriosclerosis, ocular lens angiogenesis, retinal angiogenesis and systemic wolf therapy, lupus erythematosus, polyarteritis, sickle cell anemia, venous obstruction, arterial occlusion, neck obstruction. The present invention relates to a low toxicity polysulfide cyclic compound which inhibits angiogenesis activity in vitro and in vivo, ST104P. ST104P has excellent water solubility and low cytotoxicity. ST104P inhibits the secretion of matrix metalloproteinase (MIM) by vascular endothelial cells (Mim). JT104P also blocks the movement of vascular endothelial cells and the formation of # tubular. STi〇4p inhibits angiogenesis in chicken embryo chorioallantoic membrane and this effect is dose-dependent. Furthermore, repeated administration of STio^p to mice with Lewis lung cancer can delay the growth of tumors and prolong the lifespan of cancer mice. STi〇4P administration in the form of a burial tablet or eye drops can also inhibit corneal angiogenesis in rat eyes caused by bFGF. These results show that ST104P can inhibit angiogenesis and is used to treat cancer and other diseases and conditions associated with angiogenesis. One aspect of the present invention is to provide an anticancer drug for treating a human or a ride cancer by inhibiting intratumoral blood. The pharmaceutically acceptable carrier comprises four cyclic compounds of 4,5-dioxanaphthalene-2,7-di-branched with a methylene bridge. This carrier can be applied to the mouth and tongue. Lower, rectal, nasal, vaginal, abdominal, percutaneous, epidermal, intra-articular 'in cancer, intraocular, eye surface or injection method. The carrier can be applied to make lys, capsules, granules, solutions, emulsions, suppositories, patches, eye drops, burial tablets or powders. Another aspect of the present invention provides a composition for inhibiting angiogenesis, which inhibits angiogenesis in a human or animal cancer, comprising methylene groups from four 4,5-di-cyanophthalene-2,7-di-acid sub-drugs. The ring-connected compound of the bridge can be used in medicine. This carrier can be applied to the mouth, sublingual, rectal, nasal, vaginal, abdominal, percutaneous, epidermis, intracranial, intraocular, or ocular surface or Injection method. This carrier can be formulated for use as a spinner, capsule, granule, solution, emulsion, stopper, patch, eye drop, burial tablet or powder. Another aspect of the invention provides an angiogenic inhibition to treat a non-cancer condition or disease associated with other blood stasis in a human or animal. It comprises a cyclic compound linked by four methylene bridges of 4,5-dihydroxynaphthalene-2'7-disulfonic acid steroids in a carrier which can be used in medicine. This carrier can be applied to the mouth, Sublingual, rectal, nasal cavity, L-peritoneal cavity, percutaneous, epidermis, intra-articular, intra-cancer, intraocular, or ocular surface or 200906386, Lai 峨 峨, job, granule, solution, suppository, patch, Eye drops, burial tablets or powders. The thief record includes, but depends on, diabetic eye lesions, (four) Syrian screaming ship, eye lens vascular touch, visual angiogenesis, polyoxitis, sickle cell anemia, venous / 1 and plug, arterial occlusion, neck Department resistance (four) = hardening, cis-condulitis, systemic lupus, lupus erythematosus, and retreat, that is, inflammation. Pharmaceutically acceptable carriers can be used in the oral, sublingual, rectal, nasal, vaginal, intraperitoneal, intracanal, intra-articular, intraocular, ocular surface, epidermal, transdermal' and other injection methods. Pharmaceutically acceptable pours can be applied to lozenges, capsules, granules, solutions, emulsions, stoppers, patches, eye drops, burial tablets or powder formulations. Pharmaceutically acceptable rounds can be used for oral, sublingual, rectal, nasal, vaginal, abdominal, intracranial, intra-articular, intraocular (intravitreal, intraocular), ocular surface, epidermis, work skin & Dosage forms for other injection methods can be in unit dosage form. It can be prepared by conventional mashing techniques, including combining the active ingredient with a pharmaceutical carrier or a topical agent. [Examples] Example 1 Materials and Methods 200906386 Reagents Recombinant basic fibroblast growth factor (bFGF) was purchased from the Rxian system (Minneapolis, MN). M^trigel is commercially available from BD PharMingen (La Jolla, CA). The ST104P stock solution is prepared from ST104P powder dissolved in saline or PBS (1 mg/ml or other desired concentration). (Cell culture human umbilical vein endothelial cells (HUVEC, 3-6 generation) were isolated from umbilical vein cultured in RPMI-1640 medium (Life Technologies, Gaithersburg, MD) + 15% FCS (PAA, Austria) + 20#g/ml / Ml porcine heparin (Sigma • Chemical Co.) and 100 # g/ml endothelial cell growth supplement (Calbiochem, La Jolla, CA). Bovine arterial endothelial cells (BAEC), 3TC fine l'. v cells, LL2 Lewis Lung cancer cells were cultured in DMEM (Gibco BRL, Rochvi 1 le, MD) + 10% FCS + 2 mM branamine + lOO/zg/ml penicillin + lOOyg/ml streptomycin (Gibco BRL, Rochville, MD) at 37° C, 5% C〇2 in the incubator. Human vascular endothelial cells (EA.hy926) were cultured in DMEM (GibcoBRL, Rochville, MD) + 10% FCS + 100 "M sodium hypoxanthine + 0. 4//M Amine , 16/zM thymidine (HAT, Gibco BRL, Rochville, MD) + 2 mM acetochlor + penicillin + 100 " g/ml bondin (Gibco BRL, Rochville 10 200906386 MD) at 37 ° C, 5% C〇 2 in the incubator. The cell division test ST104P toxicity to different cells was analyzed by crystal violet staining method. 2_4 χ ι〇3 cells/well cultured in 9 In a 6-well culture dish, treated with different concentrations of ST104P for 48 hours, then fixed with 2.5% glutaric acid at room temperature for 15 minutes 'stained with 0.1% crystal violet (dissolved in 2% methanol) to 50% ethanol + 〇. Dissolve 1% acetic acid, then measure the absorbance at 59 〇nm (ELISA reader, Dynatech Lab. Chantilly, VA) Cell migration assay. The endothelial cells are treated with different concentrations of ST104P for 6-12 hours, then trypsin. Remove 'centrifugation' plus DMEM + 0.1% BSA and plant it in the upper chamber of the Boyden chamber (1.2 x 105 /400# 1). Add 2〇〇# 1 DMEM to the lower chamber, containing 100 //g/ml bFGF was used as a chemical attractant. bFGF-free was used as a negative control (arbitrary shifting). The upper and lower chambers were separated by a polycarbonate membrane (8/ζιη pore size; Nucleopore, Costar, Chambridge, ΜΑ). The membrane was coated with 〇·005% gelatin and cultured in a 5% c〇2, 37 C incubator for 2-4 hours. The cells on the membrane were taken out and fixed with ethanol, stained with 10% Giemsa (Merck, Germany). Cells that moved to the opposite side counted the mean soil standard deviation at five different locations under the microscope. Tubular formation test 200906386

Matrigel以冷的，未含血清 ’用的培養基稀釋成10 mg/ml。200# 1Matrigel was diluted to 10 mg/ml in cold, serum-free medium. 200# 1

構，8-12小時即完全成形。 血管内皮細胞在2-3小時開始形成網狀結 良形。培養後，以3%三聚甲醛固定。 基質金屬蛋白酶(MMP)酵素圖 血管内皮細胞分泌MMP情況以含G1%白明膠之勝_酵素 圖來分析。血管内皮細胞在達·滿時以不含血清的培養液洗兩 次’然後以不同濃度的ST104P處理24_48小時，培養夜收集後以 Bradford方法測其蛋白質濃度，然後以含〇1%办卯a白明膠 «igma，St· Louis，M0)的10%聚丙醯胺膠體電泳進行蛋白質分 離’電泳完後，凝膠以2. 5% Triton X-100洗兩次，然後在4_Structure, fully formed in 8-12 hours. Vascular endothelial cells begin to form a reticular formation in 2-3 hours. After the cultivation, it was fixed with 3% paraformaldehyde. Matrix metalloproteinase (MMP) enzyme map The secretion of MMP by vascular endothelial cells was analyzed by the G-% gelatin-based enzyme map. Vascular endothelial cells were washed twice with serum-free medium at the time of completion. Then they were treated with different concentrations of ST104P for 24 to 48 hours. After culture night collection, the protein concentration was measured by Bradford method, and then 〇1% was used. 10% polyacrylamide colloidal electrophoresis of white gelatin «igma, St. Louis, M0) for protein separation. After electrophoresis, the gel was washed twice with 2.5% Triton X-100, then at 4_

Tris-HCl, pH8. 0，l〇mM CaCh，0. 01% NaN3 中，在 37°C 下處理 12-24小時，然後用0.25%可馬式藍R-250溶解於5〇%甲醇+ 10%乙酸中染色一小時’在10%乙酸+腦甲醇中褪染，被隱 分解的白明膠在藍色凝膠中呈白色色帶。 ***試驗 方法如 Sheu 等人於 Anticancer Res. 18: 4435-4441，1998 中所 12 200906386 述’受精來享雞蛋由台灣省動物研究所(新竹，台灣）講得。在_ 渔度’阶下培養，第三天，在蛋殼上開—個正方形小窗，取出。 2 3ml的蛋白使***脫開，小窗以玻片封起來，蛋送回培養箱， 第八天時’ 50_100#g/ml ST1〇4p注入***上，而後每日觀察 至10-12 *，血管新生在最高峰時，以Zeiss聽攝影系統 (Ze1SS，Oberkochen，Ge簡ny)在立體顯微鏡下照相，每組十個 蛋’在血管新生抑侧存在下，失去血料致雞胚死亡，這是另 一個測試指標。 動物試驗 動物試驗在高雄榮總醫院進行，雄性小鼠(6_8週大， 成功大學動物t心’台灣）每籠四隻，自由取食、飲水。小鼠以 甲氧氟院麻醉進行試驗；有些小鼠會因甲氧眺_致死劑量而 犧牲。以 σ Reilly 等人(Cell 88: 277_285，1997)的方法麵 路易士肺癌細胞。1QG"1的2.5 X 1G6LLC路好肺癌細胞/ml PBS，庄入小鼠皮下。用流標尺測量肺腫瘤塊的長、短徑，腫瘤 塊體積=她X長徑χ Q 52，當_塊體舰丨.丽3 時’小鼠隨機分於兩組，SaHne對照組1〇隻，ST1〇4p組1〇隻， 測量腫瘤塊長、短徑至錢_死亡，同時記錄存活率，以 Kaplan-Mier存活分析法進行分析。 13 200906386 t 結果ST104P在不同細胞中毒性很低 ST104P在血管内皮細胞’包括赋，，HUVEC ’ Ea · &卿及非 血管内皮細胞，包括3T3，GH3，C6，HepG2，SK_Hep i及薦中 皆沒有顯示很強的毒性(未顯示數據）。如圖一所示，Μ讀抑 制不同細胞***的程度與劑量呈正比，但毒性很低，纟K 大 於 500#g/ml (〜350#M)。 ST104P抑制血管内皮細胞移動 分析ST104P對内皮細胞移動的作用是以卿做為化學吸引劑， 在_en槽中細胞移動的程度。如圖二所示，STl〇4p處理細胞6 小時後明顯的抑制血管内皮細胞的移動，與劑量成正比，其腿 為 100#g/ml。 ST104P干擾血管内皮細胞形成管狀 如圖三所示，ST腑⑽抑制職及EAh卿在 matrigel中形成管狀的能力。 基質金屬蛋白酶(MMP)分泌 血管新生可分絲個步驟，包括基質金屬蛋自軸Mp)的分泌， 血官内皮細胞***及移動。腳是含鋅内切蛋白酶，它在血管新 14 200906386 生時是在内皮細胞移動，入侵時分泌，用來分解基質。灯丨〇妒處 理的細胞培養液内之ΜΜΡ以明膠酶圖譜(zym〇gr即如）來分析。 ST104P抑制層―2及卿_9的分泌，甚至在⑺㈣mi即有作用 (圖四）。 ST104P抑制雞胚***的血管新生 利用八天大雞胚***的血管新生來看ST着的抑制作用。雞胜 ***在50仰ST1G4P漠度下血管新生被抑制_ (圖五）。在 測仰時，血管新生幾乎全被抑制，引起_雞胚死亡。 在雞胚發育中抑制血管新生。 重複投予ST104P抑制小鼠癌腫瘤生長 路易士肺癌細胞細驗小咖，經_4P(_g)在腫 瘤塊周圍皮下注射四次，STi〇4p處# -v ^ 處理的腫瘤塊比對照組小40% (圖 ^ m碰的怖其壽命明顯的延長(Ρ<0·01，圖 輪槪應，赫贿是 讨論 的抑制劑，也是血管内皮細胞形成管 本實驗發現ST104P是顧Ρ 200906386 狀的抑制劑。ST104P經由抑制血管新生的機制來抑制雞胚*** 血管新生以及癌生長。ST104P與其他血管新生抑制，例如TNp_47〇 (Moulton et. al.， Cricu 13"tion 99: 1726~1732 1999) angiostatin (0 Reilly et.al.， Cell 79: 315-328， 1994)，或 endostatin ((0 Reilly et.al·， Cell 88: 277-285 1997)不 同在於它並不直接抑制血管内皮細胞的***，它抑制細胞移動， 管狀形成，MMP分泌來阻止企管新生。相對於tnp—470或 endostatin ’ST104P是水溶性的，可以以saline或其他緩衝液 來投予。ST104P的毒性很低，由不同途徑投予動物都被容忍而沒 有不良反應。 實施例2 方法：Tris-HCl, pH 8. 0, l mM CaCh, 0.01% NaN3, treated at 37 ° C for 12-24 hours, then dissolved in 5 % methanol + 10 with 0.25% equine blue R-250 The staining in % acetic acid for one hour was faded in 10% acetic acid + brain methanol, and the gelatin which was secreted by decomposition was white in the blue gel. The allantoic membrane test method is described by Sheu et al. in Anticancer Res. 18: 4435-4441, 1998. 12 200906386 The 'fertilization of eggs is spoken by the Taiwan Provincial Institute of Zoology (Hsinchu, Taiwan). Cultivate under the _ degree of fishing, and on the third day, open a small square window on the eggshell and take it out. 2 3ml of protein dislodges the allantoic membrane, the small window is sealed with a slide, and the egg is returned to the incubator. On the eighth day, '50_100#g/ml ST1〇4p is injected into the allantoic membrane, and then observed to 10 per day. -12 *, at the highest peak of angiogenesis, photographed under a stereo microscope with Zeiss photographic system (Ze1SS, Oberkochen, Ge Jany), each group of ten eggs 'in the presence of angiogenesis inhibition, lost blood to chicken Embryo death, this is another test indicator. Animal testing Animal experiments were conducted at Kaohsiung General Hospital. Male mice (6-8 weeks old, successful university animal t-heart 'Taiwan), four cages per cage, were free to eat and drink. Mice were tested with methoxyfluoride anesthesia; some mice were sacrificed for methoxy-lethal doses. Lewis lung cancer cells were treated as described by σ Reilly et al. (Cell 88: 277_285, 1997). 1QG"1 of 2.5 X 1G6LLC road good lung cancer cells/ml PBS, Zhuang into the mouse subcutaneous. The long and short diameters of the lung tumor mass were measured with a flow scale. The tumor mass was = her long diameter χ Q 52. When _ block 丨 丨 丽 3, the mice were randomly divided into two groups, and the SaHne control group was only 1 In the ST1〇4p group, the tumor length and short diameter were measured to the death of the tumor, and the survival rate was recorded, and analyzed by Kaplan-Mier survival analysis. 13 200906386 t Results ST104P is very toxic in different cells ST104P in vascular endothelial cells 'including Fu, HUVEC 'Ea · & Qing and non-vascular endothelial cells, including 3T3, GH3, C6, HepG2, SK_Hep i and recommended No strong toxicity was shown (data not shown). As shown in Figure 1, the degree of sputum inhibition of different cell divisions is proportional to the dose, but the toxicity is very low, 纟K is greater than 500#g/ml (~350#M). ST104P inhibits vascular endothelial cell migration The effect of ST104P on endothelial cell migration is the extent to which cells move in the _en tank as a chemical attractant. As shown in Figure 2, STl〇4p treated cells significantly inhibited the movement of vascular endothelial cells after 6 hours, which was proportional to the dose, and the leg was 100#g/ml. ST104P interferes with the formation of tubular vascular endothelial cells. As shown in Figure 3, ST腑(10) inhibits the ability of EH Qing to form a tubular shape in matrigel. Matrix metalloproteinase (MMP) secretion Angiogenesis can be divided into several steps, including the secretion of matrix metal eggs from the axis Mp), blood cell endothelial cell division and movement. The foot is a zinc-containing endoprotease, which is secreted during endothelial cell migration and is secreted during invasion to decompose the matrix. The sputum in the cell culture solution treated with the enamel was analyzed by gelatinase map (zym〇gr). The secretion of ST104P inhibitory layer-2 and _9 is even effective in (7) (iv) mi (Fig. 4). ST104P inhibits angiogenesis in chicken embryo chorioallantoic membrane The inhibition of ST is observed by angiogenesis of the eight-day large chicken embryo chorioallantoic membrane. Chicken wins The urinary vesicle membrane is inhibited by angiogenesis at 50°ST1G4P indifference _ (Fig. 5). At the time of homing, angiogenesis is almost completely inhibited, causing _chicken embryo death. Inhibition of angiogenesis in chick embryo development. Repeated administration of ST104P inhibited the growth of Lewis cancer cells in Lewis cancer cells, subcutaneous injection of _4P(_g) four times around the tumor mass, and the tumor block treated with ST-〇4p at #-v ^ was smaller than the control group. 40% (Figure ^ m touched the life of its horrible extension (Ρ <0·01, Figure 槪 槪, He Bribe is the inhibitor of discussion, but also the formation of vascular endothelial cells. The experiment found that ST104P is Gu Yu 200906386 Inhibitors. ST104P inhibits angiogenesis and cancer growth in chick chorioallanes via a mechanism that inhibits angiogenesis. ST104P and other angiogenesis inhibitions, such as TNp_47〇 (Moulton et. al., Cricu 13"tion 99: 1726~1732 1999 Angiostatin (0 Reilly et.al., Cell 79: 315-328, 1994), or endostatin ((0 Reilly et.al., Cell 88: 277-285 1997) differs in that it does not directly inhibit vascular endothelial cells. Splitting, it inhibits cell movement, tubular formation, MMP secretion to prevent angiogenesis. Relative to tnp-470 or endostatin 'ST104P is water soluble, can be administered with saline or other buffer. ST104P is very low toxicity, different Pathway They are tolerated and no adverse reactions method of Example 2:

Hydron小體 緩釋聚合小體是由含有Sucralfate，驗性纖母細胞生長因子 (bFGF， 100 ng/pellet, R & D Systems, Minneapolis, MN)，不 同濃度的 ST104P (l〇(H〇〇〇 ng/peiiet)在 jjydr〇n 聚合物 (Polyhydroxyethylmethacrylate，Sigma, St. Louis, MS)做成。 處理大鼠角膜血管新生的方法都依Associati〇n f〇I· Research in Vision and Ophthalmology Resolution on the Use of Animals in Research手則來進行。雄性Spraque_Dawiey大鼠（goo〜350克， 臺灣國科會’動物中心)在無菌狀況下進行手術，大鼠以3 % 16 200906386 tHydron small body slow-release polymeric bodies are composed of Sucralfate, assay fibroblast growth factor (bFGF, 100 ng/pellet, R & D Systems, Minneapolis, MN), different concentrations of ST104P (l〇 (H〇〇 〇ng/peiiet) is made of jjydr〇n polymer (Polyhydroxyethylmethacrylate, Sigma, St. Louis, MS). Methods for treating corneal angiogenesis in rats are based on Associati〇nf〇I· Research in Vision and Ophthalmology Resolution on the Use The Animals in Research was carried out. Male Spraque_Dawiey rats (goo~350 g, Taiwan National Science Association 'Animal Center) underwent surgery under aseptic conditions, rats were 3 % 16 200906386 t

Isof lurane於氧氣/大氣1:1的狀況下麻醉，表面麻醉劑（〇. 4 % Benoxinate hydrochloride, Ciba Vision Ltd., Hettinger, Switzerland)直接塗在角膜表面。 巧鑷子由顳骨方向結合膜緣將眼睛暴露出來，用手術刀片 (Paragon No. 11, Maersk Medical Ltd., Sheffield, England) 在周邊角膜基質中間切一個板狀小袋(0.6x1. 5x1麵/深x長x寬）， 小袋離邊緣1. 5到2mm，將保存在-20°C的緩釋聚合小體用鑷子快 , 速的置入切開的角膜基質小袋中，抗生素藥貧（0. 3 % Gentamycin，Isof lurane was anesthetized under oxygen/atmosphere 1:1 conditions and a topical anesthetic (〇. 4% Benoxinate hydrochloride, Ciba Vision Ltd., Hettinger, Switzerland) was applied directly to the corneal surface. The scorpion was exposed by the tibia in the direction of the membrane edge, and a scalpel pouch (0.6x1. 5x1 surface/deep x) was cut in the middle of the surrounding corneal stroma with a surgical blade (Paragon No. 11, Maersk Medical Ltd., Sheffield, England). Length x width), the pouch is 1.5 to 2 mm away from the edge, and the sustained-release polymer body stored at -20 ° C is quickly and quickly placed into the cut corneal stroma bag, and the antibiotic is poor (0.3%). Gentamycin,

Alcon Cusi，Spain)塗於角膜表面以減少刺激及感染。Alcon Cusi, Spain) is applied to the corneal surface to reduce irritation and infection.

Hydron小體殖入 角膜小袋殖入：bFGF 小體（100 ng/pellet)，bFGF+ST104P 小體（100 ngbFGF+500 ng 或+1000 ngST104P)，或中含 PBS 小體。Hydron corpuscles colonize the corneal pocket: bFGF bodies (100 ng/pellet), bFGF+ST104P bodies (100 ngbFGF+500 ng or +1000 ngST104P), or PBS bodies.

表面投予ST104P 大鼠眼睛殖入含bFGF (100 ng/pellet)，然後以50 ul 2 % Methocel (methylcellulose eye drops, Novartis, Ophthalmology AG，Hettinger, Switzerland)含 PBS 或 ST104P (10〜100 ug/ml最後濃度）之眼藥水投予，每日三次。 生物顯微鏡檢查 17 200906386 、旦在第三、五、七天用解剖顯微鏡來觀察結果並照相。麻醉後 、則里新生血g的長度、寬度，驗尺(脱職城Nik〇n，T〇ky〇,Surface administration of ST104P rat eyes with bFGF (100 ng/pellet), followed by 50 ul 2 % Methocel (methylcellulose eye drops, Novartis, Ophthalmology AG, Hettinger, Switzerland) with PBS or ST104P (10~100 ug/ml) The final concentration of eye drops was administered three times a day. Biological microscopy 17 200906386 On the third, fifth, and seventh days, the results were observed with a dissecting microscope and photographed. After anesthesia, the length and width of the new blood g, the test rule (disengaged city Nik〇n, T〇ky〇,

Japan)來校正’新生血官的面積是以血管生長的三角形來估計， ^^^^(c〇〇iPix 995, Nikon, Tokyo, Japan)α 640x480 Pixel來計數，由二位不知道實驗設計的操作員來觀查，含有血管 的面積在«螢絲上描記，織贿相分析軟體(—η Image 4. 02，Scion, Mainland, USA)來分析。 組織切片分析 處理後，大鼠的角膜切出來，固定，包埋在石臘中，切成5 um 的薄片放在塗過Poly L-Lycine的玻片上，以Hematoxylin/Eosin 染色。 統計分析 用雙尾Mann-Whitney U試驗來比較組間的差異，p值〈〇. 〇5 設定為顯著差異，大鼠以標準作業程序手術，然後隨機分配至各 實驗組中。 結果： 加上ST104P於Hydron小體中減輕bFGF引起的角膜血管新生 200906386Japan) to correct 'the area of the new blood official is estimated by the triangle of blood vessel growth, ^^^^(c〇〇iPix 995, Nikon, Tokyo, Japan) α 640x480 Pixel to count, by two people do not know the experimental design The operator came to see that the area containing the blood vessels was analyzed on «Fluorescence, the bribery phase analysis software (—η Image 4. 02, Scion, Mainland, USA). Tissue section analysis After treatment, the cornea of the rat was cut out, fixed, embedded in paraffin, and cut into 5 μm slices on a Poly L-Lycine-coated slide and stained with Hematoxylin/Eosin. Statistical analysis The two-tailed Mann-Whitney U test was used to compare differences between groups, p values <〇. 〇5 were set to significant differences, rats were operated under standard operating procedures and then randomly assigned to each experimental group. Results: Plus ST104P in the Hydron body to reduce corneal angiogenesis caused by bFGF 200906386

測試 ST104P 的功效，含 pbs、bFGF、ST104P 或 bFGF+ST104PTest the efficacy of ST104P with pbs, bFGF, ST104P or bFGF+ST104P

Hydron小體殖入大鼠角膜來看血管新生。殖入砂⑶小體一般在 3〜7天之内會引起血管新生（表—），殖入pBS或ST1〇4p不會引起 血管新生，但是瘦入bFGF+ST104P小體與殖入bFGF小體的比較很 明顯的減少企管新生（圖八）。在第7天，加入ST104p減少bFGF 引起新生血管的長度（1.36±〇. 4 mm bFGF; 0.17±0. 06 mm bFGF+ST104P; P&lt;〇. 〇l)及面積（i.42±〇. 28 mm2 bFGF; 0.12±0.11mm2 bFGF+ST104P; P&lt;〇. 〇l)。結果顯示加上ST1〇4P有效的干擾bFGF 引起的管新生。 表一：殖入含bFGF及PBS或ST104P Hydron小體，7天後引起大 鼠角膜新生血管之長度及面積。Hydron corpuscles colonize the rat cornea to see angiogenesis. Colonization of sand (3) bodies usually cause angiogenesis within 3 to 7 days (Table -), colonization of pBS or ST1 〇 4p does not cause angiogenesis, but thin into bFGF + ST104P bodies and colonization of bFGF bodies The comparison is obviously reducing the number of entrepreneurs (Figure 8). On day 7, the addition of ST104p reduced the length of neovascularization induced by bFGF (1.36 ± 〇. 4 mm bFGF; 0.17 ± 0.06 bFGF + ST104P; P &lt; 〇. 〇 l) and area (i.42 ± 〇. 28) Mm2 bFGF; 0.12±0.11 mm2 bFGF+ST104P; P&lt;〇. 〇l). The results showed that ST1〇4P effectively interfered with tube regeneration caused by bFGF. Table 1: Colonization with bFGF and PBS or ST104P Hydron bodies, 7 days later caused the length and area of corneal neovascularization in rats.

Pellets No. of eyes Length (mm) Area (mm2) PBS n = :8 0 0 ST104P (500 ng) n : =8 0 0 ST104P (1000 ng) n : =8 0 0 bFGF n = :16 1. 36 士 0.40 1.42 ± 0.28 bFGF + ST104 n : =8 0.17 土 0.06* 0.12 ± 0.11* 殖入小體含 bFGF 100 ng/pellet，或 bFGF 100 ng/pellet + ST104P 5G0 ng/pellet ; *P&lt;〇. 01。 200906386 表面才又予ST104P抑制bFGF引起的角膜金管新生 測試ST104P的功效，含bFGF之Hydr〇n小體殖入大鼠雙眼角 膜中而後一眼投予含PBS之眼滴液，-眼投予含ST104P (UH00 ug/ml)之眼滴液，每天三次。表面投予st騰依劑量抑制卿 引起的角赌生血官的長度及面積（圖九），眼親含射 ST104P沒有抑制bFGF引起的血管新生，但是含漏4p議^/ιη1 的眼滴液與PBS比較有明顯的抑制血管長度(Q遍.15mm相對於 1.36±0.4 mm; P&lt;0.01)及血管面積（〇·28±〇 ΐ9 -相對於 1· 42±0· 28 mm ; Ρ&lt;〇· 01) ’組織切片分析顯示ST1G4p抑制血管新 生且對眼球其他細胞沒有毒性（圖十）。總結起來，ST膽埋殖或 表面投予可以抑制眼角膜血管新生。 其他ST1G4P可以治療與血管新生有關的疾病如下： 動脈硬化（Atherosclosis) 血管新生於動脈硬化斑内的血管_是詳知的狀況，在正常血管 中’血管滋養管叢是分佈在親外虹，但是在舰硬化時，這 些管叢變多’且人侵顺化_的_上，硬化血管内常見許 多巨嗟細胞和mast CeU。這些細胞已知會刺激血管新生。引起 動脈硬化其中的-個因子是LDL氧化，這也會引起血管内皮細胞 祠亡。Endostatin給予氧化LDL處理的内皮細胞可以減少小鼠動 20 200906386 脈硬化（Ren et.al.，Methods Find Exp Clin Pharmacol 24(4): 159-199，2002)。ApoE缺乏小鼠以血管新生抑制劑Endostatin 或TNP-470處理可以減少血管内膜血管新生及硬化斑(M〇ult〇n et.al— Circulation 99: 1726-1732，1999)。 風濕性關節炎（Rheumatoid arthritis) 關節發炎的疾病例如風濕性關節炎是引起失能的主要疾病之一。 風濕性關節炎在入侵軟骨及硬骨前常見關節滑膜增厚，其内細胞 數增加。組織增大，需要血管來提供養分及氧氣，阻擾滑膜血管 新生是一個可望有效的治療法。有實驗顯示血管新生抑制劑， protease-activated kringles 1-5，可以減緩膠原蛋白引起的老 鼠關節炎，臨床上看到抗血管新生治療法可以阻止新血管形成， 減小滑膜擴大因而能治療風濕性關節炎（Sumariwalla etal.，Pellets No. of eyes Length (mm) Area (mm2) PBS n = :8 0 0 ST104P (500 ng) n : =8 0 0 ST104P (1000 ng) n : =8 0 0 bFGF n = :16 1. 36 ± 0.40 1.42 ± 0.28 bFGF + ST104 n : =8 0.17 soil 0.06* 0.12 ± 0.11* The colony contains bFGF 100 ng/pellet, or bFGF 100 ng/pellet + ST104P 5G0 ng/pellet ; *P&lt;〇. 01 . 200906386 On the surface, ST104P was also used to inhibit the effect of bFGF on the corneal tube new test ST104P. The Hydr〇n small body containing bFGF was added to the cornea of the rat's eyes, and the eye was administered with PBS-containing eye drops. Eye drops of ST104P (UH00 ug/ml) three times a day. The surface was administered with the length and area of the blood-stained blood-stained sputum caused by the dose-reducing sputum (Fig. 9). The eye-containing ST104P did not inhibit the angiogenesis caused by bFGF, but the eye drops containing the leaked 4p^^ιη1 PBS has significant inhibition of vessel length (Q.15mm vs. 1.36±0.4 mm; P&lt;0.01) and vascular area (〇·28±〇ΐ9 - relative to 1.42±0·28 mm; Ρ&lt;〇· 01) 'Tone section analysis showed that ST1G4p inhibited angiogenesis and was not toxic to other cells of the eye (Figure 10). In summary, ST bile colonization or surface administration can inhibit corneal angiogenesis. Other ST1G4P can treat diseases associated with angiogenesis as follows: Atherosclosis Angiogenesis in the atherosclerotic plaque _ is a well-known condition in which the vascular nourishing tube bundle is distributed in the pro-external rainbow, but When the ship is hardened, these tube bundles become more and more invaded, and many giant scorpion cells and mast CeU are common in the hardened blood vessels. These cells are known to stimulate angiogenesis. One of the factors that cause arteriosclerosis is LDL oxidation, which also causes the death of vascular endothelial cells. Administration of endostatin to oxidized LDL-treated endothelial cells can reduce mouse motility (Ren et. al., Methods Find Exp Clin Pharmacol 24(4): 159-199, 2002). ApoE-deficient mice treated with the angiogenesis inhibitor Endostatin or TNP-470 can reduce endovascular angiogenesis and sclerosing plaques (M〇ult〇n et.al- Circulation 99: 1726-1732, 1999). Rheumatoid arthritis Diseases of joint inflammation such as rheumatoid arthritis are one of the major diseases that cause disability. Rheumatoid arthritis is often thickened in the joint synovial membrane before invading cartilage and hard bone, and the number of cells in it increases. Tissue enlargement requires blood vessels to provide nutrients and oxygen, and obstruction of synovial blood vessels is a promising treatment. Experiments have shown that angiogenesis inhibitors, protease-activated kringles 1-5, can slow down collagen-induced arthritis in mice. Clinically, anti-angiogenic therapy can prevent the formation of new blood vessels, reduce synovial enlargement and thus treat rheumatism. Arthritis (Sumariwalla etal.,

Arthritis Res Ther 15(1): 32-39，2002)。膠原蛋白引起的老 鼠關節炎，在經投予endostatin後會抑制骨刺形成及骨絡破壞 (Kurosaka et.al·， Ann Rheum Dis 62: 677-697， 2003)。 全身性狼瘡（Systemic lupus) 抗發炎及免疫調節劑具有治療全身性狼瘡的效果。紅斑性狼瘡皮 下症狀是慢性的，不規則性的損傷。有些有全身性的疾病。皮下 症狀可分成三種（1)全身性狼瘡血管損傷處（2)慢性皮下狼瘡 21 200906386 間質損傷處（3)深度或水泡型紅斑性狼瘡之特殊損傷處。 Thalidomide, —個抗發炎，免疫調節，抗血管新生劑，明顯改盖 全身性及皮下性紅斑狼瘡（Alfadley et.al.，J Am AeadArthritis Res Ther 15(1): 32-39, 2002). Collagen-induced arthritis in rats inhibits spur formation and osteogenesis after administration of endostatin (Kurosaka et. al., Ann Rheum Dis 62: 677-697, 2003). Systemic lupus Anti-inflammatory and immunomodulatory agents have the effect of treating systemic lupus. Subcutaneous symptoms of lupus erythematosus are chronic, irregular lesions. Some have systemic diseases. Subcutaneous symptoms can be divided into three types: (1) systemic lupus vascular injury (2) chronic subcutaneous lupus 21 200906386 interstitial lesions (3) deep or blister-type lupus erythematosus special lesions. Thalidomide, an anti-inflammatory, immunomodulatory, anti-angiogenic agent, markedly modified systemic and subcutaneous lupus erythematosus (Alfadley et.al., J Am Aead

Dermatol 48(5): S89-S91, 2003; Housman et. al., Dermatol 139. 50-54, 2003; Ossandon et.al. , Clin Expt Rheumatol 2〇： 709-718, 2002; US 專利第 6, 518, 298 號）。 ’ 退化性關節炎（Osteoarthritis) 血管新生也可能與退化性關節炎有關。血管新生促進因子，例如 VEGF，與退化性關節炎的嚴重性有關，退化性關節炎的軟骨種植 於雞胚尿囊财，會吸引其血管增生人侵，表示退化性關節炎的 軟骨其抗血管新生的能力被破壞。（SmithJ. 〇. etal.，J.Dermatol 48(5): S89-S91, 2003; Housman et. al., Dermatol 139. 50-54, 2003; Ossandon et.al., Clin Expt Rheumatol 2〇: 709-718, 2002; US Patent No. 6, 518, No. 298). 'Osteoarthritis angiogenesis may also be associated with degenerative arthritis. Angiogenesis-promoting factors, such as VEGF, are associated with the severity of degenerative arthritis. Cartilage of degenerative arthritis is planted in chicken embryo urinary tract, which attracts vascular hyperplasia, which is an anti-angiogenic cartilage of degenerative arthritis. The ability of new students is destroyed. (SmithJ. 〇. etal., J.

Sci 8: 849-857, 2_)。抗血管新生劑可抑制骨路被破壞因而可 能可以抑制退化性關節炎的惡化（B〇nnet cs and %1吐臥，Sci 8: 849-857, 2_). Anti-angiogenic agents can inhibit the destruction of the bone path and may therefore inhibit the deterioration of degenerative arthritis (B〇nnet cs and %1 spit,

Rheumatol 44: 7-16，2005)。 (Age-related macular degeneration) 與年齡相_視覺退化是引起眼瞎最主要的賴之…脈絡膜上 血管新生使視網膜受損。高度近視，血管條紋（angi〇id streaks) ’其他發炎性疾病也會㈣脈、賴上的血管新生。Rheumatol 44: 7-16, 2005). (Age-related macular degeneration) and age-related visual degradation is the most important cause of eyelids... choroidal neovascularization damages the retina. High myopia, vascular streaks (angi id streaks) ‘other inflammatory diseases will also (4) veins, vascular neovascularization.

Angl〇Statin，—個已知的血管雛抑侧，纽的減小脈絡臈上 22 200906386 血管新生的損害面積（Lai et.al.，Investigative Ophthalmol and Vessel Sci 42(10): 2401-2407， 2001)。 糖尿病眼病變（Diabetic retinopathy) 許多抗血管新生藥都在臨床試驗中，例如治療視網膜疾病，包括 視網膜中央靜脈阻塞，視網膜分歧靜脈阻塞及糖尿病眼病變，例 如VEGF，它是血管新生刺激因子，也是血管滲透因子。抑制VEGF 的藥劑被用來治療糖尿病眼病變及年齡相關黃斑視覺退化 (Jampol LM，Am，Acad，of Ophthamol 2002 年年會· jjans-PeterAngl〇Statin, a known vasoconstriction, the reduction of choroidal ridges 22 200906386 The area of angiogenesis damage (Lai et. al., Investigative Ophthalmol and Vessel Sci 42(10): 2401-2407, 2001 ). Diabetic retinopathy Many anti-angiogenic drugs are used in clinical trials, such as treatment of retinal diseases, including central retinal vein occlusion, retinal venous occlusion, and diabetic eye lesions, such as VEGF, which are angiogenic stimulators and blood vessels. Osmotic factor. Agents that inhibit VEGF are used to treat diabetic eye lesions and age-related macular degeneration (Jampol LM, Am, Acad, of Ophthamol 2002 Annual Meeting jjans-Peter

Hammes，苐 59 次 Ann Sci Sessions of ADA， 1999)。 視網膜/脈絡膜血管新生（Retinal/Ch〇rOidai neovascularization) 與視網膜/脈絡膜血管新生有關的疾病包括，但不限於，糖尿病眼 病變，年齡相關黃斑視覺退化，鎌刀型細胞貧血症，肉狀瘤，梅 毒，全身性紅斑狼瘡，靜脈阻塞，動脈阻塞，頸動脈阻塞，慢性 葡萄膜炎/玻璃體炎，分枝桿菌感染等，其他疾病包括，但不限於， 眼角發紅，以及其它不正常血管纖維增生的增生性玻璃體視覺病 變(US專利第6, 518, 298號）。 【圖式簡單說明】 圖-係ST104P在不同細胞中，對於細胞***所產生之劑量相關的 23 200906386 ^響。細胞以10-500/zg/mi ST104P &lt; PBS處理48 ,h時後以結 ^紫方法決定細胞存活率。每健據代表4次重複試驗的平均值士 才下準差星號代表與對照組比較具統計顯著差異(p〈〇. 〇〇1)。 圖一係顯* ST104P會抑制血管内皮細胞的遷移。（a) sti〇4P對 於血管内皮細胞遷移所產生的影響。將BAEC養殖於6-孔培養廉中 (1 X 10細胞/孔)並以特定劑量之ST1〇4p處理12小時。胰蛋白 晦處理下來的細胞放置於BGyden槽的上孔中，下孔則加入 bFGFdOO/zg/ml)作為化學吸引劑，使細胞開始進行遷移。最後， 將在膜上移動的細胞染色並計數。（B)細胞的遷移受到ST1〇4p的 抑制，且此效果為劑量相關。每點代表3次重複試驗的平均值土樣 本平均值標準誤。 圖三係ST104P破壞血管内皮細胞形管作用。内皮細胞種植於 matrigel包膜的培養皿中，經ST104P處理8小時即在4〇〇倍顯微 鏡下觀察管狀構造。對照組為未經ST104P處理之細胞。（A) EA. hy9236 細胞。（B) BAEC 細胞。 圖四係ST104P減少血管内皮細胞分泌MMP的作用。baeC或EA、 hy926 細胞以 或 ST104P (10-500#g/ml)處理 24 小時後， 培養液内之以 gelatin-PAGE zymography 來分析。（A) 24 200906386 EA. hy9236 細胞。（B) BAEC 細胞。 圖五係STHMP對雞胚血管新生的作用。s天大雞胚***加上 saline或订猜⑽⑽㈣⑷培養仙小時’***上之血 官新生狀況在解纖魏下觀察紀錄。Saline處理的***血管 新生狀况正¥活躍’而ST104P處理的則企管網被損壞。 # 圖六係小鼠Lewis肺癌被ST104P抑制。Lewis肺癌細胞種植於 小鼠背部皮下，sti〇4P aoo/zg/o. lml PBS)從 day 〇 t〇 day 1〇 如圖所示，注入肺腫瘤塊周圍，紀錄腫瘤塊大小，每點代表隻 小鼠平均值±樣本平均值標準誤。重複試驗得到相似結果。 圖七係/主射ST104P可延長種植癌細胞小鼠的壽命。Lewjs肺癌細 • 胞種植於小鼠背部皮下，ST1〇4P (l〇0#g/〇. lml PBS)從day 0 to 《 daylO如圖所示，注入肺腫瘤塊周圍，紀錄腫瘤塊大小，每點代 表10隻小鼠平均值±樣本平均值標準誤。紀錄小鼠存活率，以 Kaplan-Meier法分析得知ST104P顯著的延長小鼠壽命（P&lt; 0. 001)。重複試驗得到相似結果。 圖八係加入ST104P之Hydron小體中抑制bFGF引起角膜血管新生 作用。（A)·殖入bFGF小體（100 ng/pellet)，7天後大鼠角膜血管 25 200906386 新生圖片（左）；殖入 bFGF+ST104P 小體（bFGF 100 ng/pellet ^ ST104P500ng/pellet)，7天後大鼠角膜血管新生圖片（右）。⑻ 殖入bFGF或bFGF+ST104P小體後，大鼠角膜血管新生長度及面 積。（a)新生血管長度；（b)新生血管面積。測量時間3〜7天，數 據是平均±8£1 (每組8隻）；*p&lt;〇. 〇1。 圖九係表面投予ST104P抑制bFGF引起角膜血管新生作用。（A). 左圖’眼滴液含PBS。中圖，眼滴液含5〇 ug/ml ST1〇4p。右圖， 眼滴液含1GG ug/ml ST黯。眼餘投予7天，錄代表Hydr〇n 小體殖入位置。⑻.表面投予ST1G4P依劑量抑制_引起之血 管新生。（a)血管長度：測量時間是第7天，劑量1〇〜1〇〇 _卜 ⑹血管面積：測量時間是第7天，劑量.⑽ug/mi。敬〇. 〇5 ; **Ρ&lt;0· 01 ;數據是平均±SEM (每組8隻）。 圖十係STHMP _液處理7天’隨引起从細血储生的組 織切片分析。大鼠角膜在處理7天後切下處理，Hemat〇xyiin/E〇sin 木色在200七放大下觀察。⑷箭頭指示之處顯現㈣殖入後 在角膜基質内有許多空管狀的新生血管構造’内含紅血球，基質 腫脹及有單贿炎細胞人侵。⑹·〜⑷絲投予議p ι〇 — ⑹；血管新生有雜卩制，表面好議p5()ug/mi⑹.；剛 ⑷.；則顯現完全抑制血f新生，角膜組織非常正常。 26Hammes, 苐 59 times Ann Sci Sessions of ADA, 1999). Retinal/Ch〇rOidai neovascularization Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic eye lesions, age-related macular degeneration, sickle cell anemia, sarcoidosis, syphilis, Systemic lupus erythematosus, venous obstruction, arterial occlusion, carotid artery occlusion, chronic uveitis/vitritis, mycobacterial infection, etc. Other diseases include, but are not limited to, redness of the corners of the eyes, and hyperplasia of abnormal vascular fibrosis Sexual vitreous visual lesions (US Patent No. 6,518,298). [Simple diagram of the diagram] Figure - is the dose-related 2310406386 of the cell division caused by ST104P in different cells. The cells were treated with 10-500/zg/mi ST104P &lt; PBS for 48 h, and then the cell viability was determined by the purple method. The average value of each of the four replicates was statistically significant (p<〇. 〇〇1) compared with the control group. Figure 1 shows that *ST104P inhibits the migration of vascular endothelial cells. (a) The effect of sti〇4P on vascular endothelial cell migration. BAECs were cultured in 6-well cultures (1 X 10 cells/well) and treated with a specific dose of ST1〇4p for 12 hours. The trypsin-treated cells were placed in the upper well of the BGyden trough, and the lower well was added to bFGFdOO/zg/ml as a chemoattractant to allow the cells to begin to migrate. Finally, the cells moving on the membrane were stained and counted. (B) Migration of cells was inhibited by ST1〇4p, and this effect was dose-related. Each point represents the average value of the three replicates. Figure 3 shows the effect of ST104P on the destruction of vascular endothelial cells. The endothelial cells were seeded in a matrigel-coated petri dish, and the tubular structure was observed under a 4 〇〇 microscope under the treatment of ST104P for 8 hours. The control group was cells that were not treated with ST104P. (A) EA. hy9236 cells. (B) BAEC cells. Figure 4 shows that ST104P reduces the secretion of MMP by vascular endothelial cells. After 24 hours of treatment with baeC or EA, hy926 cells or ST104P (10-500#g/ml), the culture medium was analyzed by gelatin-PAGE zymography. (A) 24 200906386 EA. hy9236 cells. (B) BAEC cells. Figure 5 shows the effect of STHMP on angiogenesis in chicken embryos. s day big chicken embryo chorioallantoic membrane plus saline or guess (10) (10) (four) (4) cultured celestial hours 'the blood on the urinary sac film official status in the defibrillation Wei observation record. The urinary vascular neovascularization condition of Saline-treated sputum was positively active, while the ST104P-treated vascular network was damaged. #图六系 Mouse Lewis lung cancer was inhibited by ST104P. Lewis lung cancer cells were implanted subcutaneously in the back of mice, sti〇4P aoo/zg/o.lml PBS) from day 〇t〇day 1〇, as shown in the figure, injected into the lung tumor mass, record the size of the tumor block, each point represents only Mouse mean ± sample mean standard error. Repeat the test to get similar results. Figure 7 / main shot ST104P can extend the lifespan of cancer-bearing mice. Lewjs lung cancer fine cells were implanted subcutaneously in the back of the mouse, ST1〇4P (l〇0#g/〇.lml PBS) from day 0 to “daylO as shown, injected into the lung tumor mass, record the tumor mass size, each Points represent mean values of 10 mice ± standard error of sample mean. The survival rate of the mice was recorded, and it was found by Kaplan-Meier method that ST104P significantly prolonged the lifespan of the mice (P &lt; 0. 001). Repeat the test to get similar results. Figure VIII is a method of inhibiting bFGF-induced corneal angiogenesis by adding ST104P Hydron bodies. (A) · colonization of bFGF bodies (100 ng / pellet), 7 days after rat corneal blood vessels 25 200906386 newborn pictures (left); colonization of bFGF + ST104P bodies (bFGF 100 ng / pellet ^ ST104P500ng / pellet), Photograph of rat corneal angiogenesis after 7 days (right). (8) Corneal angiogenesis length and area after colonization of bFGF or bFGF+ST104P bodies. (a) length of neovascularization; (b) area of neovascularization. The measurement time is 3 to 7 days, and the data is an average of ±8 £1 (8 per group); *p&lt;〇. 〇1. Figure 9 shows the effect of ST104P on the inhibition of bFGF on corneal angiogenesis. (A). Left image of the eye drops containing PBS. In the middle picture, the eye drops contain 5 〇 ug/ml ST1 〇 4p. On the right, the eye drops contain 1GG ug/ml ST黯. The eyes were given for 7 days, and the record represented the Hydr〇n small body colonization position. (8). Surface administration of ST1G4P is dependent on dose inhibition _ caused by angiogenesis. (a) Length of blood vessel: The measurement time is the 7th day, and the dose is 1〇~1〇〇 _b (6) Blood vessel area: The measurement time is the 7th day, the dose is (10) ug/mi. 〇. 〇5 ; **Ρ&lt;0· 01 ; data are mean ± SEM (8 per group). Figure 10 is a series of STHMP_liquid treatments for 7 days&apos; as a result of tissue sectioning from hematopoietic storage. The rat cornea was excised after 7 days of treatment, and the Hemat〇xyiin/E〇sin wood color was observed under a magnification of 200. (4) Appearance of the arrow indicates (4) After colonization There are many empty tubular neovascular structures in the corneal stroma containing red blood cells, swelling of the matrix and invasion of a single brigitis cell. (6)·~(4) silk cast to discuss p ι〇 — (6); angiogenesis has a heterozygous system, the surface is good to discuss p5 () ug / mi (6).; just (4).; appears to completely inhibit blood f newborn, corneal tissue is very normal. 26

Claims (1)

200906386 十、申請專利範圍： 種用於練與血管難之癌錢赫之組合物，包 括個4,5—殘基秦_2,7_二確酸以亞甲基橋聯所構成四 個次齡efeHeM雜化合物，及辟紅可接受之 載體。 M艮據申請專利範圍第1項之組合物，其適用於口、舌下、 直腸鼻腔、***、腹腔、經皮、表皮，關節内，癌内、 眼球内、眼球表面或注射方式投藥。 3. 根射料利範圍第！項之組合物，其係製成錠劑、膠囊、 顆粒、溶液、乳劑、塞劑、貼布、眼藥水、埋殖片或粉劑。 4. —-觀於抑制血管新生之組合物，包括—個^—二縣茶 2, 7 一喊以亞甲基橋聯所構細個次體(如聰❿） 的環狀化合物’及其上可接受之載體。 5. 根據申請專利範圍第4項之組合物，其適用於口、舌下、 直腸、鼻腔、***、腹腔、經皮、表皮，關節内，癌内、 眼球内、眼球表面或注射方式投藥。。 6·根據申請專利範圍第4項之組合物，其係製成錠劑、膠囊、 顆粒、溶液、乳劑、塞劑、貼布、眼藥水、埋殖片或粉劑。 種用於治療與血管新生有關之非癌症病況或疾病之电人 1勿’包括-個4, 5-二經基萘祭二績酸以亞甲基橋聯職 成四個次體(teferic)的環狀化合物，及其醫藥上可接 27 200906386 8. 根據申請專利範圍第7項之組合物，其中該病況或疾病是 糖尿病眼病變。 9. 根據申請專利範圍第7項之組合物，其中該病況或疾病是 鐮刀型細胞貧血症。 10. 根據申請專利範圍第7項之組合物，其中該病況或疾病是 靜脈阻塞。 11. 根據申請專利範圍第7項之組合物，其中該病況或疾病是 動脈阻塞。 12. 根據申請專利範圍第7項之組合物，其中該病況或疾病是 年齡相關黃斑視覺退化。 13. 根據申請專利範圍第7項之組合物，其中該病況或疾病是 動脈硬化。 14. 根據申請專利範圍第7項之組合物，其中該病況或疾病是 風濕性關節炎。 15. 根據申請專利範圍第7項之組合物，其中該病況或疾病是 全身性狼瘡。 16. 根據申請專利範圍第7項之組合物，其中該病況或疾病是 退化性關節炎。 17. 根據申請專利範圍第7項之組合物，其適用於口、舌下、 直腸、鼻腔、***、腹腔、經皮、表皮，關節内，癌内、 眼球内、眼球表面或注射方式投藥。 28 200906386 18.根據申請專利範圍第7項之組合物，其係製成錠劑、膠 囊、顆粒、溶液、乳劑、塞劑、貼布、埋殖片、眼藥水 或粉劑。 29200906386 X. The scope of application for patents: A composition for the treatment of vascular difficult cancer Qianhe, including a 4,5-residue Qin_2,7-di-acid consisting of methylene bridges four times Age efeHeM hetero compound, and a carrier acceptable for redness. M. The composition according to claim 1 of the patent application, which is suitable for administration in the mouth, sublingual, rectal nasal cavity, vagina, abdominal cavity, percutaneous, epidermis, intra-articular, intra-cancer, intraocular, intraocular or injection manner. 3. The scope of the root shots is the first! A composition comprising a tablet, a capsule, a granule, a solution, an emulsion, a stopper, a patch, an eye drop, a burial tablet or a powder. 4.--A composition for inhibiting angiogenesis, including -^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Acceptable carrier. 5. A composition according to claim 4 of the patent application, which is suitable for administration in the mouth, sublingual, rectal, nasal, vaginal, abdominal, percutaneous, epidermal, intra-articular, intra-cancer, intraocular, ocular surface or injection mode. . 6. A composition according to item 4 of the scope of the patent application, which is in the form of a tablet, a capsule, a granule, a solution, an emulsion, a suppository, a patch, an eye drop, a burial tablet or a powder. An electrician used to treat a non-cancer condition or disease associated with angiogenesis 1 does not include a 4, 5-di-naphthyl-naphthalene-supplemental acid with a methylene bridge to form a four-part (teferic) A cyclic compound, and a pharmaceutically acceptable composition thereof. 27 200906386 8. The composition according to claim 7, wherein the condition or disease is a diabetic eye lesion. 9. The composition according to claim 7, wherein the condition or disease is sickle cell anemia. 10. The composition according to claim 7 wherein the condition or disease is venous obstruction. 11. The composition of claim 7, wherein the condition or disease is arterial occlusion. 12. The composition according to claim 7, wherein the condition or disease is visual deterioration of age-related macular. 13. The composition of claim 7, wherein the condition or disease is arteriosclerosis. 14. The composition according to claim 7, wherein the condition or disease is rheumatoid arthritis. 15. The composition according to claim 7, wherein the condition or disease is systemic lupus. 16. The composition according to claim 7, wherein the condition or disease is degenerative arthritis. 17. The composition according to claim 7 of the patent application, which is suitable for administration in the mouth, sublingual, rectal, nasal, vaginal, abdominal, percutaneous, epidermal, intra-articular, intra-cancer, intraocular, intraocular or injection modes. 28 200906386 18. The composition according to item 7 of the patent application, which is in the form of a tablet, a capsule, a granule, a solution, an emulsion, a stopper, a patch, a burial tablet, an eye drop or a powder. 29