玖、發明說明： 【發明所屬技術"領诚】 相關申請的對照檢索 本次申請符合美國法典第35卷119條(e)款有關美國國内臨 時申請之規定，編號爲No· 60M26,363，申請提出日期爲 2〇〇2年11月I5日’中請内容通過對照檢索以並人本次公開 披露之詳細資料。 發明領域 本項發明屬於制藥學領域，特別是關於預防及治療疾 病的抗血管新生藥物領域。 、 t先前】 發明背景 血官新生是在原有毛細血管基礎上生長出新血管結 的過程。在此過財，蛋自水_的影響使基底膜對内。 細胞的吸職力減弱，導致⑽細胞麟基«。這些細 胞於是_母體血管、分裂繁殖、並且分化而成新的血管 結構(瑞薩烏啊年《自然界》第386期671 674頁；威爾；丁 等人1995年《細胞分子生物學研究》糾【4】期加加 頁）。據發現’許多不同的生物學因數都具有控制血管形成 的作用（布«諾等人㈣年《生物化學新趨勢》幻川 】期251-256頁’·弗克曼與德阿摩爾職年《細胞學》第幻 !:3=5頁)。這其中就包括生長因數、細胞表面受體蛋 白^蛋白㈣制劑和細胞外間質蛋白等具有多種功能的 蛋白負（亞琛與斯泰克觸年《國際實驗病理學期刊》第乃 /月255 265頁，迪瓦萊拉賈與裏奇蒙德〗999年《藥理學新趨 勢》第20【4】期151_156頁；哈納漢㈣年（科學》第爪 期似〇頁；梅森皮爾等人1997年《科學》第277期私6〇頁 5 ，蘇裏等人1996年《細胞學》第87期1171-1180頁；薩托等 人1995年《自然界》第376期7()-74頁；米格納提與瑞夫金 1996年《酶與蛋白》第49期117收頁；品圖奇等人觸年 《血栓和止血研究》第22【6】期517-524頁；維隆與薩奇 ^95年《美國病理學期刊》第147【4】期873_883頁；布魯 ι〇克斯等人1994年《科學》第264期569-5η頁；科赫等人測 年《自；、、i界》第376期517-519頁）。血管新生過程的複雜性 和控制因數的多樣性爲我們提供了一系列研究體内控制血 官形成治療手段的研究課題。 血笞新生正常情況下都很有規律，它是胚胎發育、生 長和創傷癒合及女性生殖周期等許多生理現象的重要步驟 15 (威爾ί丁與克裏斯特1996年《自然科學》第幻期抓⑽頁； 古奇爾與羅傑斯1995年《微循環》第2期329-343頁；奥古 斯丁等人1995年《美國病理學期刊》第147【2】期339-351 頁）。病變性血管增生過程中的某些重要步驟是一些人類疾 病發展的主要因素，包括腫瘤擴散、轉移性癌症、糖尿病 2〇視、”罔膜/病曼、風濕性關節炎和牛皮癖等其他炎症疾病（弗克 曼1995年《自然醫學》第1【1】期27-31頁；沃爾什1998年 《風濕病學》第38【2】期103-112頁；希爾利等人1998年 《人類生殖新知》第4【5】期736-740頁）。在這些情況下， 疾病的t展往往受到長期缺乏規律的血管新生所影響。例 6 =，在風濕性關節炎巾，新生成的毛細血管侵人關節，破 壞軟骨組織。而在糠尿病視網翻變中，視_中形成新 ，毛細血管’不僅侵人玻璃體，而且造成出血現象，並最 終導致失明。腫_生姊轉移在很纽度上也依賴血管 ，生。大多數原發性實體軸都要經過—段相當長的無血 管時期，在此期間腫瘤的生長大約爲直徑W毫米左右。腫 瘤生長到适樣大小之後，腫瘤細胞就可以通過簡單的被動 擴散吸收必要的氧和其他營養物質。這些只能在顯微鏡下 觀察到的Μ塊㈣會啓動Μ生成，它們彻四周的血 笞催生出新的毛細血管，形成腫瘤塊的血管化，使得腫 瘤和惡性細胞能夠擴散或轉移到身體其他部位。儘管我們 對病變性血管新生過程中發生的生物變化已經有了長足的 瞭解，但至今我們也沒有找到任何有效的藥物能夠在體内 對血管新生進行控制。能夠控制血管新生的有效治療手段 將會極大地緩解不少人類的病痛。 傳統的方法是通過篩選具有某些預期藥物特性的人造 化合物並在體内進行毒性和療效試驗以便開發出相應的藥 物。以這種方式挑選出來的化合物通常在體内具有毒性， 而且無法成功地開發出在疾病治療方面的有效血管新生抑 制劑。最近以來，分子生物學的技術已經開始應用到血管 新生抑制劑的開發上。諸如血管阻斷素angiostatin(奥萊利 等人1994年《細胞學》第79【2】期315-328頁）和血管内皮 抑素endostatin(奥萊利等人1997年《細胞學》第88【2】期 277-285頁）這樣在實驗模式下能夠控制血管形成的抗血營 200423954 新生蛋白質抑制劑已經先後被發現。不過，這類蛋白質療 法生産成本昂貴，很難形成合成並施用於治療物件。目前 血管新生蛋白質抑制劑還需要進行進一步開發才能形成用 於疾病治療的藥物産品。在這種情況下，對於那些能夠安 5 全施用於人體並有效抑制血管内皮細胞病變性生長的藥物 依然存在著較大的需求。本項發明提供了可以實現這一目 的的成分和方法以及相關的優勢。 【發明内容】 發明概要 10 根據發現，丹寧酸及其衍生物能夠抑制内皮細胞的生 長和繁殖以及血管化進程。因此，本發明提供一些抑制組 織内部内皮細胞生長的方法，尤其是那些達到病理程度的 細胞。本發明還包括一種抑制組織内部新血管化的方法。 每種方法都要求向細胞或組織施用有效數量的丹寧酸、藥 15 物鹽、衍生物或其前體藥物。 本發明還提供一種通過向個體施用有效數量的丹寧酸 、藥物鹽、衍生物或其前體藥物來治療與内皮細胞過度增 生及/或新血管化有關的疾病的方法。 此外，本項發明還提供鑒別與丹寧酸、藥物鹽、衍生 20 物或其前體藥物相比療效相同、相近或更好的新藥物製劑 的篩選方法。該篩選方法要求將丹寧酸、藥物鹽、衍生物 或其前體藥物與新製劑進行抗血管增生方面的比較。 圖式簡單說明 曲線圖1顯示在内皮細胞繁殖化驗中丹甯酸對内皮細 8 200423954 胞繁殖的抑制與濃度的對應關係。 完成本項發明的方式 在本次公佈的内容中，引用的各種文獻、專利及已發 表的專利說明都以引文的方式進行表示，讀者可以一目了 5 然。在本次公佈的内容中引用這些文獻、專利及已發表的 專利說明目的是爲了更加完整地表述本項發明涉及的最高 水準。 本項發明在實踐操作中（除非另有特別說明）使用分子 生物學（包括細胞重組技術）、微生物學、細胞生物學、生物 10 化學和免疫學的傳統技術，均未超出目前科技發展的最高 水準。這些技術將在文獻資料中予以詳細而完整的解釋。 相關定義 在本文中已對相關術語定義如下。 在專利說明及專利要求内容中，除非文中另行說明， 15 否則所有單數形式均包含複數含義。例如，“某個細胞”雖 然爲單數形式，但同樣包含該細胞的複數含義以及單數和 複數混合使用的含義。 在本文中，“含有”意指成分和方法既包括詳細列舉的 各個要素，也並沒有將其他元素排除在外。當“基本上由… 20 …組成”用於成分和方法的定義時，它的含義中不應將其他 與該化合物具有重要關係的元素排除在外。因此，一種基 本上由本文詳述的元素組成的成分不應將分離和提純方法 中的微量污染物以及磷酸緩衝液、防腐劑及其他從藥物學 理論上可以接受的載體排除在外。“由……組成”的含義中 9 200423954 外的内斜僅限於其他成分的微量污染物和本項發 矣仃化!過程中的實際操作步驟。這些術語變化所代 又、/、體内各都在本項發明的範轉之内。 2酸驗度、溫度、時間、濃度和分子量在内的所有 =及域值均爲近似值，誤差。㈣不是每次都明 但所有數值前均帶有“大約，，的字句，而且本文提 ▲的摘僅具有典㈣徵，料健均“研究領域内所 熟知。 10 分離”是指從組成物、 的成分中單獨獲取。 細胞及其他與化合物天然結合 “個體”或“宿主”爲脊椎動物，最好是哺乳動物或人類 ° ’乳動物包括(但不僅限於）鼠類、猿猴類、人類、畜類、 參賽類動物及寵物。 ‘‘癌症”和“腫瘤，，可用作單數或複數形式，它是指發生 〜14病交並對宿主機體造成病患的細胞。原代癌細胞（即惡 性病變部位附近獲得的細胞）可以通過組織檢查等先進的 檢測手段與非癌細胞區別開來。本文中涉及的癌細胞定義 不僅包括原代癌細胞’而且指源自癌細胞的任何細胞，包 括轉移癌細胞、體外培養、以及源自癌細胞的細胞株。談 到某種類型的癌症，通常是表示實體腫瘤，而“臨床可以發 現’’的腫瘤是指那些在腫瘤塊基礎上可以通過CAT掃描、磁 共振成像(MRI)、X光、超聲波或觸診等診療手段發現的腫 瘤。而單純依靠生化或免疫學手段發現的結果尚不足以符 合本定義的要求。 10 200423954 在本文中，“抑制”是指推遲、延緩或阻止内皮細胞的 生長、繁殖或細胞分裂或血管在組織内的形成。監測抑制 功效的方法包括（但不僅限於）内皮細胞繁殖化驗、通過測定 血液含量測量血管床體積、以及定量測定血管結構的密度 5 。當培養物質爲細胞混合體時，監測新血管化的過程需要 通過定量測量能夠體現内皮細胞特定指標的細胞加以完成 ，這些指標包括血管新生因數、蛋白水解酶、以及内皮細 胞的特定細胞枯附分子等。 “合成”是指活性劑與另外一種惰性（如可發現的製劑 10 或示蹤劑）或活性(如佐劑）化合物或成分的結合。 “藥物成分”包括活性劑與惰性或活性載體的結合物， 並使該成分適用於體外或體内的診斷或治療。 在本文中，“藥物學可接受載體”的含義包含磷酸緩衝 溶液、水、乳劑（如油/水乳劑或水/油乳劑）及各種濕潤劑等 15 任何標準的藥物學載體。該成分還應包括穩定劑和防腐劑 。有關載體、穩定劑和防腐劑的實例請參閱馬丁·雷明頓 1975年《藥物學標準》第15版(麥克米倫出版公司，伊斯頓 市）。 “有效數量”是指能夠對有利或預期效果産生影響的充 20 足數量。例如，治療數量指能夠獲得預期療效的數量。該 數量可能與具有預防功效的數量相同或相異，後者是指能 夠防止疾病發作或疾病症狀的必要數量。有效數量可以通 過一次或多次施用或服用得以完成。 11 200423954 丹寧酸又稱鞣酸、單寧酸或甘油，是從許多植物的表 皮和果實中使用水溶醚提煉而成的，尤其是橡樹種的五倍 子。五倍子是羅氏鹽膚木等樹種葉子及葉柄處生長的贅生 物，經常有昆蟲將其刺穿並在其内部産卵。當昆蟲卵孵化 5 成幼蟲時，五倍子的組織就包圍在幼蟲左右。這些五倍子 就富含自然生成的丹寧酸。 丹寧酸的分子式爲C76H52046，其化學性質非常複雜。 它們可分爲兩類：（a)作爲黃烷醇衍生物的濃縮類丹寧酸和 (b)作爲糖醇（即葡萄糖)並含有一種或多種三羥基苯羧酸的 10 水解類丹寧酸。它通常是以粉末狀或片狀使用，顏色爲黃 白色到淺棕色。由於其具有沈澱蛋白質的能力，因此通常 被當成收斂劑使用，不過也一直用於治療出血、腹瀉、痢 疾、關節酸痛和久咳不愈等。它還適用於多種工業用途。 有些觀點認爲，丹寧酸可能具有抗癌特性，因爲多年來的 15 各種研究都表明它能夠抑制癌細胞生長。研究還發現，丹 寧酸能夠抑制表皮生長因數(EGF)受體酪氨酸磷酸激晦(E • B·楊與P·邁克2000年《癌症檢測與預防》第竺期【增 刊1】#146摘要）。 發明人已經發現丹寧酸能夠抑制内皮細胞的生長並具 20 有抗血管新生的特性。根據這些發現，本項發明提供一種 通過向細胞施用具有生長抑制功效數量的丹寧酸或藥物學 接受的衍生物、藥物鹽或其前體藥物來抑制内皮細胞生長 的方法。本發明還包括一種抑制組織内部新血管化的方法， 12 200423954 該方法要求向組織施用具有抗新血管化功效數量的丹寧酸 或藥物學接受的衍生物、代謝物、藥物鹽或其前體藥物。 本方法可以在體内或體外使用。在體外進行時，内皮 細胞或血管化組織應在以業内熟知的技術建立的條件下進 5 行培養，或參照下文中的實例。細胞及/或組織可以來自現 有的細胞株或從個體的活組織切片樣本進行培養而成。丹 寧酸或藥物學接受的衍生物、代謝物、藥物鹽或其前體藥 物隨後可直接加入培養基或作爲藥物成分進行施用。 本項發明中使用的丹寧酸包括水解類和濃縮類兩種。 10 大批量生産的丹寧酸爲水解類，來自橡樹或漆樹結出的天 然五倍子，其標稱分子量爲1690。 藥物鹽樣本包括：醋酸鹽、己二酸鹽、藻酸鹽、天門 冬胺酸鹽、安息香酸鹽、苯磺酸鹽、硫酸氫鹽、丁酸鹽、 檸檬酸鹽、樟腦酸鹽、樟腦磺酸鹽、環戊丙酸鹽、戊二酸 15 鹽、十二烷基硫酸鹽、乙烷磺酸鹽、延胡索酸鹽、氟庚酸 鹽、甘油磷酸鹽、半硫酸鹽、庚酸鹽、辛酸鹽、氫氯化物 、氫溴酸鹽、碘化氫鹽、2-羥基乙烷磺酸鹽、乳酸鹽、馬 來酸鹽、甲基磺酸鹽、2-萘磺酸鹽、煙酸鹽、草酸鹽、雙 經萘酸鹽、果膠酸鹽、過硫酸、苯基丙酸鹽、苦味酸鹽、 20 三甲基乙酸鹽、丙酸鹽、琥珀酸鹽、酒石酸鹽、硫氰酸鹽 、甲苯磺酸鹽和十一酸鹽。其他藥物鹽包括本項發明化合 物中與Na+、NH4+和NW4+等適當陽離子（其中W屬Q_4烷基) 相結合的陰離子。 13 出於治療目的，本項發明化合物中的鹽均爲藥物學所 接受。不過，藥物學不接受的氫化物或鹽基在獲取或提純 藥物學所接受的化合物等過財也可以用到。 亚不是每種療法都任何個體都有效，因此需要進行體 5外化驗測置母位病人所反映出的療效，這樣做將比較有利 。本項發明的方法提供了相關的測定手段，能夠反映丹窗 酸在内皮細胞的病變性繁殖方面是否能夠真正對個體的某 疾病産生/α療效果。本文也提供了此類實例。例如，從 病人身上取得活組織切片之後，將其在適合細胞生長和繁 Μ $的條件下與有效數量的藥物成分接觸或施用本文介紹的 。療手&通過本文介紹的CpAE化驗等傳統程式測定的對 於病又細胞生長的抑制作用顯示，本項發明介紹的藥物成 分及/或治财奸崎紅騎有效治療。 血官新生是形成新血管的基本過程。它是生殖、發育 15和創傷癒合等許多生理現象的重要步驟。在正常情況下， 血管生成都很有規律。然而，長期缺乏規律的血管新生也 會導致許多疾病。在風濕性關節炎中，新生成的毛細血管 侵入關節，破壞軟骨組織。而在糖尿病視網膜病變中，視 網膜中形成新的毛細血管，不僅侵入玻璃體，而且造成出 20血現象’亚取終導致失明。腫瘤的生長和轉移在很大程度 上也依賴血管新生。大多數原發性實體腫瘤都要經過一段 相當長的無血管時期，在此期間腫瘤明顯處於休眠期’其 生長大、力爲直;M-2毫米左右。腫瘤生長到這樣大小之後， 腫瘤細胞就可以通過簡單的被動擴散吸收必要的氧和其他 14 200423954 營養物質。這些只能在顯微鏡下觀察到的腫瘤塊最後會“啓 動”血管生成，它們利用四周成熟的宿主血管，催生出新的 血管和毛細血管，而這些血管會朝著腫瘤塊的方向生長， 並最終滲透進去。這使得腫瘤塊和血液轉移不斷擴散，將 5 病痛的危險無情地散播到身體其他部位。血管新生的發作 最初是由腫瘤細胞産生並形成的一種稱爲“腫瘤血管新生 因數’’(TAF)的生長因數所誘發開始的。 本項發明還包括一種針對個體體内因血管增生引起的 機能紊亂的治療方法，該方法是向個體施用具有某種療效 10 的一定數量的丹寧酸或其中的一種藥物成分。在本文中，“ 治療”的含義是指緩解與病變血管化有關的症狀以及減少 血管化。這類病症包括（但不僅限於）關節炎、因血管增生引 起的皮膚病、糖尿病視網膜病變、卡波西氏肉瘤、老年黃 斑病變、毛細血管擴張、青光眼、瘢痕瘤、角膜移植排斥 15 、創傷肉芽、血管纖維瘤、遺傳性出血性毛細血管擴張症 、心肌血管新生、以及硬皮病等。作爲有代表性的關節炎 病症包括風濕性關節炎和骨關節炎。在癌症和實體腫瘤的 治療中，“治療”手段包括抑制血管的生長，從而切斷腫瘤 及/或癌細胞生長所需的營養。腫瘤將依次而縮小並有可能 20 消失。對關節炎症狀進行治療將使得軟骨組織（尤其是關節 )中的血管形成降低，從而增強這些部位的靈活性。在牛皮 癖的治療中，這樣的治療手段將緩解疥痂、銀屑和皮下血 管凸顯等皮膚病症狀。而對於糖尿病視網膜病變來說，施 用活性分餾成分將減少視網膜中的外來血管的形成，進而 15 200423954 清除視力障礙。在對卡波西氏肉瘤的治療中，施用活性八 德物可抑制血管的生長及/或進-步形成，從而抑制因此: 形成的病變及/或腫瘤。 當丹甯酸施用於老鼠或人體等個體時，可以通過藥物 5學接受的載體全身、口服、透皮或局部施用於個體。治療 數ΐ可根據經驗確定並根據不同的治療病症、治療物件以 及按照治療方法使用的分餾物形式的毒性而相應變化。活 性分餾物可以通過口服、靜脈注射、腹膜注射或透皮方式 施用。當施用于動物時，該方法可以用來進一步確定療效 ίο 以及對個體或病人最有效的劑量。 在動物實驗的實例中，裸鼠群(Balb/c核苷酸/雌性裸鼠 ，加州吉爾羅伊市西蒙森實驗室）均被皮下注射105到109個 細胞。移植完成之後’化合物通過在移植部位周圍的皮下 注射進行施用。每周使用遊標卡尺測量兩次，用兩維尺度 15 測定移植範圍的縮小。 來自傑克遜實驗室（緬因州）的MRL/lpr小鼠 (MRL/MpJ[]Faslpr)可以用來檢測或觀察在關節炎病症中的 有效性。良好的治療效果包括動物關節及後腿的腫脹減輕 以及可以通過X光觀察到的軟骨退化的緩解。 20 體内施用可能會受到劑量多少以及治療過程中施用頻 率的影響。測定最有效的施用方法和劑量需要按照業内熟 知的技術進行，並將根據不同的治療成分、治療目的、治 療的目標細胞、以及治療物件而相應變化。可以根據治療 16 面生忠擇的劑量水平及治療方式進行一次或多次施用。施 用製劑最適當的射公式和方法在Η巾另有介紹。 α、，本項發明的成分與藥物合成可用於生産藥物及保健食 涵補4，也可用於治療人類或其他動物諸如藥物中的 5活性成分那樣按照傳統程式進行。 該藥物成分可以通過口服、鼻吸、非腸道注射方式或 及入療法把用，也可以片劑、鍵劑、微粒、膠囊、藥丸、 女瓶、栓劑或氣霧劑形式使用。它們可以是活性成分在水 成或非水成稀釋劑、糖聚、微粒或粉末中的懸浮液、溶液 10及乳劑形式。除了本項發明中的化合物之外，藥物成分還 可以包括其他藥用活性成分。 更爲獨特的是，本文中也被稱爲活性成分的活性分鶴 物可以通過口服、直腸、鼻腔、局部（包括透皮、氣霧劑、 15 20 =腔和舌下）、陰道、非腸道注射（包括皮下、肌肉、靜脈和 腹膜）以及肺料任何適當的給藥途徑進行 藥途徑應根據受藥物件的條件 相應變化。 年齡和治療病症的不同而 生長或血管化受到抑制。，繩體内的病變内皮細胞 丹寧酸或藥物學接受的衍^向個體Μ具有療效數量的 藥物，以《得預期的治療㉝H射物、藥物鹽或其前體 對個體體内因血管增生314、果本項發明還包括一種針 曰王W起的機能吝禽丨a 法是向個體施用具有某種療戈、’、s勺治療方法，該方 學接受的衍生物、代的定數蕙的丹寧酸或藥物 H射物、藥物 现A/、則體藥物。這類病 17 200423954 症包括（但不僅限於）關節炎、因血管增生引起的皮膚病、糖 尿病視網膜病變、血管再狹窄、卡波西氏肉瘤、老年黃斑 病變、毛細血管擴張、青光眼、瘢痕瘤、角膜移植排斥、 創傷肉芽、血管纖維瘤、遺傳性出血性毛細血管擴張症、 5 心肌血管新生以及硬皮病等。作爲有代表性的關節炎病症 包括風濕性關節炎和骨關節炎。 當丹甯酸施用於老鼠或人體等個體時，可以通過藥物 學接受的載體全身、口服、透皮或局部施用於個體。治療 數量可根據經驗確定並根據不同的治療病症、治療物件以 10 及按照治療方法使用的分餾物形式的毒性而相應變化。 並不是每種療法都任何個體都有效’因此需要進行體 外化驗測量每位病人所反映出的療效，這樣做將比較有利 。本項發明的方法提供了相關的測定手段，能夠反映丹甯 酸在内皮細胞的病變性繁殖或血管化方面是否能夠真正對 15 個體的某種疾病産生治療效果。例如，從病人身上取得活 組織切片之後，將其在適合細胞生長和繁殖的條件下與有 效數量的藥物成分接觸或施用本文介紹的治療手段。通過 本文介紹的CPAE化驗等傳統程式測定的對於病變細胞生 長的抑制作用顯示，本項發明介紹的藥物成分及/或治療方 20 法可以對病人進行有效治療。 雖然藥物成分可以直接施用，但如果能夠將至少含有 上述一種活性成分的藥物成分與一種或多種藥物學接受的 載體及其他治療製劑結合使用，治療效果會更佳。各載體 18 200423954 必須“可以接受”，意指與合成中的其他成分相容並且不能 對病人造成傷害。 藥物合成應當包括那些可以通過口服、直腸、鼻腔、 局部（包括透皮、氣霧劑、口腔和舌下）、陰道、非腸道注射 5 (包括皮下、肌肉、靜脈和腹膜）以及肺部等途徑給藥的成分 。合成藥物應當以便利的單位劑量形式出現並以制藥業熟 知的技術水平進行生産。這些方法包括將活性成分與構成 一種或幾種助劑的載體結合起來。一般來說，合成藥物應 當將活性成分與液態載體、精細分割的固態載體或兩者均勻 10 而緊密地結合在一起，如有必要，應當對産品進行修整。 本項發明中適合口服的合成藥物可以製成膠囊、扁膠 囊或片劑等含有預先確定的活性成分數量的各自獨立的單 位、粉末或微粒、在水成或非水成液體中的溶液或懸浮液 、油/水或水/油乳液。活性成分也可以製成大藥丸、幹藥糖 15 劑或糊狀形式。 片劑可以通過加壓成型製成，也可選擇結合一種或多 種助劑。加壓片劑可以在適當的機器上對以粉末或微粒等 形式自由流動的活性成分進行加壓處理，也可選擇添加粘 合劑（例如普維酮、凝膠、羥丙基甲基纖維素等）、潤滑劑、 20 惰性稀釋液、防腐劑、分解質（例如羥基乙酸澱粉鈉、交聯 普維酮、交聯羧甲基纖維素鈉等）、表面活性劑或分散劑。 成型片劑可以在適當的機器上對粉末狀化合物與惰性稀釋 液的混合物進行成型處理。片劑表面可以塗層或刻紋，也 可以製成能夠使内部活性成分緩慢釋放的形式，例如不同 19 200423954 比例的羥丙基甲基纖維素就有助於實現預期的釋放目標。 這些片劑也可以製成腸溶形式，以保證其在腸道中（而不是 胃裏)釋放。 適合局部口服的合成藥物包括含有活性成分以及荒糖 5 、樹膠或黃芪膠等適當味道的錠劑、含有活性成分及凝膠 和甘油或蔗糖和樹膠等惰性基的喉片、以及含有活性成分 及適當液態載體的漱口水。 本項發明中可以局部施用的藥物合成可製成藥膏、乳 霜、懸浮液、乳液、粉末、溶液、藥糊、膠體、喷霧、氣 10 霧劑或藥油，也可與藥貼、繃帶或藥膏等注入活性成分的 外用品結合，可以選擇一種或多種賦形劑或稀釋液。 對於眼睛或嘴及皮膚等外部組織疾病，該藥物最好以 含有活性成分的藥膏或乳霜形式局部施用。在藥膏中，藥 物可以與含犧或水溶性藥膏基體配合使用。藥物成分也可 15 以在乳霜中與油/水霜基配合使用。 如有必要，霜基中的水相可以包括至少30% w/w的多 元醇，即丙二醇、丁烷-1、3-二醇、甘露醇、山梨糖醇、丙 三醇和聚乙二醇及其混合物等擁有兩個或兩個以上羥氫氧 基的醇。局部施用的合成藥物也可以包括能夠加強藥物成 20 分在皮膚或其他患病部位的吸收或滲透的化合物。這些能 夠加強皮膚滲透的製劑包括二甲亞砜及其他類似化合物。 本項發明的乳劑中的油相可以由任何已知成分以任何 已知方式構成。儘管油相可能只包含一種乳化劑（也稱 emulgent)，但它最好能夠含有至少一種乳化劑與油脂、油 20 200423954 或兩者組成的混合物。而吸水性乳化劑最好能夠同具有穩 定劑作用的親脂性乳化劑配合使用，並且同時包含油和油 脂。乳化劑之間或乳化劑與穩定劑之間共同組成所謂的乳 化堪，而乳化犧與油及/或油脂組成所謂的乳化藥膏基體， 5 形成乳霜中的油性分散質。 適合本項發明藥物合成使用的乳化劑和乳化穩定劑包 括Tween 60、Span 80、脂肪醇、肉豆謹醇、單硬脂酸甘油 I旨和十二烧硫酸納。 如何選擇適合藥物合成的油或油脂要根據其是否能夠 10 滿足表面調劑需要，因爲許多可能應用于藥物乳化劑合成 的活性化合物本身可溶性很低。而乳霜最好能夠以一種不 油腻、無污潰、而且可清洗的産品形式出現，並且應當具 有適當的濃度以避免從軟管或其他容器中泄漏出來。諸如 同己二酸、硬脂酸鹽、丙二醇、椰子油脂肪酸二醇、異丙 15 基肉豆蔻酸酯、油酸鹽、異丙基棕櫚酸鹽、硬脂酸丁酯等 直鏈或支鏈單基或雙基烷基酯或Crodamol CAP等一系列支 鏈酯都可以使用，最後三種酯是最佳選擇。這些酯既能夠 單獨使用，也可以根據預期的特性而相互搭配。或者使用 具有高熔點的白色軟石蠟及/或液體石蠟或其他礦物油等。 20 適合在眼部進行局部施用的合成藥物也應包括眼藥水 ，其中的活性成分可以溶解或懸浮在適當的載體中，尤其 是活性成分的水性溶劑。用於直腸送藥的合成藥物可以製 成栓劑，並選擇含有可可油或水楊酸鹽的適當基體。 21 200423954 適用于陰道送藥的合成藥物可以製成含有前體藥物的 陰道栓劑、衛生棉、乳霜、膠體、藥糊、泡沫或喷霧形式 ，並選擇業内熟知的適當載體。 適用于鼻腔送藥的合成藥物採用固態載體，包括粒度 5 爲20至500微米的粗粉末，施用方式爲吸入式，即將放置在 鼻腔前端的粉末猛然吸入鼻腔。採用液態載體的適當形式 爲鼻腔喷霧、滴鼻藥水或通過喷霧器以氣霧劑形式使用， 包括活性成分的水性或油性溶液。 適用于腸胃外注射的合成藥物包括水性或非水性等濃 10 度無菌注射液，它含有抗氧化劑、緩衝液和抑菌劑，並且 可以溶解，使藥物可以達到與施用物件血液等同的濃度； 它還包括水性和非水性無菌懸浮液，含有懸浮製劑和增稠 劑以及脂質體或其他能夠將化合物定位于血液成分或人體 器官的微粒系統。合成藥物可以放置在安瓿瓶或其他小瓶 15 等容納單位劑量或多劑量的密封容器裏，也可以儲存在冷 凍乾燥（凍幹)條件下，只是在使用前才需要注射用水等其他 無菌液態載體。可以從上文介紹的無菌粉末、微粒或片劑 獲得臨時注射液或懸浮液。 最佳的單位劑量應當是那些含有藥物成分的每日劑量 20 或單位、每曰分劑量、或適當部分的合成藥物。 我們可以這樣理解，除了上文提到的成分之外，本項 發明的合成藥物可以包含業内針對這種合成藥物通常添加 的其他傳統製劑，例如口服藥物可含有甜味劑、增稠劑和 22 200423954 調味劑等其他製劑。它也可以包含抗腫瘤、抗癌症、抗血 管新生或提高免疫力的其他活性成分。 丹寧酸及其前體藥物、代謝物、藥物鹽或衍生物及其 化學成分也可用于獸醫藥物，並可按照業内的傳統方法進 5 行製造。 此外，本項發明還提供一種鑒別能夠抑制血管化或内 皮細胞生長的治療製劑的篩選方法，包括： (a) 用適當的細胞或組織接觸該製劑； (b) 用另外的細胞或組織樣本接觸具有療效數量的 10 丹寧酸或藥物學接受的衍生物、代謝物、藥物鹽或其前體 藥物； (c) 對步驟A和步驟B的樣本生長進行比較，發現步 驟A中任何與步驟B中的樣本相同或相似的能夠延緩細胞 生長速度的製劑都是一種能夠抑制血管化或内皮細胞生長 15 的製劑。 本文介紹的其他療法和製劑可以與該樣本配合使用。 本項發明包括一整套針對個體體内因病變性血管增生引起 的機能紊亂的治療方案，該方案是按照使用指南向個體施 用具有某種療效的一定數量的丹寧酸或藥物學接受的衍生 20 物、代謝物、藥物鹽或其前體藥物。該方案可以用來治療 的病症包括關節炎、因血管增生引起的皮膚病、糖尿病視 網膜病變、血管再狹窄、卡波西氏肉瘤、老年黃斑病變、 毛細血管擴張、青光眼、瘢痕瘤、角膜移植排斥、創傷肉 芽、血管纖維瘤、遺傳性出血性毛細血管擴張症、心肌血 23 200423954 管新生、硬皮病、風濕性關節炎、銀屑病關節炎和骨關節 炎。 【實施方式】 較佳實施例之詳細說明 5 下列例證對本項發明做以進一步說明，而不應被視爲 對本發明的限制。 例證1 丹寧酸的分離和提純 大批里生産的丹寧酸或單寧酸可以通過一系列分離法 10進行提純(例如K·青木等人91979《分析生物化學》第95期 575-578頁）。美國化學學會高純度(Reagent Grade)丹寧酸也 可以向美國Sigma公司購買。 例證2 使用丹甯酸進行内皮細胞抑制化驗 15 内皮細胞繁殖化驗： 該化驗根據康諾利等人1986年《分析生物化學》第152 期136-4頁提出的化驗程式以及相關的改進（梁與王2〇〇〇年 《血管新生：從分子到綜合藥物學》2〇9_223頁/編輯：馬拉 多達基斯/紐約克魯維爾學術/充實出版社）而進行的。從美 2〇國菌種保存中心(ATCC)取得的新生牛心肺動脈内皮(CPAE) 細胞在MEM-10E時出現了接近95%的匯合。細胞與濃度爲 0.25%的胰島素溶液從組織培養瓶中釋放出來，並爲放置在 24孔的組織培養盤中，培養盤中的培養基完全相同，密度 爲每個培養孔裏10,000個細胞。培養盤在5%的二氧化^和 24 200423954 37 C的溫度環境下培盲 卜t 月〜J、蛉。加入化驗樣本和控制樣本。 母個樣本都裝在兩個 M的孔裏，每個培養孔100毫升以確 保其具有繁殖能力。樣 尽培育60小時之後，抽取培養基， 在細胞酸性ί粦酸酶的多 結果與討論： I基礎上測疋細胞的數目° 表I·抑制内皮細胞繁殖 丹寧酸濃度(毫克/毫升）： '丄3 6.25 12.5 25 50 内皮細胞繁殖抑制率(％) · — 0 〇 0 19.3 84.4 ίο 15 表1”、、員不我們在内皮細胞繁殖抑制化驗中獲得的結果 。：寧酸的確能夠抑制内皮細胞繁瘦。在每毫升5_ 低派度下丹f酸使内皮細胞的生長降低了 8 4 · 4 %。 …我]對丹丁酉夂/辰度與抑制内皮細胞繁殖之間的對應關 係進灯了n驗結果也絲i巾顯*。非常清楚，在低 濃度下，丹寧蚊—種内皮細胞繁___。而且丹 用I對:皮細胞繁殖的抑制能力與其濃度有直接關係。 20 + g上返么明已經通過說明和實例進行了詳細的描 述，但在這-領域内其他專業人士的眼裏，還需進一步的 優化和改進。例如’本項發明介紹的方法完全可以同一種 或^種已知的k腫瘤、抗血管新生或提高免疫力的療法和 策魚軟骨、_胺醇和鞘㈣等化學成分配合❹。因此， 本文所做的糾和例㈣顧視錢本項發料及範圍的 限制，這-點我們也在附加的中請中進行了必要的閣述。 【圖式簡單說明】 曲線圖1顯示在内皮細胞繁殖化驗中丹富酸對内皮細 25 200423954 胞繁殖的抑制與濃度的對應關係。 【圖式之主要元件代表符號表】 (無)玖, Description of the invention: [Invented technology " Leadership] Cross-reference search of related applications This application complies with the provisions of Section 35, Part 119 (e) of the United States Code on provisional applications in the United States. No. 60M26, 363, The application was filed on November 5th, 2002, and the content was requested to be retrieved through a cross-reference search to incorporate the detailed information disclosed this time. FIELD OF THE INVENTION This invention belongs to the field of pharmacy, Especially in the field of antiangiogenic drugs for the prevention and treatment of diseases. , Previously [Background of the Invention] A new blood vessel is a process in which new blood vessels grow on the basis of the original capillaries. Spend money here, The effect of eggs from the water makes the basement membrane inward. Cell's suction is weakened, Lead to ⑽ cells Rinki «. These cells then _ maternal blood vessels, Split breeding, And differentiate into new vascular structures (Ressau, Nature, 386, 671, 674; Will Ding et al. 1995 "Research on Cell and Molecular Biology" [4] plus plus pages). It was found that 'many different biological factors have a role in controlling angiogenesis (Buhno et al. "New Trends in Biochemistry" Kawagawa] Issue 251-256. Cytology Chapter I! : 3 = 5 pages). This includes growth factors, Cell surface receptor protein peptone preparations and extracellular interstitial proteins have multiple functions of protein negative (Aachen and Stark hit the International Journal of Experimental Pathology, page 255/265, Divale Raja and Richmond〗 999 "New Trends in Pharmacology" pp. 20 [4] 151_156; Hanahan's Leap Year (Science), claw issue, page 0; Mason Peel et al., Science, 1997, No. 277, p. 60. 5 Suri et al., 1996, "Cytology", 87, 1171-1180; Sato et al. 1995 Nature 376 (7) -74; Mignati and Rifkin 1996, Enzymes and Proteins, Issue 49, 117; Pintuqi et al. "Thrombosis and Hemostasis Research" 22 [6] 517-524 pages; Weilon and Saatchi ^ American Journal of Pathology, pp. 147 [4] 873_883, 1995; Brukers et al., Science, 1994, 264, 569-5η; Koch et al. "Date from; , , i World, 376, pp. 517-519). The complexity of the angiogenesis process and the diversity of control factors provide us with a series of research topics to study the treatment of blood organ formation in vivo. Blood pupa newborns are very regular under normal circumstances, It is embryonic development, Important steps in many physiological phenomena such as growth and wound healing and female reproductive cycle 15 (Weilding and Krister, 1996, Nature Science, scratch page title; Guccier and Rogers 1995 Microcirculation, Vol. 2, pp. 329-343; Augustine et al. 1995 American Journal of Pathology 147 [2] pp. 339-351). Some important steps in the process of pathological angiogenesis are the main factors in the development of some human diseases, Including tumor spread, Metastatic cancer, Diabetes 20 罔 membrane / sick man, Rheumatoid arthritis and psoriasis and other inflammatory diseases (Falkman 1995, Natural Medicine, Issue 1 [1], pp. 27-31; Walsh 1998 Rheumatology 38 [2] 103-112; Hillley et al., 1998, "New Knowledge in Human Reproduction," 4 [5], pp. 736-740). In these cases, The t-spread of the disease is often affected by chronic angiogenesis. Example 6 =, In rheumatoid arthritis towels, Newly generated capillaries invade joints, Broken cartilage tissue. In the transformation of branuria vision, As new Capillaries ’not only invade the vitreous, And cause bleeding, And ultimately blindness. Swelling is also dependent on blood vessels to a great extent. Raw. Most primary solid axes go through a fairly long angioperiod, During this period, tumor growth was about W mm in diameter. After the tumor has grown to the right size, Tumor cells can absorb the necessary oxygen and other nutrients through simple passive diffusion. These M blocks that can only be observed under the microscope will start the M generation, New blood capillaries spawned from the blood around them, Vascularization of the tumor mass, Allows tumors and malignant cells to spread or metastasize to other parts of the body. Although we already know a lot about the biological changes that occur during pathological angiogenesis, But so far we have not found any effective drugs to control angiogenesis in the body. Effective treatments that can control angiogenesis will greatly ease the pain of many humans. The traditional method is to develop artificial drugs by screening for artificial compounds with certain expected drug properties and conducting toxicity and efficacy tests in vivo. Compounds picked in this way are usually toxic in the body, Moreover, effective angiogenesis inhibitors for the treatment of diseases have not been successfully developed. Recently, Molecular biology techniques have begun to be applied to the development of angiogenesis inhibitors. Such as the angiostatin angiostatin (O'Reilly et al. 1994, Cytology 79 [2] pp. 315-328) and the endostatin endostatin (O'Reilly et al. 1997 Cytology 88 [ 2] Issues 277-285) Anti-blood camp 200423954 neonatal protein inhibitors that can control angiogenesis in the experimental mode have been found. but, Such protein therapies are expensive to produce, It is difficult to form a composition and apply it to a therapeutic article. At present, further development of angiogenesis protein inhibitors is needed to form pharmaceutical products for disease treatment. under these circumstances, There is still a great demand for drugs that can be safely applied to the human body and effectively inhibit the vascular endothelial cell pathological growth. This invention provides the ingredients and methods and related advantages that can accomplish this. [Summary of the Invention] Summary of the Invention 10 According to the findings, Tannic acid and its derivatives can inhibit the growth and reproduction of endothelial cells and the process of vascularization. therefore, The present invention provides methods for inhibiting the growth of endothelial cells in tissues. Especially those cells that have reached a pathological level. The invention also includes a method for inhibiting neovascularization inside the tissue. Each method requires the administration of an effective amount of tannin to the cell or tissue, Medicine 15 salt, Derivatives or their prodrugs. The invention also provides an effective amount of tannic acid by administering to an individual, Drug salt, Derivatives or prodrugs thereof. A method for treating diseases associated with excessive proliferation of endothelial cells and / or neovascularization. In addition, This invention also provides identification with tannins, Drug salt, Derivatives 20 or their prodrugs have the same efficacy compared to Similar or better screening methods for new pharmaceutical preparations. This screening method requires that tannic acid, Drug salt, Derivatives or prodrugs are compared with new formulations in terms of antiangiogenic effects. Brief description of the figure Graph 1 shows the corresponding relationship between tannic acid's inhibition of endothelial cell proliferation and endothelial cell proliferation in endothelial cell proliferation assay. Ways to accomplish this invention In this announcement, Various references cited, Both patents and issued patent specifications are indicated by citations, Readers can see at a glance 5. Citing these references in the contents of this publication, The purpose of patents and published patent specifications is to provide a more complete picture of the highest level of the invention. This invention uses molecular biology (including cell recombination technology) in practice (unless otherwise specified), microbiology, Cell Biology, Biology 10 traditional techniques of chemistry and immunology, None exceed the current highest level of scientific and technological development. These techniques are explained in detail and in the literature. Related definitions Related terms have been defined in this article as follows. In the patent description and patent requirements, Unless otherwise stated in the text, 15 Otherwise, all singular forms include the plural. E.g, "Single cell" is singular, However, it also includes the plural meaning of the cell and the meaning of a mixture of singular and plural. in the text, “Containing” means that the ingredients and methods include both the elements listed in detail, Nor does it exclude other elements. When "consisting essentially of ... 20" is used in the definition of ingredients and methods, Its meaning should not exclude other elements that have a significant relationship with the compound. therefore, An ingredient consisting essentially of the elements detailed herein should not separate trace contaminants from the separation and purification methods, as well as phosphate buffers, Preservatives and other carriers that are pharmaceutically acceptable are excluded. In the meaning of "consisting of" 9 200423954, the inclination outside is limited to trace pollutants of other components and the development of this item! Practical steps in the process. These terms have changed, /, Each in the body is within the scope of the invention. 2 acid test, temperature, time, All = and domain values, including concentration and molecular weight, are approximate. error. ㈣ is not always clear, but all values are preceded by "about, , Words of And the extracts mentioned in this article only have allusions, Jianjun Jun is well known in the research field. "Separation" means separating from the composition, The ingredients are obtained separately. Cells and others naturally bind to compounds "individual" or "host" are vertebrates, Preferably mammals or humans ° ’Dairy animals include (but are not limited to) rodents, Apes, Humanity, Livestock, Participating animals and pets. ‘Cancer’ and “Tumor, , Available as singular or plural, It refers to cells that have ~ 14 disease interactions and cause disease in the host body. Primary cancer cells (ie, cells obtained near the site of a malignant lesion) can be distinguished from non-cancer cells by advanced detection methods such as tissue examination. The definition of cancer cell referred to herein includes not only primary cancer cells ’but also any cell derived from cancer cells, Including metastatic cancer cells, In vitro culture, And cell lines derived from cancer cells. When it comes to some type of cancer, Usually indicates a solid tumor, And "clinically detectable tumors" are those tumors that can be scanned by CAT, Magnetic resonance imaging (MRI), X-ray, Tumors detected by ultrasound or palpation. However, the findings based on biochemical or immunological methods alone are not enough to meet the requirements of this definition. 10 200423954 In this article, "Inhibit" means to postpone, Delay or prevent the growth of endothelial cells, Reproduction or cell division or formation of blood vessels in tissues. Methods for monitoring inhibition include (but are not limited to) endothelial cell proliferation assays, Vascular bed volume by measuring blood content, And quantitative determination of the density of vascular structures 5. When the culture is a mixture of cells, Monitoring the process of neovascularization needs to be done by quantitatively measuring cells that can reflect specific indicators of endothelial cells. These indicators include the angiogenesis factor, Proteolytic enzymes, As well as specific cell desorption molecules of endothelial cells. "Synthetic" refers to the combination of an active agent with another inert (eg, discoverable formulation 10 or tracer) or active (eg, adjuvant) compound or ingredient. "Pharmaceutical ingredient" includes a combination of an active agent and an inert or active carrier, And make this component suitable for diagnosis or treatment in vitro or in vivo. in the text, "Pharmaceutically acceptable carrier" means phosphate buffered solution, water, Emulsions (such as oil / water emulsions or water / oil emulsions) and various wetting agents 15 Any standard pharmaceutical carrier. This ingredient should also include stabilizers and preservatives. About the carrier, For examples of stabilizers and preservatives, see Martin Remington, 1975, Pharmacological Standards, 15th Edition (Macmillan Publishing Company, Easton). "Effective quantity" means a sufficient quantity that has an impact on a beneficial or expected effect. E.g, The number of treatments is the number that will achieve the desired effect. This amount may be the same or different from the amount that has a preventive effect, The latter is the amount necessary to prevent the onset of symptoms or symptoms of the disease. An effective amount can be accomplished by one or more administrations or administrations. 11 200423954 Tannic acid is also known as tannic acid, Tannic acid or glycerol, Are extracted from the epidermis and fruits of many plants using water-soluble ethers, Especially the Galla gallica. Galla Chinensis is a neoplasm that grows on the leaves and petioles of tree species Insects often pierce them and lay eggs inside them. When insect eggs hatch into 5 larvae, Gallbladder tissue is surrounded by larvae. These gallics are rich in naturally occurring tannins. The molecular formula of tannin is C76H52046, Its chemical properties are very complex. They can be divided into two categories: (A) Concentrated tannic acid as a flavanol derivative and (b) Hydrolyzed tannic acid as a sugar alcohol (ie, glucose) and containing one or more trihydroxybenzenecarboxylic acids. It is usually used in powder or flake form, Color is yellow white to light brown. Because of its ability to precipitate proteins, So it is usually used as an astringent, But it has also been used to treat bleeding, diarrhea, Dysentery, Joint soreness and prolonged cough. It is also suitable for many industrial uses. Some argue that Tannic acid may have anti-cancer properties, Because over the years 15 various studies have shown that it can inhibit cancer cell growth. The study also found that Tannic acid can inhibit epidermal growth factor (EGF) receptor tyrosine phosphorylation (E • B. Young and P. Mike 2000, Cancer Detection and Prevention, Issue 1 [Suppl. 1] # 146 Abstract). The inventors have discovered that tannic acid can inhibit the growth of endothelial cells and has antiangiogenic properties. Based on these findings, This invention provides a tannic acid or a pharmaceutically acceptable derivative by administering to a cell an amount having a growth-inhibiting effect, A method of inhibiting the growth of endothelial cells by using a drug salt or a prodrug thereof. The invention also includes a method for inhibiting neovascularization inside the tissue, 12 200423954 The method requires administering to the tissue an amount of tannic acid or a pharmaceutically acceptable derivative with an anti-neovascularization effect, Metabolites, A drug salt or a prodrug thereof. This method can be used in vivo or in vitro. When performed in vitro, Endothelial cells or vascularized tissues should be cultured under conditions established using techniques well known in the art, Or refer to the examples below. Cells and / or tissues can be derived from existing cell lines or cultured from individual biopsy samples. Tannic acid or a pharmaceutically acceptable derivative, Metabolites, The drug salt or its prodrug can then be added directly to the culture medium or administered as a pharmaceutical ingredient. The tannins used in this invention include two types, hydrolyzed and concentrated. 10 Mass produced tannins are hydrolyzed, Natural gallic seeds from oak or lacquer trees, Its nominal molecular weight is 1690. Drug salt samples include: Acetate, Adipic acid, Alginate, Asparagine Benzoate, Besylate, Bisulfate, Butyrate, Citrate, Camphor salt, Camphor sulfonate, Cyclopentapropionate, Glutaric acid 15 salt, Dodecyl sulfate, Ethane sulfonate, Fumarate, Fluoroheptanoate, Glycerophosphate, Hemisulfate, Heptanoate, Octanoate, Hydrochloride Hydrobromide, Hydrogen iodide, 2-hydroxyethane sulfonate, Lactate, Maleate, Mesylate, 2-naphthalenesulfonate, Nicotinate, Oxalate, Double naphthoate, Pectate, Persulfuric acid, Phenylpropionate, Picrates, 20 trimethyl acetate, Propionate, Succinate, Tartrate, Thiocyanate Tosylate and undecanoate. Other pharmaceutical salts include the compounds of this invention with Na +, An anion combining appropriate cations such as NH4 + and NW4 + (W is a Q_4 alkyl group). 13 For therapeutic purposes, The salts in the compounds of this invention are all pharmaceutically acceptable. but, Pharmaceutically unacceptable hydrides or bases can also be used to obtain or purify pharmacologically acceptable compounds. Ya not every treatment is effective for any individual, Therefore, an external body test is needed to measure the effect of the mother patient. It would be advantageous to do so. The method of this invention provides related measuring means, It can reflect whether salicylic acid can actually produce / α treatment effect on an individual's disease in terms of the diseased reproduction of endothelial cells. This article also provides such examples. E.g, After taking a biopsy from a patient, It is contacted or administered with an effective amount of a pharmaceutical ingredient under conditions suitable for cell growth and proliferation. Healing & The inhibition of disease and cell growth measured by traditional procedures such as the CpAE assay described in this article shows that The pharmaceutical composition and / or treatment of the traitorous traitorous red horse riding described in this invention is effective. The regeneration of hemorrhage is the basic process of forming new blood vessels. It is reproduction, Development 15 and wound healing are important steps in many physiological phenomena. under normal circumstances, Angiogenesis is very regular. however, Prolonged lack of regular angiogenesis can also cause many diseases. In rheumatoid arthritis, Newly generated capillaries invade the joint, Destruction of cartilage tissue. And in diabetic retinopathy, New capillaries form in the omentum, Not only invades the vitreous body, It also caused the phenomenon of 20 blood ′, which resulted in blindness. Tumor growth and metastasis also depend to a large extent on angiogenesis. Most primary solid tumors go through a fairly long period of angioplasty, During this period, the tumor is obviously in the dormant stage ’, its growth is large, Force is straight; M-2 mm. After the tumor has grown to this size, Tumor cells can then absorb the necessary oxygen and other nutrients through simple passive diffusion 14 200423954. These tumor masses, which can only be observed under a microscope, will eventually “initiate” angiogenesis, They take advantage of the mature host blood vessels that surround them, Spawn new blood vessels and capillaries, And these blood vessels will grow towards the tumor mass, And eventually penetrate into it. This allows the tumor mass and blood metastasis to continue to spread, Relentlessly spread the danger of 5 illnesses to other parts of the body. The onset of angiogenesis is initiated by a growth factor called "tumor angiogenesis factor '" (TAF), which is produced and formed by tumor cells. This invention also includes a method for treating functional disorders caused by vascular proliferation in an individual, The method involves administering to a subject a certain amount of tannic acid or one of its pharmaceutical ingredients. in the text, "Treatment" means alleviating the symptoms associated with vascularization of the lesion and reducing vascularization. Such conditions include (but are not limited to) arthritis, Skin diseases caused by angiogenesis, Diabetic retinopathy, Kaposi's sarcoma, Age-related macular degeneration, Capillary dilation, glaucoma, Keloids, Corneal transplant rejection 15 Wound granulation, Angiofibromas, Hereditary hemorrhagic telangiectasia, Myocardial angiogenesis, And scleroderma. Representative arthritis conditions include rheumatoid arthritis and osteoarthritis. In the treatment of cancer and solid tumors, "Treatment" means inhibiting the growth of blood vessels, This cuts off the nutrients needed for tumor and / or cancer cell growth. The tumor will shrink in sequence and may disappear. Treating arthritis symptoms will reduce blood vessel formation in cartilage tissue, especially joints, Thereby enhancing the flexibility of these parts. In the treatment of psoriasis, This kind of treatment will relieve 疥 痂, Dermatological symptoms such as psoriasis and subcutaneous blood vessels are prominent. For diabetic retinopathy, Application of active fractionation will reduce the formation of foreign blood vessels in the retina, Further 15 200423954 Eliminate visual impairment. In the treatment of Kaposi's sarcoma, Administration of active octadecants can inhibit blood vessel growth and / or further formation, Thus suppressing therefore: Formation of lesions and / or tumors. When tannic acid is administered to individuals such as rats or humans, Carriers that can be accepted through pharmacology oral, Transdermal or topical application to an individual. The number of treatments can be determined empirically and according to different treatment conditions, The toxicity of the treatment articles and the fractions used in accordance with the method of treatment varies. Active fractions can be taken orally, Intravenous injection, Intraperitoneal or transdermal administration. When applied to animals, This method can be used to further determine efficacy and the most effective dose for an individual or patient. In the case of animal experiments, Nude mouse population (Balb / c nucleotide / female nude mice, (Simonson Laboratories, Gilroy, California) were injected subcutaneously with 105 to 109 cells. After the completion of the transplantation, the 'compound is administered by subcutaneous injection around the transplantation site. Measured twice a week with a vernier caliper, The two-dimensional scale 15 was used to determine the reduction in transplantation range. MRL / lpr mice (MRL / MpJ [] Faslpr) from Jackson Labs (Maine) can be used to detect or observe effectiveness in arthritic conditions. Good treatment results include reduction of swelling in the joints and hind legs of animals and reduction of cartilage degradation that can be observed by X-rays. 20 In vivo administration may be affected by the dose and the frequency of administration during treatment. Determining the most effective method of application and dosage needs to be performed according to techniques well known in the industry, And will be based on different therapeutic ingredients, Purpose of treatment, Target cells for treatment, And treatment items. One or more administrations can be performed depending on the dosage level and treatment method for treating the 16 facials. The most appropriate formulas and methods for applying the formulations are described elsewhere in the wipes. α, , The composition of this invention and the synthesis of medicine can be used to produce medicine and health food. It can also be used to treat humans or other animals such as the 5 active ingredients in medicine according to traditional procedures. The medicinal composition can be taken orally, Sniffing, For parenteral or parenteral use, Also tablets, Bonding agent, particle, capsule, pill, Female bottle, Used as a suppository or aerosol. They can be active or non-aqueous diluents, Glucose, Suspensions in particles or powders, Solution 10 and emulsion form. In addition to the compounds in this invention, Pharmaceutical ingredients may also include other pharmaceutically active ingredients. More uniquely, Active ingredients, also referred to herein as active ingredients, can be taken orally, rectum, nasal cavity, Local (including transdermal, aerosol, 15 20 = cavity and sublingual), vaginal, Parenteral (including subcutaneous, muscle, Intravenous and peritoneal) and any appropriate route of administration of the lung material. The route of administration should be changed according to the conditions of the drug receiving device. Growth or vascularization is inhibited depending on age and condition being treated. , Diseased endothelial cells in the rope. Tannin or a pharmacologically acceptable derivative has a therapeutically effective amount of drug. Based on "Getting the Expected Treatment ㉝H Projectile, Drug salts or their precursors If this invention also includes a kind of functional bird from the king, a method is to administer to an individual ’, s spoon treatment, Derivatives accepted by this method, A fixed number of tannins or drugs Drugs now A /, The body drugs. 17 200423954 Symptoms include (but are not limited to) arthritis, Skin diseases caused by angiogenesis, Diabetic retinopathy, Restenosis, Kaposi's sarcoma, Age-related macular degeneration, Capillary dilation, glaucoma, Keloids, Corneal transplant rejection, Wound granulation, Angiofibromas, Hereditary bleeding telangiectasia, 5 Myocardial angiogenesis and scleroderma. Typical arthritic conditions include rheumatoid arthritis and osteoarthritis. When tannic acid is administered to individuals such as rats or humans, Pharmacologically acceptable carriers throughout the body, oral, Transdermal or topical application to an individual. The number of treatments can be determined empirically and according to different treatment conditions, The therapeutic article varies with the toxicity of 10 and the fractions used in accordance with the method of treatment. Not every treatment is effective for any individual ’so an in vitro test is needed to measure the efficacy of each patient, It would be advantageous to do so. The method of this invention provides related measuring means, It can reflect whether tannic acid can really produce a therapeutic effect on a certain disease in 15 individuals in the pathological reproduction or vascularization of endothelial cells. E.g, After taking a biopsy from a patient, It is contacted with an effective amount of a pharmaceutical ingredient under conditions suitable for cell growth and reproduction or the treatments described herein are administered. The inhibitory effect on the growth of diseased cells measured by traditional procedures such as the CPAE assay described in this article shows that The pharmaceutical composition and / or treatment method described in this invention can effectively treat patients. Although the pharmaceutical ingredients can be administered directly, However, if a pharmaceutical ingredient containing at least one of the above active ingredients can be used in combination with one or more pharmaceutically acceptable carriers and other therapeutic agents, The effect will be better. Each carrier 18 200423954 must be "acceptable", This means that it is compatible with other ingredients in the synthesis and cannot cause harm to the patient. Drug synthesis should include those that can be taken orally, rectum, nasal cavity, Local (including transdermal, aerosol, Oral and sublingual), vaginal, Parenteral injection 5 (including subcutaneous, muscle, Intravenous and peritoneal) and lungs. Synthetic drugs should be presented in convenient unit dosage forms and produced at a level of technology known to the pharmaceutical industry. These methods include combining the active ingredient with a carrier that constitutes one or more adjuvants. Generally speaking, Synthetic drugs should combine the active ingredient with a liquid carrier, Finely divided solid carriers or both are evenly and tightly combined, If necessary, The product should be trimmed. The synthetic drugs suitable for oral administration in this invention can be made into capsules, Separate units such as flat capsules or tablets containing a predetermined amount of active ingredient, Powder or particles, A solution or suspension in an aqueous or non-aqueous liquid, Oil / water or water / oil emulsion. The active ingredients can also be made into large pills, 15 doses of dried sugar or pasty form. Tablets can be made by compression molding, Optionally, one or more auxiliaries can be combined. Pressurized tablets can pressurize active ingredients that are free-flowing in the form of powder or granules, etc. on a suitable machine, You can also choose to add binders (e.g., povidone, gel, Hydroxypropyl methyl cellulose, etc.), Lubricant, 20 inert diluent, preservative, Decomposing substances (such as sodium starch glycolate, Cross-linked buprene, Croscarmellose sodium, etc.), Surfactant or dispersant. The shaped tablets can be shaped on a suitable machine with a mixture of a powdery compound and an inert diluent. The surface of the tablet can be coated or engraved, It can also be made into a form that can release the internal active ingredients slowly, For example, different 19 200423954 ratios of hydroxypropyl methylcellulose can help achieve the desired release target. These tablets can also be made enteric, To ensure it is released in the intestine, not in the stomach. Synthetic drugs suitable for topical oral administration include active ingredients and sucrose 5, Lozenges such as gum or tragacanth, Pastilles containing active ingredients and gels and inert bases such as glycerin or sucrose and gums, And mouthwash containing active ingredients and a suitable liquid carrier. The drug which can be applied topically in this invention can be synthesized into ointment, Cream, suspension, Lotion, powder, Solution, Medicated paste, colloid, spray, Aerosol or medicated oil, Can also be used with medicine patches, Bandages or ointments infused with active ingredients, One or more excipients or diluents can be selected. For diseases of the external tissues such as eyes or mouth and skin, The medicament is preferably applied topically in the form of an ointment or cream containing the active ingredient. In the ointment, The drug can be used with sacrificial or water-soluble ointment bases. Pharmaceutical ingredients can also be used in creams with oil / water cream bases. If necessary, The water phase in the cream base may include at least 30% w / w polyol, Namely propylene glycol, Butane-1, 3-diol, Mannitol, Sorbitol, Glycerols and polyethylene glycols and mixtures thereof include alcohols having two or more hydroxyl groups. Topical synthetic drugs may also include compounds that enhance the absorption or penetration of the drug into the skin or other affected areas. These agents that enhance skin penetration include dimethyl sulfoxide and other similar compounds. The oily phase in the emulsion of the invention may be composed of any known ingredients in any known manner. Although the oil phase may contain only one emulsifier (also known as emulgent), But it should be able to contain at least one emulsifier and oil, Oil 20 200423954 or a mixture of both. The water-absorbing emulsifier is best used in combination with a lipophilic emulsifier that has a stabilizing effect. It also contains both oil and grease. Emulsifiers or emulsifiers and stabilizers together form a so-called emulsion, Emulsification sacrifices oil and / or grease to form a so-called emulsified ointment base 5 Forms an oily dispersion in the cream. Emulsifiers and emulsifiers suitable for the synthesis of the drug of this invention include Tween 60, Span 80, Fatty alcohol, Myrtle, Glyceryl monostearate and sodium dodecyl sulfate. How to choose an oil or grease suitable for pharmaceutical synthesis depends on whether it can meet the needs of surface conditioning, Because many active compounds that may be used in the synthesis of pharmaceutical emulsifiers are inherently poorly soluble. Creams are best used in a non-greasy, No fouling, And in the form of washable products, It should also be of a suitable concentration to prevent leakage from hoses or other containers. Such as adipic acid, Stearates, Propylene glycol, Coconut oil fatty acid diols, 15 isopropyl myristate, Oleate, Isopropyl palmitate, A series of branched chain esters such as butyl stearate, such as linear or branched mono or dialkyl alkyl esters, or Crodamol CAP, The last three esters are the best choice. These esters can be used alone, It can also be matched with each other according to the expected characteristics. Or use white soft paraffin with high melting point and / or liquid paraffin or other mineral oil. 20 Synthetic drugs suitable for topical application to the eye should also include eye drops, The active ingredients can be dissolved or suspended in a suitable carrier, Especially aqueous solvents of active ingredients. Synthetic drugs used for rectal delivery can be made into suppositories, And choose the appropriate matrix containing cocoa butter or salicylate. 21 200423954 Synthetic drugs suitable for vaginal delivery can be made into vaginal suppositories containing prodrugs, Tampon, Cream, colloid, Medicated paste, In foam or spray form, And choose the appropriate carrier known in the industry. Synthetic drugs suitable for nasal delivery use solid carriers, Including coarse powders with a particle size 5 of 20 to 500 microns, Application method is inhalation, The powder to be placed on the front of the nasal cavity is suddenly inhaled. Suitable forms using liquid carriers are nasal sprays, Nasal drops or as an aerosol via a sprayer, Includes aqueous or oily solutions of the active ingredients. Synthetic drugs suitable for parenteral injection include aqueous or non-aqueous 10-degree sterile injections, It contains antioxidants, Buffers and bacteriostatic agents, And can dissolve, So that the drug can reach the same concentration as the blood of the application object; It also includes aqueous and non-aqueous sterile suspensions, Contains suspension formulations and thickeners as well as liposomes or other particulate systems capable of localizing compounds to blood components or human organs. Synthetic drugs can be placed in sealed or single-dose containers such as ampoules or other vials, Can also be stored under freeze-drying (lyophilization) conditions, Other sterile liquid carriers such as water for injection are only required before use. You can choose from the sterile powders described above, Particles or tablets Obtain temporary injections or suspensions. Optimal unit doses should be those containing medicinal ingredients in daily doses of 20 or units, Divided dose, Or an appropriate portion of a synthetic drug. We can understand it this way, In addition to the ingredients mentioned above, The synthetic drug of this invention may include other traditional preparations commonly added to this synthetic drug in the industry, For example, oral medications may contain sweeteners, Thickeners and other formulations such as 22 200423954 flavorings. It can also contain antitumor, Anti-cancer, Anti-angiogenesis or other active ingredients to improve immunity. Tannic acid and its prodrugs, Metabolites, Pharmaceutical salts or derivatives and their chemical ingredients can also be used in veterinary medicine, It can be manufactured in accordance with the traditional methods in the industry. In addition, This invention also provides a screening method for identifying a therapeutic agent capable of inhibiting vascularization or endothelial cell growth, include: (a) contact the preparation with appropriate cells or tissues; (b) contact another cell or tissue sample with a therapeutically effective amount of 10 tannic acid or a pharmaceutically acceptable derivative, Metabolites, Drug salts or their prodrugs; (c) comparing the sample growth in steps A and B, It was found that any agent in step A that is the same as or similar to the sample in step B and that slows the growth of cells is a agent that inhibits vascularization or endothelial cell growth 15. Other therapies and preparations described herein can be used with this sample. This invention includes a complete set of treatments for functional disorders caused by pathological angiogenesis in an individual. The protocol is to apply to a subject a certain amount of tannic acid or a pharmacologically acceptable derivative 20 with a certain effect according to the guidelines for use, Metabolites, A drug salt or a prodrug thereof. Conditions that this regimen can treat include arthritis, Skin diseases caused by angiogenesis, Diabetic retinopathy, Restenosis, Kaposi's sarcoma, Age-related macular degeneration, Capillary dilation, glaucoma, Keloids, Corneal transplant rejection, Wound granulation, Angiofibromas, Hereditary bleeding telangiectasia, Myocardial blood 23 200423954 tube neonatal, scleroderma, Rheumatoid arthritis, Psoriatic arthritis and osteoarthritis. [Embodiment] A detailed description of the preferred embodiment 5 The following examples further illustrate this invention, It should not be construed as limiting the invention. Example 1 Isolation and purification of tannic acid Tannic acid or tannic acid produced in large batches can be purified by a series of separation methods 10 (for example, K. Aoki et al. 91979 Analytical Biochemistry 95: 575-578) . The American Chemical Society's Reagent Grade tannins can also be purchased from Sigma, USA. Example 2 Endothelial Cell Inhibition Assay Using Tannic Acid 15 Endothelial Cell Reproduction Assay: The test is based on the test procedure proposed by Connolly et al., 1986, Analytical Biochemistry, 152, 136-4, and related improvements. From Molecule to Comprehensive Pharmacy, 209-223 pages / edit: Mara Dockies / Crouville Academic / Fulfillment Press). Neonatal bovine heart and pulmonary artery endothelial (CPAE) cells obtained from the United States National Strain Collection Center (ATCC) showed a confluence of nearly 95% at MEM-10E. Cells with a concentration of 0. The 25% insulin solution was released from the tissue culture flask and placed in a 24-well tissue culture plate. The culture medium in the culture plate was exactly the same, with a density of 10,000 cells in each culture well. The culture plate was cultured under the environment of 5% CO2 and 24 200423954 37 C. t ~ J, 蛉. Add test and control samples. The mother samples were placed in two M wells, and each culture well was 100 ml to ensure that it had the ability to reproduce. After 60 hours of incubation, the medium was extracted, and the results and discussions of cell acid enzymes were measured: I. The number of tritium cells was measured. Table I · Tannic acid concentration (mg / ml) that inhibits endothelial cell proliferation: '丄 3 6. 25 12. 5 25 50 Inhibition rate of endothelial cell proliferation (%) ·-0 〇 0 19. 3 84. 4 ίο 15 Table 1 ", the results we obtained in the endothelial cell proliferation inhibition assay .: Ning acid can indeed inhibit endothelial cell leanness. Danfic acid reduces endothelial cell growth at a low level of 5_ per milliliter It is 8 4 · 4%.… I] The correspondence between Dantinx / Chendu and inhibition of endothelial cell proliferation was tested. The results were also shown. It is very clear that at low concentrations, tannin Mosquito—species of endothelial cells multiply ___. And Dan I's ability to inhibit the proliferation of skin cells is directly related to its concentration. 20+ g Shanghuimingming has been described in detail through descriptions and examples, but in this area In the eyes of other professionals, further optimization and improvement are needed. For example, the method described in this invention can be completely the same or one of the known k tumors, anti-angiogenesis or immune-improving therapies and strategies for cartilage. Chemical compounds such as amines, sphingosine, and sphincter are combined with 因此. Therefore, the corrections made in this article ignore the limitations of the materials and scope of this item, and this point-we have also made the necessary comments in the attached request. [Schematic description] Graph 1 shows the corresponding relationship between the inhibition of endothelial cell proliferation and the concentration of dansolic acid in endothelial cell proliferation assays. [The main elements of the figure represent the symbol table] (none)
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