經濟部智慧財產局員工消費合作社印製 550074 A7 __ B7 五、發明說明（） 的非免疫往因子，鉑高血壓，高161脂症，高並贍固醇症等 也扮演一角β 慢性排斥作周所顯現之不寬容饬稆不受控制是因爲目 前沒有已知有效之治療或預防模式。因此，現仍繼績存在 對於預防、控制或是逆轉慢性移植物血管疾病症狀的有效 處理方式之需要。 同時也繼續存在的需要爲預防或治療當血管內膜平滑 肌細胞的增殖及移動所造成的再狹窄或血管閉塞之結 杲，例如藉由血管外科所誘導諸釦血管成形術之類。 依照本發明，目前已令人驚訝地發現式I化合物會抑制 諸如血管重建之類的血管病態，且特別地顯示可預防或對 抗在被移植器官所生的慢性排斥作闱。 依照本發明的特別發現，本發明提供： 1 · 一種.用於預防或治療患者中新血管內膜之增殖和增厚 的方法’其包括施藥給該患者有效治療量之式I化合物。 在一系列進一步特異性或選擇性具體事中，本發明 也提供： 本紙張尺度適用中國國豕ί示準（CNS)A4規格（210 X 297公釐） ---------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 550074 A7 B7 五、發明說明（） •2-1. —種用於預防或對抗在一個接受器宫或組織移植构 的接受者身上所顯示出的慢性排斥作用之方法，其包括之 步驟爲施藥給該接受者有效治療量之式I化合物。 2-2· —種用於預防或對抗移植物血管疾病的方法，例如 在一個接受器官或組織移植的接受者所產生的移植物血 管病態、動脈硬化或動脈粥樣硬化，或是局部缺血或再灌 流的傷害，其包括之步驟爲施藥給該接受者有效治療童之 式I化合物。 藉由慢性排斥作用症狀一詞意欲指對移植物所產生的 免疫反應，以及在如上述所移植的器官或組織之血管壁的 反應而造成的病況。式I化合物可有效減低慢性排斥作用 症狀或是改善因慢性排斥作周所造成的病況。 可以從一個捐贈者至一個相同或不同物種的受贈者進 行器官或組織移植。在這類被移植的器官或組織之中既有 的示範例是心臟、肝臟、腎臟、脾臟、肺贜、小腸稆胰贜， 或是前述任何的組合。 在一個進一步或選擇性具體事實中，本發明提俟： 3 · —種用於在有此需要之患者內預防或治療|管內膜 平滑肌細胞之增殖和移動的方法，例如再狹窄，以及或血 9 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ---------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 550074 A7 B7 五、發明說明（） 管閉塞隨後造成的血管傷害’例如並管成形術，其包括施 藥給該患者之有效治療量之式1化合物。 在一個進一步或選擇惶具體事實中，本發明边提俟： 4· 一種用於預防或對抗在一個接受器官或組織異種移 植的接受者所發生的急性或慢性排斥作用的方法，其包括 施藥給該接受者有效治療童之式I化合物。 異種器官或組織的移植例如包括心臟、肝臟、腎臟、脾 臟、肺臟、小腸和駿臟的（完整或部份，例如Langerhans 氏島），皮膚和骨髓的異種移植。 本發明另外也提烘： 5. —種式I化合物，其係用於任何如上述1至4點所 定義的方法；或是 6· —種式I化合物，其係用於一種藥物組成物的製備， 此藥物組成物係用於任何如同上述1至4點所定義之方 法；或是 7 ·—種於任何如上述1至4點所定義之方法之藥物組 成物，其包括一種式I化合物以及一種或多種的製藥上可 10 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------訂---------線 (請先閱讀背面之注意事項再填寫本頁) A7 B7 550074 五、發明說明（） 接受稀釋劑或載體。 式I化合物在治療上述的疾病和病況之用途可以在動 物試驗中獲得證钥，例釦依照以下所描述的方法。 A.慢性同種異體移植的排斥作用 將一雄性DA(RTla)大白鼠的腎臟以同樣位置移植入〜 雄性LewiKRT1)的受贈者內。全部有24隻動物被移植。 戶斤有的動物從移植的那天起1 4天中以每隻動物每天每公 斤體重口服7.5毫克的環胞黴素A來治療，以便預防急性 細胞的排斥作周°對側的腎切除術沒有執行β每一個實驗 組由6隻動物來組成，分別使用不同劑量的式I化合物或 安慰劑來處理。Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 550074 A7 __ B7 V. The non-immune factor of the invention (platinum hypertension, high 161 lipid, high steroids, etc.) also plays a role in chronic chronic rejection The manifestation of intolerance and uncontrol is because there are currently no known effective treatment or prevention models. Therefore, there is still a need for effective treatments to prevent, control or reverse the symptoms of chronic graft vascular disease. There also continues to be a need to prevent or treat crusting of the restenosis or vascular occlusion caused by the proliferation and movement of vascular intimal smooth muscle cells, such as angioplasty induced by vascular surgery. In accordance with the present invention, it has now surprisingly been found that compounds of formula I inhibit vascular pathologies such as vascular remodeling, and have particularly been shown to prevent or counteract chronic rejection in transplanted organs. In accordance with the particular findings of the present invention, the present invention provides: 1. A method for preventing or treating the proliferation and thickening of neovascular intima in a patient ', which comprises administering to the patient a therapeutically effective amount of a compound of formula I. In a series of further specific or selective matters, the present invention also provides: This paper size is applicable to China National Standards (CNS) A4 (210 X 297 mm) ----------- ---------- Order --------- line (Please read the notes on the back before filling out this page) Printed by the Intellectual Property Bureau Employee Consumer Cooperative of the Ministry of Economic Affairs 550074 A7 B7 V. Invention Instructions () • 2-1. — A method for preventing or combating chronic rejection shown on a recipient in a recipient house or tissue transplantation structure, including the steps of applying the drug to the recipient effectively A therapeutic amount of a compound of formula I. 2-2 · — A method for preventing or combating graft vascular disease, such as graft vascular pathology, arteriosclerosis or atherosclerosis, or ischemia in a recipient receiving an organ or tissue transplant Or reperfusion injury, comprising the step of administering to the recipient an effective treatment of a compound of formula I in a child. The term by the symptoms of chronic rejection is intended to refer to a condition caused by an immune response to a graft and a reaction in the vascular wall of an organ or tissue transplanted as described above. The compounds of formula I are effective in reducing the symptoms of chronic rejection or improving the conditions caused by chronic rejection. Organ or tissue transplants can be performed from one donor to a recipient of the same or different species. Exemplary examples of such transplanted organs or tissues are the heart, liver, kidney, spleen, lung, small intestine, pancreas, or any combination of the foregoing. In a further or optional specific fact, the present invention provides: 3-a method for preventing or treating in patients in need | endothelial smooth muscle cell proliferation and migration, such as restenosis, and or Blood 9 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) --------------------- Order ------- --Line (Please read the notes on the back before filling this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 550074 A7 B7 V. Description of the invention Includes a therapeutically effective amount of a compound of formula 1 administered to the patient. In a further or alternative specific fact, the present invention provides: 4. A method for preventing or combating acute or chronic rejection in a recipient of an organ or tissue xenograft, which includes the administration of The recipient is effectively treated with a compound of formula I in a child. Xenotransplantation of xenogeneic organs or tissues include, for example, heart, liver, kidney, spleen, lung, small intestine and viscera (whole or partial, such as Langerhans Island), xenografts of skin and bone marrow. The present invention also provides: 5.-a compound of formula I, which is used in any method as defined in points 1 to 4 above; or 6. · a compound of formula I, which is used in a pharmaceutical composition For preparation, the pharmaceutical composition is used in any method as defined in points 1 to 4 above; or 7. A pharmaceutical composition in any method as defined in points 1 to 4 above, which includes a compound of formula I And one or more pharmaceuticals, 10 paper sizes are applicable to China National Standard (CNS) A4 (210 X 297 mm) -------- Order --------- Line (please first Read the notes on the back and fill in this page) A7 B7 550074 5. Description of the invention () Accept thinner or carrier. The use of compounds of formula I for the treatment of the aforementioned diseases and conditions can be obtained in animal experiments, for example by following the methods described below. A. Rejection effect of chronic allograft The kidneys of a male DA (RTla) rat were transplanted in the same position into a recipient of ~ Male LewisKRT1). A total of 24 animals were transplanted. Some animals in the household have been treated with 7.5 mg of cyclosporin A orally per kg body weight per animal per day for 14 days from the day of transplantation to prevent acute cell rejection. Contralateral nephrectomy did not Each beta group was performed with 6 animals, each treated with a different dose of a compound of formula I or a placebo.
從移植手術後第53_64天開始，每隻受贈的動物口服式 ί化合掏或接受安慰劑來治療另外69-72天。在進行，移_ 手術後第1 4天，動物被置於藉囱核磁共振影像與腎臟的 灌流測量（將被移植的腎臟和自身對側腎臟作出較進 行移植物的評估。這樣的評估會重複至移植手術後_ 5 3 _ 64天以及至實驗結束。隨後將動物殺死並做屍體_ ^ σ 將排斥作用的參數（如MRI評分，被移植腎臟的相4對灌流 速率以及腎臟同種異體移植對於細胞的排斥作用$ $ _ 評分）和血管變異進行測定並做統計的分析。式I ϋ ---------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）From the 53th to the 64th day after transplantation, each of the recipient animals was treated orally or received a placebo for another 69-72 days. On the 14th day after the operation, the animals were placed on the MRI and the perfusion measurement of the kidneys (the transplanted kidney and the contralateral kidney will be evaluated more than the graft. Such an evaluation will be repeated. _ 5 3 _ 64 days after transplantation and until the end of the experiment. Animals were subsequently killed and carcass _ ^ σ parameters of rejection (such as MRI score, phase 4 perfusion rate of the transplanted kidney, and renal allograft For cell rejection ($ _ scoring) and vascular variation are measured and statistically analyzed. Formula I ϋ --------------------- 订----- Line (Please read the notes on the back before filling this page) Printed by the Intellectual Property Bureau Employee Consumer Cooperatives of the Ministry of Economic Affairs This paper is sized for China National Standard (CNS) A4 (210 X 297 mm)
550074 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（） (如化合物A)以每公斤體重0· 5至2·5毫克劑量施用在此 種大白鼠腎臟同種異體移植的模式上，在所有上述的排斥 作用參數倶造成降低效果。在這個試驗中，每隻動物每天 每公斤體重周2· 5毫克的化合物A來治療使其具有在排斥 作用上顯著較低的MRI評分、對於細胞排斥作用的組織 評分和血管變異：且一種藉由MRI所評鑑的灌注速率與 使周安慰劑實驗動物組比較具有顯著較低的降低效果。 B.主動脈移植手術 在大白鼠-內主動脈移植手術之模式中，對移植物的同種 異體反應不會破壞移植物，但會激發與臨床移植手術中慢 性排斥作用的症狀相像之病理變異。這些包括單核細胞 (淋巴細胞，巨噬細胞，部份的漿細胞）滲透進入動脈外 膜，以及血管內膜的增厚作用。 從一雄性的DA(RT1a)大白鼠內獲得捐贈者介於腎臟動 脈的分枝和尾部腸系膜主動脈起始之間長度大約1公分 之主動脈，並旦同位地移植在一雄性Lewis (RT11)大白鼠 內。在移植手術後的每一週，將大白鼠的體重記錄下來。 在解剖屍體時，將移植物和受贈者的移植物以及正好在其 上面及下面的主動脈之部份移出。將此部份在活體外用內 含2%聚甲醛和2.5 %戊二醛的磷酸鹽緩衝溶液完全浸入 約2分鐘，然後在栢同溶液中進行浸入固定作厍固定24 12 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 550074 經濟部智慧財產局員工消費合作社印製 A7 B7 1、發明說明（） 小時，之後放在4%緩衝性福馬林內固定。將移植物的片 段包埋入石鐵中’以此方法製備是由移植物的主動脈和受 贈者本身的主動脈的橫切面和縱切面。 將這些4微米厚的切面藉由蘇木精-伊紅，elastica-von-Gieson 及 periodic-acid-schiff 染色。除傳統性光學顯 微鏡之外，影像可藉S同焦距雷射掃瞄顯微鏡來記錄。在 每一個切面中，有四個區域被掃瞄，且甴每一個區域中血 管內膜和血管內膜加上介質的厚度以五個位置進行測 量σ · 在屍體解剖中，對於胸腺、脾臟、肝臟、腎臟、睪九和 精囊進行體重與組織分析。 在首先的實驗中包括四組動物，每組甴4隻動物組成。 在一組中進行同基因異體移植手術（Lewis對 Lewis)， 且該動物接受一種安慰劑之微乳化劑；其他組中包括同種 異體移植手術，且該每隻動物口服接受每天每公斤體重 2.5毫克安慰劑之微乳化劑或是含式I化合物的微乳化 劑，此實驗在移植手術後第七週予以終止。 第二個實驗中包括四組動物，每組由4隻動物組成。在 所有的實施例中進行同種異體移植手術，並且每隻動物口 服接受每天每公斤體重0.63，1.2 5, 2.5或5.0毫克的安慰 13 ^紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱^ --------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 550074 A7 ______ B7 五、發明說明（） 劑之微乳化劑或是含式I化合物的微乳化劑，此實驗在移 植手術後第十一週予以終止。 在這兩個實驗中，式I化合物（特別是化合物A )明顯 地抑制移植物浸潤作用和新血管內膜的生成。 C·血管成形術 血管成形術的硏究在氣球導管損害的模式中被完成：氣 球導管***法在第零天起進行，其中的精要如Powell et ai. (19S9)所描述。在異氟烷麻醉法之下，一支Fogarty 2F導 管經由外部的頸動脈***左邊共通的頸動脈孔道並且使 其膨脹（膨脹約使用10微升的水）。將膨脹像氣球的導 管沿著兵通的頸動脈之長度被收回三次，當後兩次時予以 溫和的搓柔以便獲得一種均勻的去內皮化作兩。然後將導 管移走，沿著外部的頸動脈予以縫合以便預防出血並且讓 動物恢復健康。 二組12隻RoRo大白鼠（400公克重，約24週大）被 使周於本硏究：一個控制組和另一組接受式I化合物。大 白鼠在m有的操作，實驗的步驟和分析之中是完全隨機。 被測試的化合物是利用口服（胃管灌食法）於每隻 動物從產生氣球狀傷害之前的第3天（-3天）直到本硏究 14 本紙張尺度適用中國國家標準（CNS)A4規格（210 x 297公釐） --------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 550074 A7 B7 五、發明說明（） 結束，即在產生氣球狀傷害之後第14天（+14天）。大 白鼠被放在獨立之鐵籠內並且無限制給予食物及水。 大白鼠隨後使闱異氟烷來麻醉，一個灌流導管經由心臟 的左心室***並且緊_主動脈弓，然後甩一個抽吸套管插 入心臟的右心室。將動物在灌流壓爲1 50mmHg下被灌 流，起始時用0.1M之磷酸鹽緩衝溶液（PBS，ρΗ7·4)灌 流1分鐘，隨後用含有2.5%的戊二醛溶液之磷酸鹽緩衝 溶液（ΡΗ7.4)灌流15分鐘。在套管尖端灌流壓爲150mmHg (約等於主動脈內灌流壓lOOmmHg，其係在一個初步實 驗中藉由在外頸部動脈中***一個連接壓力轉導器之套 管玦定）。隨後將頸動脈管切除，將其從週圍的組織中分 離並且浸入在內含7%蔗糖之0.1M的二甲砷酸鹽緩衝液 (ρΗ7·4)並且在4T：下培養整夜。隔天將頸動脈在室溫下 放入內含0.05%過錳酸鉀之0.1Μ的二甲砷酸鹽緩衝液內 予以浸濕和振盪。隨後將組織用不同濃度的乙醇系列予以 脫水；在75%的乙醇內兩次，每次10分鐘；在85%的乙 醇內雨次，每次10分鐘；在95%昀乙醇內三次，每次10 分鐘以及在100%的乙醇內三次，每次10分鐘…依照廠商 的建議將脫水的頸動脈隨後包埋入Technovit 7100內。因 爲氧被發現會抑制合適堅硬度的達成，故將包埋介質放在 乾燥器內於氬氣下進行聚合整晚。 使用硬金屬小刀在一個旋轉切片機中，從每一個頸動脈 15 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------^--------- (請先閱讀背面之注意事項再填寫本頁) 55〇〇7 4 經濟部智慧財產局員工消費合作社印製 A7 B7 $、發明說明（） 的中段部份切出1-2微米厚的切斷面並且用金沙氏染料染 色2分鐘。從每一個頸動脈中如此製備大約5個切斷面， 且介質，新血管內膜以及內腔之橫切面區域藉由一影像分 桥系統（MCID,多偷多，加拿大）進行形態測定評估。 在這個檢驗中，當每隻動物每天口服劑量在每公斤體重 0.5至2.5毫克式I化合物時，會抑制肌血管內膜的增殖。 在接受化合物A之大白鼠的血管內血管內膜的增厚較控 制組動物明顯地較少，例如，在接受每公斤體重〇. 5毫克 的動物中有50%的新血管內膜形成在統計上有抑制作 用，在接受每公斤體重2.5毫克的動物中75%有明顯抑制 作用。 D·活體心臟異種移植（甶田鼠至大白鼠） 將田鼠移植至大白鼠之異種移植的組合是一種所謂困 難一致性的組合。在大白鼠內不具有天然抗田鼠抗體，此 種抗體在足量下便可產生直接的超急性排斥作用，如同在 一致性組台中所觀察到的情形；儘管如此·在3-4天內排 斥作甩在未治療的受贈者中會產生，這是藉由抗體以及補 體而產生。這些反應在組織內藉囱血管的破壞，紅¢1球的 » * 滲出和外滲作用，以及藉由多形核白血球的顆粒球的流入 而顯現出來；通常那些是出血和血栓形成的徵兆。一旦此 種排斥作周已經藉由有效抑制抗體形成或是補體不活化 16 ^紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------------------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 550074 A7 __ B7 五、發明說明（） 來克服，隨後則可見一種細胞性排斥作用。這些反應在組 織內藉由單核細胞包括淋巴球、類淋巴母細胞和巨噬細胞 的流入，以及肌細胞實質部份的破壞而顯現。對於細胞排 斤作周的卻制需要較多的免疫抑制。先天性胸腺缺失的大 白鼠（mii/mu)缺乏完整細胞免疫系統（胸腺依賴性）並且 通常不能排斥同種異體移植物。此種動物能在3-4天內排 斥田鼠的異種移植物，就如同在胸腺正常的大白鼠內所產 生的相似反應，顯示（至少有部份的）抗田鼠抗體在大白 鼠內的合成是循胸腺依賴性的B-細胞反應而產生。此種 受贈者在田鼠異種移植手術中有助於藉由非胸腺依賴性 抗體媒介性排斥作用來評估排斥作用。 將一種敘利亞田鼠的心臟異位地移植在一種雄 性 Lewis(RTl’）大白鼠的腹部內並將捐贈者和受贈者的 主動脈接通，Μ且将捐贈者的右肺動脈接至受贈者的下靜 脈腔內。每天藉由腹部的觸診來觀察移植物。在動物的心 跳停止的情形下排斥作闱就終止。每週都將動物稱重。在 這系列的實驗中，終止是設定在第 28天。將動物殺死並 予以解剖；取出移植物，對胸腺、脾臟、肝臟、小腸和睪 丸檢驗其重量和組織。將動物的血液取出並處理成爲血 iW *羯於測定細胞溶解性抗-田鼠紅血球抗體和溶血丨生補 體活性。 在此種檢測中，式I化合物（化合物A )，當以例 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） --------訂---------線 (請先閱讀背面之注意事項再填寫本頁) 550074 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明說明（） 如每天每公斤體重2·5毫克施藥時，可延長無胸腺受贈 者內異種移植物的存活時間。 每日所需之劑量在實施本發明的方法洌釦將篏照所使 用的式I化合物、宿主的種類、施藥的方弍以及被治療 時之狀況嚴重性而改變。較佳的每日劑量範圍是大約從 0.25至25毫克，其爲單一劑量或分次劑量。 像對患者而言適合的每日劑量從例如每隻動物0.2至 25毫克，較佳者爲5至25毫克。式I化合物可藉由任 何傳統的途徑來施藥，特別是經過腸部的方式，例如經口 部的方式，例如以錠劑、膠囊、藥水的形式；經過鼻部的、 肺部的（藉由吸藥的方式）或非經腸部的方式，例如以可注 射周溶液或懸浮液的形式。做爲口部施薬的適當劑量其組 成大約〇.〇5至1J5毫克，通常爲1至1〇毫克的活惶成 份，例如化合物 A，與一種或多種製藥上可接受的稀釋 劑或載體一起使用。 當使闱如上述所指定之式I化合物於預防或治療慢性 排斥作用或異種移植的排斥作周可被施藥爲單獨的活倥 成份或與其它的藥物一起周在免疫調節攝生。舉例來說， 式I化合物可以用在與環胞黴素或ascomycins的組合， 或是這些藥物的免疫抑制類似物，例如環胞黴素A ’環胞 鐵素G，FK506等等；腎上腺皮脂類固醇；環磷醯胺；硫 18 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公爱） --------------------訂---------線^^ (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 550074 A7 B7 五、發明說明（） 嗤嘿玲；甲氣碟 Π令；brequinar ; leflunomide ; mizoribine ; 黴酉分酸（mycophenolic acid) ； mvcophenolate mofetil ； 15· deoxyspergualine，免疫抑制單株抗體，例如針對白血球受 體的單株抗體，例如 MHC，CD2，CD3，CD4，CD7，CD25， CD28，B7，CD45或CD5 8或是這些受體的配體；或是這 些受體免疫調節的化合物，例如CTLA4Ig。 在式I化合物與其它免疫抑制/免疫調節的治療共同施 藥的場合下，例如如上述所指定用於預防或治療慢性排斥 作甩或異種移植的排斥作甩，其與免疫抑制劑或免疫調節 的化合物兵同施藥的劑量將必然的依照所共同使用之藥 物的型式而改變，例如，是一種類固醇抑或是一種環胞黴 素，所使用之特殊藥物，以及在所治療時的狀況等等。依 照前述，本發明提侯更進一步的觀點： 一種如上述所定義其包括爲共同施藥的方法，例如伴隨 地或是按照順序的一種式I化合物之有效治療量和一霉另 外的薬物物貢，所述之另外的薬物物質是一種免疫抑詞劑 或是免疫調節的藥物，如同上面所述。 處方實例：膠囊 乙醇 20.0毫克 1，2-丙二醇 81.0毫克 ^ 精製浊 121.5毫克550074 A7 B7 printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the invention (such as Compound A) is administered at a dosage of 0.5 to 2.5 mg per kg of body weight on this model of rat kidney allograft In all the above-mentioned repulsion parameters 倶 caused a reduction effect. In this test, 2.5 mg of compound A per kilogram of body weight per day per animal was treated to give it a significantly lower MRI score for rejection, a tissue score for cell rejection, and vascular variation: Perfusion rates evaluated by MRI have a significantly lower reduction effect compared to the weekly placebo group of experimental animals. B. Aortic transplantation In the rat-inner aortic transplantation model, an allogeneic response to the graft will not destroy the graft, but it will trigger pathological variations similar to the symptoms of chronic rejection in clinical transplantation. These include the penetration of monocytes (lymphocytes, macrophages, and some plasma cells) into the adventitia of the arteries, and the thickening of the lining of the blood vessels. A male aorta with a length of about 1 cm between the branch of the renal artery and the start of the tail mesenteric aorta was obtained from a male DA (RT1a) rat and transplanted in the same position in a male Lewis (RT11). Inside the rat. The weight of the rats was recorded every week after the transplantation. During the dissection of the cadaver, the graft and the recipient's graft and the aorta just above and below it were removed. This part was completely immersed in vitro with a phosphate buffer solution containing 2% paraformaldehyde and 2.5% glutaraldehyde for about 2 minutes, and then immersed and fixed in Baitong solution for fixation. 24 12 This paper size applies to China Standard (CNS) A4 Specification (210 X 297 mm) -------- Order --------- Line (Please read the precautions on the back before filling this page) 550074 Intellectual Property of the Ministry of Economic Affairs A7 B7 printed by the Bureau ’s Consumer Cooperatives 1. Description of the invention () hours, and then fixed in 4% buffered formalin. Embedding pieces of the graft into stone iron 'was prepared in this way from the cross section and longitudinal section of the aorta of the graft and the aorta of the recipient itself. These 4 micron-thick sections were stained with hematoxylin-eosin, elastica-von-Gieson, and periodic-acid-schiff. In addition to traditional optical microscopes, images can be recorded with S-focus laser scanning microscopes. In each section, four areas were scanned, and the thickness of the vascular intima and vascular intima plus media in each area was measured at five locations. Σ In autopsy, for the thymus, spleen, Liver, kidney, 睪 Jiu and seminal vesicles for body weight and tissue analysis. The first experiment consisted of four groups of animals, each consisting of 4 animals. Allogeneic transplantation was performed in one group (Lewis vs. Lewis), and the animal received a microemulsifier of a placebo; the other group included allograft surgery, and each animal received 2.5 mg per kilogram of body weight per day orally Microemulsifiers of placebo or microemulsifiers containing compounds of formula I were terminated at the seventh week after transplantation. The second experiment included four groups of animals, each group consisting of 4 animals. Allogeneic surgery was performed in all the examples, and each animal received oral comfort of 0.63, 1.2 5, 2.5 or 5.0 mg per kg of body weight per day. 13 ^ Paper size applies Chinese National Standard (CNS) A4 (210 X 297) Public Love ^ -------------------- Order --------- Line (Please read the precautions on the back before filling in this page) Ministry of Economy Wisdom Printed by the Consumer Cooperative of the Property Bureau 550074 A7 ______ B7 V. Description of the invention () Microemulsifier of the agent or microemulsifier containing the compound of formula I, this experiment was terminated in the eleventh week after transplantation. In the experiment, the compound of formula I (especially compound A) significantly inhibited the infiltration of the graft and the formation of neovascular intima. C. Angioplasty The investigation of angioplasty was done in the mode of balloon catheter damage: balloon catheter The insertion method was performed from day zero, the essence of which is described by Powell et ai. (19S9). Under isoflurane anesthesia, a Fogarty 2F catheter was inserted into the common carotid artery port on the left through the external carotid artery. And make it swell (swell with about 10 microliters of water The balloon-like balloon was retracted three times along the length of the piercing carotid artery, and gently rubbed in the latter two times to obtain a uniform de-endothelialization into two. Then the catheter was removed and along the outside Carotid arteries were sutured to prevent bleeding and restore the animals' health. Two groups of 12 RoRo rats (400 g, about 24 weeks old) were studied in this study: one control group and the other received compounds of formula I In the operation of rats, the experimental procedures and analysis are completely random. The compounds tested were taken orally (gastric tube feeding) on the 3rd day before each animal's balloon injury (- 3 days) Until this study 14 This paper size applies to China National Standard (CNS) A4 (210 x 297 mm) -------------------- Order- ------- line (Please read the notes on the back before filling this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 550074 A7 B7 V. Description of the invention () After the balloon-like injury, 14 days (+14 days). Rats are placed in an independent iron cage and infinite Food and water were given. The rats were then anesthetized with isoflurane, a perfusion catheter was inserted through the left ventricle of the heart and the aortic arch was tightened, and then a suction cannula was inserted into the right ventricle of the heart. The animals were perfused at a pressure of 1 Perfused at 50mmHg, initially perfused with 0.1M phosphate buffer solution (PBS, ρ ， 7.4) for 1 minute, and then perfused with phosphate buffer solution (P 溶液 7.4) containing 2.5% glutaraldehyde solution for 15 minutes The perfusion pressure at the tip of the cannula is 150 mmHg (approximately equal to the intra-aortic perfusion pressure of 100 mmHg, which was determined in a preliminary experiment by inserting a cannula connected to a pressure transducer into the external cervical artery). The carotid artery tube was subsequently excised, separated from the surrounding tissue and immersed in 0.1 M dimethylarsenate buffer (ρΗ7.4) containing 7% sucrose and cultured overnight at 4T :. The next day, the carotid artery was placed in a 0.1 M dimethylarsenate buffer solution containing 0.05% potassium permanganate at room temperature, soaked and shaken. The tissue was subsequently dehydrated with a series of ethanol of different concentrations; twice in 75% ethanol for 10 minutes each; rain in 85% ethanol for 10 minutes each; three times in 95% ethanol For 10 minutes and three times in 100% ethanol for 10 minutes each time ... Follow the manufacturer's recommendation to embed the dehydrated carotid artery into the Technovit 7100. Since oxygen was found to inhibit the achievement of a suitable hardness, the embedding medium was placed in a desiccator under argon for polymerization overnight. Using a hard metal knife in a rotary microtome, 15 papers from each carotid artery are applicable to Chinese National Standard (CNS) A4 (210 X 297 mm) -------- ^ ----- ---- (Please read the notes on the back before filling this page) 55〇07 4 Printed A7 B7 $ printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, the middle section of the invention description () cut out 1-2 microns Thick cut surfaces and stained with golden sand's dye for 2 minutes. Approximately 5 sections were prepared in this way from each carotid artery, and the media, neovascular intima, and cross-sectional area of the lumen were assessed by a morphological bridge system (MCID, Steal More, Canada). In this test, when the daily oral dose of each animal is 0.5 to 2.5 mg of the compound of formula I per kilogram of body weight, the myointimal proliferation is inhibited. The thickening of the vascular intima in the blood vessels of rats receiving Compound A was significantly less than that in the control group. For example, in the animals receiving 0.5 mg per kilogram of body weight, 50% of the neovascular intima was formed. Inhibition effect was observed in 75% of animals receiving 2.5 mg per kg body weight. D. Living Heart Xenotransplantation (Mole Voles to Rats) The combination of xenograft transplanting Voles to rats is a so-called difficult combination. There is no natural anti-vole antibody in rats, and this kind of antibody can produce direct hyperacute rejection in sufficient amount, as observed in the consistent group; however, rejection within 3-4 days Action rejection occurs in untreated recipients, which is produced by antibodies and complement. These reactions are manifested in the destruction of blood vessels in the tissues, the exudation and extravasation of red ¢ 1 spheres, and the influx of granulocytes from polymorphonuclear leukocytes; usually those are signs of bleeding and thrombosis. Once such rejection has been achieved by effectively inhibiting the formation of antibodies or inactivation of complement 16 ^ paper size applies Chinese National Standard (CNS) A4 specifications (210 X 297 mm) ------------ -------- Order --------- line (please read the notes on the back before filling this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 550074 A7 __ B7 V. Description of the invention () To overcome, then a cellular rejection can be seen. These reactions are manifested in tissues by the influx of monocytes, including lymphoblasts, lymphoblasts, and macrophages, and the destruction of substantial portions of muscle cells. For cell killing, it requires more immunosuppression. Rats with congenital thymus deficiency (mii / mu) lack an intact cellular immune system (thymus-dependent) and usually cannot reject allografts. This animal can reject voles xenografts in 3-4 days, similar to the response in normal thymus rats, showing that (at least part of) the synthesis of anti-voles antibodies in rats is Produced by a thymus-dependent B-cell response. Such recipients can help evaluate rejection by thymus-independent antibody-mediated rejection during vole xenograft surgery. A Syrian voles heart was ectopically transplanted into the abdomen of a male Lewis (RTl ') rat and the aorta of the donor and the recipient was connected, and the donor's right pulmonary artery was connected to the recipient's lower Intravenous cavity. The graft is observed daily by palpation of the abdomen. Rejection ends when the animal's heartbeat stops. Animals are weighed every week. In this series of experiments, termination was set at day 28. The animals were killed and dissected; the grafts were removed and their weights and tissues were examined on the thymus, spleen, liver, small intestine and testes. The blood of the animal was taken out and processed into blood iW *, and the cytolytic anti-vole red blood cell antibody and hemolytic complement activity were measured. In this kind of test, the compound of formula I (compound A), when the paper size is exemplified, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) is applied. -------- Order ----- ---- Line (Please read the precautions on the back before filling this page) 550074 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention () When 2.5 mg per kg of body weight per day, Prolongs survival of xenografts in athymic recipients. The daily dose required to implement the method of the invention will vary depending on the compound of formula I used, the type of host, the method of administration, and the severity of the condition at the time of treatment. The preferred daily dose range is from about 0.25 to 25 mg, which is a single dose or a divided dose. A daily dose which is suitable for the patient is, for example, 0.2 to 25 mg per animal, preferably 5 to 25 mg. The compounds of formula I can be administered by any conventional route, in particular via the intestines, such as via the oral route, for example in the form of tablets, capsules, potions; via the nasal, pulmonary (by By inhalation) or parenterally, for example in the form of a weekly injectable solution or suspension. A suitable dose for oral administration has a composition of about 0.05 to 1 J5 mg, usually 1 to 10 mg of a live tincture, such as Compound A, for use with one or more pharmaceutically acceptable diluents or carriers. . When the compound of formula I as specified above is used in the prevention or treatment of chronic rejection or rejection of xenograft, it can be administered as a separate active ingredient or together with other drugs to immunomodulate. For example, compounds of formula I can be used in combination with cyclosporine or ascomycins, or immunosuppressive analogs of these drugs, such as cyclosporin A 'cyclosporin G, FK506, etc .; adrenal sebaceous steroids ; Cyclophosphamide; Sulfur 18 This paper size applies to China National Standard (CNS) A4 (210 X 297 public love) -------------------- Order- ------- line ^^ (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 550074 A7 B7 V. Description of the invention () Let; brequinar; leflunomide; mizoribine; mycophenolic acid; mvcophenolate mofetil; 15. deoxyspergualine, immunosuppressive monoclonal antibodies, such as monoclonal antibodies against leukocyte receptors, such as MHC, CD2, CD3, CD4, CD7 CD25, CD28, B7, CD45 or CD5 8 or ligands for these receptors; or compounds that immunoregulate these receptors, such as CTLA4Ig. Where the compound of formula I is co-administered with other immunosuppressive / immunomodulatory treatments, such as those specified above for the prevention or treatment of chronic rejection or xenograft rejection, it is associated with immunosuppressive or immunomodulatory The dosage of the compound administered will necessarily change depending on the type of drug used together, such as a steroid or a cyclosporine, the special drug used, and the condition under treatment, etc. . According to the foregoing, the present invention provides a further perspective: a method, as defined above, that includes co-administration, such as, concomitantly or sequentially, a therapeutically effective amount of a compound of formula I The other mash material is an immunosuppressant or an immunomodulatory drug, as described above. Prescription example: Capsules ethanol 20.0 mg 1,2-propanediol 81.0 mg ^ refined turbidity 121.5 mg
Cremophor RH40 202.5 毫克 化合物A 20.0毫克 19 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ---------------------訂---------線^ (請先閱讀背面之注意事項再填寫本頁) 550074 A7 _B7 五、發明說明（） 合計 500毫克 式I化合物對依照本發明所需的劑量具有良好的耐受 性。舉例來說，NTEL對於化合物A而言在一個4適的毒 性硏究中是在大白鼠內每天每公斤體重〇. 5毫克以及在猴 子內每天每公斤體重1.5毫克的劑量。 --------------------^--------- (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 20 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）Cremophor RH40 202.5 mg Compound A 20.0 mg 19 This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) --------------------- Order --------- Line ^ (Please read the notes on the back before filling out this page) 550074 A7 _B7 V. Description of the invention () A total of 500 mg of the compound of formula I has a good dose according to the present invention. Tolerance. For example, NTEL for Compound A in a 4 toxicity study was a dose of 0.5 mg / kg body weight per day in rats and 1.5 mg / kg body weight per day in monkeys. -------------------- ^ --------- (Please read the precautions on the back before filling out this page) Employees of the Intellectual Property Bureau of the Ministry of Economy Consumption Printed by the cooperative 20 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)