TW200824699A - Arsenic compounds for the treatment of the arsenic-sensitive blast-cell related diseases - Google Patents
Arsenic compounds for the treatment of the arsenic-sensitive blast-cell related diseases Download PDFInfo
- Publication number
- TW200824699A TW200824699A TW095145082A TW95145082A TW200824699A TW 200824699 A TW200824699 A TW 200824699A TW 095145082 A TW095145082 A TW 095145082A TW 95145082 A TW95145082 A TW 95145082A TW 200824699 A TW200824699 A TW 200824699A
- Authority
- TW
- Taiwan
- Prior art keywords
- group
- arsenic trioxide
- disease
- mice
- test
- Prior art date
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
200824699 九、發明說明: 【發明所屬之技術領域】 本發明涉及使用砷化物,如三氧化二砷處理胚胞細胞疾病的方法。 【先前技術】 近年氣喘病的基本病因發現仍是以慢性發炎(Chronic inflammation)爲 主軸。由於基因遺傳與外界環境的雙重影響,導致過敏發炎反應的發生, 影響的發炎細胞,包括··樹突細胞、上皮細胞、T細胞、β細胞、肥胖細胞、 顆粒細胞及嗜伊紅白血球皆捲入這場戰爭,如何早期發現、早期預防或早 期治療氣喘病是努力的目標。 氣喘的發明原因可能爲各種刺激(誘因)引發Τ細胞的免疫反應控制失 常而產生的氣管炎症(ΑΙ),其誘發或加重氣管高反應性並導致支氣管收縮' 粘膜充血水腫及分泌亢進、微血液滲漏並麵有氣管重塑,最終引起氣管 狹窄、氣流受限,臨床症狀發作。 全身性紅斑狼瘡(systemic lupus erythematosus,SLE)是一種進行性 結締組織病,其特徵爲體內產生多種自身抗體,能對自身許多標的組織成 分造成損害。本病屬于多系統性自身免疫病,臨床主要表現爲:關節炎、 腎小球腎炎、癲癇樣發作、貧血或血小板減少等。本病常有發作和緩解相 交替。過去認爲SLE治療困難,預後甚差;近來由於醫學科學的進展,對 SLE已有了較深的認識,在早期診斷和及時治療情況下,使重要器官受損 減少,提升存活率,使S丨E的預後大爲改觀。 近30多年由於免疫病理學的發展,證實SLE患者體液免疫和細胞免 疫均有不同程度異常改變。然而對于SLE的確切病因至今仍未完全闡明, 5 200824699 唯與下列某些因素可能有關:遺傳原素、性激素原素、病毒感染'藥物。 瘧疾(malaria)在我國古代稱爲瘴氣,國外稱malaria,爲義大利文 mala(不良)與aria(空氣)兩字合成。該病是由雌性瘧蚊叮咬人體時將其體內 寄生的瘧原虫傳入人體而引起。臨床表現爲間歇性、定時性、發作性寒戰、 高熱和大汗,以及貧血和脾腫大。間日瘧和三日瘧常有復發,惡性瘡發熱 不規則,的病患表現爲中樞神經系統功能失常,呈現凶險發作。 人類瘧疾有4種,由4種不同瘧原虫引起,即間日瘧(vivax malaria, benign tertian),病原爲間日瘡原虫(Plasmodium vivax);三日瘡(quartan φ malaria, malariae malaria),病原爲三日瘧原虫(Ρ· malariae);卵开多瘧(ovale malariae),病原爲卵形瘡原虫(Ρ· ovale);惡性瘧(falciparum malaria, malignant tertian),病原爲惡性瘧原虫(Ρ· falciparum)。 錐蟲約有20多種,作爲人類致病原的僅2種。錐蟲病是錐蟲寄生人 體引起的疾病,包括美洲錐蟲病(American trypanosomiasis)和非洲錐蟲 病,前者又稱恰加斯病(chagas disease〉,後者又稱睡眠病(African sleeping sickness)〇 非洲錐蟲病病原爲布氏錐蟲,其3個亞種對人均致病。布氏羅得西亞 • 錐蟲(T· b. rhodesiense)引起羅得西亞錐蟲病(東非睡眠病);布氏甘比亞錐 蟲(T· b, gambiense)引起·岡比亞錐蟲病沖西非睡眠病);布氏布氏錐蟲(T. b· brucei)引起的病例,臨床雖有報告,但較少,主要引起牛發病。 非洲錐蟲病(African trypanosomiasis)又稱昏睡病(sleeping sickness),是非洲人畜共患的嚴重疾病之一。該病由一種屬于布氏錐蟲複 合組(T. brucei Complex)帶有鞭毛的寄生原蟲弓丨起的疾病,采采蠅爲傳播媒 介。疾病早期表現爲不規則發熱、淋巴結炎等,後期爲中樞神經系統受損 表現,有嚴重頭痛、反應遲鈍、嗜睡直至昏睡,終至死亡。 200824699 傳統中藥因爲採擷於天地自然,許多天然藥物用於人體時不免產生_ 性等副作用。中藥炮製技術長時間以來一直是減低中藥毒性副作用的主要 方法之一。砒霜爲一種至毒藥物代表,傳統中藥炮製技術雖可將其毒性減 至一定程度,彳旦長久以來仍給人不良印象,並令人聞之色變,因此限制應 用砒霜的發展。砒霜內所含的化學成分種類眾多,然其主要成分爲三氧化 二砷,目前已知的文獻中,對於砸霜的應用,亦以其主要成分三氧化二砷 爲主。由於中草藥逐日受到世界各國的重視,對於砥霜的瞭解及應用與日 • 俱增,因此亦使得三氧化二砷的使用受到重視,舉例而言,利用三氧化二 砷治療如子宮頸癌、皮膚癌、食道癌等癌症,或皮膚疾病如俗稱雞眼。然 . 而,關於三氧化二砷的其他應用,仍有待醫藥硏究者進一步開發與硏究。 【發明内容】 本發明涉及使用砷化物,如三氧化二砷處理胚胞細胞疾病的方法。本 發明涉及以三氧化二砷在製備治療對過敏性疾病、免疫性疾病、纖維母細 胞疾病、發炎疾病和感染性疾病,特別是瘧疾、非洲錐蟲病、氣喘和全身 性紅斑狼瘡的藥物中的應用。 本發明解決其技術問麵採用的技術方案詳述如下: 本發明涉及提供以三氧化二砷在製備治療對砷化物敏感的胚胞細胞疾 病的藥物中的應用。 較佳的是,藥物以注射方式使用。 較佳的是,對砷化物敏感的胚胞細胞疾病選自如下:過敏性疾病、免 疫性疾病、纖維母細胞疾病、發炎疾病' 感染疾病和触蟲疾病。 7 200824699 更佳的是,免疫性疾病選自如下:結締組織疾病、自體免疫甲狀腺疾 病、神經肌肉自體免疫疾病、腸胃道自體免疫疾病、自體免疫肝炎、原發 型肝膽硬化症、原發型硬化性膽管炎、自體免疫心臟炎和動脈炎。 最佳的是,免疫疾病選自如下:全身性紅斑狼瘡、類風濕性關節炎、 硬化症、格雷氏病、重症肌無力、多重硬化症、潰瘍性結腸炎和克隆氏症 (Crohn’s disease)〇 較佳的是,過敏性疾病選自如下:氣喘、過敏性肺炎、擴散性肺間纖 φ 維病變、過敏性鼻炎、嗜伊紅關聯性鼻炎、春天結膜炎和馨痲疹。 較佳的是,發炎疾病選自如下:肺炎、骨髓炎、痲瘋病、梅毒、結核 • 病、肝炎、腫瘤形成和慢性阻塞性肺部疾病。 . 較佳的是,纖維母細胞疾病選自如下:肝纖維化、肺纖維化、皮膚纖 維化、全身性纖維化、塵肺病、結核病、非典型肺炎(S AR S)、成人呼 吸困厄綜合症狀、慢性阻塞性肺部疾病、疤痕、疤腫、乾癖、侵襲囊性纖 維變性和神經纖維瘤病。 • 較佳的是,感染疾病爲寄生蟲疾病。 雛的是,寄生蟲疾病爲瘧疾雜蟲病。 較佳的是,對砷化物敏感的胚胞細胞選自如下:白血球、週邊血液單 核球和纖維母細胞。 較佳的是,藥物包括約〇·〇〇1μΜ至約20μΜ的三氧化二砷。 更加的是,藥物包括約〇·1μΜ至約15μΜ的三氧化二砷。 最佳的是,藥物包括約0.1 μΜ至約10μΜ的三氧化二砷。 200824699 在此所使用的專有名詞『對砷化物敏感的胚胞細胞』意指由白血球、 週邊血液單核球麵維母細胞。 亦吐所使用的專有名詞F哺乳動物』意指包含人類的哺乳動物。 下面結合附圖和實施例對本實用新型做進一步地詳細說明: 實施例一:以三氧化二砷治療支氣管氣喘的動物試驗 1·1材料 試驗動物:選用常用的Balb/c雌鼠,6-β週大,餵食六週,每組15 隻。自台大動物中心購進SPF六週大的BALB/c雌鼠,每隻小鼠分別飼養, • 室溫控制於22±2t:,光暗循環時間各爲12小時(早上六點亮,下午六點 暗),自由攝食飲水及AIN_76配方粉末飼料,每週記錄攝食量及體重二次, “ 隔周以眼窩採血一次;小鼠八週大時則隨機分組爲四組,致敏步驟與接受 治療的方法如後述。 藥物:三氧化二砷注射液(Asadin®, 10mg/10ml/vial),以PBS solution 稀釋。 試劑:不含銘鎂離子之平衡鹽水(balanced salt solution ; HBSS)灌洗 液、Trypan blue、蘇木青-伊紅(hematoxylin-eosin、碳酸覆蓋緩衝液 (carbonate coating buffer)、含有 0.05%Tween 20 之 1X PBS 緩衝液、阻 斷液(blocking solution ;含有 1% BSA-0.05% Tween 20 之 1X PBS 緩沖 液)、[3H] TdR(lf 確認)。 其他:卵白蛋白(ovalbumin)、臘膜(paraffin)、eotaxin EUSA 套組(旧L, Hamburg, Germany)、抗老鼠 IgE 抗體。 主要儀器:霧化器(DeVilbiss, Somerset, PA)、離心機、血球計數器、 顯微鏡、eotaxln ELISA讀取機、培養盤讀取機(microplate reader)、液體 200824699 閃爍計數器、Plethymography。 ‘ 1·2試驗方法 1·2·1建立呼吸道發炎動物模式 選用常用的BALB/C雌鼠,6-8週大,老鼠致敏步驟先以卵白蛋白腹 腔注射置老鼠體內,分別於第〇、14、21、和28天,以相同劑量之卵白蛋 白重複注射老鼠。將6到8隻老鼠同時置於密閉容器中,以霧化器將OVA 抗原製作成可吸入粒子型態後,讓老鼠在此密閉容器內吸入抗原超過20分 • 鐘。霧化器會以〇.3ml/min的速率送出0.5-5m⑺的抗原顆粒分子。 1_2·2測定卵白蛋白(ovalb_in,_專一性抗體的EUSA測量法 " 將〇.5mg抗原溶於100ml碳酸覆蓋緩衝液後,加入微量培養盤于4 — 靜置過夜。第2天以含有a〇5%Tween 20之1XPBS緩衝液清洗微量培養 盤。然後以阻斷液阻斷微量培養盤,再清洗3次。接著每個微孔加入100 μΙ 待測樣本-血清似阻斷液稀釋)反應,反應後再清洗β次。加入100m丨抗老 鼠丨g[抗體進行反應再清洗6次。上述反應皆在37。(:進行45分鐘,在移 _ 至室溫反應15分鐘。最後,每個微孔再加入100μ丨ABTS( 2.2,-Azino- bis-3-200824699 IX. INSTRUCTIONS: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method of treating blast cell disease using an arsenide such as arsenic trioxide. [Prior Art] The basic cause of asthma in recent years is still the main cause of chronic inflammation (Chronic inflammation). Due to the dual effects of genetic inheritance and the external environment, allergic inflammatory reactions occur, affecting inflammatory cells, including dendritic cells, epithelial cells, T cells, beta cells, obese cells, granulosa cells, and eosinophils. In the war, how to detect early, prevent early or treat asthma early is the goal of hard work. The cause of asthma may be caused by various stimuli (inducing) to induce tracheal inflammation (ΑΙ) caused by abnormal control of immune response of sputum cells, which induce or aggravate tracheal hyperresponsiveness and cause bronchoconstriction' mucosal congestion and edema, hypersecretion, micro blood Leakage and tracheal remodeling in the end, resulting in tracheal stenosis, airflow limitation, clinical symptoms. Systemic lupus erythematosus (SLE) is a progressive connective tissue disease characterized by the production of a variety of autoantibodies in the body that can cause damage to many of its own constituent components. The disease is a multi-system autoimmune disease, the main clinical manifestations are: arthritis, glomerulonephritis, epileptic seizures, anemia or thrombocytopenia. The disease often has an episode and an alternating phase of remission. In the past, it was considered that SLE was difficult to treat and the prognosis was very poor. Recently, due to the advancement of medical science, SLE has a deeper understanding. In the case of early diagnosis and timely treatment, the damage of vital organs is reduced, and the survival rate is improved. The prognosis of 丨E is greatly improved. Due to the development of immunopathology in the past 30 years, it has been confirmed that the humoral immunity and cellular immunity of SLE patients have abnormal changes in different degrees. However, the exact cause of SLE has not yet been fully elucidated. 5 200824699 may be related to some of the following factors: genetic elements, sex hormones, viral infections. Malaria is called hernia in ancient China. It is called malaria in foreign countries. It is a synonym for Mala mala (bad) and aria (air). The disease is caused by the female malaria mosquito biting the parasitic malaria parasite into the human body when biting the human body. Clinical manifestations include intermittent, timed, episodic chills, high fever and sweating, as well as anemia and splenomegaly. There are frequent recurrences between vivax malaria and three-day malaria, and irregular malignant sores are characterized by a malfunction of the central nervous system and a dangerous episode. There are four types of human malaria caused by four different malaria parasites, namely vivax malaria (benign tertian), the pathogen is Plasmodium vivax, and the third disease (quartan φ malaria, malariae malaria), pathogen It is Plasmodium malariae; ovale malariae, the pathogen is Ovary ovale; falciparum malaria (malignant tertian), the pathogen is Plasmodium falciparum (Ρ· Falciparum). There are about 20 species of trypanosomes, and only two species are pathogenic to humans. Trypanosomiasis is a disease caused by parasitic parasites, including American trypanosomiasis and African trypanosomiasis. The former is also known as chagas disease, which is also known as African sleeping sickness. The pathogen of African trypanosomiasis is Trypanosoma brucei, and its three subspecies are pathogenic to humans. T. b. rhodesiense causes Rhodesia trypanosomiasis (eastern sleep disease); Trypanosoma chinensis (T·b, gambiense) caused by Gambia trypanosomiasis (Western non-sleep disease); cases caused by T. b. brucei, although clinically reported, but less It mainly causes the onset of cattle. African trypanosomiasis, also known as sleeping sickness, is one of the serious diseases of African zoonosis. The disease is caused by a parasitic protozoan that is associated with the flagella of the T. brucei Complex, which is the vector of transmission. Early manifestations of the disease are irregular fever, lymphadenitis, etc., and later manifested as damage to the central nervous system, severe headache, unresponsiveness, lethargy until drowsiness, and eventually death. 200824699 Traditional Chinese medicine is naturally natural, and many natural medicines are used in humans, which can cause side effects such as _ sex. Traditional Chinese medicine processing technology has long been one of the main methods to reduce the side effects of traditional Chinese medicine. The arsenic is a representative of the toxic drugs. Although the traditional Chinese medicine processing technology can reduce its toxicity to a certain extent, it has been a bad impression for a long time, and it has become a fascinating change, thus limiting the development of arsenic. There are many kinds of chemical components contained in arsenic, but its main component is arsenic trioxide. In the current known literature, the application of arsenic is mainly based on its main component, arsenic trioxide. Since Chinese herbal medicines have been highly valued by countries all over the world, the understanding and application of arsenic have increased, so the use of arsenic trioxide has also been paid attention to. For example, the use of arsenic trioxide for the treatment of cancers such as cervical cancer, skin cancer, and esophageal cancer. Or skin diseases such as corns. However, other applications of arsenic trioxide have yet to be further developed and studied by pharmaceutical researchers. SUMMARY OF THE INVENTION The present invention relates to a method of treating blast cell disease using an arsenide such as arsenic trioxide. The present invention relates to the use of arsenic trioxide for the preparation of a medicament for the treatment of allergic diseases, immune diseases, fibroblast diseases, inflammatory diseases and infectious diseases, particularly malaria, African trypanosomiasis, asthma and systemic lupus erythematosus. DETAILED DESCRIPTION OF THE INVENTION The technical solution employed by the present invention to address its technical aspects is as follows: The present invention relates to the use of arsenic trioxide for the preparation of a medicament for the treatment of arsenic-sensitive blast cell diseases. Preferably, the drug is used by injection. Preferably, the arsenic-sensitive blast cell disease is selected from the group consisting of an allergic disease, an immunological disease, a fibroblast disease, an inflammatory disease, an infectious disease, and a caterpillar disease. 7 200824699 More preferably, the immune disease is selected from the following: connective tissue disease, autoimmune thyroid disease, neuromuscular autoimmune disease, gastrointestinal autoimmune disease, autoimmune hepatitis, primary hepatobiliary sclerosis, Primary sclerosing cholangitis, autoimmune carditis and arteritis. Most preferably, the immune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, sclerosis, Gracies disease, myasthenia gravis, multiple sclerosis, ulcerative colitis, and Crohn's disease. Preferably, the allergic disease is selected from the group consisting of asthma, hypersensitivity pneumonitis, diffuse interstitial fibrosis, allergic rhinitis, eosinophilic rhinitis, spring conjunctivitis, and succulent measles. Preferably, the inflammatory disease is selected from the group consisting of pneumonia, osteomyelitis, leprosy, syphilis, tuberculosis, hepatitis, tumor formation, and chronic obstructive pulmonary disease. Preferably, the fibroblastic disease is selected from the group consisting of liver fibrosis, pulmonary fibrosis, skin fibrosis, systemic fibrosis, pneumoconiosis, tuberculosis, atypical pneumonia (S AR S), and adult respiratory distress syndrome. Chronic obstructive pulmonary disease, scarring, swelling, dryness, invasive cystic fibrosis and neurofibromatosis. • Preferably, the infectious disease is a parasitic disease. The younger one is that the parasitic disease is malaria. Preferably, the arsenic-sensitive blast cells are selected from the group consisting of white blood cells, peripheral blood mononuclear cells, and fibroblasts. Preferably, the drug comprises arsenic trioxide from about 1 μM to about 20 μM. More specifically, the drug comprises arsenic trioxide from about 1 μΜ to about 15 μΜ. Most preferably, the drug comprises from about 0.1 μΜ to about 10 μΜ of arsenic trioxide. 200824699 The proper term "arsenic-sensitive blast cells" as used herein means mononuclear spheroid cells from white blood cells and peripheral blood. The term "mammal" as used in vomiting means mammals containing humans. The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Example 1: Animal test for treating bronchial asthma with arsenic trioxide 1.1 Materials Test animals: commonly used Balb/c female rats, 6-β weeks old, Feed for six weeks, 15 in each group. BALF/c female rats of SPF six weeks old were purchased from National Taiwan University Animal Center, and each mouse was raised separately. • Room temperature was controlled at 22±2t: and light and dark cycle time was 12 hours each (6 in the morning, 6 in the afternoon) Point dark), free access to drinking water and AIN_76 formula powder feed, weekly food intake and body weight twice, "week blood collection once every other week; mice eight weeks old are randomly divided into four groups, sensitization steps and treatment The method is as follows. Drug: arsenic trioxide injection (Asadin®, 10mg/10ml/vial), diluted with PBS solution Reagent: Balanced salt solution (HBSS) lavage solution, Trypan blue, Hematoxylin-eosin, carbonate coating buffer, 1X PBS buffer containing 0.05% Tween 20, blocking solution (1X PBS buffer containing 1% BSA-0.05% Tween 20) Liquid), [3H] TdR (lf confirmed) Other: oval albumin (ovalbumin), wax film (paraffin), eotaxin EUSA kit (old L, Hamburg, Germany), anti-mouse IgE antibody. Main instrument: nebulizer (DeVilbiss, Somerset, PA), centrifuge, hemocytometer, microscope, eotaxln ELISA reader, microplate reader, liquid 200824699 scintillation counter, Plethymography. '1·2 test method 1·2·1 establishes respiratory tract inflammation The animal model was selected from the commonly used BALB/C female rats, 6-8 weeks old. The mouse sensitization step was first intraperitoneally injected into the mice with ovalbumin, and the same dose of ovalbumin was obtained on the second, 14, 21, and 28 days, respectively. The mice were repeatedly injected. Six to eight mice were simultaneously placed in a closed container, and the OVA antigen was made into an inhalable particle type by a nebulizer, and the mice were inhaled in the closed container for more than 20 minutes. The device will send 0.5-5m (7) of antigen particle molecules at a rate of ml3ml/min. 1_2·2 Determination of ovalbumin (ovab_in, _specific antibody EUSA measurement " 〇.5mg antigen dissolved in 100ml carbonate coverage buffer After that, the microplate was added to the mixture overnight. On the second day, the microplate was washed with 1×PBS buffer containing a〇5% Tween 20. Then the microplate was blocked with blocking solution and washed three times. Each well was added with 100 μΙ of the sample to be tested - diluted with serum-like blocking solution), and washed for β times after the reaction. Add 100 m 丨 anti-aging mouse 丨 g [antibody for reaction and wash 6 times. The above reactions are all at 37. (: For 45 minutes, transfer to _ to room temperature for 15 minutes. Finally, add 100 μ丨 ABTS to each well (2.2, -Azino-bis-3-
Ethylbenzthiazoline各Sulfonic Acid)受質溶液於室溫呈色約30分鐘,以每 微孔100μΙ的5%SDS終止反應,並以培養盤讀取機讀取吸光値。 1·2·3支氣管肺泡職液與巨曬細胞、淋巴球、嗜中性球與嗜伊紅性 白血球之數目計算 最後一次吸入抗原24小時後,將所有的老鼠眼窩採血後犧牲。然後 立即進行支氣管插管,以1ml不含鈣鎂離子之HBSS灌洗老鼠肺部四次, 並收集沖洗液。將所收集的肺泡沖洗液以400xg的轉速在4°C下離心1 〇分 鐘。離心所得之細胞懸浮於1 ml HBSS中,然後以血球計數劑計算細胞總 200824699 數。接著以細胞離心沉激法(Cytocentrifuged preparations)將細胞固定在玻 片上,再以劉氏染色(Liu's stain)計算各類細胞數目。各類細胞數目以300 顆細胞中巨嘯細胞、淋巴球、嗜中性球與嗜伊紅性白血球各自所占之數目 來計算,並且根據其標準型態來分類。 1 ·2·4測定支氣管肺泡沖洗液中eotaxin的含量 支氣管肺泡沖洗液(BAL)中eotax丨η含量,以eotaxin ELISA套組測 定,操作步驟則依ELISA套組製造商所指示。 1.2.5小鼠的呼吸道阻力測試 φ 以增加methacholine (Mch.)濃度的方法(分別爲6·25 mg/m卜12·5 mg/m卜 25 mg/mh 50 mg/ml)刺激小鼠,並且利用 plethymography 的方 法來測定小鼠的呼吸道阻力(airway enhanced pause (PenH) values),以了 解小鼠的呼吸道收縮是否受到改善。 1.3試驗分組 小鼠八週大時隨機分成四組,每組15隻,四組小鼠接受治療的方法 如後述。A組小鼠僅用PBS致敏與治療。B組小鼠用卵白蛋白致敏,但僅 用PBS治療。C組小鼠用卵白蛋白致敏,用劑量爲2.5mg/kg的三氧化二 • 砷治療。D組小鼠用卵白蛋白致敏,用劑量爲5.0mg/kg的三氧化二砷治療。 表1·1 致敏抗原 處理 Α組 PBS PBS B組 OVA PBS c組 1 OVA ΑΤΟ 2.5 mg/kg D組 OVA ΑΤΟ 5 mg/kg π 200824699 1·4試驗步驟 選用8週大的BALB/c雌鼠,老鼠飼養方法及分組方式如同前述。老 鼠致敏的方法及途徑如前述,老鼠致敏後每週以眼窩採血法測定抗原專一 性抗體的效價。 1.5處理方法 先以卵白蛋白(ovalbumin ,OVA)腹腔注射置老鼠體內,分別于第〇、 14、21及28天,以相同劑量之卵白蛋白重複注射老鼠完成致敏步驟後, 第38天以PBS和劑量爲2.5mg/kg與5.0mg/kg的三氧化二砷腹腔連續注 φ 射七日治療4遍。再於第42、43及44天以吸入卵白蛋白抗原刺激小鼠。 第45天進行小鼠的呼吸道阻力測試。呼吸道阻力測試結果如表1 ·2所示。 表1.2小鼠的呼麵阻力(氣管反應)測試 呼吸道阻力 測試 Mch(mg/ml) 6.25 mg/ml 12.5 mg/ml 25 rng/rnl 50 mg/ml A組 2.20 ± 0.28# 8.22 ± 1.22 11.99 ± 1.50 14_21 ± 1,06 B組 2.50 ±0.40 8.00 ±0.93 14.52 ±1_42 19·15±3·35 C組 3·62±0·47 7·83± 1.13 10.15 ±0·841 9·83±1·171 D組 2.55 ±0.35 8.82 ±0.88 11.57 ±1.52 12.51 ± 1.90Ethylbenzthiazoline each Sulfonic Acid) was subjected to a coloring solution at room temperature for about 30 minutes, and the reaction was terminated with 100 μΙ of 5% SDS per micropore and the absorbance was read with a plate reader. 1·2·3 Calculation of the number of bronchoalveolar fluids and giant sun cells, lymphocytes, neutrophils and eosinophils White blood cells 24 hours after the last inhalation of antigen, all the mouse eye sockets were sacrificed after blood collection. Bronchial intubation was then performed immediately, and the lungs of the mice were lavaged four times with 1 ml of HBSS containing no calcium and magnesium ions, and the rinse was collected. The collected alveolar lavage fluid was centrifuged at 400 x g for 1 Torr at 4 °C. The cells obtained by centrifugation were suspended in 1 ml of HBSS, and then the total number of cells was counted as 200824699 by the blood cell counting agent. The cells were then fixed on a glass slide by Cytocentrifuged preparations, and the number of cells was counted by Liu's stain. The number of cells in each cell is calculated as the number of squamous cells, lymphocytes, neutrophils, and eosinophils in 300 cells, and is classified according to their standard type. 1 ·2·4 Determination of eotaxin content in bronchoalveolar lavage fluid The content of eotax丨η in bronchoalveolar lavage fluid (BAL) was determined by eotaxin ELISA kit, and the procedure was as directed by the ELISA kit manufacturer. 1.2.5 Respiratory resistance test in mice φ The mice were stimulated by increasing the concentration of methacholine (Mch.) (6·25 mg/m Bu 12·5 mg/m b 25 mg/mh 50 mg/ml, respectively). The plethymography method was used to determine the airway enhanced pause (PenH) values of the mice to see if the airway contraction of the mice was improved. 1.3 Experimental group The mice were randomly divided into four groups at the age of eight weeks, and each group of 15 patients received treatment in four groups as described later. Group A mice were sensitized and treated with only PBS. Group B mice were sensitized with ovalbumin but treated with PBS only. Group C mice were sensitized with ovalbumin and treated with arsenic trioxide at a dose of 2.5 mg/kg. Group D mice were sensitized with ovalbumin and treated with arsenic trioxide at a dose of 5.0 mg/kg. Table 1-1 sensitizing antigen treatment Α group PBS PBS group B OVA PBS c group 1 OVA ΑΤΟ 2.5 mg / kg D group OVA ΑΤΟ 5 mg / kg π 200824699 1 · 4 test steps 8 weeks old BALB / c female rats The method of feeding and grouping the mice is as described above. Methods and Routes for Sensitization in Old Rats As described above, the titer of antigen-specific antibodies was determined weekly by eye socket blood sampling after sensitization in mice. 1.5 treatment method was first intraperitoneal injection of ovalbumin (OVA) in mice, respectively, on the third, 14, 21 and 28 days, the same dose of ovalbumin repeated injection of mice to complete the sensitization step, the 38th day with PBS And the dose of 2.5mg / kg and 5.0mg / kg of arsenic trioxide in the peritoneal cavity continuous injection φ shot seven days of treatment 4 times. Mice were stimulated with inhaled ovalbumin antigen on days 42, 43 and 44. The airway resistance test of the mice was carried out on the 45th day. The results of the airway resistance test are shown in Table 1-2. Table 1.2: Call surface resistance (tracheal reaction) of the test. Airway resistance test Mch (mg/ml) 6.25 mg/ml 12.5 mg/ml 25 rng/rnl 50 mg/ml Group A 2.20 ± 0.28# 8.22 ± 1.22 11.99 ± 1.50 14_21 ± 1,06 Group B 2.50 ±0.40 8.00 ±0.93 14.52 ±1_42 19·15±3·35 Group C 3.62±0·47 7·83± 1.13 10.15 ±0·841 9·83±1·171 D Group 2.55 ± 0.35 8.82 ± 0.88 11.57 ± 1.52 12.51 ± 1.90
#數値爲平均± SD Μ 1 與Β組比較,ρ< 0·05。 由表 1·2 顯示增加 methacholine (Mch·)濃度(6·25 mg/m卜 12.5 mg/m卜25 mg/mh 50 mg/ml)朿隞小鼠後,plethymography測定小鼠的呼 吸道阻力,以氣管過敏反應-PenH比値(AHR ; airway 200824699 hyper-reactivity-PenH(airway enhanced pause))表示。與未接受三氧化二 砷治療的B組小鼠比較,劑量2.5 mg/kg三氧化二砷治療成功地阻斷C組 小鼠的氣管高免疫反應性而減低小鼠的呼吸道阻力 最後一次吸入卵白蛋白抗原刺激48小時後,所有的老鼠眼窩採血後 犧牲。然後立即進行支氣管插管,以1ml不含讎離子之HBSS灌洗老鼠 肺部四次,收集肺泡沖洗液。所收集的肺泡沖洗液進行組織病理學硏究、 計算各類細胞之數目(巨噬細胞、淋巴球、嗜中性球與嗜伊紅性白血球)並測 定支氣管肺泡沖洗液中eotaxin的含量,結果如表1 ·3及表1 ·4所示。 • 表1.3支氣管肺泡沖洗液巨噬細胞、淋巴球、嗜中性球與嗜伊紅性白 血球數目計算 BALF小鼠白 血球計數 (χ1000 cells/ml) 巨噬細胞 嗜伊紅性白血 球 嗜中性球 淋巴球* A組 135.6±54.5# 3·2±2·1 8.4±4.9 12.9±8.2 B組 243.8±69.3 105.3±60·7 77.1 ±32.8 Ί 37.8±16.3 C組 621.0±199.6 28.6±13.0 200.7±75.2 37.0±9.4 D組 499.1±131.6 33.2±12.5 244.0±72.6 28.7±13.6 *嗜鹼性白血球數目稀少,在此並未表示0 表1.4顯示在小鼠支氣管肺泡沖洗液的eotaxin含量的平均濃度與標 準差。與未接受三氧化二砷治療的B組小Μ比較,C、D組小鼠接受三氧化 二砷治療使其支氣管肺泡沖洗液之的eotaxin含量減少,尤其是接受 2.5mg/kg三氧化二砷治療的C組小鼠較未接受三氧化二砷治療的罐小鼠 }3 200824699 有顯著明顯差異(Ρ <〇·〇5)。#数値 is the average ± SD Μ 1 Compared with the Β group, ρ < 0·05. After increasing the concentration of methacholine (Mch·) (6·25 mg/m b 12.5 mg/m b 25 mg/mh 50 mg/ml) in mice shown in Table 1. 2, the respiratory resistance of the mice was determined by plethymography. The tracheal allergic reaction-PenH is indicated by AHR; airway 200824699 hyper-reactivity-PenH (airway enhanced pause). Compared with group B mice not treated with arsenic trioxide, treatment with 2.5 mg/kg arsenic trioxide successfully blocked tracheal hyperimmune response in group C mice and reduced respiratory resistance in mice. 48 hours after the last inhalation of ovalbumin antigen stimulation All the mouse eye sockets were sacrificed after blood collection. Immediately after the bronchial intubation, the lungs of the rats were lavaged four times with 1 ml of HBSS containing no strontium ions, and the alveolar lavage fluid was collected. The collected alveolar lavage fluid was subjected to histopathological investigation, the number of various cells (macrophages, lymphocytes, neutrophils and eosinophils) was measured, and the content of eotaxin in the bronchoalveolar lavage fluid was determined. As shown in Table 1 · 3 and Table 1 · 4. • Table 1.3 Bronchoalveolar lavage fluid macrophage, lymphocytes, neutrophils and eosinophilic white blood cell count BALF mouse white blood cell count (χ1000 cells/ml) Macrophage eosinophilic white blood cell neutrophil lymph Ball * Group A 135.6 ± 54.5 # 3 · 2 ± 2 · 1 8.4 ± 4.9 12.9 ± 8.2 Group B 243.8 ± 69.3 105.3 ± 60 · 7 77.1 ± 32.8 Ί 37.8 ± 16.3 Group C 621.0 ± 199.6 28.6 ± 13.0 200.7 ± 75.2 37.0 ±9.4 D group 499.1±131.6 33.2±12.5 244.0±72.6 28.7±13.6 * The number of basophilic white blood cells is sparse and is not indicated here. Table 1.4 shows the average concentration and standard deviation of eotaxin content in mouse bronchoalveolar lavage fluid. Compared with group B sputum that was not treated with arsenic trioxide, mice in groups C and D received arsenic trioxide treatment, which reduced the amount of eotaxin in bronchoalveolar lavage fluid, especially in group C mice treated with 2.5 mg/kg arsenic trioxide. There was a significant difference in the arsenic trioxide-treated pot mice}3 200824699 (Ρ <〇·〇5).
表1.4支氣管肺泡沖洗液中eotaxin的含量測定Table 1.4 Determination of eotaxin in bronchoalveolar lavage fluid
Eotaxin (pg/ml) 平均 SD A組 14.42 8.39 B組 20.90 4.97 C組 12.38* 3.92 D組 16.00 4.06 *與B組比較,ρ<0·05 在/J、鼠接受卵白蛋白抗原刺激後,採取小鼠血液並測量血清中卵白蛋 白專一性抗體含量(θνΆ-specific IgE values),作爲致敏反應(immunization) 的證據。表1.5顯示B、C、D組小鼠之血清中卵白蛋白專一性抗體含量比 控制組A組小鼠明顯升高,表示B、C、D組小鼠接受卵白蛋白抗原刺激致 敏成功。 表1.5卵白蛋白朿[I激指數(OVAstimulatiorHndex) 0.專一性IgE 平均 SD 抗體(S1) A組 0.30 0.09 B組 2.06 0.81 C組 2.20 0.70 D組 1.96 0.31 實施例二:以三氧化二砷治療系統性紅斑狼瘡之試驗Eotaxin (pg/ml) Average SD A group 14.42 8.39 Group B 20.90 4.97 Group C 12.38* 3.92 Group D 16.00 4.06 *Compared with Group B, ρ<0·05 was taken after /J, mouse received egg albumin antigen stimulation Rat blood and serum levels of ovalbumin-specific IgE values were measured as evidence of sensitization. Table 1.5 shows that the serum albumin-specific antibody content in the B, C, and D groups was significantly higher than that in the control group A mice, indicating that the mice in groups B, C, and D were successfully sensitized by ovalbumin antigen stimulation. Table 1.5 Ovalbumin 朿 [I-Amazing Index (OVAstimulatiorHndex) 0. Specific IgE Average SD Antibody (S1) Group A 0.30 0.09 Group B 2.06 0.81 Group C 2.20 0.70 Group D 1.96 0.31 Example 2: Treatment of systemic lupus erythematosus with arsenic trioxide Test
u 200824699 2·1試驗材料 2·1_1試驗動物 ISIZBxNZW F1雌性小鼠,約7個月大;NZB (H-2d)雌性小鼠;NZW (H-2Z)雄性小鼠 2· 1.2藥物 三氧化二砷注射液(Asadin ®,10mg/10ml/vial),以PBS稀釋。 2·1·3試劑 (1) 甲基化牛血清白蛋白溶液(mBSA ; methylated Bovine Serum AIbumin)working solution) (2) 小牛胸線 DNA 處理液(calf thymus DNA working solution) (3) 阻斷液(Geletin-PBS 溶液) (4) 血清及正控制組:血清由採血後之血液經離心(5000rpm,10分 鐘,4)而取得。使用時血清以阻斷液稀釋。正控制組則由融合瘤細胞 (hybridoma)分泌的抗-DNA IgG或IgM,於每一個培養盤取抗體並稀釋成 250倍的500μΙ溶液,再作連續稀釋。 (5) 2 級抗體(2Ab; Horserad丨sh peroxldase(HRP)-conjugated 抗小老 鼠 Y 鏈或 μ 鏈抗體;Horseradish peroxidase(HRP)-conjugated anti-mouse γ chain or μ chain Ab): (6) 2,2,-azino-bisC3-ethylbenzthiazoline-6-suIphonic acid (ABTS) solution ⑺5X檸檬酸緩衝液 (8)SDS溶液 2_1 _4主要儀器:測餓體的ELISA讀取機 200824699 2·2試驗方法 2·2·1建立人類系統性紅斑野麵的動物模式 約7個月大MZBxNZW F1雌性小鼠及啦8燜^配種之小鼠會自然 發生一種類似人類系統性IBS麵的自體免疫徵候群(請確認)。NZB/NZW 配種之小鼠的自體免疫徵候群在四個月大時會產生讎的抗體(antinuclear antibodies (丨gG)),九個月大時會發生腎小球腎炎與蛋白尿,腎臟衰竭引發 尿毒症是死亡的主要原因。 _ 2.2.2測定抗-dsDNA和抗-ssDNA抗體的ELISA S!量法.u 200824699 2·1 test material 2·1_1 test animal ISIZBxNZW F1 female mouse, about 7 months old; NZB (H-2d) female mouse; NZW (H-2Z) male mouse 2. 1.2 drug arsenic trioxide injection (Asadin®, 10 mg/10 ml/vial), diluted in PBS. 2·1·3 reagents (1) methylated bovine Serum AIbumin working solution) (2) calf thymus DNA working solution (3) blocking Liquid (Geletin-PBS solution) (4) Serum and positive control group: Serum was obtained by centrifugation (5000 rpm, 10 minutes, 4) of blood after blood collection. Serum was diluted with blocking solution at the time of use. The positive control group was anti-DNA IgG or IgM secreted by the fusionoma cells, and the antibody was taken from each culture plate and diluted to a 250-fold 500 μM solution, followed by serial dilution. (5) Level 2 antibody (2Ab; Horserad丨sh peroxldase (HRP)-conjugated anti-mice mouse Y chain or μ chain antibody; Horserapish peroxidase (HRP)-conjugated anti-mouse γ chain or μ chain Ab): (6) 2 , 2,-azino-bisC3-ethylbenzthiazoline-6-suIphonic acid (ABTS) solution (7) 5X citrate buffer (8) SDS solution 2_1 _4 main instrument: ELISA reader for measuring hungry body 200824699 2·2 test method 2·2 ·1 Establishing an animal model of human systemic erythema field. About 7 months old MZBxNZW F1 female mice and 8 焖^ breeding mice naturally develop an autoimmune syndrome similar to human systemic IBS surface (please confirm ). The autoimmune syndrome of NZB/NZW-matched mice produces antinuclear antibodies (丨gG) at four months of age, and glomerulonephritis and proteinuria occur at nine months of age, kidney failure Inducing uremia is the leading cause of death. _ 2.2.2 Determination of anti-dsDNA and anti-ssDNA antibodies ELISA S!
運用標準ELISA測量法測定血清中抗dsDNA和ssDNA的抗-DNA - 抗體含量 > 其步驟如下: (1) 以mBSA覆蓋(coating):於每微孔中覆蓋100 μ丨的 mBSA(10Mg/mL),於4°C下靜置隔夜,之後以PBS清洗二次。 (2) dsDNA和ssDNA覆蓋:於每微孔中覆蓋100μΙ的dsDNA及 ssDNA(分別爲7.5ijg/mL),於4°C下靜置隔夜,之後以PBS清洗二次。 • (3似阻斷液阻斷:於每微孔中覆蓋2〇〇μ1的阻斷液,於4代靜置隔 夜(或室溫下2小時或37°C下靜置45分鐘後室溫15分鐘),以PBS-tween 20清洗三次。 ⑷添加血清或正控制組或負控制組:每微孔中分別添加1〇〇μΙ血清或 正控制組或負控制組,於37。。靜置45分鐘後室溫15分鐘(或室溫2小時), 以PBS-tween 20清洗六次。 (5)添加前述2級抗體:於每微孔中添加100μΙ的二級抗體,於37°C靜 /6 200824699 置45分鐘後室溫15分鐘,以PBS-tween 20清洗六次。 ⑼添加呈色劑:每微孔中添加10ΟμΙ的呈色劑(ABTS溶液),5-10分鐘 避光反應,加入終止液(5%SDS溶液)並於吸光値405酬下進行0.D測量。 ⑺抗-DNA IgG的量係似ELISA分析法的單位表示(Eli/ml) 其與mAb 10F10相較,1〇「1〇抗體在74叩加丨濃度下所產生的〇0 値定義爲1 EU/ml的抗DNA丨gG。抗核醣體IgG濃度係根據10F10抗體濃 度以EU計量。由231ng/ml的10F10抗體所產生的吸收値,則定義爲1 φ EU/ml的抗核醣體丨gG。 2.2.3小鼠的腎小球腎炎檢測 . 小鼠的腎小球腎炎評估,係蓮用尿液測試沾片(Multistix®, Bayer)比色 „ 測定尿蛋白含量。比色測定尿蛋白含量大於1g/L(2+)定義爲嚴重腎小球腎 炎。於小鼠第37週大與第40週大時檢測。 2.2.4小鼠的存活率評估 每組6隻小鼠,紀錄小鼠存活率至第43週大。 _ 2·3試驗步驟 2.3.1試驗分組 小鼠24週大時隨機分爲三組,每組6隻:(1)Α組:僅用PBS(1〇ml/kg) 治療;(2)B組:用劑量爲2.5mg/kg的三氧化二砷治療;(3)C組:用劑量爲 5.0mg/kg的三氧化二砷治療。 2·3·2試驗流程 小鼠24週大時則隨機分爲三組,每組6隻,接受治療的方法每個星 /7 200824699 期一、三、五各以腹腔注射打一次,持續1個月,治療之前與治療停止後, 每四週老鼠以眼嵩採血法採取麵測抗-SSDNA和抗-dsDMA抗體,持續3 個月。 2,3.3結果 運用EUSA測定小鼠血清中抗_dsDNA和抗-ssDNA抗體的含量:抗 •dsDNA的測量結果如表Z1所示。 表2.1丨gG抗-dsDNA效價(ELISA單位) 試驗時間(週) 試驗前 4 8 12 PBS(A 組) 0-280 0.623 0.679 0.381 2.5mg/kg ΑΤΟ (Β組) 0.262 0.572 0.464 0.442 5mg/kg ΑΤΟ (C組) 0.362 0.285 0.357 0.702 試驗結果顯示,接受三氧化二砷治療的B、C組小鼠比未接受三氧化 • 二砷治療的A組小鼠的血清中抗-DNA抗體含量較低;並且較高劑量三氧化 二砷效果較強。因爲抗-DNA抗體的含量與SLE疾病活性有極強的關聯性, 此試驗結果顯示三氧化二砷可能成爲SLE新的治療藥劑。 2·3·4小鼠的腎小球腎炎檢測結果 小鼠的腎小球腎炎評估系運用尿液測試沾片比色測定尿蛋白含量。比 色測定尿蛋白含量大於1g/L (2+)定義爲嚴重腎小球腎炎。 表2·2小鼠具有嚴重腎小球腎炎之比例 18 200824699 時間(週) 37 40 PBS (A 組) 25 too 2.5mg/kgATO(B 組) 16.67 40 5mg/kg ΑΤΌ (C _ 25 25 表2.2的試驗數據顯示接受三氧化二砷治療可以顯著延緩β、C組小 鼠嚴重腎/J碌腎炎的發生,有一些尙未發生嚴重腎4錄腎炎的小鼠,其中 一部分甚至仍有正常的腎臟功能也無蛋白尿的現象。而未接受三氧化二砷 • 治療的A組小鼠則全部發生了嚴重腎小球腎炎。與未接受三氧化二砷治療 的A組小鼠比較,三氧化二砷治療可以顯著降低B、C組小鼠嚴重腎小球 - 腎炎的發生與進行。 2.3_5小鼠存活率評估 表Z3小鼠存活率評估(%) 時間 ㈣ 22 31 38 39 40 I 41 42 43 PBS (A 組) 100 100 66.7 50 50 16J 0 0 2.5mg/k gATO (B觸 100 100 100 83.3 66.7 66.7 66.7 50 5mg/kg ΑΤΟ (C 組) 100 100 66.7 66.7 66.7 66.7 66.7 66.7 表2·3顯示接受三氧化二砷治療的B、C組小鼠,其43周的存活率 爲50%,未接受三氧化二砷治療的Α組小鼠貝牷部死亡。與未接受三氧化 /9 200824699 二砷治療的A組小鼠比較,三氧化二砷治療可以顯著地延長B、。組小鼠 的存活。 實施例三:以三氧化二砷毒殺惡性瘧疾瘧原虫的體外試驗 3.1試驗材料 3-11藥物:三氧化二砷注射液(Asadin®,10mg/10ml/vial) 〇三氧砷化 物,分子量197.64,試驗藥物劑型濃度爲1mg/ml (5054·6μΜ),試驗前將 其稀釋爲160_,然後再對半稀釋,終濃度按ng/ml計算。 3.1 _2 原蟲 惡性瘧原虫FCC1/_引自海南,試驗室連續培養或冷藏保種5年以 - 上。 3.1.3 • 培養液:常規體外連續培養惡性瘧原蟲用的培養基(液),主要成分爲 RPMM640 (GBCO),HEPES (GBCO),NaHC03 和 AB 血清等。 3-1.4其他材料 血清、紅細胞:廣州血液中心提供。紅細胞(RBC)爲ACD抗凝B 型全血,4°C保存,不超過一個月,臨用前用無血清培養基洗滌2-3次。 3·2試驗方法 連續培養FCC1/HN。參照管惟濱【73】、陳林[74】等體外微量測定抗瘧 藥藥效的方法,在培養盤上每孔加藥液20μΙ,再加入感染率1 %左右的蟲血 180μΙ,加蓋,$莖輕振盪數秒鐘,於37°C,5%C02,培養48小時,去上 清,取RBC塗一層薄血膜,經吉氏染色及鏡檢,計算原蟲感染率,對照組 作爲100,求出各劑量組的減蟲率【減蟲率=(對照組感染率-試驗組感染率)The anti-DNA-antibody content of anti-dsDNA and ssDNA in serum was determined by standard ELISA method. The steps were as follows: (1) Covering with mBSA: covering 100 μM of mBSA per microwell (10 Mg/mL) ), allowed to stand overnight at 4 ° C, and then washed twice with PBS. (2) dsDNA and ssDNA coverage: 100 μM of dsDNA and ssDNA (7.5 ijg/mL, respectively) were placed in each well, and allowed to stand overnight at 4 ° C, followed by washing twice with PBS. • (3 blocking blockage: cover 2 μl of blocking solution per microwell, and stand overnight in 4th generation (or room temperature for 2 hours or 37 ° C for 45 minutes) 15 minutes), wash three times with PBS-tween 20. (4) Add serum or positive control group or negative control group: Add 1 μμΙ serum or positive control group or negative control group to each well, at 37. After 45 minutes, room temperature for 15 minutes (or room temperature for 2 hours), washed six times with PBS-tween 20. (5) Add the above-mentioned grade 2 antibody: add 100 μΙ of secondary antibody per microwell, and let stand at 37 ° C /6 200824699 After 45 minutes at room temperature for 15 minutes, wash with PBS-tween 20 six times. (9) Add coloring agent: Add 10 μμ of coloring agent (ABTS solution) to each micropore, avoiding light reaction for 5-10 minutes. The stop solution (5% SDS solution) was added and the 0.D measurement was performed under the absorbance 405. (7) The amount of anti-DNA IgG is expressed in units of ELISA assay (Eli/ml) compared with mAb 10F10, 1 〇 “1〇 antibody produced at 74叩 plus concentration is defined as 1 EU/ml anti-DNA丨gG. Anti-ribosomal IgG concentration is measured in EU according to 10F10 antibody concentration. 10F10 from 231ng/ml The absorption enthalpy produced by the body is defined as 1 φ EU/ml anti-ribosomal 丨gG. 2.2.3 Detection of glomerulonephritis in mice. Evaluation of glomerulonephritis in mice, urine test with urine Tablet (Multistix®, Bayer) colorimetric Measure urine protein content. Colorimetric determination of urine protein content greater than 1g / L (2+) is defined as severe glomerulonephritis. When mice are 37th week old and 40th week old 2.2.4 Survival rate of mice The evaluation of mouse survival rate of each group of 6 mice was recorded to the 43th week. _ 2·3 test steps 2.3.1 test group mice were randomly divided into three at 24 weeks of age. Group, 6 rats in each group: (1) sputum group: treated with PBS only (1 〇 ml/kg); (2) group B: treated with arsenic trioxide at a dose of 2.5 mg/kg; (3) group C: dose Treatment with 5.0 mg/kg of arsenic trioxide. 2·3·2 test procedure mice were randomly divided into three groups at the age of 24 weeks, each group of 6 patients, each method of treatment / 7 200824699 one, three, five Each was intraperitoneally injected once for 1 month. Before treatment and after treatment was stopped, anti-SSDNA and anti-dsDMA antibodies were surface-measured every four weeks for 3 months. 2,3.3 EUSA was used to determine the content of anti-dsDNA and anti-ssDNA antibodies in mouse serum: the anti-dsDNA measurement results are shown in Table Z1. Table 2.1 丨gG anti-dsDNA titer (ELISA unit) Test time (week) test The first 4 8 12 PBS (Group A) 0-280 0.623 0.679 0.381 2.5mg/kg ΑΤΟ (Β group) 0.262 0.572 0.464 0.442 5mg/kg ΑΤΟ (Group C) 0.362 0.285 0.357 0.702 Test results show that B, treated with arsenic trioxide Group C mice had lower anti-DNA antibody levels in the serum of Group A mice that were not treated with arsenic and arsenic; and higher doses of arsenic trioxide were more potent. Because the anti-DNA antibody content is strongly correlated with SLE disease activity, the results of this trial show that arsenic trioxide may become a new therapeutic agent for SLE. Glomerulonephritis test results in mice in 2·3·4 The evaluation of glomerulonephritis in mice was performed by urine colorimetry using urine test. Colorimetric determination of urinary protein content greater than 1 g/L (2+) is defined as severe glomerulonephritis. Table 2. 2 The proportion of mice with severe glomerulonephritis 18 200824699 Time (week) 37 40 PBS (Group A) 25 too 2.5 mg/kg ATO (Group B) 16.67 40 5 mg/kg ΑΤΌ (C _ 25 25 Table 2.2 The trial data showed that treatment with arsenic trioxide significantly delayed the onset of severe kidney/J nephritis in mice in the β and C groups. Some mice that did not have severe renal nephritis did not even have normal renal function. The phenomenon of proteinuria. In group A mice that were not treated with arsenic trioxide, all of them developed severe glomerulonephritis. Compared with group A mice that were not treated with arsenic trioxide, arsenic trioxide treatment significantly reduced mice in group B and C. Glomerulone-nephritis occurrence and progression. 2.3_5 Mouse survival rate evaluation Table Z3 mouse survival rate evaluation (%) Time (4) 22 31 38 39 40 I 41 42 43 PBS (Group A) 100 100 66.7 50 50 16J 0 0 2.5mg/k gATO (B-touch 100 100 100 83.3 66.7 66.7 66.7 50 5mg/kg ΑΤΟ (Group C) 100 100 66.7 66.7 66.7 66.7 66.7 66.7 Table 2.6 shows groups B and C treated with arsenic trioxide, 43-week survival rate is 50 %, the caries in the sputum group that did not receive arsenic trioxide died. Compared with the group A mice that did not receive the oxidized/9 200824699 arsenic treatment, the arsenic trioxide treatment significantly prolonged the survival of the mice in group B. Example 3: In vitro test of Plasmodium falciparum poisoning by arsenic trioxide 3.1 Test material 3-11 Drug: Arsenic trioxide injection (Asadin®, 10mg/10ml/vial) 〇trioxide arsenide, molecular weight 197.64, test drug dosage form concentration 1mg/ Ml (5054·6μΜ), dilute it to 160_ before the test, and then semi-dilution, the final concentration is calculated as ng/ml 3.1 _2 Protozoa Plasmodium falciparum FCC1/_ cited from Hainan, laboratory continuous culture or cold storage 5 years to -3. 3.1.3 • Culture medium: The medium (liquid) used for continuous culture of P. falciparum in vitro, the main components are RPMM640 (GBCO), HEPES (GBCO), NaHC03 and AB serum, etc. 3- 1.4 Other materials Serum, red blood cells: supplied by Guangzhou Blood Center. Red blood cells (RBC) are ACD anticoagulated type B whole blood, stored at 4 ° C, no more than one month, washed 2-3 times with serum-free medium before use. 2 test methods Continuously culture FCC1/HN. Refer to Tube Weibin [73], Chen Lin [74] and other methods for micro-measurement of anti-malarial drug in vitro, add 20 μΙ per well to the culture plate, and add about 1% infection rate. Insect blood 180μΙ, capped, $ stem lightly shake for a few seconds, at 37 ° C, 5% CO 2 , cultured for 48 hours, go to the supernatant, take RBC coated with a thin film of blood, Gyr stain and microscopic examination, calculate the protozoa The infection rate was determined as 100 in the control group, and the worm reduction rate of each dose group was determined [worm reduction rate = (control group infection rate - test group infection rate)
2Q 200824699 /對照組感染率x 100%],以劑量對數爲橫座標,以減蟲率的機率單位爲縱座 標作回歸方程,計算出使原蟲增殖比對照組減少50%和90%的用藥量。 3·3試驗步驟與結果㈠ 3_3_1藥物配製 滅菌青黴素瓶8個,按下表3·1分別加入RPMM640完全培養基和 三氧化二砷(ΑΤΟ) 表3.1三氧化二砷於培養液中之濃度 ΑΤΟ (μΙ) 0 0.5 10 15 2.0 2.5 3.0 0 RPMI-1 640培 養基 (ml) 1 1 1 1 1 1 1 1 濃度 (uM) 0 2.5 5 7.5 10 12.5 15 0 3.3.2蟲血稀釋 取連續培養的FCC1/HN,塗片,染色,計算感染率,以Β型RBC 調節感染率至1 %左右。 3·3·3測試 參照陳林等的方法,在3599培養盤上每微孔加入藥液2_,再加入 含蟲血180μΙ,試驗4行。之後加蓋振盪數秒鐘,置37。(:於5%。〇2箱培 養48小時,塗薄片染色計算5000個RBC內感染有瘧原蟲的RBC數。 3·3·4結果 開始培養時原蟲感染率爲0.98%,表3.2中爲培養後每5000個紅血 200824699 球中的原蟲數 表3.2培養後每5000個紅血球中的原蟲數 Α(對 照) B C D E F G 晒 照) ΑΤΟ 劑量 (βΜ) 0 2.5 5 7.5 10 12.5 15 0 1 172 82 79 81 60 53 39 199 2 306 163 48 33 47 49 33 294 3 251 125 36 29 46 32 17 213 4 198 74 57 55 53 48 26 208 從上表可見,最高劑量(15μΜ) <3#未能完全抑制瘧原虫發育。 3·4試驗步驟與結果㈡ 3.4.1藥物配製 無菌青黴素瓶Θ個,按下表3·3分別加入RPiyn-1640完全培養基和 三氧化二砷(ΑΤΟ)。 表3.3三氧化二砷於培養液中的濃度 AJO ⑽ 0 1 2 3 4 5 6 7 8 RPM1- 1640 (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 濃度 0 10 20 30 40 50 60 70 80 (μΜ) 3.4.2蟲_稀釋2Q 200824699 / control group infection rate x 100%], with the logarithm of the dose as the abscissa, the probability unit of the worm reduction rate as the ordinate as the regression equation, calculate the drug that reduces the proliferation of protozoa by 50% and 90% compared with the control group. the amount. 3. 3 Test Procedures and Results (1) 3_3_1 Drugs were prepared and sterilized with 8 penicillin bottles. Add RPMM640 complete medium and arsenic trioxide (ΑΤΟ) according to Table 3-1. Table 3.1 Concentration of arsenic trioxide in culture solution Ι (μΙ) 0 0.5 10 15 2.0 2.5 3.0 0 RPMI-1 640 medium (ml) 1 1 1 1 1 1 1 1 Concentration (uM) 0 2.5 5 7.5 10 12.5 15 0 3.3.2 Wet blood dilution Take continuous culture of FCC1/HN, smear, stain The infection rate was calculated, and the infection rate was adjusted to about 1% by sputum type RBC. 3·3·3 test According to the method of Chen Lin et al., the liquid medicine 2_ was added to each micropore on the 3599 culture plate, and then the blood containing 180 μΙ was added, and the test was performed for 4 lines. Then cover and shake for a few seconds, set 37. (: 5% in 〇2 boxes for 48 hours, and the number of RBCs infected with Plasmodium in 5000 RBCs was calculated by thin-staining. The protozoal infection rate was 0.98% when the results were started. Table 3.2 For the number of protozoa in the 200824699 ball per 5000 red blood in the table, the number of protozoa per 5000 red blood cells after cultivation 3.2 (control) BCDEFG sun exposure) 剂量 dose (βΜ) 0 2.5 5 7.5 10 12.5 15 0 1 172 82 79 81 60 53 39 199 2 306 163 48 33 47 49 33 294 3 251 125 36 29 46 32 17 213 4 198 74 57 55 53 48 26 208 As can be seen from the above table, the highest dose (15 μΜ) <3# not Can completely inhibit the development of Plasmodium. 3. 4 Test Procedures and Results (2) 3.4.1 Preparation of Drugs One sterile penicillin bottle is added, and RPiyn-1640 complete medium and arsenic trioxide (ΑΤΟ) are added separately according to Table 3. 3 below. Table 3.3 Concentration of arsenic trioxide in culture solution AJO (10) 0 1 2 3 4 5 6 7 8 RPM1- 1640 (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Concentration 0 10 20 30 40 50 60 70 80 (μΜ) 3.4. 2 insects_dilution
取連續培養的FCC1/HN,塗片,染色,計算感染率,以Β型RBC 調節感染率至1 %左右。 22 200824699 3.4.3 mm 參照管惟濱、陳林等的方法,在40孔的培養盤上先於每微孔加入藥 液20μΙ,再加入含蟲血180μ卜做4行。之後加蓋振盪數秒鐘,置37t:、5 %C02箱培養48小時,去上清液,取RBC塗薄片,染色及鏡檢,計算每 5000個RBC內感染有瘧原蟲的紅血球數。 3.4_4結果 開始培養時原虫感染率爲1 ·6%,表3·4中爲培養後每5000個紅血球 φ 中的原蟲數 表3.4培養後每5000個紅血球中的原蟲數 顺 照) 2 3 4 5 6 7 8 9 /VTO 三|旦 βΜ 0 10 20 30 40 50 60 70 80 A 227 96 49 46 45 45 50 40 25 B ’ 209 50 68 63 64 60 70 56 19 C 188 70 79 90 61 87 67 「57 |23 D 229 71 77 77 67 74 69 66 30 從上表可見,80μΜ#原蟲密度有所下降,但還未能完全抑制其發育。 3.5試驗步驟與結果㈢ 3.5.1藥物配製 取原液20μ丨用完全培養基,在青黴素瓶內對半稀釋共11個劑量組。 分別爲:2522、1216、633、316、158、79、40、20、10、5、2.5、ΟμΜ。 3.5.2蟲血稀釋 ίο 200824699 取連續培養的FCC1/HN,塗片及染色,計算感染率,以B型RBC 調節感染率至1 %左右。 3_5·3測試: 參照管惟濱、陳林等的方法,在3599培養盤上每微孔加入藥液20μΙ, 再加入含蟲血180μΙ,做4行。之後加蓋,振盪數秒鐘,置37。(:及5%G02 箱培養48小時,去上清液,取RBC塗薄片,染色及鏡檢,計算每5000 個RBC內感染有瘧原蟲的RBC數。 • 3·5·4結果 開始培養時原蟲感染率爲1.8%,表3.5中爲培養後每5000個紅血球 中的原蟲數 表3.5培養後每5000個紅血球中的原蟲數 1 2 3 4 5 6 ΑΤΟ計 量//Μ 2522 1216 633 316 158 79 A 0 0 0 0 0 38 B 0 0 0 0 0 28 C 0 0 0 0 0 33 D 0 0 0 0 0 25 四行混 勻 0 0 0 0 0 32 績上表) 7 8 9 10 11 12 ΑΤΟ計 量Μ μ 40 20 10 5 2.5 〇對照 A 63 75 88 108 206 503 24 200824699 Β 63 75 88 108 206 503 C 52 69 81 160 236 450 D 43 60 77 108 186 392 四行混 44 65 92 116 200 473 勻 上表可見,158uM可以完全抑制FCC1發育。 3·5·5結果統計: 三氧化二砷(ΑΤΟ)對FCC1抑制作用結果統計表如表3.6 表3.6三氧化二砷(ΑΤΟ)對FCC1抑制作用結果統計表 劑量 劑量對數 感染率 減虫率 機率戰 (uM) (X) (%) (%) (Y) 0.25 0.6021 42.3 57.7 5.1942 0.5 -0.3010 24.5 75.5 5.6903 1.0 0 19.5 80.5 5.8596 20 0.3010 13.7 86.3 6.0939 4.0 0.6021 9.3 90.7 6.3225 8.0 0.9031 6.8 93.2 6.4909 3·6試驗步驟與結果㈣ 3.6.1藥物配製: 取原液100μΙ,加完全培養基3059μΙ,混勻,此爲最高濃度藥液 (160μΜ ),取最高濃度藥液200μΙ,另取200μΙ培養基在青黴素瓶內混勻, 進行對半稀釋,得一系歹!I濃度的藥液爲:、160、80、40、20、10、5、 2.5、1·25、0.625、0.3125、ΟμΜ。(共做 4 行,11 列爲最高劑量孔 160UM)。 3.6.2蟲血稀釋:Continuously cultured FCC1/HN, smear, stain, calculate the infection rate, and adjust the infection rate to about 1% with sputum type RBC. 22 200824699 3.4.3 mm Refer to the method of Weibin, Chen Lin, etc., on the 40-well culture plate, add 20 μM of the solution to each micropore, and then add 180 μ of the blood containing bacteria to do 4 lines. After shaking for a few seconds, set 37t:, 5% C02 box for 48 hours, remove the supernatant, take RBC smears, stain and microscopic examination, calculate the number of red blood cells infected with Plasmodium every 5000 RBC. 3.4_4 The protozoal infection rate was 1.6% when the culture started, and the number of protozoa in every 5000 red blood cells φ after culture in Table 3.4. Table 3.4 The number of protozoa per 5000 red blood cells after culture was observed) 2 3 4 5 6 7 8 9 /VTO III | ΜβΜ 0 10 20 30 40 50 60 70 80 A 227 96 49 46 45 45 50 40 25 B ' 209 50 68 63 64 60 70 56 19 C 188 70 79 90 61 87 67 "57 |23 D 229 71 77 77 67 74 69 66 30 As can be seen from the above table, the density of 80 μΜ# protozoa has decreased, but its development has not been completely inhibited. 3.5 Test procedure and results (3) 3.5.1 Drug preparation The stock solution was 20 μM in complete medium and half-diluted in a penicillin bottle for a total of 11 dose groups: 2522, 1216, 633, 316, 158, 79, 40, 20, 10, 5, 2.5, ΟμΜ. Insect blood dilution ίο 200824699 Take continuous culture of FCC1/HN, smear and stain, calculate the infection rate, and adjust the infection rate to about 1% with B-type RBC. 3_5·3 test: Refer to the methods of Guan Weibin, Chen Lin, etc. Add 20 μΙ of the drug solution to each micropore on the 3599 culture plate, and add 180 μM containing the blood of the insects to make 4 rows. Then cover and shake for a few seconds and set 37. : and 5% G02 box culture for 48 hours, remove the supernatant, take RBC smear, stain and microscopic examination, calculate the number of RBC infected with Plasmodium in 5000 RBC. • 3. 5 · 4 results when starting culture Protozoa infection rate was 1.8%. Table 3.5 shows the number of protozoa per 5000 red blood cells after culture. Table 3.5 Number of protozoa per 5000 red blood cells after culture 1 2 3 4 5 6 ΑΤΟ Measured / / 2522 1216 633 316 158 79 A 0 0 0 0 0 38 B 0 0 0 0 0 28 C 0 0 0 0 0 33 D 0 0 0 0 0 25 Four lines of mixing 0 0 0 0 0 32 Performance table) 7 8 9 10 11 12 ΑΤΟMeasuring Μ μ 40 20 10 5 2.5 〇Control A 63 75 88 108 206 503 24 200824699 Β 63 75 88 108 206 503 C 52 69 81 160 236 450 D 43 60 77 108 186 392 Four lines mixed 44 65 92 116 200 473 Even on the table, 158uM can completely inhibit FCC1 development. 3·5·5 Results Statistics: The results of the inhibition of FCC1 by arsenic trioxide (ΑΤΟ) are shown in Table 3.6 Table 3.6 Arsenic Trioxide (ΑΤΟ) on FCC1 inhibition results Statistical Table Dose-dose log-infection rate worm-reducing rate probability (uM) (X (%) (%) (Y) 0.25 0.6021 42.3 57.7 5.1942 0.5 -0.3010 24.5 75.5 5.6903 1.0 0 19.5 80.5 5.8596 20 0.3010 13.7 86.3 6.0939 4.0 0.6021 9.3 90.7 6.3225 8.0 0.9031 6.8 93.2 6.4909 3·6 Test procedure and results (4) 3.6 .1 Preparation of the drug: Take the stock solution 100μΙ, add the complete medium 3059μΙ, mix, this is the highest concentration liquid (160μΜ), take the highest concentration of the solution 200μΙ, and take 200μΙ medium in the penicillin bottle to mix, half-dilution, A series of solutions are available: I, 160, 80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, ΟμΜ. (Total 4 rows, 11 columns for the highest dose hole 160UM). 3.6.2 Dilution of blood:
取連續培養的FCC1/HN,塗片,染色,計算感染率,以Β型RBC 25 200824699 調節感染率約1¾ 〇 3·6·3測試: 參照管惟濱、陳林等的方法,在3599培養盤每微孔加入藥液20|jh 再加入含蟲血180μΙ,做4行。之後加蓋,振邊數秒鐘,置37t:於5%C02 箱培養48小時,去上清液,取RBC塗片及染色,鏡檢,計算每5000個 RBC內感染有瘧原蟲的RBC數(只有核細胞沒有細胞質的死蟲不計算)。 3_6.4結果: 開始培養時原蟲感染率爲1.5%,表37中培養後每5000個紅血球中 的原蟲數Continuous culture of FCC1/HN, smear, staining, calculation of infection rate, sputum type RBC 25 200824699 regulation of infection rate of about 13⁄4 〇3·6·3 test: according to the method of tube Weibin, Chen Lin, etc., cultured in 3599 Add the drug solution 20|jh to each micropore and add 180 μM containing insect blood to make 4 rows. Then cover, shake the side for a few seconds, set 37t: incubated in 5% C02 box for 48 hours, remove the supernatant, take RBC smear and stain, microscopic examination, calculate the number of RBC infected with Plasmodium per 5000 RBC (Only nuclear cells without cytoplasmic dead insects are not counted). 3_6.4 Results: The protozoal infection rate was 1.5% at the start of culture, and the number of protozoa per 5000 red blood cells after culture in Table 37.
表3.7培養後每5000個紅血球中的原蟲數 1 2 3 4 5 6 ΑΤΟ劑 |—-t 對照 0.3125 0.625 1.25 2.5 5 .*—*-L· 里 Μμ Ε 394 384 371 328 279 71 F 410 407 401 361 302 72 G 457 414 364 276 240 67 H 454 385 339 308 235 98 混合4 448 358 330 306 246 99 行 釀上表) 7 8 9 10 11 ΑΤΟ劑 里 MM 10 20 40 80 160 Ε 70 50 24 19 0 200824699 F 45 ^22 34 25 0 G 55 36 28 24 0 Η 69 47 18 0 混合4行 80 58 50 23 0 3.6.5統計結果 三氧化二砷(ΑΤΟ)對FCC1抑制作用結果統計,表示如表3.8。 表3.8三氧化二砷(ΑΤΟ)對FCC1抑制作用結果統計 ΑΤΟ劑量劑量對數 感染率 減虫率 機率雜 (uM) (X) (%) (%) (Υ) 0.031 -1.509 79.9 20.1 4.1619 0.063 -1.201 73.7 26.3 4.3659 0.125 -0.903 68.3 317 4.5239 0.25 -0.6021 54.9 45.1 4.8743 0.5 -0.3010 44.4 55.6 5.1408 1.0 0 17.9 82.1 5.9192 2.0 0.3010 12.9 87.1 6.1311 4.0 0.6021 11.2 88.8 6.2160 8.0 0.9031 5.1 94.9 6.6352 Y=10904x+5.6582 Γ2=0.9706 IC50=0.25uM=49.28ng/ml 95% 信賴極限(confidence limit) (28.00-86.73ng/ml) IC90=3.73uM=737.94ng/ml 95% 信賴極限(confidence limit) (419.28-1298.77ng/ml) 3·7試驗步驟與結果㈤ 3·7·1藥物配製: 取原液32μΙ,加完全培養基0,5m丨,混勻,此爲最高濃度藥液 (320μΜ ),在青髓瓶內進行對半稀釋,得一系歹〇濃度的藥液爲:〇、320、 160、80、40、20、10、5μΜ。(共做 12 行,11 歹[|爲最高劑量孔 32〇um) 27 200824699 3.7.2 蟲血_ : 取連續培養的FCC1/HN,塗片,染色,計算感染率,以B型RBC 調整感染率至1 %左右。 3.7.3測試: 參照管惟濱、陳林等的方法,在3599培養盤上每微孔加入藥液20μΙ, 再加入含蟲血180μΙ,做12行。之後加蓋,振盪數秒鐘,置37。(:並於5% C02箱培養48小時,去上清液,混勻,各取RBC 5μΙ,混勻,塗片且染 φ 色鏡檢,計算每5000個RBC內感染有瘧原蟲的RBC數(只有細胞核沒有 細胞質的死蟲不計算)。 • 3.7.4結果Table 3.7 Number of protozoa per 5000 red blood cells after culture 1 2 3 4 5 6 Tanning agent|--t Control 0.3125 0.625 1.25 2.5 5 .*—*-L· 里Μμ Ε 394 384 371 328 279 71 F 410 407 401 361 302 72 G 457 414 364 276 240 67 H 454 385 339 308 235 98 Mix 4 448 358 330 306 246 99 Row Stuffed) 7 8 9 10 11 Ingredients MM 10 20 40 80 160 Ε 70 50 24 19 0 200824699 F 45 ^22 34 25 0 G 55 36 28 24 0 Η 69 47 18 0 Mixed 4 rows 80 58 50 23 0 3.6.5 Statistical results The results of inhibition of FCC1 by arsenic trioxide (ΑΤΟ) are shown in Table 3.8. Table 3.8 Results of inhibition of FCC1 by arsenic trioxide (ΑΤΟ) Statistics ΑΤΟ dose dose logarithmic infection rate worm reduction rate probability (uM) (X) (%) (%) (Υ) 0.031 -1.509 79.9 20.1 4.1619 0.063 -1.201 73.7 26.3 4.3659 0.125 -0.903 68.3 317 4.5239 0.25 -0.6021 54.9 45.1 4.8743 0.5 -0.3010 44.4 55.6 5.1408 1.0 0 17.9 82.1 5.9192 2.0 0.3010 12.9 87.1 6.1311 4.0 0.6021 11.2 88.8 6.2160 8.0 0.9031 5.1 94.9 6.6352 Y=10904x+5.6582 Γ2=0.9706 IC50=0.25uM =49.28ng/ml 95% confidence limit (28.00-86.73ng/ml) IC90=3.73uM=737.94ng/ml 95% confidence limit (419.28-1298.77ng/ml) 3·7 test Steps and results (5) 3·7·1 drug preparation: Take 32μΙ of the original solution, add complete medium 0,5m丨, mix, this is the highest concentration of liquid (320μΜ), half-dilute in the marrow bottle, get a series The concentration of the drug solution is: 〇, 320, 160, 80, 40, 20, 10, 5 μΜ. (Total 12 rows, 11 歹[| is the highest dose hole 32〇um) 27 200824699 3.7.2 Worm blood _ : Take continuous culture of FCC1/HN, smear, stain, calculate infection rate, adjust infection with type B RBC The rate is around 1%. 3.7.3 Test: According to the method of Guan Weibin, Chen Lin, etc., add 20 μΙ of the drug solution per micropore on the 3599 culture plate, and then add 180 μM containing insect blood to make 12 rows. Then cover, shake for a few seconds, set 37. (: and cultured in 5% C02 box for 48 hours, remove the supernatant, mix, take RBC 5μΙ, mix, smear and stain φ color microscopy, calculate RBC infected with Plasmodium every 5000 RBC Number (only dead cells with no cytoplasm in the nucleus are not counted) • 3.7.4 Results
開始培養時原蟲感染率爲1.1%,表3.Θ中爲培養後每5000個RBC 中的原蟲數。 表3.9培養後每5000個RBC中的原蟲數 [ I A B C D E F G Η ΑΤΟ ft里 (μΜ) 0 5 10 20 40 80 160 320 混合 256 75 34 21 9 5 0 0 混合 247 72 39 25 10 4 0 0 混合 231 69 28 20 9 6 0 0 混合 258 57 26 17 8 4 0 0 28 200824699 3.7.5三氧化二砷(ΑΤΟ)對FCC1抑制作用: 表3·10三氧化二砷(ΑΤΟ)對FCC1抑制作用結果統計表 ΑΤΟ劑量劑量對數 感染率 減虫率 機率單位 (uM) (X) (%) (%) (Y) 0.5 -0.3010 1.0 0 2.0 0.3010 4.0 0.6021 8.0 0.9031 5.6.0 8.32. 4 9 7· 2· 2 1 72.6 5.6008 87.1 5.1311 91.5 6.3722 96.3 6.7866 98.0 7.0537 Y=1.1832x+6.0328 r2=0.9849 _ IC50=0.13uM=25.72ng/ml 95%信賴極限(confidence limit) (18.91-34.9ng/ml) - IC90=0.96uM=189.93ng/ml 95%信賴極限(confidence limit) (139.65-258.3ng/mi) 3.8試驗步驟與結果(六) 3·8·1藥物配製: 取原液32μΙ,加完全培養基0.5ml,混勻,此爲最高濃度藥液 _ ( 320μΜ ),在青黴素瓶內進行對半稀釋,得一系歹{]濃度的藥液爲:〇、320、 160、80、40、20、10、5μΜ 撇 1-6 列,Η 行爲最高劑量孔 32〇uM)。 3_8·2蟲血稀釋: 取連續培養的FCC1/HN,塗片,染色,計算感染率,以Β型RBC 調整感染率至1 %左右。 3-8.3 mm · 參照管惟濱、陳林等的方法,在3599培養盤上每微孔加入藥液20μΙ, 29 200824699 再加入含蟲血180μΙ,做12行(1-6爲ΑΤΟ,7-12爲ATS)。之後加蓋振盪數 秒鐘,置37°C並於5%C02箱培養48小時,去上清液,混勻,各取RBC 5μ卜混勻,塗片並染色鏡檢,計算每5000個RBC內感染有瘧原蟲的RBC 數(只有細胞核沒有細胞質的死蟲不計算)。 3·8·4結果: 開始培養時原蟲感染率爲1·39%,表3· 11爲培養後每5000個RBC 中的原蟲數。The protozoal infection rate at the start of culture was 1.1%. Table 3. The number of protozoa per 5000 RBCs after culture. Table 3.9 Number of protozoa per 5000 RBCs after culture [ IABCDEFG Η ΑΤΟ ft (μΜ) 0 5 10 20 40 80 160 320 Mix 256 75 34 21 9 5 0 0 Mix 247 72 39 25 10 4 0 0 Mix 231 69 28 20 9 6 0 0 Mix 258 57 26 17 8 4 0 0 28 200824699 3.7.5 Inhibition of FCC1 by arsenic trioxide (ΑΤΟ): Table 3.10 Inhibition of FCC1 by arsenic trioxide (ΑΤΟ) results Statistical data ΑΤΟ dose dose logarithmic infection Rate of worm reduction rate unit (uM) (X) (%) (%) (Y) 0.5 -0.3010 1.0 0 2.0 0.3010 4.0 0.6021 8.0 0.9031 5.6.0 8.32. 4 9 7· 2· 2 1 72.6 5.6008 87.1 5.1311 91.5 6.3722 96.3 6.7866 98.0 7.0537 Y=1.1832x+6.0328 r2=0.9849 _ IC50=0.13uM=25.72ng/ml 95% confidence limit (18.91-34.9ng/ml) - IC90=0.96uM=189.93ng/ml 95% confidence limit (139.65-258.3ng/mi) 3.8 Test procedure and results (6) 3·8·1 drug preparation: Take the stock solution 32μΙ, add complete medium 0.5ml, mix, this is the highest concentration drug Liquid _ (320μΜ), half-dilution in a penicillin bottle, to obtain a system of 歹{] concentration of liquid medicine: 〇, 320, 1 60, 80, 40, 20, 10, 5μΜ 1-6 1-6 columns, Η behavior highest dose hole 32〇uM). 3_8·2 dilution of worm blood: Take continuous culture of FCC1/HN, smear, stain, calculate the infection rate, adjust the infection rate to about 1% with sputum type RBC. 3-8.3 mm · According to the method of tube Weibin, Chen Lin, etc., add 20 μΙ per micropore on the 3599 culture plate, 29 200824699 and then add 180 μΙ containing insect blood, do 12 lines (1-6 is ΑΤΟ, 7- 12 is ATS). Then, cover and shake for several seconds, set at 37 ° C and incubate in 5% CO 2 box for 48 hours, remove the supernatant, mix, take RBC 5μ each, mix, smear and stain microscopy, calculate every 5000 RBC The number of RBCs infected with Plasmodium (only dead cells with no cytoplasm in the nucleus are not counted). 3·8·4 Results: The protozoan infection rate was 1.39% at the start of culture, and Table 3·11 was the number of protozoa per 5000 RBCs after culture.
表3· 11培養後每5000個RBC中的原蟲數 A B C D E F G Η ΑΤΟ 計量 (uM ) 0 5 10 20 40 80 160 320 混合 420 103 66 38 17 8 0 0 3_8_5結果統計: 三氧化二砷(/VFO)對FCC1抑制作用結果統計表,如表3.12所示。 表3.12三氧化二砷(ΑΤΟ)對FCC1抑制作用結果 ΑΤΟ 劑量 (uM) 劑量對數 (X) 感染率 (%) 減虫率 (%) 機率單位 (Y) 0.5 -0.3010 24.5 75.5 5.6903 1.0 0 15.7 84.3 6.0027 2.0 0.3010 9.0 91.0 6.3408 4.0 0.6021 4.0 96.0 6.7507 8.0 0.9031 1.9 98.1 7.0749 200824699 Y=1.5032X+5.8726 r2=0.9495 lC50=0.32uM=63.31ng/mI 95%信賴極限(confidence limit) (40.32-99.4ng/mI) IC90=2.09uM=413.48ng/ml 95% 信賴極限(confidence limit) (263.06-258.30ng/ml) 3·9· 經由多次試驗,結果發現丨C50在19.78-63.31ng/m丨之間,平均爲 35.57ng/m卜 IC90 在 185.97-737.94ng/ml 之間,平均 451O7ng/m卜此劑量 較現有的抗瘧藥磷酸氯喹大10倍左右,較憐酸哌喹的劑量相當。 實施例四:三氧化二砷毒殺非洲錐虫病的體外試驗 4.1試驗材料 4.11藥物: 三氧化二砷注射液(八33<^取10〇19/10111副31)。 4.1.2原蟲:Table 3.11 Number of protozoa per 5000 RBCs after culture ABCDEFG Η 计量 Metering (uM) 0 5 10 20 40 80 160 320 Mixing 420 103 66 38 17 8 0 0 3_8_5 Result statistic: Arsenic trioxide (/VFO) vs. FCC1 The statistical table of inhibition results is shown in Table 3.12. Table 3.12 Results of inhibition of FCC1 by arsenic trioxide (ΑΤΟ) 剂量 dose (uM) dose log (X) infection rate (%) worm reduction rate (%) probability unit (Y) 0.5 -0.3010 24.5 75.5 5.6903 1.0 0 15.7 84.3 6.0027 2.0 0.3010 9.0 91.0 6.3408 4.0 0.6021 4.0 96.0 6.7507 8.0 0.9031 1.9 98.1 7.0749 200824699 Y=1.5032X+5.8726 r2=0.9495 lC50=0.32uM=63.31ng/mI 95% confidence limit (40.32-99.4ng/mI) IC90= 2.09uM=413.48ng/ml 95% confidence limit (263.06-258.30ng/ml) 3·9· Through several experiments, it was found that 丨C50 is between 19.78-63.31ng/m丨, with an average of 35.57. The ng/m Bu IC90 is between 185.97-737.94 ng/ml, and the average dose is 451O7 ng/m. This dose is about 10 times larger than the existing antimalarial drug chloroquine, which is equivalent to the dose of pigoquine. Example 4: In vitro test of arsenic trioxide poisoning African trypanosomiasis 4.1 Test material 4.11 Drug: Arsenic trioxide injection (eight 33 < ^ 10 19/10111 pair 31). 4.1.2 Protozoa:
Trypanosoma brucei標定爲”427”。該原蟲株係來自於紐約大學李美 麗博士(Dr_ Mary Lee卜 41.3培養液: 含有胎牛血清的SDM79 (JRH 57453)培養液。 4_2試驗方法 以顯微鏡觀察活細胞數量與型態變化。 4·3試驗步驟Trypanosoma brucei is labeled "427". The protozoan strain was obtained from Dr. Li Mei from New York University (Dr_ Mary Lee Bu 41.3 medium: SDM79 (JRH 57453) medium containing fetal bovine serum. 4_2 Test method The number and type of living cells were observed by microscope. 3 test steps
V 200824699 以三氧化二砷aimg/ml/day處理3天,觀察7日的毒殺作用。 4.4試驗結果 細胞學發現蟲體死亡呈現線狀而非一般的小圓形。暫時的觀察顯示三 氧化二砷對培養之Trypanosoma brucei的毒殺作用與Melarsoprol相當。 實施例五:三氧化二砷對人類周邊血液單核白血球關於細胞凋亡作用 的體外試驗 5.1試驗材料 5.1·1細胞株: 自行分離之新鮮人類周邊血液單核白血球。 5.1 ·2藥物: 三氧化二砷注射液(Asadin®,10mg/10ml/vial)。 5.13試劑:白血球分離試劑。 5_2試驗方法及步驟 5·2_1細胞株分離: 由人類血液分離出人類周馳液單核白血球,運用Ficoll Plaque方 法,利用其它血球與人類周邊血液單核白血球的密度不同而將其分離。 5.2.2細胞株培養: 分離出之人類周邊血液單核白血球培養于RPMI1640並加入10%胎 牛血清,在5%C02及95%空氣、37。(:下培養。 5.2.3細胞凋亡檢測: 以P丨染色法染色後,由流式細胞儀(flowcytometry)檢測。 32 200824699 5_2·4人類特定周邊血液單核白血球的細胞凋亡測試: 結合單株抗體-營光串聯染色法(mAb-fluorescein tandem staining)和 Phiphilux處理後,再由流式細胞儀檢測分類。 5·3實驗結果 5.3.1三氧化二砷誘發人類周馳液職白血球細胞凋亡作用 由實驗中顯示,發現三氧化二砷誘發人類周邊血液單核白血球細胞凋 亡作用。 • 當以不同Μ度之三氧化二砷(分別爲〇、〇·5、2.5、5、或10μΜ),對 全部人類周邊血液單核白血球細胞分別處理24小時和48小時後,再以ΡΙ • staining將人類周邊血液單核白血球細胞染色,由流式細胞儀檢測全部人類 周邊血液單核白血球細胞的細胞凋亡百分比。 由實驗中顯示,在處理24小時和48小時的兩組中,三氧化二砷在濃 度2.5至10 μΜ之間,三氧化二砷誘發人類周邊血液單核白血球細胞凋亡 作用並無劑量依存關係(dose-dependent);在48小時組有稍高的死亡比 φ 率,也許一些特定Μ的人類周邊血液單核白血球細胞對三氧化二砷比其 它細胞敏感。 5.3·2運用caspase_3活性區別對三氧化二砷敏感的白血球種類 配合特殊種類細胞染色模式區別對三氧化二砷所誘發之凋亡反應最 明顯的人類周邊血液單核白血球細胞。結果如表5.2所示。 運用caspase-3活性測試,配合特殊種類細胞染色模式,以人類周邊 血液單核白血球細胞表面記號綱如T淋巴球以“CD3+”辨識,B淋巴球以 33 200824699 冗019+,’辨識)。由表5.2之試驗結果顯示,以濃度5.0口1\/1的三氧化二砷處 理48小時後,T淋巴賴96%死亡。因此,在三氧化二砷誘發特定人類周 邊血液單核白血球細胞的凋亡作用中,Τ淋巴球具有一定的敏感度。 表5.2經48小時三氧化二砷處理後,所誘發特定人類周邊血液單核 白血球細胞凋亡作用 控制組 34.5(%) 1μΜ 36.1(%) 5μΜ 96.2(%) 實施例六:三氧化二砷對人類纖維母細胞之細胞凋亡作用與影響細胞 週期的體外試驗 ‘ 6.1試驗材料 . 6·1·1細胞株: 購買自ATCC的人類纖維母細胞關R-90[丨MR90]。。 6·1·2藥物: 三氧化二砷注射液(Asadin®,10mg/10ml/vial)。 # 6·1·3 試劑:V 200824699 was treated with arsenic trioxide aimg/ml/day for 3 days, and the poisoning effect on the 7th was observed. 4.4 Test results Cytology found that the death of the worms was linear rather than a generally small circle. A temporary observation showed that the toxic effect of arsenic trioxide on the cultured Trypanosoma brucei was comparable to that of Melarsoprol. Example 5: In vitro test of arsenic trioxide on apoptosis of human peripheral blood mononuclear leukocytes. 5.1 Test materials 5.1·1 cell line: Fresh human peripheral blood mononuclear leukocytes isolated by self. 5.1 · 2 drugs: arsenic trioxide injection (Asadin®, 10mg/10ml/vial). 5.13 Reagent: White blood cell separation reagent. 5_2 Test methods and procedures 5·2_1 cell line isolation: Human centrifugal mononuclear leukocytes were isolated from human blood, and the Ficoll Plaque method was used to separate the other blood cells from the peripheral blood mononuclear leukocytes. 5.2.2 Cell culture: The isolated human peripheral blood mononuclear leukocytes were cultured in RPMI 1640 and added with 10% fetal bovine serum at 5% CO 2 and 95% air, 37. (: Next culture. 5.2.3 Apoptosis detection: After staining by P丨 staining, it is detected by flowcytometry. 32 200824699 5_2·4 Apoptosis test of human peripheral blood mononuclear leukocytes: combination After single-body antibody-mum-fluorescein tandem staining and Phiphilux treatment, the classification was detected by flow cytometry. 5.3 Experimental results 5.3.1 Arsenic trioxide induced apoptosis of human leukocyte cells in Zhouchi liquid It has been shown in experiments that it is found that arsenic trioxide induces apoptosis of human peripheral blood mononuclear leukocytes. • When arsenic trioxide (different 〇, 〇·5, 2.5, 5, or 10 μΜ, respectively) with different concentrations, all human peripheral blood singles The nucleated white blood cells were treated for 24 hours and 48 hours respectively, and the peripheral blood mononuclear white blood cells were stained with sputum staining, and the percentage of apoptosis of all human peripheral blood mononuclear white blood cells was detected by flow cytometry. It was shown that in the two groups treated for 24 hours and 48 hours, the arsenic trioxide was between 2.5 and 10 μΜ, and the arsenic trioxide was induced. There is no dose-dependent relationship between the apoptosis of human peripheral blood mononuclear leukocytes; there is a slightly higher rate of death than φ in the 48-hour group, and perhaps some specific sputum peripheral blood mononuclear leukocytes to arsenic trioxide ratio Other cells are sensitive. 5.3·2 Use caspase_3 activity to distinguish arsenic trioxide-sensitive white blood cell types with special cell staining patterns to distinguish human peripheral blood mononuclear leukocytes from the arsenic trioxide-induced apoptosis. The results are shown in Table 5.2. Using the caspase-3 activity test, combined with a special type of cell staining mode, the surface markers of human peripheral blood mononuclear white blood cells are identified as "CD3+" by the T lymphocytes, and the B lymphocytes are identified as "20082008". From the test results in Table 5.2, after treatment with arsenic trioxide at a concentration of 5.0 and 1/1 for 48 hours, 96% of T lymphoids died. Therefore, in the arsenic trioxide-induced apoptosis of specific human peripheral blood mononuclear leukocytes, the axillary lymphocytes have certain sensitivity. Table 5.2 After treatment with arsenic trioxide for 48 hours, the apoptosis of mononuclear leukocytes induced by specific human peripheral blood was controlled by 34.5 (%) 1 μΜ 36.1 (%) 5 μΜ 96.2 (%) Example 6: cells of arsenic trioxide against human fibroblasts Apoptosis and in vitro assays affecting the cell cycle' 6.1 Test Materials. 6.1 Cell Line: Human fibroblasts purchased from ATCC, R-90 [丨MR90]. . 6.1.2 Drugs: Arsenic Trioxide Injection (Asadin®, 10 mg/10 ml/vial). # 6·1·3 Reagents:
Triton Χ-100、氯化鈉、EGTA、ΝΡ-40、氟化鈉、Na3V04、aprotinin、 leupeptln、PBS、phenylmethyl-sulfonylfluoridepMSF)。 6.2試驗方法 6·2·1三氧化二砷抑制規成纖維母細胞細胞株增殖作用的敏感性測 試 6·2·1·1細胞賴測試 τ4 200824699 以不同濃度之三氧化二砷(分別爲〇、1.0、2·5、5、或10μΜ),對人 類成纖維母細胞細胞株處理24小時後,利用碘化物染色法(propidium iocHde staining)染色後,以流式細胞儀量測細胞週期停滞。 6-2,1_2利用MTT比色法測量細胞活性 在96-微孔的培養盤中,每個微孔加入5x103個細胞。細胞在37°C下 培養後,加入30μΙ MTT溶液,之後避光培養4小時。Formazan grain溶 解在DMSO後,利用EL]SA麵機於570nm下,測量吸光値。 β_2.1.3觀察凋亡小體的產生 細胞凋亡小體的型態是利用Hoechst染色觀察。去掉培養液,利用在 PBS中4% formaldehyde室溢下固定5分鐘。然后加入Hoechst dye 33258 5pg/mMn PBS作用5分鐘,洗去之後,加入試劑(PBS:glycerol=3:1)。利 用熒光顯微鏡觀察被染成螢光的細胞核。利用Hoechst 33258染色來觀察 細胞凋亡的形態。 6·2·2實驗結果 6_2.2.1三氧化二砷引發人類成纖維母細胞之細胞週期停滯 以不同濃度之三氧化二砷(分別爲0、1·0、2.5、5、10μΜ),對人類成 纖維母細胞細胞株處理24小時後,利用碘化物染色法及流式細胞儀分析細 胞週期,發現三氧化二砷弓丨發人類成纖維母細胞之細胞週期停滞于G2/M 〇 6·2·2·2三氧化二砷引發人類成纖維母細胞之細胞死亡 以不同濃度之三氧化二砷份別爲0、1·0、2.5、5、10μΜ),對人類成 纖維母細胞細胞株處理24小時後,利用从11比色法測量細胞活性,發現 三氧化二砷對人類成纖維母細胞具細胞毒性。 55* 200824699 同時,以不同濃度之三氧化二砷(分別爲〇、1·〇、2·5、5、10μΜ),對 人類成纖維母細胞細胞株處理24小時後,並以DAPI staining測試細胞凋 亡小體。實驗結果發現控制組並無細胞凋亡現象,1·〇和2·5 μΜ三氧化二 砷誘發細胞小體;5.0和10 μΜ出現明顯的細胞凋亡小體,且人類成纖維 母細胞發生細胞凋亡與壞死現象。 6.Ζ2.3分析方法 試驗採用兩原素析因設計數據的方差分析。數據均採用SPSS套裝軟 件完成。 根據本發明可作夕不同修正及變化對於熟習該項技術者而言 然不會碰本發明的範圍與精神、雖然本發明已鍵特定的較_ 體事實,必嫌解的是本發明不麵不當地限制於該等特定具触 實上。專眚卜,在眚施本發明之已流樽式方面,對於熟習該項11¾Triton Χ-100, sodium chloride, EGTA, ΝΡ-40, sodium fluoride, Na3V04, aprotinin, leupeptln, PBS, phenylmethyl-sulfonylfluoridepMSF). 6.2 Test method 6.11 sensitization of arsenic trioxide inhibiting the proliferation of fibroblastic cell lines 6.2·1·1 cell lysing test τ4 200824699 Different concentrations of arsenic trioxide ((, 1.0, 2·5, respectively) 5, or 10 μΜ), after treatment with human fibroblastic cell lines for 24 hours, staining with iodide staining (propidium iocHde staining), cell cycle arrest was measured by flow cytometry. 6-2, 1_2 Measurement of cell viability by MTT colorimetry In a 96-well microplate, 5 x 103 cells were added to each well. After the cells were cultured at 37 ° C, 30 μM of MTT solution was added, followed by incubation for 4 hours in the dark. After the Formazan grain was dissolved in DMSO, the absorbance enthalpy was measured at 570 nm using an EL]SA machine. Observation of apoptotic bodies by β_2.1.3 The type of apoptotic bodies was observed by Hoechst staining. The culture solution was removed and fixed by a 4% formaldehyde chamber in PBS for 5 minutes. Then, Hoechst dye 33258 5pg/mMn PBS was added for 5 minutes, and after washing, a reagent (PBS: glycerol = 3:1) was added. The nuclei stained with fluorescence were observed using a fluorescence microscope. Hoechst 33258 staining was used to observe the morphology of apoptosis. 6·2·2 Experimental results 6_2.2.1 Arsenic trioxide induced cell cycle arrest in human fibroblasts with different concentrations of arsenic trioxide (0, 1·0, 2.5, 5, 10 μΜ, respectively) for human fibroblastic cell lines After 24 hours of treatment, the cell cycle was analyzed by iodide staining and flow cytometry. It was found that the cell cycle of human fibroblasts arrested by arsenic trioxide was arrested at G2/M 〇6·2·2·2 arsenic trioxide induced human fibroblasts. The cell death of the mother cells was 0, 1·0, 2.5, 5, 10 μΜ with different concentrations of arsenic trioxide. After treatment with human fibroblastic cell lines for 24 hours, the cell activity was measured by 11 colorimetry. Arsenic trioxide is cytotoxic to human fibroblasts. 55* 200824699 At the same time, human fibroblastic cell lines were treated with different concentrations of arsenic trioxide (〇, 1·〇, 2·5, 5, 10 μΜ, respectively) for 24 hours, and the apoptosis was small by DAPI staining. body. The results showed that there was no apoptosis in the control group, 1·〇 and 2·5 μΜ arsenic trioxide induced cell bodies; 5.0 and 10 μΜ showed obvious apoptotic bodies, and apoptosis of human fibroblasts occurred. Necrosis. 6.Ζ2.3 Analytical method The experiment uses the variance analysis of the two elemental factorial design data. The data is done using the SPSS suite of software. According to the present invention, various modifications and changes may be made without departing from the scope and spirit of the invention, and although the invention has been specific to the key, it is undoubted that the present invention is not Unduly limited to these specific touches. Specialized in the application of the present invention, for the familiarity of the item 113⁄4
涵著於下列權利要求之內。 者而言顯而易知之不同修正 : 【實施方式】 【圖式簡單說明】 【主要元件符號說明】 36It is intended to be within the following claims. Different amendments that are obvious to the user: [Embodiment] [Simple description of the diagram] [Explanation of main component symbols] 36
Claims (1)
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11/165,454 US20060292243A1 (en) | 2005-06-24 | 2005-06-24 | Arsenic compounds for the treatment of the arsenic-sensitive blast-cell related diseases |
CNA2005101032388A CN1931186A (en) | 2005-09-16 | 2005-09-16 | Application of arsenous oxide in preparing medicine for treating embryo cell diseases sensitive to arsenide |
EP06012727A EP1736161A3 (en) | 2005-06-24 | 2006-06-21 | Use of an arsenic compound for the manufacture of a medicament for the treatment of arsenic susceptible blast-like cell related diseases |
Publications (1)
Publication Number | Publication Date |
---|---|
TW200824699A true TW200824699A (en) | 2008-06-16 |
Family
ID=44802554
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
TW095145082A TW200824699A (en) | 2005-06-24 | 2006-12-05 | Arsenic compounds for the treatment of the arsenic-sensitive blast-cell related diseases |
Country Status (1)
Country | Link |
---|---|
TW (1) | TW200824699A (en) |
-
2006
- 2006-12-05 TW TW095145082A patent/TW200824699A/en unknown
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Escamilla-Ramírez et al. | Autophagy as a potential therapy for malignant glioma | |
US11344545B2 (en) | Use of levocetirizine and montelukast in the treatment of autoimmune disorders | |
CN108472280A (en) | Compounds and methods for for treating or preventing hematologic cancers | |
WO2016014859A1 (en) | Bet inhibitor and bruton's tyrosine kinase inhibitor combinations | |
CN103002896A (en) | Use of levocetirizine and montelukast in the treatment of influenza, common cold and inflammation | |
CN101370783B (en) | Salts of 1-alkylamino-1-desoxypolyols with 9-oxoacridine-10-acetic acid, medicinal preparations on base, use, preventive measures and treatment thereof | |
TW201840537A (en) | Kdm4 inhibitors | |
UA125892C2 (en) | An aurora a kinase inhibitor for use in the treatment of neuroblastoma | |
CN113876946B (en) | Combined pharmaceutical application of PD-1 antibody and pseudomonas aeruginosa and pharmaceutical composition | |
WO2023138630A1 (en) | Pharmaceutical combination for treating tumors and use thereof | |
WO2023104151A1 (en) | Pharmaceutical composition for treating tumors and use | |
CN107441092A (en) | Qinghaosu and hydroxychloroquine combine the purposes for treating IgA nephrosis | |
TW200824699A (en) | Arsenic compounds for the treatment of the arsenic-sensitive blast-cell related diseases | |
CN101048160B (en) | Purine and pyrimidine cdk inhibitors and their use for the treatment of autoimmune diseases | |
TW202140005A (en) | Novel medicament for treating inflammatory disease | |
RU2731535C1 (en) | Combination, use thereof and method of treating | |
WO2008008841A2 (en) | Use of benzimidazole derivatives for the treatment and/or prevention of autoimmune disorders | |
WO2024067631A1 (en) | Pharmaceutical combination of fak inhibitor and substance for inducing immunogenic cell death and use | |
Chen et al. | RETRACTED ARTICLE: Experimental evidence of shikonin as a novel intervention for anti-inflammatory effects | |
WO2024041527A1 (en) | Pharmaceutical combination of fak inhibitor and microtubule inhibitor and use thereof | |
CN107349191A (en) | Application of the Vorinostat in the medicine for preparing treatment lung cancer | |
US20240285627A1 (en) | Drug combination of fak inhibitor and immunogenic cell death inducer and use thereof | |
WO2024140295A1 (en) | Pharmaceutical combination for treating tumors and use thereof | |
CN114569559B (en) | New use of wuling fungus powder | |
WO2023246869A1 (en) | Pharmaceutical combination for treating tumors and use thereof |