200838567 九、發明說明： 【發明所屬之技術領域】 由放射藥物與化學治療藥物組合而成雙功能與雙效奈米標輕套組 (kit)〇 【先前技術】 微脂體（LiP〇some)被認為是被動標減運藥物的好劑型， 下 幾項優點： ⑴放射性同位素與化療藥物包埋在微脂體内，會改變藥物的 動力學，延長藥品在血液半衰期，而且這樣大小（〜ι〇〇 的奈米微脂體透過強化滲透與滯留（enhaneed p_eabilityandretenti0n，EPR )效用能穿過腫瘤新生血管壁 較鬆散的癌细胞組織、，使包埋放射性同位素與抗癌藥物的奈米標 乾微脂體能大量累積（10 —觸倍）在腫瘤部位，增進治療效果 並降低對正常細胞傷害，此奈米微賴屬於鶴式餘（押― targeting) 〇 (2) 毒性高的藥物包裹在奈米錄微脂體内，由於藥物之濃縮與 局限釋藥可以減少不良的副作用。 (3) 奈米標嫌脂體的脂質組成、顆粒大小、結構、製備的方法 與包裹藥物的選擇性报大，能夠符合各種不同的情況，進行 各種應用。 (4) 奈米標綠脂體是由鱗脂質構成，其與細胞膜成分相同，在 生物體内能被分解，所以不具毒性，而且不像蛋白質會引起 免疫反應，所以能夠多次使用。 鍊-m、銖携為放射出陳線，可做為標乾治療腫瘤放射性同位素， 因它們同時含服eV及·v ’故科賤加,_彡麟功能。它 6 200838567 的物理性質如 放射核種 (Radio -nuclide) 物理半衰 期 (Physical half-life) (Tl/2) 衰減模 式 (Mode of decay) γ-ray 3-ray 能量 (Energy) (MeV) 豐裕度 Abundance (%) 能量(Energy) (MeV) 組織内之範 圍(Range in tissue)(mm) ax. Ave· Max. Ave· 16.98 h β·(100) 0.155 14.9 2.12 0.765 11 3.5 186Re 3.8 d β·(92) EC (8) 0.139 9 1.075 0.323 3.6 1.8 _ 先前技術，Bao發表以186Re，99mTc標幟BMEDA，以及包埋在微脂體 内探討放射診斷造影劑或放射治療在正常老鼠之基礎研究（Bao et al. J.200838567 IX. Description of the invention: [Technical field of invention] A combination of radiopharmaceuticals and chemotherapeutic drugs to form a dual-function and double-effect nanometer kit (previous technique) Liliposome (LiP〇some) It is considered to be a good dosage form for passive standard reduction drugs. The following advantages: (1) Radioisotopes and chemotherapeutic drugs are embedded in the microlipids, which will change the kinetics of the drug, prolong the half-life of the drug, and the size (~ι The nano-lipids of sputum can penetrate the loose cancer cell tissues of the neovascular wall of the tumor through the effect of enhanced penetration and retention (EPR), so that the radiolabels of radioisotopes and anticancer drugs can be dried. The body of fat can accumulate a large amount (10 - touch) in the tumor site, improve the therapeutic effect and reduce the damage to normal cells. This nano-relation belongs to the crane-type (target) 〇 (2) The highly toxic drug is wrapped in the nanometer. In the liposome, due to the concentration of the drug and the limited release of drugs, it can reduce adverse side effects. (3) The lipid composition, particle size, structure, and The preparation method and the selectivity of the packaged drug are large, and can be used in various applications according to various conditions. (4) The nanometer green body is composed of squamous lipid, which is the same as the cell membrane component and can be Decomposed, so it is not toxic, and unlike protein, it can cause immune response, so it can be used multiple times. Chain-m, sputum is used to emit the aging line, which can be used as a standard for the treatment of tumor radioisotopes, because they also contain eV and · v '故科贱加, _ 彡 功能 function. It 6 physical properties of 200838567 such as radionucleus (Radio-nuclide) physical half-life (Tl/2) Mode of decay γ-ray 3-ray Energy (MeV) Abundance (%) Energy (MeV) Range in tissue (mm) ax. Ave· Max. Ave· 16.98 h β·(100) 0.155 14.9 2.12 0.765 11 3.5 186Re 3.8 d β·(92) EC (8) 0.139 9 1.075 0.323 3.6 1.8 _ Prior art, Bao published 186Re, 99mTc flag BMEDA, and embedded in liposome to investigate radiodiagnosis Agent or radiation therapy based on normal mice Study (Bao et al. J.
Pharm· Sci (2003) 92, 1893-1904 and J· Nucl· Med(2003)，44, 1992-1999)。該 項技術僅具單功能放射診斷或放射治療，缺少放射及化學治療雙功能與雙 效技術。 【發明内容】 本發明是由放射藥物與化學治療藥物組合而成雙功能與雙效奈米標靶 套組（kit)，此套組有A、B、C參瓶藥物，A瓶其配方成分包括BMEDA， gluconate acetate，SnCl2 等，B 瓶其配方成分包括 DSPC，cholesterol， ♦ DSPE-PEG，Doxorubicin 或 Daunorubidn 或 vinolbine 等，C 瓶是 188Re〇4- (或186Re〇4 )溶液。使用此奈米標乾套組方法，將c瓶放射同位素鍊-188 或銖-186液體，放入A瓶，此A瓶經過加熱反應後，瓶内液體轉移至B瓶， 接著B瓶加紐，則得到組合式雙功能與雙效放射診斷與治療及化學治療 之奈米標嫌脂體藥物，可做為轉與治療腫瘤或惡性腫瘤腹水之應用。 本發明優點：⑴方便（2)操作簡單（3)具有放射診斷與治療雙功能 (4)具放射及化學治療加成效果特性。 7 200838567 【實施方式】 .縮寫列表如下： BMEDA : N，N-bis(2-mercaptoethyl)-N，,N，-diethylethylenediamine DSPC : Distearoyl phosphatidylcholine PEG Polyethylene glycol DSPE ： Distearyl phosphatidylethanolamine 實施例一··放射性治療藥物配方製備（A瓶）及品管分析 祥取 5mg BMEDA 置於 A 瓶中，加入 0.5ml 的 0.17 mol/L Gluconate-_ acetatate溶液，隨後灌入氮氣約1分鐘，隨即加入以〇·〇ΐΝΗα新鮮配製的 SnCl2溶液120pL_g/mL)，灌入氮氣約1分鐘，以避免SnCl2氧化，以塑 膠軟塞及鋁蓋將瓶蓋密封。接著進行標幟效率品管分析，取A瓶液體與銖 -188溶液置於80°C恆溫水浴槽内以200rpm震盪搖晃作用反應一小時，隨 後取出並置於室溫自然冷卻，以mdio-ITLC/SG (移動相為normal saline)分 析標幟效率(labeling efficiency )，標幟效率達 99土 1.73%。（Rf: 1，free 188Re ; Rf: 0,188Re-BMEDA) _ 實施例二：化學治療藥物配方製備（B瓶）及品管分析 分別秤取 DSPC (70 pmole)/Cholesterol/DSPE-PEG2_ (3:2:0.3莫耳比)於250 mL圓底燒瓶内，分別加入8毫升氯仿並使其均勻溶 解。利用旋轉減壓濃縮儀於60°C下真空抽除有機溶液，待氯仿完全抽除後 可於瓶壁上形成脂質薄膜。抽乾後，再加入5此250 mM硫酸銨溶液(250 ^(^^(^^•(^(^(^至已形成脂質薄膜的圓底燒瓶内’於⑼艺 水浴中震盪搖晃至瓶壁上脂質薄膜全部分散於硫酸銨溶液中，即可得到多 層微脂體(MLV)。再將多層微脂體懸浮液以液態氮及6〇〇c水浴進行反覆冷 凍與解康六次。讀，私X紐義猶线(Lipex Bi_mbtane， 8 200838567 -Canada)進行過濾擠壓得到單一脂雙層微脂體。 • 接著進行doxorubicin包覆，每Ipmole之磷脂質加入14(^§之 doxorubicin的比例將事先配製之doxorubicin stock (10 mg/mL)加入微脂體懸 浮液中，並於60°C，100 rpm下反應30分鐘。反應完成後立刻至於冰水浴 中將之冷卻。再將包覆doxorubicin之微脂體懸浮液通過Sephadex G50 gel filtration column並以0·9 % NaCl為沖提液，藉以移除未被包覆之 doxorubicin。收集通過column之微脂體懸浮液，再利用超高速離心機於 150000xg下離心90分鐘，移除大部分上清液，留住少量上清液將沉殿之微 脂體再均勻懸浮。以0·22μιη filter過濾微脂體懸浮液，得到最後產品，裝入 • B瓶進行化學治療藥物品管分析·· ⑴利用N4 Phis粒徑分析儀，測得微脂體平均粒徑為75-95nm之常態分 佈。 (2)利用 Spectrofluorometer (JASCO, FP6200)於激發光 475nm 和發射光 580nm下測定，包覆doxorubicin之微脂體，其濃度為2mg/ml。 實施例三:放射治療藥物與化學治療藥物組合式奈米標把藥物製備 將C瓶放射性同位素鍊-188溶液加入A瓶（BMEDA，SnCl2, φ Gluconate-acetate)，置於80°C恆溫水浴槽内震盪一小時，待冷卻後，將標 幟完成的銖-188-BMEDA加入B瓶内（Lip〇_DXR)，放入60°C水浴槽中反 應30分鐘。完成銖-188-BMEDA包覆進入Lip〇-DXR中後，以PD-10 column 進行純化’首先以20 mL的生理食鹽水(Normal Saline)平衡PD-10 column， 接著使用生理食鹽水當沖提液。每〇·5 mL收取一個fraction共收取12個 fraction，並計算各個fracti〇n活度及殘留在c〇lmnn的活度。Lip〇_DXR由於 包覆doxorubicin可見其紅色顏色，而紅色顏色集中在第6〜9個fracti〇n，且 這幾個fraction的活度也較高，包覆效率40 - 60%。 9 200838567 實施例四：銖-188_8]\1£0人-1^〇8〇111©1111«*〇_8?£0^造影及影像定量 分析 以 3% isoflurane (in 100% oxygen)麻醉老鼠後，將 g-25 Column 純化後 的銖-188-BMEDA -Liposome以尾靜脈注射(IV injection)方式打入老鼠體 内，分別於注射後1小時、4小時、24小時、48小時、72小時，以3% isoflurane (in 100% oxygen)麻醉老鼠，且造影期間仍持續以3% is〇flurane (in 1〇〇% oxygen)麻醉老鼠；將老鼠四肢拉直固定於動物檯上，以利用低能量，高解 析度的準值儀(collimator)進行micrc-SPECT造影，能量視窗(energy window) 设疋為 155±10% keV ’ 影像大小設定為 64 x 64，ROR : 1·〇 cm，FOV : 1.37 _ cm。此外’為了進行後續的影像定量分析，因此於上述每個時間進行已知 放射活度的銖-188-參考射源micro-SPECT/CT造影；影像分析結果以標準吸 收值(standard uptake value ; SUV)呈現。 圈取影像中腫瘤組織ROI (region 〇f interest )的區域，依據參考射源， 計算出每一克的腫瘤組織具有多少放射活度(//Ci/g)，帶入下列公式計算腫 瘤組織標準吸收值(SUV): (measured activity concentration (μ€ί/δ) / [Injected Dose (μ〇ί) / body weight (g)] 由弟一圖可看到鍊_188-BMEDA_Lip〇some注射24小時後，右後腿腫 φ瘤位置有明顯的吸收，直至72小時仍有明顯的影像；miciO-SPECT影像定 虿分析結果顯示，在注射後24小時腫瘤組織的吸收量(Standardised tum〇r uptake value; SUV)高達 2.81 土 0.36。 實施例五：奈米標靶組合式藥物銖_188_BMEDA/DXR-Lip〇s〇me腫瘤模式 及療效評估（尾靜脈注射） 將2xl〇5 cells/5〇 μΐ之C26大腸腫瘤細胞以皮下注射方式將腫瘤細胞接 種於6週大BALB/c小鼠的右後腿，待生長約一週後，腫瘤大小約 50 mm3 〜 100mm3即可進行動物實驗。本實驗將動物隨機分成以下4組，每組八隻： 200838567 188-BMEDAA)XR-Lip〇s〇me > 188-BMEDA- Liposome' DXR-Liposome -及Normal Saline。其療程如下： 1·第一組：由尾靜脈注射銖-188-BMEDA/DXR-LiP〇some，内含 500//Ci 放射活度及2mg/Kg的Doxombicin(DXR)，共給藥三次，每次給藥劑量 均相同，第一次給藥後間隔三天再進行第二次給藥，於第二次給藥後七 天再進行第三次給藥。 2·第二組：由尾靜脈注射給予500 //Ci放射活度的銖_188_bmeda_ Liposome，共給藥二次，每次給藥劑量均相同，第一次給藥後間隔三天 再進行第二次給藥，於第二次給藥後七天再進行第三次給藥。 ® 3·第三組：由尾靜脈注射給予DXR-LiP〇some ，Doxorubicin濃度為 2mg/Kg，共給藥二次，每次給藥劑量均相同，第一次給藥後間隔三天再 進行第二次給藥，於第二次給藥後七天再進行第三次給藥。 4·弟四組：由尾靜脈注射給予Normal Saline，共注射三次，第一次注射後 間隔三天再進行第二次注射，於第二次給藥後七天再進行第三次注射。 研究進行期間，每個禮拜以電子游標尺(digital caliper)進行兩次腫瘤量測， 每曰進行存活率紀錄。 由第二圖顯示（註：圖式數據代表平均正負值），在注射後第27天， #只注射Normal Saline為給予藥物治療的老鼠腫瘤體積急遽生長達2220.60土 431.35 mm3，施予銖-188-BMEDA/DXR_LiP〇some治療的老鼠腫瘤體積最小 ^(^。々別腿^其次依序為施予銖以邱规^丄中咖咖治療的老鼠 (298·14± 157.25 mm3)及施予 DXR_Liposome 治療的老鼠(9172〇士 17759 mm3)。由第三圖顯示，只注射Normal Saline為給予藥物治療的老鼠在注射 後的第48天全數死亡；在注射後的第5〇天，施予鍊 -188-BMEDA/DXR-Liposome治療的老鼠其存活率最高，達87·5%，其次依 序為施予銖-lSS-BMEDA/Liposome治療的老鼠（62·5%)及施予 DXR-LiP〇S〇me治療的老鼠(37·5%)。以上實驗結果展示了奈米標靶放射與 11 200838567 化學組合式雙效藥物對腫瘤細胞治療之加成與有效性。 實施例六··奈米標靶放射與化學雙效藥物銖-188-BMEDA/ DXR-Liposome惡性腫瘤腹水模式及療效評估試驗（腹腔注射） 將 C26 cell line (colon carcinoma) 2 X 1〇5 cells/500 μί PBS 以腹腔注射 方式，接種於六週齡的BALB/c mice。待腫瘤生長1〇天後，可見其腹部腫 脹，表示腫瘤細胞在腹腔内擴散及腹水產生，取體重相近之BALB/c mice 進行療效評估試驗。 將腹腔接種C26腫瘤細胞之BALB/c mice分為鍊-188-BMEDA _ Liposome、Lipo-DXR、銖-188-BMEDA/DXR-Liposome 及生理食鹽水(Normal Saline)四組，每組 8〜10 隻 BALB/cmice。銖-188-BMEDALiposome 組以腹 腔注射方式給予 200 μί (400 〜600 μ(：〇的銖_188-BMEDA_Liposome， Lipo_DXR組以腹腔注射方式給予200 pL (5 mg/kg的doxorubicin)的 碑〇»〇\11’銖-188"6]\^〇^/〇}〇1-1^〇8〇11^組以腹腔注射方式給予20(^[ (400 〜600 μα，5 mg/kg 的 doxorubicin)的鍊-188· BMEDA/DXR-Liposome， 生理食鹽水(Normal Saline)組以腹腔注射方式給予200 μ]：的生理食鹽水。這 四組老鼠每日監控、紀錄其體重及存活率。每日監控及紀錄之老鼠死亡或 φ直到120天後還存活，即停止繼續紀錄，並將存活之老鼠解剖觀察是否還 有殘存的腫瘤小節(tumornodules)。由下表可知其mediansurvivaltime之銖 -188-BMEDA-Liposome 組為 21 天，Lip〇-DXR 組為 18 天，銖-188-BMEDA/DXR-Liposome組為27天，且與Normal saline組比較有顯著差異 (P value < 0.05)，Normal saline 組為 17.67 天。Pharm Sci (2003) 92, 1893-1904 and J. Nucl. Med (2003), 44, 1992-1999). This technique is only available for single-function radiodiagnosis or radiation therapy, and lacks dual- and dual-effect techniques for radiation and chemotherapy. SUMMARY OF THE INVENTION The present invention is a combination of a radiopharmaceutical and a chemotherapeutic drug to form a dual-function and double-effect nano-target kit. The kit has A, B, and C bottles, and the A bottle has a formula. Including BMEDA, gluconate acetate, SnCl2, etc., the composition of the B bottle includes DSPC, cholesterol, ♦ DSPE-PEG, Doxorubicin or Daunorubidn or vinolbine, etc. The C bottle is 188Re〇4- (or 186Re〇4) solution. Using this nanometer dry set method, a bottle of radioisotope chain -188 or 铢-186 liquid is placed in the A bottle. After the A bottle is heated, the liquid in the bottle is transferred to the B bottle, and then the B bottle is added. The combination of dual-function and double-effect radiological diagnosis and treatment and chemotherapy for nanometer-labeled liposome drugs can be used for the treatment of tumor or malignant tumor ascites. The invention has the advantages of: (1) convenience, (2) simple operation, (3) dual function of radiological diagnosis and treatment, and (4) addition and radiation treatment effects. 7 200838567 [Embodiment] The list of abbreviations is as follows: BMEDA : N, N-bis (2-mercaptoethyl)-N,, N,-diethylethylenediamine DSPC : Distearoyl phosphatidylcholine PEG Polyethylene glycol DSPE : Distearyl phosphatidylethanolamine Example 1 · Radiotherapy Formulation preparation (A bottle) and quality control analysis Take 5mg BMEDA in A bottle, add 0.5ml 0.17 mol/L Gluconate-_ acetatate solution, then inject nitrogen for about 1 minute, then add 〇·〇ΐΝΗα fresh The prepared SnCl2 solution was 120 pL_g/mL), and nitrogen gas was poured for about 1 minute to avoid oxidation of SnCl2, and the cap was sealed with a plastic soft plug and an aluminum cap. Next, the label efficiency tube analysis was carried out, and the A bottle liquid and the 铢-188 solution were placed in a constant temperature water bath at 80 ° C for shaking at 200 rpm for one hour, then taken out and allowed to cool at room temperature, to mdio-ITLC/ SG (mobile phase is normal saline) analyzes the labeling efficiency, and the flag efficiency is 99 soil and 1.73%. (Rf: 1, free 188Re; Rf: 0, 188Re-BMEDA) _ Example 2: Preparation of chemotherapeutic drug formulation (B bottle) and quality control analysis DSPC (70 pmole) / Cholesterol / DSPE-PEG2_ (3: 2: 0.3 molar ratio) In a 250 mL round bottom flask, 8 ml of chloroform was added and dissolved uniformly. The organic solution was vacuum-extracted at 60 ° C using a rotary vacuum concentrator, and a lipid film was formed on the wall of the bottle after the chloroform was completely removed. After draining, add 5 250 mM ammonium sulphate solution (250 ^(^^(^^•(^(^(^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ The upper lipid film is completely dispersed in the ammonium sulfate solution to obtain a multi-layered liposome (MLV), and the multi-layered liposome suspension is repeatedly frozen and repelled six times in a liquid nitrogen and a 6 〇〇c water bath. The private X-nucleus line (Lipex Bi_mbtane, 8 200838567 - Canada) was subjected to filtration and extrusion to obtain a single lipid bilayer liposome. • Next, doxorubicin coating was carried out, and the ratio of phospholipid per Ipmole was 14 (the proportion of doxorubicin in ^§) The pre-formulated doxorubicin stock (10 mg/mL) was added to the liposome suspension and allowed to react at 60 ° C, 100 rpm for 30 minutes. Immediately after the reaction was completed, it was cooled in an ice water bath, and then coated with doxorubicin. The microlipid suspension was passed through a Sephadex G50 gel filtration column with 0. 9 % NaCl as the extract to remove the uncoated doxorubicin. The microlipid suspension was collected through the column and then subjected to an ultra-high speed centrifuge. Centrifuge at 150,000 xg for 90 minutes to remove most of the supernatant Retain a small amount of supernatant and suspend the micro-lipids of Shen Dian again. Filter the micro-lipid suspension with 0·22μιη filter to obtain the final product, and load it into the B bottle for chemical treatment drug analysis. (1) Use N4 The Phis particle size analyzer measured the normal distribution of the average particle size of the micro-lipids at 75-95 nm. (2) The microspheres coated with doxorubicin were measured by Spectrofluorometer (JASCO, FP6200) at 475 nm excitation light and 580 nm emission light. The concentration is 2 mg / ml. Example 3: radiotherapy drug and chemotherapeutic drug combination nanometer standard drug preparation C bottle of radioisotope chain -188 solution is added to the A bottle (BMEDA, SnCl2, φ Gluconate-acetate), The mixture was shaken in a constant temperature water bath at 80 ° C for one hour. After cooling, the labeled 铢-188-BMEDA was added to the B bottle (Lip〇_DXR) and placed in a 60 ° C water bath for 30 minutes. After completion of the 铢-188-BMEDA coating into the Lip〇-DXR, purify with PD-10 column. First, balance the PD-10 column with 20 mL of normal saline (Normal Saline), then use physiological saline to sip. Liquid. Each 〇·5 mL receives a fraction of a total of 1 2 fractions, and calculate the activity of each fracti〇n and the activity remaining in c〇lmnn. Lip〇_DXR is visible in red color due to coating of doxorubicin, while the red color is concentrated in the 6th to 9th fracti〇n, and the activity of these fractions is also high, and the coating efficiency is 40 - 60%. 9 200838567 Example 4: 铢-188_8]\1£0人-1^〇8〇111©1111«*〇_8?£0^ Contrast and image quantitative analysis anesthetized mice with 3% isoflurane (in 100% oxygen) Thereafter, the g-25 Column purified 铢-188-BMEDA-Liposome was injected into the mice by IV injection, 1 hour, 4 hours, 24 hours, 48 hours, 72 hours after the injection. The rats were anesthetized with 3% isoflurane (in 100% oxygen), and the rats were anesthetized with 3% is〇flurane (in 1%% oxygen) during the angiography; the rats were straightened and fixed on the animal table to make use of low Energy, high-resolution collimator for micrc-SPECT imaging, energy window setting 155±10% keV ' Image size is set to 64 x 64, ROR: 1·〇cm, FOV: 1.37 _ cm. In addition, for the subsequent quantitative analysis of the image, the 铢-188-reference source micro-SPECT/CT angiography with known radioactivity was performed at each of the above times; the image analysis results were based on standard uptake value (SUV). ) Presentation. Circle the region of the tumor tissue ROI (region 〇f interest), calculate how much radioactivity (//Ci/g) per gram of tumor tissue based on the reference source, and bring the following formula to calculate the tumor tissue standard. Absorption value (SUV): (measured activity concentration (μ€ί/δ) / [Injected Dose (μ〇ί) / body weight (g)] From the brother's picture, you can see the chain _188-BMEDA_Lip〇some injection for 24 hours. After the right hind leg swelling φ tumor position has obvious absorption, until 72 hours still have obvious images; miciO-SPECT imaging 虿 analysis results show that the amount of tumor tissue absorption 24 hours after injection (Standardised tum〇r uptake value SUV) up to 2.81 soil 0.36. Example 5: Nano-target combination drug 铢 _188_BMEDA/DXR-Lip〇s〇me tumor model and efficacy evaluation (tail vein injection) 2xl〇5 cells/5〇μΐ C26 colorectal tumor cells were inoculated subcutaneously into tumor cells in the right hind leg of 6-week-old BALB/c mice. After about one week of growth, the tumor size was about 50 mm3 ~ 100 mm3. Animal experiments were performed. Divided into the following 4 groups, each group of eight 200838567 188-BMEDAA) XR-Lip〇s〇me > 188-BMEDA- Liposome 'DXR-Liposome - and Normal Saline. The course of treatment is as follows: 1. The first group: 铢-188-BMEDA/DXR-LiP〇some was injected from the tail vein, containing 500//Ci radioactivity and 2 mg/Kg of Doxombicin (DXR), which were administered three times. The dose was the same every time, the second administration was performed three days after the first administration, and the third administration was performed seven days after the second administration. 2. The second group: 铢_188_bmeda_ Liposome with 500/Ci radioactivity was injected from the tail vein, and the dose was the same twice. The dose was the same every time. The interval was three days after the first dose. For the second administration, a third administration was performed seven days after the second administration. ® 3· The third group: DXR-LiP〇some was administered by tail vein injection, and the concentration of Doxorubicin was 2 mg/Kg, which was administered twice, each dose was the same, and the interval was three days after the first administration. For the second administration, a third administration was performed seven days after the second administration. 4·Testar 4: Normal Saline was given by tail vein injection for a total of three injections. The second injection was performed three days after the first injection, and the third injection was performed seven days after the second administration. During the study, tumor measurements were performed twice a week using a digital caliper, and survival rates were recorded for each sputum. As shown in the second figure (Note: the schema data represents the average positive and negative values), on the 27th day after the injection, #only injection of Normal Saline for the drug-treated mice, the tumor volume rapidly increased to 2220.60 soil 431.35 mm3, administered 铢-188 -BMEDA/DXR_LiP〇some treatment of mice with the smallest tumor volume ^ (^. 々 腿 leg ^ followed by the application of 铢 铢 邱 邱 邱 丄 丄 咖 咖 298 298 298 298 298 298 298 298 298 298 298 298 298 298 298 298 298 298 298 298 298 DX DX DX DX DX DX DX DX DX DX DX DX DX Treated mice (9172 gentlemen 17759 mm3). As shown in the third figure, only the rats injected with Normal Saline for drug treatment died on the 48th day after the injection; on the 5th day after the injection, the chain was administered - 188-BMEDA/DXR-Liposome-treated mice had the highest survival rate of 87.5%, followed by sputum-lSS-BMEDA/Liposome-treated mice (62.5%) and DXR-LiP〇 S〇me-treated mice (37.5%). The above results demonstrate the addition and effectiveness of nano-targeted radiation and 11 200838567 chemical combination of double-acting drugs for tumor cell therapy. Example 6·Nylon Target radiation and chemical double-effect drug 铢-188-BMEDA/ DXR-Liposome malignant tumor Mode and efficacy evaluation test (intraperitoneal injection) C26 cell line (colon carcinoma) 2 X 1〇5 cells/500 μί PBS was inoculated intraperitoneally into six-week-old BALB/c mice. After tumor growth for 1 day It can be seen that the abdomen is swollen, indicating that the tumor cells spread in the abdominal cavity and the ascites is produced, and the BALB/c mice with similar body weight are taken for the efficacy evaluation test. The BALB/c mice inoculated with C26 tumor cells in the peritoneal cavity are divided into chains-188-BMEDA _ Liposome , Lipo-DXR, 铢-188-BMEDA/DXR-Liposome and normal saline (Normal Saline) in groups of 8 to 10 BALB/cmice. The 铢-188-BMEDALiposome group was given 200 μί (400) by intraperitoneal injection. ~600 μ(:〇铢_188-BMEDA_Liposome, Lipo_DXR group was given 200 pL (5 mg/kg doxorubicin) by intraperitoneal injection »〇\11'铢-188"6]\^〇^/〇 }〇1-1^〇8〇11^ group was given intraperitoneal injection of 20(^[(400 ～600 μα, 5 mg/kg doxorubicin) chain-188· BMEDA/DXR-Liposome, normal saline (Normal) The Saline group was given 200 μ]: physiological saline by intraperitoneal injection. The four groups of mice monitored and recorded their body weight and survival rate daily. Daily monitoring and recording of mouse deaths or φ survived after 120 days, ie, the recording was stopped and the surviving mice were dissected to see if there were residual tumors (tumornodules). The following table shows that the mediansurvivaltime 铢-188-BMEDA-Liposome group is 21 days, the Lip〇-DXR group is 18 days, and the 铢-188-BMEDA/DXR-Liposome group is 27 days, and it is significantly more significant than the Normal saline group. The difference was (P value < 0.05) and the Normal saline group was 17.67 days.
Group Median survival time P value (d) (Compare with NS) Re-188-BMEDA 21 0.8715 liposomes Lipo-DXR 18 0.7558 12 200838567Group Median survival time P value (d) (Compare with NS) Re-188-BMEDA 21 0.8715 liposomes Lipo-DXR 18 0.7558 12 200838567
Re-188-BMEDA/ DXR-liposomes 27 0.0014 Normal saline 17.67 P values were determined by used of log-rank test.Re-188-BMEDA/ DXR-liposomes 27 0.0014 Normal saline 17.67 P values were determined by used of log-rank test.
The level of statistical significance was set at a P value of <0.05. 由第四圖可以看出施打放射治療與化學治療藥物（銖 DXR-LipOS〇me)老鼠在12〇天後，還有10%存活，宜^ m田- ，、他二組老鼠均死亡。 此結果顯示了奈米標靶放射與化學組合式雙效藥物 加成與有效性 a二、性腫瘤腹水治療之 , 【圖式簡單說明】 小時後之造影結果。 弟一圖係銖-lSB-BMEDA-Liposome 注射 24 第二圖係腫瘤生長曲線結果。 第三圖係尾靜脈注射之腫瘤治療生存率結果。 第四圖係腹腔注射之腫瘤治療生存率結果。 【主要元件符號說明】 Μ. 13The level of statistical significance was set at a P value of <0.05. From the fourth figure, it can be seen that the radiation therapy and chemotherapeutic drugs (铢DXR-LipOS〇me) mice are still 10% after 12 days. Survival, should be ^ m Tian -, and his two groups of mice died. This result shows the addition and effectiveness of nano-targeted radiation and chemical combination double-effect drugs. A. Sexual tumor ascites treatment, [Simplified illustration] The angiographic results after hours. The first picture is the 肿瘤-lSB-BMEDA-Liposome injection 24 The second picture shows the tumor growth curve results. The third panel is the tumor treatment survival rate of the tail vein injection. The fourth panel is the result of tumor treatment survival rate by intraperitoneal injection. [Main component symbol description] Μ. 13