TW201021801A - A pharmaceutical composition for the treatment of cancers - Google Patents

Abstract

Disclosed herein is a pharmaceutical composition for treating cancers, comprising a synergistic effective amount of an O6-Methylguanine DNA methyltransferase (MGMT) inhibitor and an alkylating antineoplastic agent, wherein a combination index of the pharmaceutical composition is less than 1. IN one example, the MGMT inhibitor is n-Butulidenephthalide and the alkylating antineoplastic agent is carmustine (BCNU). The pharmaceutical composition is particularly useful for the treatment of human solid cancers, such as liver cancers, lung cancers, brain tumors, ovarian cancers, and colorectal cancers.

Description

201021801 九、發明說明： 【發明所屬之技術領域】 本發明係關於一種用於治療癌症之醫藥組合物，且 特別關於一種包含有人類06-甲基鳥嘌呤-DNA甲基轉移 酶（MGMT)抑制劑及一烷化類抗癌藥物的醫藥組合物，其 可增強治療人類實質癌，如肺癌、肝癌、腦瘤、印巢癌 及大腸直腸癌等的效果，而不會增加毒性。 ®【先前技術】 肝癌是世界上最常見的惡性腫瘤之一，目前是國人 十大死亡原因的第一位，在西方以開發國家中，每十萬 人約有1-4人罹患肝癌，而在中國大陸、台灣和韓國等亞 洲地區則每十萬人約有 20-150、（Dominguez-Malagon H ei α/.，ραί/ιο/ 2001，25:497-516)。在疾病早 期發現時以手術切除是唯一可能提供治療的希望，然而 ❹ 由於肝癌病患多半合併嚴重的肝硬化、腹水、肝腫瘤過 大或是肝癌轉移至肝外等症狀，因此只有9_27%的患 者是合施以手術，當病患發生轉移現象時，全身性化學 療法（化療）是唯一的治療選擇。最常使用之傳統化療 藥物5-FU在細胞作用的主要機制，是經由存在腫瘤細 胞的酵素胸線嘧啶磷酸酶（thymidine phosphorylase，ΤΡ) 活化成具有具有抗癌活性性的5-氟-2’-去氧尿密啶-5，-磷 酸鹽（5-fluoro_2，_deoxyuridine-5，-phosphate，FDUMP)， 再和胸腺嚷。定合成酶（Thymidine synthetase，TS)結合後 201021801 抑制了體内去氧核糖單磷酸胸腺嘧啶d_TMp的產生， 以抑制DNA的合成。雖然5_FU對癌組織之親和性比 正常組織高，以及副作用比較低，可是肝癌病患大多伴 隨程度不等之肝硬化，對於抗癌藥物代謝的能力較差， 所以較容易因藥物在體内堆積而產生毒性。 另外’成人的惡性腦瘤約占成人惡性腫瘤的2 %， 雖然比例很低，但是因為腫瘤生長的部位，可說是我們 的中樞指揮中心，所以腦瘤在腦部所引起的臨床症狀， 響會對病患造成嚴重的|果，且惡性腦瘤的病人癒後非常 不好，平均存活率不到一年，在沒有治療的情況下，平 均活不到半年（資料來源：陶聲洋防癌基金會， http://www.sydao.org.tw /cure/cancer)。stupp 等人於 ΙΧ1ίη.〇η〇.，20 (5) :1375 _ 1382 (2〇〇2)中提到腦瘤約 涵蓋所有惡性腫瘤疾病之2% ^然而，經描述每 100,000人中有5人之發生率，在美國每年約診斷出 參超過）7,000之案例，其約有13，_和死亡有關。在成人 中，Stupp等人提出，最常見之組織為第3級之退行性 星狀細胞和第4級之多形性膠質母細胞瘤（"GBM")。根 據Stupp等人所提出，惡性神經膠質瘤之標準治療管理 涉及經由手術切除之細胞減量，如可行，接著利用輕射 冶療（RT) ’並g&合或沒有配合輔助性化學療法。然而， Stupp等人提到，儘管利用該各種專業之方法，罹患 GBM之病人的預後情況仍然不佳。研究報告指出， GBM之平均存活率典型上在9 i 12個月之範圍，2 201021801 年存活率則僅在8%至12%之範圍。亞硝基脲類 (Nitrosoureas)為用於治療惡性腦瘤、肝癌、淋巴癌、 原發性中樞神經系統腫瘤等之主要化學治療藥物，歸麵 為烷化類抗癌藥物。主要是利用與DNA結合，在結構 上扭曲DNA雙股結構以防止DNA轉錄和轉譯而造成細 胞死亡，在正常細胞中，院,化類抗癌藥物之損害作用可 利用像Ο 6 _曱基烏嘌呤_ DNA曱基轉移酶 等酵素將之移除。儘管在美國經常會開立處方，然而單 一藥劑卡莫司汀（carmustine ; BCNU)或洛莫司、; (lomustine)或組合配方配方丙卡巴（pr〇carbazine)、洛 莫司汀（lomustine)和長春新驗（Vincristine)的協同作 用從未得到最終之證實。由上述得知單一藥物或臨床藥 物的合併使用對於肝癌及腦瘤的治療效果有限，因此找 尋合適的化合物進行合併療法 (combination chemotherapy)是目前臨床試驗致力的方向。此法除了 減少腫瘤對抗癌藥物之抗藥性，同時也期避免不良反應 產生的加成作用。 近年來的研究結果發現，甲基化（methylation)是 重要的上位基因現象（epigenetic phenomena)，通常會導 致基因沉寂 （gene silence)現象。腫瘤的發生與多基因 的異常有關，其中 DNA曱基化是基因表達調控的一種 方式’其曱基化狀態的改變與基因的異常表達相關。肝 癌的發生常是多因素、多階段、多基因異常累積的過 程’且肝臟細胞是最容易受到烷化類抗癌藥物的傷害而 201021801201021801 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD The present invention relates to a pharmaceutical composition for treating cancer, and in particular to a method comprising human 06-methylguanine-DNA methyltransferase (MGMT) inhibition And a pharmaceutical composition of an alkylating anticancer drug, which can enhance the treatment of human parenchymal cancer, such as lung cancer, liver cancer, brain tumor, Indian cancer and colorectal cancer, without increasing toxicity. ® [Prior Art] Liver cancer is one of the most common malignant tumors in the world. It is currently the number one cause of death among the top ten people in the country. In the developing countries of the West, about 1-4 people per 100,000 people suffer from liver cancer. In Asia, such as China, Taiwan, and South Korea, there are about 20-150 per 100,000 people (Dominguez-Malagon H ei α/., ραί/ιο/ 2001, 25: 497-516). Surgical resection is the only possible treatment for early detection of the disease, however, because most patients with liver cancer have severe cirrhosis, ascites, liver tumors, or liver cancer metastasis to the liver, only 9-27% of patients It is a combination of surgery, when the patient metastasis, systemic chemotherapy (chemotherapy) is the only treatment option. The main mechanism of the most commonly used traditional chemotherapy drug 5-FU in cell action is activation of 5-fluoro-2 - Deoxyuridine-5,-phosphate (5-fluoro_2, _deoxyuridine-5,-phosphate, FDUMP), and thymus gland. After the binding of Thymidine synthetase (TS), 201021801 inhibited the production of deoxyribose monophosphate thymidine d_TMp in vivo to inhibit DNA synthesis. Although the affinity of 5_FU for cancer tissues is higher than that of normal tissues, and the side effects are relatively low, most patients with liver cancer are accompanied by cirrhosis of varying degrees, and the ability to metabolize anticancer drugs is poor, so it is easier to accumulate drugs in the body. Produces toxicity. In addition, 'malignant brain tumors of adults account for about 2% of adult malignant tumors. Although the proportion is very low, because the site of tumor growth can be said to be our central command center, the clinical symptoms caused by brain tumors in the brain are ringing. It will cause serious consequences for patients, and the patients with malignant brain tumors are very bad afterwards. The average survival rate is less than one year. In the absence of treatment, the average survival rate is less than half a year. (Source: Tao Shengyang Cancer Cancer Fund Yes, http://www.sydao.org.tw /cure/cancer). Stupp et al., ΙΧ1ίη.〇η〇., 20 (5): 1375 _ 1382 (2〇〇2) mention that brain tumors cover approximately 2% of all malignant tumor diseases. However, it is described that there are 5 out of every 100,000 people. The incidence of human beings is about 7,000 cases per year in the United States, and about 13% of them are related to death. In adults, Stupp et al. suggest that the most common tissues are grade 3 degenerative stellate cells and grade 4 pleomorphic glioblastoma ("GBM"). According to Stupp et al., standard treatment management for malignant glioma involves cell reduction via surgical resection, if feasible, followed by light-radiation therapy (RT)' and g& or with or without adjuvant chemotherapy. However, Stupp et al. mentioned that despite the various professional methods, the prognosis of patients with GBM is still poor. According to the study, the average survival rate of GBM is typically in the range of 9 i 12 months, and the survival rate in 2010 21801 is only in the range of 8% to 12%. Nitrosoureas are the main chemotherapeutic drugs for the treatment of malignant brain tumors, liver cancer, lymphoma, primary central nervous system tumors, etc., and are classified as alkylating anticancer drugs. Mainly by using DNA binding, structurally distorting the DNA double-strand structure to prevent DNA transcription and translation and causing cell death. In normal cells, the damage of the anticancer drugs can be utilized like Ο6 _曱基乌嘌呤_DNA thiotransferase and other enzymes remove it. Although prescriptions are often prescribed in the United States, single-agent carmustine (BCNU) or lomomox, (lomustine) or combination formula pr〇carbazine, lomustine and The synergy of Vincristine has never been confirmed. From the above, it is known that the combined use of a single drug or a clinical drug has limited therapeutic effects on liver cancer and brain tumors, so finding a suitable compound for combination chemotherapy is the current direction of clinical trials. In addition to reducing the resistance of cancer anticancer drugs, this method also avoids the additive effect of adverse reactions. Recent studies have found that methylation is an important epigenetic phenomenon that usually leads to gene silence. The occurrence of tumors is associated with abnormalities in multiple genes, of which DNA thiolation is a way of regulating gene expression. The change in its thiolation state is associated with abnormal expression of genes. The occurrence of liver cancer is often a multi-factor, multi-stage, multi-gene abnormal accumulation process and liver cells are most vulnerable to alkylation-based anticancer drugs. 201021801

導致 DNA 的斷裂及複製障礙。在生物體内，Ο6-曱基 鳥嘌吟-DNA甲基轉移酶 （06-Methylguanine DNA methyltransferase, MGMT)負責 DNA烧基化損傷的切 除修復，其功能的喪失將導致 DNA加合物 06-MedG 不能被修復而累積，由於〇6-MedG具有高突變性，在 DNA修復之前如不能及時地切除修復，就可能會有突變 的發生，這些突變如果發生在癌基因或抑癌基因則可能 導致細胞發生癌變。所以，MGMT啟動子高曱基化 (MGMT promoter hypermethylation)而失活可能是肝瘤 形成過程的重要事件 （Huang YC et al., ·/· 〇/ Hepatobiliary Pancreat Surg 1998, 5:355-366;圖一）。 而MGMT基因表達缺陷很少是因為基因的缺失、突變重 排或 mRNA不穩定所致，其CpG島曱基化可能為其轉錄 調節的主要方式，許多腫瘤細胞株缺乏 MGMT，就是因 為該基因啟動子高曱基化而喪失表達功能。目前對於腦 瘤和 MGMT 的表現相關性了解比較透徹，Lausanne 大學醫院的Dr· Monika E. Hegi等人的研究結果發現， 曱基化 MGMT 啟動子與使用烷化類藥物替莫唑胺 (temozolomide; TMZ)的膠質母細胞瘤 （glioma)患者的 生存期延長有關。研究者使用曱基化特異性 PCR技術 (Methylation-specific PCR, MSP)測定了 206個腫瘤標本 MGMT啟動子的曱基化程度。結果發現，有曱基化的 患者中位生存期為 18.2月，而無甲基化的患者中位生 存期為 12.2月。在有甲基化啟動子的 TMZ治療組患 201021801 者中，最長的中位生存期為21.7月。TMZ對MGMT 啟動子未發生甲基化的腫瘤患_者無效[N Engl J Med. 2005, 352:997-1003.]。另外，在 MGMT 轉入肝癌細 胞的實驗中發現，MGMT蛋白質活性的回復會抑制 BCNU等烧化劑類藥物所造成的蛋白質泛素化降解途徑 作用 （Ubiquitin-Proteosome degradation pathway) (Grombacher T et al.，1998，17:845-851)。值得 注意的是’ MGMT蛋白質的過度表現是腫瘤產生抗性的 主要原因之一 ’由於腫瘤抗藥性的產生是目前臨床藥物 普遍反應率不佳的主要原因，因此利用找到特定化合物 抑制MGMT的表現’可能有助於臨床藥物的合併治 療。 【發明内容】Causes DNA breakage and replication disorders. In vivo, -6-Methylguanine DNA methyltransferase (MGMT) is responsible for the excision repair of DNA alkylation damage, and its loss of function will lead to DNA adduct 06-MedG Can not be repaired and accumulated, because 〇6-MedG has high mutation, if it can not be repaired in time before DNA repair, there may be mutations, these mutations may lead to cells if they occur in oncogenes or tumor suppressor genes Carcinogenesis occurs. Therefore, inactivation of MGMT promoter hypermethylation may be an important event in the formation of hepatoma (Huang YC et al., ·/· 〇/ Hepatobiliary Pancreat Surg 1998, 5:355-366; Figure 1) . However, MGMT gene expression defects are rarely caused by gene deletion, mutation rearrangement or mRNA instability, and its CpG island thiolation may be the main mode of transcriptional regulation. Many tumor cell lines lack MGMT because the gene is activated. The sorghum is basalized and loses its expression function. At present, the correlation between brain tumor and MGMT is well understood. Dr. Monika E. Hegi et al. at Lausanne University Hospital found that the thiolated MGMT promoter is linked to the alkylating drug temozolomide (TMZ). Prolonged survival in patients with glioblastoma (glioma). The researchers used Methylation-specific PCR (MSP) to determine the degree of thiolation of the MGMT promoter in 206 tumor specimens. The median survival of patients with thiolation was 18.2 months, while the median survival of patients without methylation was 12.2 months. Among the patients with the methylated promoter in the TMZ treatment group with 201021801, the longest median survival was 21.7 months. TMZ is ineffective against tumor patients whose MGMT promoter has not been methylated [N Engl J Med. 2005, 352:997-1003.]. In addition, in the experiment of transferring MGMT into liver cancer cells, it was found that the recovery of MGMT protein activity inhibits the Ubiquitin-Proteosome degradation pathway (Grombacher T et al.) caused by CNTNU and other burning agents. 1998, 17: 845-851). It is worth noting that 'overexpression of MGMT protein is one of the main causes of tumor resistance'. Because the development of tumor resistance is the main reason for the poor general response rate of clinical drugs, it is necessary to find specific compounds to inhibit the performance of MGMT. May contribute to the combined treatment of clinical drugs. [Summary of the Invention]

本申請案發明人發現，將人類〇6_甲基鳥嘌呤_DNA 甲基轉移酶的抑制劑與及烷化類抗癌藥物合併使用時， 可以協同地作用而達到增強的癌症治療效果，且不會增 加毒性。 因此，本發明之主要目的在於提供一種用於治療癌 症’例如較佳為肝癌、肺癌、腦瘤、卵巢癌及大腸直腸 癌等之醫藥組合物，其包含一協同有效量之人類Ο6甲基 鳥嘌呤-DNA甲基轉移酶（MGMT)抑制劑和一烷化類抗癌 藥物；以及一藥學上可接受的載體，其中該MGMT抑制 劑與該貌化類抗癌藥物的濃度比在約U至約1:8間且該醫 201021801 藥組合物具有一小於1的組合指數（c〇mbinati〇n in(jex， CI)。 在一實例中’當該MGMT抑制劑與該烷化類抗癌藥物 的瀵度比為約1:2時，該組合指數在約〇 3至約0.35間。在另 一實例令，當該MGMT抑制劑與該烷化類抗癌藥物的濃度 比為約1:4時，該組合指數約為0.22。在另一實例中，當該 MGMT抑制劑與該烷化類抗癌藥物的濃度比為約1:8時，該 組合指數在約〇.〇6至約〇.〇8間。 在一實例中，該MGMT抑制劑為正亞丁基苯酚且該烷 化類抗癌藥物為卡莫司丨丁。 在另一實施方式中’該MGMT抑制劑與該烷化類抗癌 藥物是同時或依序經由靜脈注射而施用至活體内至少5 天，該MGMT抑制劑係以每天1〇〇毫克/公斤至每天5〇〇毫克/ △斤間的量施用；該烷化類抗癌藥物則是以每天10毫克/公 斤至每天100毫克/公斤的量施用。 【實施方式】 如上过：本發明目地在組合使用目前臨床上已經在 使用的MGMT抑制劑和烧化類抗癌藥㊈，以提供協同性 的癌症治療效果，且不會增加毒性。 因此，以下姐_ _ 词不一種用於治療癌症的醫藥組合物， ^ 協同有政量之—人類06-曱基鳥嘌呤-DNA曱基轉移 酶（MGMT)抑制劑和—烷化類抗癌藥物；以及一藥學上可 接受的載體；复 、宁該MGMT抑制劑與該烷化類抗癌藥物的 201021801 濃度比在約1:2至約1:8間，且該醫藥組合物具有一小於1的 組合指數（combination index, CI)。 可用於本發明之MGMT抑制劑可為此技藝中任何已知 衍生自葡萄糖的MGMT抑制劑，包括，但不限於，Ο6-苄 基鳥嗓吟（06-benzylguanine, 06-BG)、8-偶氮-Ο6-节基鳥 嗓0令（8-aza-06-benzylguanine，8-aza-BG)、06-(4-漠曱基） 鳥嗓吟（06-(4-bromothenyl)guanine，4-BTG)，以及另一非 φ 衍生自葡萄糖的化合物，如正亞丁基苯酚。 在一較佳實施方式中，可用於本發明之該MGMT抑制 劑為正亞丁基苯酚，其自然界中包含 Z構型或 E構 型之雜環化合物，其係揭示於Harn等人之美國及台灣專 利申請案中（歐盟專利案號·· 0540165.5;美國案號： US/08.10.04/USP 616636;台灣公告號：1298259 及 200616656)，具有以下之化學結構式： n-Butylidenephthalide ©(BP; BdPh, a natural compound, MW: 188.23)The inventors of the present application found that when an inhibitor of human 〇6-methylguanine_DNA methyltransferase is used in combination with an alkylating anticancer drug, synergistic action can be achieved to achieve an enhanced cancer therapeutic effect, and Does not increase toxicity. Accordingly, it is a primary object of the present invention to provide a pharmaceutical composition for treating cancer, such as preferably liver cancer, lung cancer, brain tumor, ovarian cancer, colorectal cancer, etc., comprising a synergistically effective amount of human Ο6 methyl bird a 嘌呤-DNA methyltransferase (MGMT) inhibitor and an alkylating anticancer drug; and a pharmaceutically acceptable carrier, wherein the concentration ratio of the MGMT inhibitor to the profiling anticancer drug is about U to Approximately 1:8 and the medical 201021801 pharmaceutical composition has a combination index of less than 1 (c〇mbinati〇n in (jex, CI). In one example 'when the MGMT inhibitor and the alkylating anticancer drug When the twist ratio is about 1:2, the combination index is between about 3 and about 0.35. In another example, when the concentration ratio of the MGMT inhibitor to the alkylating anticancer drug is about 1:4 The combination index is about 0.22. In another example, when the concentration ratio of the MGMT inhibitor to the alkylating anticancer drug is about 1:8, the combination index is about 〇.〇6 to about 〇. In one example, the MGMT inhibitor is n-butylene phenol and the alkylating anticancer drug is In another embodiment, the MGMT inhibitor is administered to the living body at least 5 days simultaneously or sequentially via intravenous injection, and the MGMT inhibitor is administered once a day. The amount is from 〇mg/kg to 5〇〇mg/△kg per day; the alkylating anticancer drug is administered in an amount of 10 mg/kg per day to 100 mg/kg per day. The present invention aims to provide a synergistic cancer therapeutic effect without using a MGMT inhibitor and a burnt-out anticancer drug 9 which are currently in clinical use, and thus does not increase toxicity. Therefore, the following sister _ _ word is not used for a pharmaceutical composition for treating cancer, ^ synergistically administrated - human 06-mercaptoguanine-DNA thiol transferase (MGMT) inhibitor and - alkylating anticancer drug; and a pharmaceutically acceptable carrier; The concentration ratio of the MGMT inhibitor to the alkylating anticancer drug of 201021801 is between about 1:2 and about 1:8, and the pharmaceutical composition has a combination index (CI) of less than 1. MGMT inhibitors useful in the present invention Any MGMT inhibitor known to be derived from glucose in the art, including, but not limited to, 6-benzylguanine (06-BG), 8-azo-purine 6-membered guanine 0 Order (8-aza-06-benzylguanine, 8-aza-BG), 06-(4-momothenyl)guanine (4-BTG), and another non-φ derivative a compound derived from glucose, such as n-butylene phenol. In a preferred embodiment, the MGMT inhibitor useful in the present invention is n-butylene phenol, which naturally contains a heterocyclic compound in a Z configuration or an E configuration. It is disclosed in the US and Taiwan patent applications of Harn et al. (EU Patent Case No. 0540165.5; US Case No.: US/08.10.04/USP 616636; Taiwan Bulletin No.: 1298259 and 200616656), having the following chemistry Structural formula: n-Butylidenephthalide ©(BP; BdPh, a natural compound, MW: 188.23)

正亞丁基苯酚對於腦瘤及肝癌具有抗腫瘤活性，經 由造成細胞凋亡 （apoptosis)達到抑制細胞株生長的情 形。以皮下方式給藥，其對人類惡性腦瘤及肝癌細胞之 11 201021801 動物裸鼠異位移植模式具有明顯的腫瘤抑制效果，且觀 察給藥後生化數值及組織病理切片，不會有骨髓抑制或 其他器官的損傷，這可能是由於正亞丁基笨酚對人類肝 癌、肺癌、腦瘤、卵巢癌及大腸直腸癌產生高度專一性 (high specificity)的毒殺能力所致。 適用於本發明之正亞丁基苯酚可包含z構型或E 構型之雜環化合物，化合物的所有異構形式都包括在本 φ發明中。本發明之一具體例中，正—亞丁基苯酞之E —及Z —幾何異構物可以使用管柱層析加以單離且使 用HPLC及NMR加以特徵化，亦可利用有機方式合 成’或選自超臨界萃取所得。 在本發明中，可與正亞丁基苯酚結合使用於抗癌療 法中之抗癌藥物選擇目前臨床上已經在使用且具有價值 的藥物’主要為造成DNA曱基化的烷化類抗癌藥物。 由此所製成的醫藥組合物係為有效之抗癌藥物，可以協 ❹ 同地作用以有效抑制腫瘤細胞生長，具有增強的抗腫瘤 效果’且能降低抗腫瘤所產生之副作用。 適用於本發明中之烷化類抗癌藥物可為技藝中任何 已知適合用於治療癌症之烧化藥物，包括，但不限於， 卡莫司汀、洛莫司汀、替莫唾胺，或其水合物、同電子 排列體（isosteres)及其醫藥上可接受之鹽類。 在一較佳實施方式中，該烷化類抗癌藥物為卡莫司 汀。 12 201021801 此處所謂之「抗癌療法」係指用 癌（solid tumor) ’包括肝癌、肺癌、聪才 實質 斶瘤、卵巢痣 , 直腸癌、黑色素瘤、乳癌、攝護腺癌、胃癌 、大腸 腎臟癌及中樞神經系統腫瘤之所有類型療法胰臟癌' 據本發明之醫藥組合物使用於上述抗癌療、亦可將根 之較佳實施態樣中，醫藥組合物可以用认法在本發明 ή ^ 以用於治療肝癌、腦 瘤、肺癌，特別是非小細胞肺癌，包杠^ & _ I栝初期、臨床藥物 _ 治療無效及已經轉移之肺癌，或是對卡 、』汀或替莫0^ 胺具有抗藥性的腦癌及肝癌。 此處所謂之「增強抗腫瘤效果」係指於女Μ 」诹钿鲍抑制腫瘤生 長，或是完全使腫瘤消退。此處所謂之r降 v低抗腫瘤所 產生之副作用」係指沒有明顯的生理毒性或是骨趙抑 制、產生神經毒性等相關現象。 使用本發明之醫藥組合物除含有上述的活性成分 外’可視需要進一步包含此技術領域中具有通常知識者 ❹所熟知之其他組份，例如，但不限於醫藥上可接受之載 體、賦形劑、緩衝液或防腐劑’或其他相關抗癌藥物。 本發明醫藥組合物中的活性成分可以同時、分開或 以任意次序連續方式給藥。在一實例中，MGMT抑制劑 與烷化類抗癌藥物是連續從第1至5天同時投予。 在一較佳實施方式中，當該MGMT抑制劑與該烷化類 抗癌藥物的濃度比為約1:2時，該組合指數在約0.3至約0.35 間。在另一較隹實施方式中，當該MGMT抑制劑與該烧 化類抗癌藥物的濃度比為約1:4時，該組合指數約為0.22。 13 201021801 在另-較佳實施方式中，當該MGMT抑制劑與該烷化類 抗癌藥物的濃度比為約1:8時，該組合指數在約〇〇6至約 0.08 間。 此處所謂之「給藥」或「投予」係指醫藥組合物中 的活性成分可經由醫學上認定之任何方式投予病患，例 如以口服、非腸道、鼻腔或局部給藥等。給藥的實際較 佳方法及次序需根據活性成分之特殊配方的利用情況、 接受治療的遽瘤移植模型及患者的個別差異而調整。 本發明醫藥組合物的形式可配成如口服的懸浮片、 錠劑、膠囊、糖錠、乳劑、粉末、溶液等。非腸道給藥 包括經由皮下、靜脈内或肌肉注射而投予組成成分，例 如將活性成分溶於生理可接受的稀釋液中製成可注射的 形式’再加上藥用載體’如水、生理食鹽水、水性糊精 或相關糖溶液、乙醇、聚乙埽甘油4〇〇或油類等。藥物也 可製成鼻腔吸入劑或懸浮用藥經由皮膚或黏膜吸收。 在本發明之具體實施態樣中，經由注射方式給藥可 以精確地控制給藥的時間及劑量，是較佳的給藥途徑。 本發明醫藥組合物可由此技術領域中具有通常知識者所 慣用的方法或其改良加以配製，並無特殊限制，可依一 般的方法混合調配而成’且各組份的添加順序可以任奄 改變。 ^ 在本發明之一則具體實施態樣中，醫藥組合物係經 配方成’例如’可將適當劑量之正亞丁基苯酚預溶於— 溶劑，如二甲亞砜（DMSO)中，搭配適當比例之助溶劑， 14 201021801 如氫化t麻油聚氧乙烯（cremophor EL)經由稀釋劑，如 生理食鹽水稀釋成適當劑量後成為皮下注射用藥。 醫藥組合物中活性成_分的用量及給藥頻率會根據接 受治療的患者的嚴重度及個別差異有所不同，如活性& 分組合後的代謝穩定度及作用長度、患者年齡、體重、 健康狀況、性別、飲食、給藥途徑、給藥時間、***速 度及賦形劑等因素，需由此技術領域中具有通常知識者 做決定》 在本發明一實例中，正亞丁基苯齡的給藥方式，— 般是以介於每天100毫克/公斤至每天500毫克/公斤 間的治療有效量，經由靜脈注射方式給予，若藥物合併 治療時’正亞丁基苯酚較佳自約50毫克/公斤會有效抑 制腫瘤。 在本發明中烷化基藥的給藥方式，一般是以介於每 天毫克/公斤至每天100毫克/公斤間的治療有效 量，經由靜脈注射方式給予，若藥物合併治療時，卡莫 司江較佳自約4毫克/公斤會有效抑制腫瘤。 以下實施例係用於對本發明作進一步說明，唯非用 以限制本發明之範圍。任何熟悉此項技藝之人士可輕易 達成之修飾及改變均包括於本案說明書揭示内容及所附 申請專利範圍之範圍内。 實施例 下列實施例係敘述以已知技術來分析本發明醫藥組 15 201021801 制能力及結果， 為所屬技術領域 σ物對肺癌、肝癌及腦瘤細胞生長的抑 所涉與此分析方法相關的方法和步驟均 中具有通常知識者所知悉者。 以治療HepG2、J5肝癌細胞株以裸小鼠的皮下腫 瘤細胞生長抑制的情形作為藥物效用的有效模型，來研 究本發明醫藥組合物之有效性。 赢 MIU：物紐厶公析 9 用MTT細胞存活分析（pavweis等著，】Virol Methods，1988, 20, 309_321)來研究醫藥組合物。醫藥組 合物中活性成分的添加方法是將異柴胡内酯沿％孔 (well)微量孔盤内壁滴入，然後將抗癌藥物連續滴下，每 次分析要用4個微量孔盤，而每種藥物組合要用4個孔。 藥物以兩倍或對半稀釋，第一排放入溶劑做為對照組。 通常每個孔中培養3xl〇3個HepG2及J5肝癌細 φ胞，培養至隔天再加入單獨或不同組合形式的藥物培養2 天’然後將培養液吸出後改加入含5 〇〇毫微克（^g)/毫升 MTT的200微毫升（μ)培養液繼續培養4小時，將培養液吸 出後加入200微毫升二甲亞砜，然後用微量孔盤分光光度 計測量其在5 70奈米（nm)的吸光值。各個藥物組合的 平均值可以异出不同藥物劑量的反在曲線。以接受最高 藥量孔的讀值當作正向控制組（活細胞的生長不受抑 制），第一排只加溶劑的孔讀值當作負向控制組。由此可 得藥物的IC50(抑制一半細胞生長的濃度）。其中，負向 16 201021801 控制組的存活率為100%，致死率（lethal rate)為〇% (100%-存活率=致死率）。N-butylene phenol has antitumor activity against brain tumors and liver cancer, and causes apoptosis to inhibit the growth of cell lines. It is administered subcutaneously, which has obvious tumor suppressive effect on human ectopic transplantation model of human malignant brain tumor and liver cancer cells. The biochemical and histopathological sections after administration are observed, and there is no bone marrow suppression or Other organ damage, probably due to the high specificity of the toxic ability of n-butylene phenol to human liver cancer, lung cancer, brain tumor, ovarian cancer and colorectal cancer. The n-butylene phenol suitable for use in the present invention may comprise a heterocyclic compound in the z configuration or the E configuration, and all isomeric forms of the compound are included in the invention. In one embodiment of the present invention, the E- and Z- geometric isomers of n-butylidene benzoquinone can be isolated by column chromatography and characterized by HPLC and NMR, or can be synthesized organically' or It is selected from supercritical extraction. In the present invention, an anticancer drug which can be used in combination with n-butylene phenol in an anticancer therapy is a drug which is currently clinically useful and valuable, and is mainly an alkylating anticancer drug which causes DNA thiolation. The pharmaceutical composition thus prepared is an effective anticancer drug which can synergistically act to effectively inhibit tumor cell growth, has an enhanced antitumor effect and can reduce side effects caused by antitumor. The alkylating anticancer drug suitable for use in the present invention may be any burning agent known in the art to be suitable for treating cancer, including, but not limited to, carmustine, lomustine, temostanide, or Its hydrates, isosteres and their pharmaceutically acceptable salts. In a preferred embodiment, the alkylating anticancer drug is carmustine. 12 201021801 The term "anti-cancer therapy" as used herein refers to the use of solid tumors including liver cancer, lung cancer, Congcai parenchyma, ovarian fistula, rectal cancer, melanoma, breast cancer, prostate cancer, stomach cancer, large intestine. All types of cancer of the kidney cancer and central nervous system tumors of the pancreatic cancer. The pharmaceutical composition of the present invention is used in the above anti-cancer therapy, and in the preferred embodiment of the root, the pharmaceutical composition can be used in the present invention. Invention ή ^ For the treatment of liver cancer, brain tumors, lung cancer, especially non-small cell lung cancer, including the initial stage, clinical drug _ treatment is ineffective and has metastasized lung cancer, or on the card, 〗 or Moxamine is resistant to brain cancer and liver cancer. The term "enhanced anti-tumor effect" as used herein refers to the inhibition of tumor growth by the female mites, or the complete regression of the tumor. Here, the term "r-lower v-lower anti-tumor side effects" means that there is no obvious physiological toxicity or bone inflammation, neurotoxicity and the like. The pharmaceutical composition of the present invention, in addition to the above-mentioned active ingredient, may further comprise other components well known to those of ordinary skill in the art, such as, but not limited to, pharmaceutically acceptable carriers, excipients. , buffer or preservatives' or other related anticancer drugs. The active ingredients of the pharmaceutical compositions of the invention may be administered simultaneously, separately or in a continuous manner in any order. In one example, the MGMT inhibitor and the alkylating anticancer drug are administered simultaneously from day 1 to day 5. In a preferred embodiment, when the concentration ratio of the MGMT inhibitor to the alkylating anticancer drug is about 1:2, the combination index is between about 0.3 and about 0.35. In another comparative embodiment, when the concentration ratio of the MGMT inhibitor to the burn-in anticancer drug is about 1:4, the combination index is about 0.22. 13 201021801 In another preferred embodiment, when the concentration ratio of the MGMT inhibitor to the alkylating anticancer drug is about 1:8, the combination index is between about 6 and about 0.08. The term "administration" or "administration" as used herein means that the active ingredient in the pharmaceutical composition can be administered to a patient by any means medically recognized, for example, orally, parenterally, nasally or topically. The actual preferred method and sequence of administration will be adjusted based on the particular formulation of the active ingredient, the oncoming tumor transplant model, and individual differences in the patient. The pharmaceutical composition of the present invention may be formulated into a suspension tablet, a tablet, a capsule, a lozenge, an emulsion, a powder, a solution or the like, for example, orally. Parenteral administration includes administration of a component by subcutaneous, intravenous or intramuscular injection, for example, by dissolving the active ingredient in a physiologically acceptable diluent in an injectable form, together with a pharmaceutical carrier such as water, physiological Saline, aqueous dextrin or related sugar solution, ethanol, polyglycerin 4 or oil. The drug can also be made into a nasal inhalation or suspension drug absorbed through the skin or mucous membranes. In a specific embodiment of the present invention, administration by injection can precisely control the timing and dosage of administration, and is a preferred route of administration. The pharmaceutical composition of the present invention can be formulated by a method conventionally used by those skilled in the art or a modification thereof, and is not particularly limited, and can be mixed and prepared according to a general method, and the order of addition of each component can be changed. . ^ In a specific embodiment of the present invention, the pharmaceutical composition is formulated as 'for example' to pre-dissolve an appropriate amount of n-butylene phenol in a solvent such as dimethyl sulfoxide (DMSO) in an appropriate ratio. Cosolvent, 14 201021801 If the hydrogenated sesame oil cremophor EL is diluted to a suitable dose via a diluent such as physiological saline, it becomes a subcutaneous injection. The amount of active ingredient in the pharmaceutical composition and the frequency of administration will vary depending on the severity and individual differences of the patient being treated, such as the activity & the metabolic stability and length of the combination, the age and weight of the patient, Factors such as health status, gender, diet, route of administration, time of administration, rate of excretion, and excipients are determined by those of ordinary skill in the art. In an example of the present invention, n-butylene benzoate The mode of administration, generally, is administered intravenously at a therapeutically effective amount between 100 mg/kg per day to 500 mg/kg per day. If the drug is combined, 'n-butylene phenol is preferably from about 50 mg/ Kilograms will effectively inhibit tumors. In the present invention, the mode of administration of the alkylating base drug is generally administered by intravenous injection in a therapeutically effective amount between mg/kg per day to 100 mg/kg per day, and if the drug is combined, the Camus River is treated. Preferably, about 4 mg/kg will effectively inhibit the tumor. The following examples are intended to further illustrate the invention and are not intended to limit the scope of the invention. Modifications and variations that may be readily made by those skilled in the art are included within the scope of the disclosure and the scope of the appended claims. EXAMPLES The following examples describe the ability and results of the pharmaceutical group 15 201021801 of the present invention to be analyzed by known techniques, and the method for analyzing the growth of lung cancer, liver cancer and brain tumor cells in the prior art is related to the method of analysis. And the steps are all known to those who have the usual knowledge. The effectiveness of the pharmaceutical composition of the present invention was investigated by treating HepG2 and J5 hepatoma cell lines as an effective model for drug efficacy in the inhibition of subcutaneous tumor cell growth in nude mice. Win MIU: 厶 厶 9 9 The pharmaceutical composition was studied using MTT cell survival assay (pavweis et al., Virol Methods, 1988, 20, 309_321). The active ingredient in the pharmaceutical composition is added by dropping isobutadione along the inner wall of the microwell plate, and then continuously dropping the anticancer drug, and using 4 microplates for each analysis, and each Four wells are used for the drug combination. The drug was diluted twice or half, and the first was discharged into the solvent as a control group. Usually, 3xl〇3 HepG2 and J5 liver cancer fine φ cells were cultured in each well, cultured until the next day, and the drug was cultured for 2 days alone or in different combinations. Then the culture solution was aspirated and then added to contain 5 〇〇 nanograms ( The culture medium of ^g)/ml MTT was further cultured for 4 hours, the culture solution was aspirated, and 200 μml of dimethyl sulfoxide was added, and then it was measured at 5 70 nm by a microplate spectrophotometer ( The absorbance of nm). The average of each drug combination can be different from the inverse of the different drug doses. The reading of the highest dose well was used as the positive control group (the growth of living cells was not inhibited), and the first row of solvent-only well readings was used as the negative control group. Thus, the IC50 of the drug (concentration for inhibiting the growth of half of the cells) can be obtained. Among them, the negative 16 201021801 control group survival rate was 100%, and the lethal rate was 〇% (100% - survival rate = lethal rate).

Calcug.xp軟體法分析醫藥組合物之協同作用 本申請案中主要是利用CalcuSyn軟體來繪製藥物劑 量反應曲線，將各藥物組合的IC50，不論是單獨或以固 定濃度的其他藥物組合的數據，都計算並紀錄在等權圖 上。CalcuSyn軟體主要應用在癌症或愛滋病的混合藥物 #治療時之協同作用或拮抗作用的分析，其能執行多種藥 物之劑量效果（dose-effect)的演算運用，而這項運算方法 係由T-C Chou和P. Talalay等人共同開發出來。首先需先 計算出兩種藥物單獨使用於癌細胞各自IC50的濃度，將 此濃度定義為1 ’加上取此濃度之0.25、0.5、2及4倍的濃 度共5個濃度一起添加到癌細胞後進行MTT藥物組合 分析，之後將其中一種藥物分別以上述5個濃度減半 後進行重覆的MTT藥物組合分析後，再計算致死率，觀 φ 察三條曲線並計算醫藥組合物的組合指數，可由此決定 藥物是否具有協同性關係、加成性效果或有拮抗性。 (Chou, T. C. and Talalay, P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul., 22:27-55, 1984) ° 由這些資料及下式可決定組合指數（Cl): α_ (D)t t (Ρ)2 | α(Ό\Φ\ (DX), (DX)2 (DX)1(DX)2 17 201021801 其中of對相容的試劑為1 (DX)!=上述藥物1之1(：50 (DX)2 =上述藥物2之1(：50 (DhA(D)2 =藥物丨及藥物2組合後得到50%抑制時的 濃度 若此值<1表具協同性 CI = 1表互無作用 > 1表互為拮抗 實施例1 亞丁基苯酚和卡葸司汁的抗癌活性 將院化類抗癌藥物卡莫司汀（Carinustine; BCNU)(表 1A及 1B)和正亞丁 基苯酚（n_Butylidenephthalide)一同组 合後研究。試驗組合物有效性的方法是用前述的細胞存 活分析測驗不同濃度藥物組合的抗腫瘤細胞活性。研究 這些活性成分的方法可由分析資料數據的組合指數而決 定是否有協同性。 加藥48小時到J5和HepG2細胞株後，測得各下 列活性成分的 IC50 : —----- IC50(mM) 活性成分 ---------------- J5 —------ 25 100 HepG2 丁烯基苯酚 25 ~ ------- 卡莫司汀 100 表1A、1B、1C分別為加藥48小時到J5細胞株，正 18 201021801 亞丁苯酚：卡莫司汀=1:2、1:4、1:8的MTT實驗結果，CI 值分別為0.296、0.224及 0.064，表示各劑量比為均具 有協同性。Calcug.xp software analysis of the synergy of pharmaceutical compositions. In this application, the CalcuSyn software is used to draw the drug dose response curve. The IC50 of each drug combination, whether alone or at a fixed concentration of other drug combinations, is Calculated and recorded on the equal weight map. CalcuSyn software is mainly used in the analysis of synergy or antagonism in the treatment of cancer or AIDS. It can perform the dose-effect calculation of a variety of drugs, and this algorithm is performed by TC Chou and P. Talalay and others jointly developed. First, we need to calculate the concentration of each drug in the IC50 of each cancer cell. The concentration is defined as 1 ' plus the concentration of 0.25, 0.5, 2 and 4 times of this concentration is added to the cancer cells together. After the MTT drug combination analysis, and then one of the drugs was halved by the above five concentrations and then repeated MTT drug combination analysis, then calculate the lethal rate, observe the three curves and calculate the combination index of the pharmaceutical composition, It can be determined whether the drug has a synergistic relationship, an additive effect or an antagonistic effect. (Chou, TC and Talalay, P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul., 22:27-55, 1984) ° can be determined by these data and the following formula Combination index (Cl): α_ (D)tt (Ρ)2 | α(Ό\Φ\ (DX), (DX)2 (DX)1(DX)2 17 201021801 where of the compatible reagent is 1 ( DX)!=1 of the above drug 1 (:50 (DX)2 = 1 of the above drug 2 (:50 (DhA(D)2 = concentration of 50% inhibition after drug combination and drug 2 combination, if this value is <; 1 table with synergistic CI = 1 table has no effect on each side > 1 table mutually antagonistic Example 1 anti-cancer activity of butylene phenol and caloside juice will be hospitalized anticancer drug carmustine (Carinustine; BCNU) (Tables 1A and 1B) were studied in combination with n-buttylophenol (n_Butylidenephthalide). The method for testing the effectiveness of the composition is to test the anti-tumor cell activity of different concentrations of the drug combination by the aforementioned cell survival assay. Analyze the combined index of the data to determine whether there is synergy. After 48 hours of dosing to J5 and HepG2 cell lines , IC50 of each of the following active ingredients was measured: —----- IC50 (mM) Active ingredient ----------------- J5 —------ 25 100 HepG2 Alkenylphenol 25 ~ ------- Carmustine 100 Tables 1A, 1B, 1C are dosing for 48 hours to J5 cell line, positive 18 201021801 butylene phenol: carmustine = 1: 2 : 4, 1:8 MTT test results, CI values were 0.296, 0.224 and 0.064, respectively, indicating that each dose ratio is synergistic.

表1A 丁烯基苯酚(μΜ) 卡莫司汀(μΜ) 致死率 (%) 200 400 76.1 100 200 73.8 50 100 62.3 25 50 64 12.5 25 56.7 CI=0.296，表示具有協同性Table 1A Butenylphenol (μΜ) Carmustine (μΜ) Mortality rate (%) 200 400 76.1 100 200 73.8 50 100 62.3 25 50 64 12.5 25 56.7 CI=0.296, indicating synergy

表1B 丁烯基苯酚(μΜ) 卡莫司汀(μΜ) 致死率（％) 100 400 71.2 50 200 69.5 25 100 64 12.5 50 60.6 6.25 25 53.2Table 1B Butenylphenol (μΜ) Carmustine (μΜ) Mortality rate (%) 100 400 71.2 50 200 69.5 25 100 64 12.5 50 60.6 6.25 25 53.2

φ CI=0.224，表示具有協同性φ CI=0.224, indicating synergy

表1C 丁烯基苯酚(μΜ) 卡莫司汀(μΜ) 致死率（％) 100 800 70 50 400 67.8 25 200 60.9 12.5 100 58.3 6.25 50 53.3 CI=0.06，表示具有協同性 19 201021801 表2A、2B、2C分別為加藥48小時到 HepG2細胞 株，正亞丁苯酚：卡莫司汀=1:2、1:4、1:8的MTT實驗結果，CI 值分別為0.346、0.222、0.080，表示各劑量比均具有協同 性。Table 1C Butenylphenol (μΜ) Carmustine (μΜ) Mortality rate (%) 100 800 70 50 400 67.8 25 200 60.9 12.5 100 58.3 6.25 50 50.3 CI=0.06, indicating synergy 19 201021801 Table 2A, 2B 2C was the result of MTT test for 48 hours to HepG2 cell line, gamma butyrate: carmustine = 1:2, 1:4, 1:8, CI values were 0.346, 0.222, 0.080, respectively. The dose ratios are all synergistic.

表2A 丁烯基苯酚(/χΜ) 卡莫司、;丁(μΜ) 致死率（％) 200 400 76.2 100 200 74.1 50 100 623 25 50 64.2 12.5 25 56.6 C卜0.346，表示具有協同性Table 2A Butenylphenol (/χΜ) Caromos, Ding (μΜ) Mortality rate (%) 200 400 76.2 100 200 74.1 50 100 623 25 50 64.2 12.5 25 56.6 C Bu 0.346, indicating synergy

表2B 丁烯基苯酚(μΜ) 卡莫司汀(μΜ) 致死率（％) 100 400 71.2 50 200 68.7 25 100 64 12.5 50 59.7 6.25 25 51.2Table 2B Butenylphenol (μΜ) Carmustine (μΜ) Mortality rate (%) 100 400 71.2 50 200 68.7 25 100 64 12.5 50 59.7 6.25 25 51.2

CI=0.022表示具有協同性 ❿ 表2C 丁烯基苯酚(μΜ) 卡莫司汀(μΜ) 致死率（％) 100 800 78.8 50 400 70.6 25 200 70.2 12.5 100 66.2 6.25 50 65.2 CI=0.080表示具有協同性 實施例2 正亞丁基笨酚和卡莫司汀的體内Wvo)抗癌活性 20 201021801 卡莫司汀是一種烷化基類的化療藥物，其和正亞丁基苯酚 一同組合後是有效的代表醫藥組合物。試驗醫藥組合物有效 性方法是用皮下腫瘤移植模式觀察醫藥組合物的體内抗腫瘤 活性。主要觀察較低藥物組合劑量時是否達成跟單獨使用藥 劑具有同等的腫瘤抑制效果。表3和4為太平洋卡莫司汀與正 亞丁基苯酚結合後對抗裸小鼠皮下HepG2和 J5肝癌的活性 分析。 ❹ 表 3 (J5) 藥物 治療方式 時間（1，2) 劑量(毫克/ 公斤/天） 腫瘤相對大小 (倍數)⑶ 統計分析 (4) 溶劑 i.p.連續5天 0 6.30 P=0.45 正亞丁基苯酚 i.p.連續5天 50 6.16 P=0.17 正亞丁基苯酚 i.p.連續5天 100 2.74 P=0.30 正亞丁基苯酚 i.p.連續5天 300 1.86 P=0.23 正亞丁基苯酚 i.p.連續5天 500 0.03 P=0.03 卡莫司汀 i.p.連續5天 4 6.37 P=0.21 卡莫司汀 i.p.連續5天 8 3.46 P=0.35 正亞丁基苯酚 + 卡莫司汀 i.p.連續5天 50 4 0.43 P=0.01 表 4 (HepG2) 藥物 治療方式 時間（1,2) 劑量(毫克/ 公斤/天） 腫瘤相對大小 (倍數)⑶ 統計分析 (4) 溶劑 i.p.連續5天 0 9.55 P=0.38 正亞丁基苯酚 i.p.連續5天 50 4.68 P=0.19 正亞丁基苯盼 i.p.連續5天 100 0.21 Ρ=0·04 正亞丁基苯酚 i.p.連續5天 300 0.10 Ρ=0·03 正亞丁基苯酚 i.p.連續5天 500 0.04 Ρ=0.03 卡莫司汀 i.p.連續5天 4 8.25 Ρ=0.34 卡莫司汀 i.p.連續5天 8 6.59 Ρ=0·32 21 201021801 正亞丁基笨酚 50 4 + 卡莫司汀 i.p.連續5天 2.25 P=0.30 (1) 分別取經無菌培養5xl06個HepG2及1又1〇7個J5 癌細胞注射至Balb/c免疫不全裸小鼠皮下進行體内腫瘤移 植實驗。 (2) 在腫瘤生長至100 mm3 (長叉寬又寬/2)後開始皮下 (i.p.)給藥治療’在腫瘤對侧超過15公分處給藥，連續$天， ❹ 每天1次。合併治療組先給藥正亞丁基苯酚，兩小時後再給藥 卡莫司汀。 (3) 第5天給藥後連續觀察28天，每3天測量腫瘤大小及 體重，每隻裸鼠第28天測得腫瘤大小除以第！天所得倍數為 腫瘤相對大小值（relative tumor size)，本試驗以腫瘤相對大 小值做為抑制腫瘤生長效果的活性評估結果，數值愈小抑 制效果愈好。 (4) 觀察單獨或合併治療後的腫瘤相對大小值與對照組 (只打溶劑）有無顯著差異，p值小於〇 5時表示有顯著差異\ 表3和4數據清楚顯示相較於單獨給藥，低劑量的正亞丁基 苯紛㈣）與卡莫司彳（BCNU)合併給藥可以更有效地抑制 腫瘤細胞生長，增強抗腫瘤效果。治療前與藥物治療後的腫瘤 組織進订免疫染色分析’第1圖⑷及第]圖⑻，顯示合併使 用正亞丁基苯盼與卡莫司汁來處理HepG2及J5人類肝癌細 胞株時’其協同之抗癌效果。 第2圖顯示合併使用正亞丁基苯酚與卡莫司汀來處理 HepG2及J5人類肝癌細胞株時，其協同之抗癌效果可能 22 201021801 是透過抑制 MGMT蛋白的表現及經過***卡斯蛋白 (cleaved caspase-3)所致 ° 另外，第3圖顯示單獨使用正亞丁基苯酚（n-BP)來處理 HepG2及 J5人類肝癌細胞株時，會造成mgmi基因啟動 子的高度曱基化，這種抑現象與隨藥物劑量及添加時間成正 相關。第3圖顯示單獨使用正亞丁基苯酚（n-BP)來處理HepG2 及J5人類肝癌細胞株時，會造成mgmi基因啟動子的高度 曱基化，這種抑現象與隨藥物劑量及添加時間成正相關（第3 ® 圖（A))，另外，此曱基化會導致MGMT mRNA及蛋白質的 表現量降低（第3圖（B))。 第4圖顯示合併使用正亞丁基苯酚（n-BP)與卡莫司汀 (BCNU)來處理 HepG2及 J5人類肝癌細胞株時，與單 獨給藥n-BP比較，合併給藥會明顯地增強 mgmi基因啟 動子的高度甲基化，進而抑制 MGMT蛋白質的表現（第 4(A))並導致細胞凋亡的大量產生（第4圖（B))。 雖然本發明已以較佳實施例揭露如上，然其並非用以限 參 定本發明，任何熟習此技藝者，在不脫離本發明之精神與範 圍内，當可做些許之更動與潤飾，因此本發明之保護範圍當 視後附之申請專利範圍所界定者為準。 【圖式簡單說明】 第1圖顯示合併使用正亞丁基苯酚（n-BP)與卡莫司汀 (BCNU)來處理HepG2及 J5人類肝癌細胞株時，其協同 之抗癌效果。 23 201021801 第2圖顯示合併使用正亞丁基苯酚與卡莫司汀來處理 HepG2及 J5人類肝癌細胞株時，其協同之抗癌效果可能 是透過抑制 MGMT蛋白的表現及經過***卡斯蛋白 (cleaved caspase-3)所致。 第3圖顯示單獨使用正亞丁基苯酚（η·ΒΡ)來處理HepG2及 J5人類肝癌細胞株時，會造成mgmi基因啟動子的高度曱 基化，這種抑現象與隨藥物劑量及添加時間成正相關（第3圖 (A))，另外，此甲基化會導致MGMT mRNA及蛋白質的表 參現量降低（第3圖⑻）。 第4圖顯示合併使用正亞丁基苯酚（n-BP)與卡莫司汀 (BCNU)來處理HepG2及J5人類肝癌細胞株時，與單獨給藥 n-BP比較，合併給藥會明顯地增強mgmi基因啟動子的高 度曱基化，進而抑制MGMT蛋白質的表現（第4圖（A))並導 致細胞凋亡的大量產生（第4圖（B))。 24CI=0.022 indicates synergy ❿ Table 2C Butenylphenol (μΜ) Carmustine (μΜ) Mortality rate (%) 100 800 78.8 50 400 70.6 25 200 70.2 12.5 100 66.2 6.25 50 65.2 CI=0.080 indicates synergy Example 2 In vivo Wvo) anticancer activity of n-butylene phenol and carmustine 20 201021801 Carmustine is an alkylating group of chemotherapeutic drugs which are effective combination with n-butylene phenol. Pharmaceutical composition. An effective method for testing a pharmaceutical composition is to observe the in vivo antitumor activity of a pharmaceutical composition using a subcutaneous tumor transplantation mode. It is mainly observed whether the lower drug combination dose achieves the same tumor suppressing effect as the drug alone. Tables 3 and 4 show the activity of Pacific Cammostin in combination with n-butylphenol to counteract the activity of subcutaneous HepG2 and J5 liver cancer in nude mice. ❹ Table 3 (J5) Time of drug treatment (1,2) Dose (mg/kg/day) Relative tumor size (multiple) (3) Statistical analysis (4) Solvent ip for 5 consecutive days 0 6.30 P=0.45 n-butylene phenol ip 5 consecutive days 50 6.16 P=0.17 n-butylene phenol ip continuous 5 days 100 2.74 P=0.30 n-butylene phenol ip continuous 5 days 300 1.86 P=0.23 n-butylene phenol ip continuous 5 days 500 0.03 P=0.03 Camo Ting ip for 5 consecutive days 4. 6.37 P=0.21 carmustine ip for 5 consecutive days 8.46 P=0.35 n-butylene phenol + carmustine ip for 5 consecutive days 50 4 0.43 P=0.01 Table 4 (HepG2) drug treatment Time (1,2) Dose (mg/kg/day) Tumor relative size (multiple) (3) Statistical analysis (4) Solvent ip for 5 consecutive days 0 9.55 P=0.38 n-butylene phenol ip for 5 consecutive days 50 4.68 P=0.19 Butylene Benzene ip for 5 consecutive days 100 0.21 Ρ=0·04 n-Butyl phenol ip for 5 consecutive days 300 0.10 Ρ=0·03 n-butylene phenol ip for 5 consecutive days 500 0.04 Ρ=0.03 Carmustine ip continuous 5 Day 4 8.25 Ρ=0.34 Carmustine ip for 5 consecutive days 8.59 Ρ=0·32 21 201021801 Zhengya Base phenol 50 4 + carmustine ip for 5 consecutive days 2.25 P=0.30 (1) Separately cultured 5xl06 HepG2 and 1 〇7 J5 cancer cells were injected subcutaneously into Balb/c immunocompromised nude mice. In vivo tumor transplantation experiments. (2) The subcutaneous (i.p.) administration was started after the tumor grew to 100 mm3 (long fork width and width/2). The drug was administered more than 15 cm on the opposite side of the tumor for $100 consecutive days, ❹ once a day. The combined treatment group was first administered with n-butylene phenol, and two hours later, carmustine was administered. (3) On the 5th day, the patient was continuously observed for 28 days, and the tumor size and body weight were measured every 3 days. The tumor size was measured on the 28th day of each nude mouse divided by the first! The multiplication of the day is the relative tumor size of the tumor. In this test, the relative tumor size is used as the activity evaluation for inhibiting the tumor growth effect. The smaller the value, the better the inhibition effect. (4) Observing whether the relative size of tumors after treatment alone or in combination was significantly different from that of the control group (solvent only), the p value was less than 〇5, indicating significant difference. Tables 3 and 4 clearly show that compared with the single administration The combination of low-dose n-butylene benzene (IV) and carmine (BCNU) can inhibit tumor cell growth and enhance anti-tumor effect. Pre- and post-treatment tumor tissue sequencing immunostaining analysis 'Fig. 1 (4) and Fig. 8 (8), showing the use of n-butylidene phenyl and kamos juice in combination with HepG2 and J5 human hepatoma cell lines Synergistic anti-cancer effect. Figure 2 shows that the synergistic anticancer effect of HepG2 and J5 human hepatoma cell lines when combined with n-butylene phenol and carmustine may be 22 201021801 by inhibiting the expression of MGMT protein and cleaving the caspase protein (cleaved) Caspase-3) In addition, Figure 3 shows that the use of n-butylene phenol (n-BP) alone to treat HepG2 and J5 human hepatoma cell lines results in a high degree of thiolation of the mgmi gene promoter. The phenomenon is positively correlated with the dose of the drug and the time of addition. Figure 3 shows that the use of n-butylene phenol (n-BP) alone to treat HepG2 and J5 human hepatoma cell lines results in a highly thiolated promoter of the mgmi gene, which is positive with drug dose and time of addition. Correlation (Fig. 3 ® Figure (A)), in addition, this thiolation results in a decrease in the expression of MGMT mRNA and protein (Fig. 3(B)). Figure 4 shows that when n-butyl phenol (n-BP) and carmustine (BCNU) were used in combination to treat HepG2 and J5 human hepatoma cell lines, the combined administration was significantly enhanced compared with n-BP alone. High methylation of the mgmi gene promoter, which in turn inhibits the expression of MGMT protein (4(A)) and leads to a large number of apoptosis (Fig. 4(B)). While the present invention has been described above in terms of the preferred embodiments thereof, it is not intended to limit the invention, and it is to be understood that those skilled in the art can make some modifications and refinements without departing from the spirit and scope of the invention. The scope of the invention is defined by the scope of the appended claims. [Simplified Schematic] Fig. 1 shows the synergistic anticancer effect of the combination of n-butylphenol (n-BP) and carmustine (BCNU) to treat HepG2 and J5 human hepatoma cell lines. 23 201021801 Figure 2 shows that when combined with n-butyl phenol and carmustine to treat HepG2 and J5 human hepatoma cell lines, the synergistic anti-cancer effect may be through inhibition of MGMT protein expression and cleavage of caspase Caused by caspase-3). Figure 3 shows that the use of n-butylene phenol (η·ΒΡ) alone to treat HepG2 and J5 human hepatoma cell lines results in a high degree of thiolation of the mgmi gene promoter, which is consistent with drug dosage and addition time. Correlation (Fig. 3(A)), in addition, this methylation leads to a decrease in the amount of MGMT mRNA and protein (Fig. 3 (8)). Figure 4 shows that when n-butyl phenol (n-BP) and carmustine (BCNU) were used in combination to treat HepG2 and J5 human hepatoma cell lines, the combined administration was significantly enhanced compared with n-BP alone. High glycosylation of the mgmi gene promoter, which in turn inhibits the expression of MGMT proteins (Fig. 4 (A)) and leads to a large number of apoptosis (Fig. 4(B)). twenty four

Claims (1)

201021801 十、申請專利範困： ! 一種用於治療癌症之醫藥組合物，包含一協同 有效量之一人類〇1 2 3·甲基烏嘌呤-DNA甲基轉移酶（MGMT) 抑制劑和一烷化類抗癌藥物，以及一藥學上可接受的載 體；其中該MGMT抑制劑與該烷化類抗癌藥物的濃度比 在約1:2至約1間，且此醫藥組合物具有一小於1的組合 指數。 ® 2.如請求項1所述之醫藥組合物，其中該烧化類抗 癌藥物係選自由卡莫司汀、洛莫司汀、替莫唑胺，或其 水合物、同電子排列體（iS0Steres)及醫藥上可接受之鹽 類所組成的群組中。 3. 如請求項1所述之醫藥組合物，其中該MGMT抑 制劑是選自由〇3_苄基鳥嗓呤（〇3-benzylguanine，03· BG)、8-偶氮-〇3_ 节基烏嗓呤（8-aza-03-benzylguanine，8-aza-BG) 、 〇3_(4溴甲基）烏嘌呤（03-(4- bromothenyl)guanine，4-BTG)及正亞丁基苯酚所組成的群 組中。 25 1 · 如請求項1所述之醫藥組合物，其中當該MGMT 2 抑制劑與該烷化類抗癌藥物的濃度比為約U時，該組合 3 指數在約0.3至〇. 3 5間。 201021801 5. 如請求項1所述之醫藥組合物，其中該MGMT抑 制劑與該烧化類抗癌藥物的濃度比為約1 4時，該組合指 數約為0.22。 6. 如請求項1所述之醫藥組合物，其中該MGMT抑 制劑與該烷化類抗癌藥物的濃度比為約卜8時，該組合指 數在約0.06至約〇.〇8之間。 1'如請求項1所述之醫藥組合物，其中該癌症是肝 癌、肺癌、腦瘤、卵巢癌或大腸直腸癌。 8· 如請求項7所述之醫藥組合物，其中該癌症是肺 癌、具有抗藥性的腦癌或肝癌。 9· 如請求項1所述之醫藥組合物，其中該MGMT抑 ❹制劑及該抗癌藥物係以同時、分開或連續方式經由非經 腸胃道的方式施用至少5天。 iO·如請求項9所述之醫藥組合物，其中該非經腸胃 道的方式包含經由皮下注射方式給藥。 u.如請求項10所述之醫藥組合物，其中該MGMT 抑制劑是正亞丁基苯酚’該烷化類抗癌藥物是卡莫司 ^，且正亞丁基苯酚的用量是介於每天100毫克/公斤至 26 201021801 每天500毫克/公斤間，卡莫司汀的用量則是介於每天10 毫克/公斤至每天100毫克/公斤間。 十一、圖式： 如次頁201021801 X. Applying for patents: A pharmaceutical composition for the treatment of cancer, comprising a synergistically effective amount of human 〇1 2 3·methyl 嘌呤-DNA methyltransferase (MGMT) inhibitor and mono a chemical anticancer drug, and a pharmaceutically acceptable carrier; wherein the concentration ratio of the MGMT inhibitor to the alkylating anticancer drug is between about 1:2 and about 1, and the pharmaceutical composition has a less than one Combination index. The pharmaceutical composition according to claim 1, wherein the burn-in anticancer drug is selected from the group consisting of carmustine, lomustine, temozolomide, or a hydrate thereof, an isoelectronic array (iS0 Steres), and a pharmaceutical In a group consisting of acceptable salts. 3. The pharmaceutical composition according to claim 1, wherein the MGMT inhibitor is selected from the group consisting of 〇3_benzylguanine (〇3-benzylguanine, 03·BG), 8-azo-〇3_jiejiu嗓呤(8-aza-03-benzylguanine, 8-aza-BG), 〇3_(4 bromomethyl) uranium (03-(4- bromothenyl) guanine, 4-BTG) and n-butylene phenol In the group. The pharmaceutical composition according to claim 1, wherein when the concentration ratio of the MGMT 2 inhibitor to the alkylating anticancer drug is about U, the combination 3 index is between about 0.3 and 〇. . The pharmaceutical composition according to claim 1, wherein the combination index of the MGMT inhibitor and the burnt-in anticancer drug is about 12, and the combination index is about 0.22. 6. The pharmaceutical composition according to claim 1, wherein the concentration ratio of the MGMT inhibitor to the alkylating anticancer drug is about 8, and the combination index is between about 0.06 and about 〇. The pharmaceutical composition according to claim 1, wherein the cancer is liver cancer, lung cancer, brain tumor, ovarian cancer or colorectal cancer. The pharmaceutical composition according to claim 7, wherein the cancer is lung cancer, drug-resistant brain cancer or liver cancer. The pharmaceutical composition according to claim 1, wherein the MGMT inhibitory preparation and the anticancer drug are administered parenterally in a simultaneous, separate or continuous manner for at least 5 days. The pharmaceutical composition according to claim 9, wherein the parenteral route comprises administration via subcutaneous injection. The pharmaceutical composition according to claim 10, wherein the MGMT inhibitor is n-butylene phenol. The alkylating anticancer drug is carmust, and the amount of n-butylene phenol is 100 mg per day/ Kg to 26 201021801 500 mg / kg per day, the amount of carmustine is between 10 mg / kg per day to 100 mg / kg per day. XI. Schema: as the next page 2727