201247208 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種醫藥組成物，尤指一種用於抑制肝癌復發、惡 . 化或轉移之醫藥組成物。本發明另關於一種製備藥物的方法，尤指一 種製備供抑制肝癌復發、惡化或轉移之藥物的方法。 【先前技術】 依據癌症國際雜tfe(Intemational Journal of Cancer)之資訊，肝癌 (LiverCancer)是全球發生率第五高的惡性腫瘤，肝癌大部份乃屬肝細 胞癌（Hepatocellular Carcinoma，HCC)，約占所有肝癌患者的 70%〜85% ’目前多透過手術切除(Resecti〇n)、局部消融療法(L〇cal Ablation)及肝臟移植進行治療，但僅對15〜2〇%的患者適用◊雖然肝癌 早期發現與治療能提高存活率，但即使是接受手術切除的病人，往往 在治療後肝癌仍會復發。外科手術年報(Annals of Surgery)之資料顯示 60%的肝癌病患在手術後12個月内會復發，而肝癌的微小轉移 (micro-metastasis)能在手術時透過靈敏的檢測技術測得，其發現患者在 術後一年時仍有30〜40%的復發率。此外’手術後餘留下來的病變肝組 織亦有可能進一步重新形成肝癌。肝癌的復發將大幅縮短患者的整體 存活期，因此，如何避免或延遲肝癌患者在接受手術治療後再度復發， 仍是一有待解決的大問題。 過去有許多試驗在於尋找降低術後肝癌復發率的有效方式，例如 動脈化學治療(trans-arterial chemotherapy)、類視色素(retin〇ids)、輔助性 干擾素(adjuvant interferon)、過繼性免疫療法(adoptive immun〇therapy) 及動脈内放射性蛾油(intra-arterial radioactive lipiodol)等。雖然上述方法 具有降低肝癌復發風險或提高患者存活率的潛力，卻都未通過臨床試 ，的驗，而尚未能成為肝癌術後輔助治療的標準療法，因此對於新 藥物或新治療方法仍然有迫切的需求。 201247208 從肝癌形成的角度進行分析，肝癌具有高度血管化的特性，且其 發展與血管新生因子(angi〇genic fact〇rs)有很大的關聯。包括研究血管 新生之權威科學家Judah Folkman在内之研究人員，已從學理上以及實 驗上指出抗血管新生藥物合併其他療法可以在癌症治療上得到更好的 效果(Christopher Rice，L· Eric Huang. From amiangiogenesis to hypoxia current research and future directions. Cancer Management and Research. 2011:3 9-16)。此外，細胞外基質(extraceuuiar matrix，ECM)中的成分之 一，硫酸乙醯肝素(heparan suifate，HS)的降解已被證實為腫瘤的侵犯 與轉移之_因素。參與此-降解過㈣主要酵素之—即為類肝素酶 (heparanase)。 類肝素酶是一内源性葡萄糖醛酸酶，能降解細胞外基質中的硫酸 乙醯肝素側鏈，並且是惟一能降解硫酸乙醯肝素蛋白聚糖的内源性葡 萄糖醛酸酶，其可在特定部位裂解硫酸乙醯肝素以促進腫瘤的浸潤和 轉移。類肝素酶透過參與細胞外基質的降解、血管生長因子的釋放以 及血管重構等過程，在腫瘤轉移與血管生成中產生關鍵影響。此外， 硫酸乙醯肝素經過類肝素酶的裂解後會釋放具有生物活性的血管生長 因子於細胞外基質中，這些血管生長因子會促成血管生長進而促進癌 細胞增生、轉移與癌症的惡化◊因此，抑制類肝素酶可能對抑制腫瘤 生長及轉移產生效果，類肝素轉的過量表現與否成為預防或治療惡性 腫瘤的重要標的。 過去的相關研究發現類肝素酶在許多類型的腫瘤中有較高程度的 表現，且與致病過程的發展有所關連，導致許多研究報告係以抑制此 一酵素作為癌症治療的策略，包括小分子藥物、經化學修飾的天然物， 以及中和性抗體(neutralizing antibodies)的開發等等。 美國第6，143,730號專利係揭露一種硫酸化寡糖的製備和應用，該 寡糖具有如通式I: R1 -(Rx)n-R2之結構’其中R1和幻以及每個肽為 單糖單元，其全部可相同或不同，相鄰的單糖單元由1—2、3、丨―4 及/或1—6糖苷鍵所連接，且η為1至6的整數，以及該寡糖作為抗血 管生成、抗轉移及/或抗發炎劑的用途。前述抗血管生成功效已經體外 實驗及臨床試驗數據佐證，而抗轉移功效則僅有動物實驗數據，皆未 4 201247208 有人體臨床試驗數據驗證。 =上所述’雜目前已有部分麟降低肝癌復發風險或提高肝癌 、、子=率的方法’卻都未有足觸人體臨床數據加以驗證，因此， 對具有安全性及有錄崎醫藥組祕及治療方法的需求仍然存在。 【發明内容】 就，案的一方面而言，本案提出一種用於抑制肝癌復發、惡化或轉 移之醫藥組成物’其包含—治療有效量之至少—通式⑴化合物及一醫藥 上可接受之稀釋劑、賦形劑或載劑； ’、201247208 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition, and more particularly to a pharmaceutical composition for inhibiting recurrence, malignant transformation or metastasis of liver cancer. The invention further relates to a method of preparing a medicament, and more particularly to a method of preparing a medicament for inhibiting recurrence, progression or metastasis of liver cancer. [Prior Art] According to the information of the Cancer Journal, the liver cancer (LiverCancer) is the fifth most common malignant tumor in the world. Most of the liver cancer is Hepatocellular Carcinoma (HCC). It accounts for 70%~85% of all liver cancer patients. Currently, it is treated by resection (Resecti〇n), local ablation (L〇cal Ablation) and liver transplantation, but only for 15~2% of patients. Early detection and treatment of liver cancer can improve survival rate, but even in patients undergoing surgical resection, liver cancer often recurs after treatment. According to the Annals of Surgery, 60% of liver cancer patients relapse within 12 months after surgery, and micro-metastasis of liver cancer can be measured by sensitive detection techniques during surgery. The patient was found to have a recurrence rate of 30 to 40% at one year after surgery. In addition, the diseased liver tissue left after the operation may further re-form liver cancer. The recurrence of liver cancer will greatly shorten the overall survival of patients. Therefore, how to avoid or delay the recurrence of liver cancer patients after surgery is still a big problem to be solved. There have been many trials in the past to find effective ways to reduce the recurrence rate of postoperative liver cancer, such as trans-arterial chemotherapy, retin〇ids, adjuvant interferon, and adoptive immunotherapy ( Adoptive immun〇therapy) and intra-arterial radioactive lipiodol. Although the above methods have the potential to reduce the risk of liver cancer recurrence or improve the survival rate of patients, they have not passed the clinical test, but have not become the standard treatment for postoperative adjuvant therapy of liver cancer, so there are still new drugs or new treatment methods. Urgent needs. 201247208 From the perspective of liver cancer formation, liver cancer is highly vascularized and its development is strongly associated with angiogenic factors. Researchers including Judah Folkman, an authoritative scientist who studies angiogenesis, have theoretically and experimentally pointed out that anti-angiogenic drugs combined with other therapies can achieve better results in cancer treatment (Christopher Rice, L. Eric Huang. From Amiangiogenesis to hypoxia current research and future directions. Cancer Management and Research. 2011:3 9-16). In addition, degradation of heparan suifate (HS), one of the components in the extracellular matrix (ECM), has been shown to be a factor of tumor invasion and metastasis. Participate in this - degradation of (four) the main enzyme - that is heparanase (heparanase). Heparanase is an endogenous glucuronidase that degrades the side chain of heparin sulfate in the extracellular matrix and is the only endogenous glucuronidase that degrades the heparin heparin proteoglycan. Heparin sulfate can be cleaved at specific sites to promote tumor infiltration and metastasis. Heparanase has a key influence on tumor metastasis and angiogenesis by participating in the degradation of extracellular matrix, release of angiogenic factors, and vascular remodeling. In addition, heparin sulfate is cleaved by heparanase to release biologically active angiogenic factors in the extracellular matrix. These angiogenic factors promote blood vessel growth and promote cancer cell proliferation, metastasis and cancer deterioration. Inhibition of heparanase may have an effect on inhibiting tumor growth and metastasis, and excessive expression of heparin transfection is an important target for preventing or treating malignant tumors. Previous studies have found that heparanase has a high degree of performance in many types of tumors and is associated with the development of pathogenic processes, leading to many studies reporting strategies to inhibit this enzyme as a treatment for cancer, including Small molecule drugs, chemically modified natural substances, and the development of neutralizing antibodies. U.S. Patent No. 6,143,730 discloses the preparation and use of a sulfated oligosaccharide having the structure of formula I: R1 -(Rx)n-R2 wherein R1 and phantom and each peptide is a monosaccharide Units, all of which may be the same or different, adjacent monosaccharide units are linked by a 1-2, 3, 丨-4, and/or 1-6 glycosidic linkage, and η is an integer from 1 to 6, and the oligosaccharide is Anti-angiogenic, anti-metastatic and/or anti-inflammatory agents. The aforementioned anti-angiogenic effects have been confirmed by in vitro experiments and clinical trial data, while the anti-metastatic efficacy is only animal experimental data, none of which has been verified by human clinical trial data. = The above-mentioned "Mixed lining has reduced the risk of liver cancer recurrence or the method of improving liver cancer, and the rate of sub-subjects" has not been verified by human clinical data. Therefore, it is safe and has a recorded drug group. The need for secrets and treatments still exists. SUMMARY OF THE INVENTION In one aspect, the present invention provides a pharmaceutical composition for inhibiting recurrence, exacerbation or metastasis of liver cancer, which comprises - a therapeutically effective amount of at least - a compound of the formula (1) and a pharmaceutically acceptable Thinner, excipient or carrier; ',
其中該通式(I)化合物之n為〇至3的整數，且有3n+6或3n+7個R 代表S〇3H，其餘R皆代表η。 根據上述構想，其中該通式①化合物中有知+⑽尺代表犯识者 係為主要組分。 根據上述構想，纟中該通式⑴化合物之η為2或3且有3η+7個r 代表S〇3H者係為主要組分。 根據上述構想，其中該通式(I)化合物之η為3且有3n+7個R代表 S〇3H者之姆含私料。 Μ 根據上述構想，其中邊治療有效量係介於每日8〇 mg至每日315 mg 根據上述構想，其中該治療有效量較佳係為每日160mg。 於本案所提出之醫藥組成物中，作為活性成分之通式(I)化合物係以 納鹽形式存在與伽，惟亦可以其他醫藥上可接受之鹽，如触或胺鹽 之I式存在與使用。因此，本案醫藥組成物之活性成分係包括了通式(I) 化合物之納鹽或其他醫藥上可接受之鹽。 201247208 前述醫藥上可接受之稀釋劑、賦形劑或載劑係包括溶劑、分散介 質、填充劑、固體載體、水溶液、抗細菌劑、抗黴菌劑及延遲吸收劑或 前述之類似物。除了與活性成份不相容的傳統介質或試劑外，本發明之 醫藥組成物中係使用可相容的介質或試劑。 前述本案所提出之醫藥組成物係可與至少一治療方式合併用於抑 制肝癌之復發、惡化或轉移《前述治療方式係包括栓塞治療、標靶藥物 治療、化學治療、放射治療及肝臟切除手術。 根據上述構想’本案醫藥組成物係用於降低肝癌患者之術後復發率 或延長肝癌患者術後復發所需之時間。 就本案的另一方面而言’本案提出一種使用通式⑴化合物製備供抑 制肝癌復發、惡化或轉移之藥物的方法，包括將一治療有效量之通式(1) 化合物與一醫藥上可接受之稀釋劑、賦形劑或載劑相結合。 根據上述構想，該通式(I)化合物之η為〇至3的整數，且有3n+6 或3n+7個R代表s〇3H，其餘R皆代表η。 根據上述構想’其中該通式(I)化合物中有3η+7個R代表S〇3h者 係為主要組分。 根據上述構想，其中該通式(I)化合物之η為2或3且有3n+7個R 代表S〇3H者係為主要組分。 根據上述構想，其中該通式(I)化合物之η為3且有3n+7個R代表 S〇3H者之相對含量為最高。 根據上述構想，該醫藥上可接受之獅劑、賦糊或_係為水或 以水為溶劑之溶液。 根據上述構想，該醫藥上可接受之稀釋劑、賦形劑或載劑係為生理 食鹽水或葡萄糖水溶液。 本案得藉由以下圖式與實施方式說明而更易於讓在此領域具通常 知識者瞭解本案的精神。 【實施方式】 本案「用於抑制肝癌復發、惡化或轉移之醫藥組成物」將可透過以 6 201247208 下的實施例說明而讓在此領域具通常知識者瞭解其創作精神並可據 以兀成。然本案的實施並非由下列實施例而限制其實施型態。以下分 就本案醫藥組成物之製備、確認及用途之實施方式分別說明於下。 本案醫藥組成物之製備 本案醫藥組成物活性成分之原料係來自於酵母菌疳， 其製備主要可分鱗母H培養與發酵、產物水解、純化及績化等步驟， 分別敘述如下： ,將菌株名為NRRL Y-2448之酵母菌幼&疳於有氧氣且氮源 又限制之培養基中培養，以右旋葡萄糖(D-gluc〇se)作為碳源並提供過量 之正磷酸鹽（ortho-phosphate)，將產出細胞外之磷酸甘露聚醣 (ph〇Sph〇mannan ’ PS) ’其包含多種多醣結構，並以此作為本案醫藥組 成物活成分之半合成(Semi_synthesis)基本原料。填酸甘露聚醣係由分 子結構兩度分枝且具有高分子量(約5 χ 1G6~39 X丨心⑽⑽)的鱗酸甘 露聚醣核(phosphomannan core，PC)所組成。p.妬/幼).之培養、發酵方 法及磷酸甘露聚紅分離雜據A ; pittsley, L E ; ⑽，p. r.; Dimler，R. J. Arch. Biochem Bi〇phys 1961，92,343_35〇 及此他_，r K.; Kaczorowski，G. J·; Weise，M. J. Biochemistry 1973, 12, 1251-1256 所 揭示之資訊。-般情況下之絲約為働L之發酵規模可產出2〇公斤 之磷酸甘露聚醣粗產物。 在一較佳實施例中，磷酸甘露聚醣之水解反應係在1〇(rc下實行6 至10小時，磷酸甘露聚醣之濃度約控制在40〜50 之間。水解環境 之pH值介於2.2〜2.5 ’以1M之HC1溶液作為催化劑並在KC1的存在 下進行水解。在水解反應的初始丨小時期間，當磷酸甘露聚醣漸與溶 液混合時’ pH值將稍微升高約至3，因此必須再加入1Μ2Ηα溶液 以將酸鹼值回ΡΗ 2·2〜2.5伞欠水解反應所處理之雜甘露聚聰約 為2.5 a斤(屬重）’水解後之產物包括分子量較低之寡醣填酸鹽級分 fligosaccharide phosphate fraction，0PF)，將水解產物以 1MiNa〇H 溶液進行中和，使酸鹼值調整至pH 9〜9.5。 201247208 在一更佳實施例中’用於進行水解之溶液配製係將600克之KCl 溶於60L水中，加入1M之HC1溶液至酸鹼值調整為pH 2.4，並將此 溶液置於75L之不銹鋼反應容器中。將發酵所得之磷酸甘露聚醣粗產 物(濕重2.49公斤)經由反應容器之添加口分次加入至溶液中，並將混合 後之溶液加熱至l〇〇°C ’於劇烈攪拌狀態下維持7小時。反應環境之 pH值每小時檢測一次，並在反應開始後1小時加入1M之HC1使酸驗 值調整回pH 2.3。水解反應終止後，將水解產物冷卻至室溫並加入1M 之NaOH，使酸鹼值調整至pH 9.5。 水解反應後之純化步驟係利用分子孔徑為1〇,〇〇〇 Dalton之滤膜 (nominal molecular weight cut off membrane，NMWCO membrane)進行超 過濾(ultrafiltration) ’水解產物中之寡醣磷酸鹽級分、未磷酸化之寡糖 及鹽類將通過該濾膜，而分子量較高之磷酸甘露聚醣核則留置於濾膜 上’從而使寡醣磷酸鹽級分及未磷酸化之寡糖與磷酸甘露聚醣核相分 離。前述分離過程係在滲濾(diafiltration)模式下以切流式(tangential)或 掃流式(cross flow)過濾系統完成，此一系統能夠透過提高可用濾膜面積 及流速而輕易地增加處理量，其中濾膜之分子孔徑較佳係介於3,〇〇〇至 100,000 Dalton 間，而最佳則為 l〇,〇〇〇 Dalton。 在一較佳實施例中’係將水解產物稀釋至7〇L並置於一 i5〇L之不 鏽鋼槽内，接著使用連續兩組Sartorius Hydrosart 10K (NMWCO 10,000) 渡膜匣(每一濾、膜匣之滤膜面積為0.6 m2)所組成之超過濾系統，以八倍 渗濾體積之純水進行滲濾。前述滲濾之參數設定為：進氣壓力(inlet pressure)為 200 kPa ’ 排氣壓力(〇utlet pressure)為 i5〇kpa，在渗滤過程中 未能通過濾膜而被保留下的滯留物(retentate)之掃流流速(cr〇ss fl〇w rate) 為 15.6 L/min，通透物(permeate)之流速則為 66〜π L/h/m2(16〜2rc)。 經過/參滤後之通透物(630L，導電度為1 1 ms/cm)被分成六批以進行後 續之離子交換層析(ion-exchange chromatography^。 接著進一步對前述之濾出物進行二次純化，二次純化係利用離子交 換層析法。在一較佳實施例中，離子交換層析法係先使用3〇 L之 DEAE-Spherilose管柱以0.01M之溶液在流速為丨5 L / _ 下達到平衡’接著將前述濾出物(每次約励L，共六次)由管柱上端加 201247208Wherein n of the compound of the formula (I) is an integer from 〇 to 3, and 3n+6 or 3n+7 R represents S〇3H, and the remaining R represent η. According to the above concept, wherein the compound of the formula 1 has a known + (10) scale representing the guilty person as a main component. According to the above concept, the η of the compound of the formula (1) in the oxime is 2 or 3 and 3 η + 7 r represents the S 〇 3H as the main component. According to the above concept, wherein the compound of the formula (I) has a η of 3 and 3n+7 of R represents S〇3H. Μ According to the above concept, the therapeutically effective amount is between 8 〇 mg per day and 315 mg per day. According to the above concept, the therapeutically effective amount is preferably 160 mg per day. In the pharmaceutical composition proposed in the present invention, the compound of the formula (I) as an active ingredient is present in the form of a sodium salt, but may also be present in other pharmaceutically acceptable salts, such as a contact or amine salt. use. Accordingly, the active ingredient of the pharmaceutical composition of the present invention comprises a sodium salt of a compound of the formula (I) or other pharmaceutically acceptable salt. 201247208 The aforementioned pharmaceutically acceptable diluents, excipients or carriers include solvents, dispersion media, fillers, solid carriers, aqueous solutions, antibacterial agents, antifungal agents, and delayed absorption agents or the like. Compatible media or agents are employed in the pharmaceutical compositions of the present invention in addition to conventional media or agents which are incompatible with the active ingredient. The pharmaceutical composition proposed in the above-mentioned case can be combined with at least one treatment mode for inhibiting recurrence, deterioration or metastasis of liver cancer. The foregoing treatment methods include embolization treatment, targeted drug treatment, chemotherapy, radiation therapy and liver resection. According to the above concept, the pharmaceutical composition of the present invention is for reducing the postoperative recurrence rate of liver cancer patients or prolonging the time required for postoperative recurrence of liver cancer patients. In another aspect of the present invention, the present invention provides a method of using a compound of the general formula (1) for the preparation of a medicament for inhibiting recurrence, exacerbation or metastasis of liver cancer comprising administering a therapeutically effective amount of a compound of the formula (1) with a pharmaceutically acceptable The diluent, excipient or carrier is combined. According to the above concept, η of the compound of the formula (I) is an integer from 〇 to 3, and 3n+6 or 3n+7 R represents s〇3H, and the remaining R represents η. According to the above concept, wherein the compound of the formula (I) has 3? + 7 R represents S? 3h as a main component. According to the above concept, wherein the compound of the formula (I) has η of 2 or 3 and 3n+7 of R represents S〇3H as a main component. According to the above concept, the relative content of the compound of the formula (I) wherein n is 3 and 3n + 7 R represents S〇3H is the highest. According to the above concept, the pharmaceutically acceptable lion, paste or _ is a solution of water or water. According to the above concept, the pharmaceutically acceptable diluent, excipient or carrier is physiological saline or aqueous dextrose. This case can be easily explained by the following figures and implementation descriptions to let the general knowledge in this field understand the spirit of the case. [Embodiment] The present invention "medicine composition for inhibiting recurrence, deterioration or metastasis of liver cancer" will be explained by the example of 6 201247208, so that those who have ordinary knowledge in this field can understand the spirit of creation and can . However, the implementation of the present invention is not limited by the following embodiments. The following is a description of the preparation, confirmation and use of the pharmaceutical composition in this case. Preparation of the pharmaceutical composition of the present invention The raw material of the active ingredient of the pharmaceutical composition of the present invention is derived from yeast mash, and the preparation thereof can be mainly divided into the steps of culturing and fermentation of the squamous H, hydrolysis, purification and characterization of the product, respectively, as follows: A yeast called NRRL Y-2448 is cultured in a medium with oxygen and a limited nitrogen source, using D-gluc〇se as a carbon source and providing excess orthophosphate (ortho- Phosphate) will produce extracellular phosphate mannan (ph〇Sph〇mannan 'PS)' which contains a variety of polysaccharide structures and serves as a semi-synthesis basic material for the active constituents of the pharmaceutical composition of the present invention. The acid-loaded mannan is composed of a phosphomannan core (PC) which is branched twice and has a high molecular weight (about 5 χ 1G6~39 X丨(10)(10)). p.妒/幼). Culture, fermentation method and mannose phosphate red separation impurity A; pittsley, LE; (10), pr; Dimler, RJ Arch. Biochem Bi〇phys 1961, 92, 343_35 and this _, r K.; Kaczorowski, G. J.; Weise, MJ Biochemistry 1973, 12, 1251-1256. In general, the silk is about 働L. The fermentation scale can produce 2 〇 kg of the crude phosphate mannan product. In a preferred embodiment, the hydrolysis reaction of phosphomannan is carried out at 1 Torr for 6 to 10 hours, and the concentration of phosphomannan is controlled between 40 and 50. The pH of the hydrolysis environment is between 2.2~2.5 'The hydrolysis is carried out with 1M of HCl solution as catalyst and in the presence of KC1. During the initial enthalpy of the hydrolysis reaction, the pH will increase slightly to about 3 when the phosphate mannan is gradually mixed with the solution. Therefore, it is necessary to add 1Μ2Ηα solution to return the acid value to the ΡΗ2·2.5~2.5 umbrella under hydrolysis reaction. The product is about 2.5 a kg (weight). The product after hydrolysis includes the lower molecular weight oligosaccharide. The hydrolyzate was neutralized with a solution of 1 MiNa〇H to adjust the pH to pH 9 to 9.5. 201247208 In a preferred embodiment, 'solution preparation for hydrolysis is 600 g of KCl dissolved in 60 L of water, 1 M of HCl solution is added to adjust the pH to pH 2.4, and the solution is placed in 75 L of stainless steel. In the container. The crude product of mannomannan obtained by fermentation (wet weight 2.49 kg) is added to the solution in portions through the addition port of the reaction vessel, and the mixed solution is heated to 10 ° C to maintain 7 under vigorous stirring. hour. The pH of the reaction environment was measured every hour, and 1 M of HCl was added 1 hour after the start of the reaction to adjust the acid value back to pH 2.3. After the hydrolysis reaction was terminated, the hydrolyzate was cooled to room temperature and 1 M NaOH was added to adjust the pH to pH 9.5. The purification step after the hydrolysis reaction is carried out by ultrafiltration of the oligosaccharide phosphate fraction in the hydrolyzed product by using a molecular pore size of off membrane (NMWCO membrane) having a molecular pore size of 1 Å. The unphosphorylated oligosaccharides and salts will pass through the filter, while the higher molecular weight phosphomannan core will remain on the filter', thereby allowing the oligosaccharide phosphate fraction and the unphosphorylated oligosaccharide and phosphate mannose. The glycan core phase is separated. The separation process described above is accomplished in a diafiltration mode with a tangential or cross flow filtration system that can easily increase throughput by increasing the available filter area and flow rate. The molecular pore size of the filter membrane is preferably between 3 and , to 100,000 Dalton, and the best is l〇, 〇〇〇Dalton. In a preferred embodiment, the hydrolysate is diluted to 7 〇L and placed in an i5 〇L stainless steel tank, followed by two successive Sartorius Hydrosart 10K (NMWCO 10,000) membranes (each filter, membrane enthalpy) The ultrafiltration system consisting of a filter membrane area of 0.6 m2) is percolated with eight times the percolation volume of pure water. The parameter of the aforementioned percolation is set as: the inlet pressure is 200 kPa' The 排气utlet pressure is i5〇kpa, and the retentate that is retained by the filter membrane during the percolation process ( The flow rate of the retentate (cr〇ss fl〇w rate) is 15.6 L/min, and the flow rate of the permeate is 66 to π L/h/m 2 (16 to 2 rc). The permeated/filtered permeate (630 L, conductivity 1 1 ms/cm) was divided into six batches for subsequent ion exchange chromatography (ion-exchange chromatography^. Further, the above-mentioned filtrate was subjected to two Secondary purification, secondary purification by ion exchange chromatography. In a preferred embodiment, the ion exchange chromatography method first uses a 3 〇 DE DE-Spherilose column with a 0.01 M solution at a flow rate of 丨 5 L / _ under the balance 'then the previous filtrate (each time about L, a total of six times) from the upper end of the column plus 201247208
入，以0.01M之NH4HC〇3溶液進行沖提直到流出物(effluent)之導電度 在0.2 mS / cm之基線以内時，中性級分(neutrai丘3出〇11)則被沖提出。 接下來使用0.25M之NH4HC〇3溶液將寡醣磷酸鹽級分沖提出，收集 比之流出物進行高效液相層析儀(111>1^)分析。將前述六次沖提所得之 適當級分結合並以逆滲透法濃縮至2〇L同時去除鹽類。逆滲透係在滲 濾模式下持續進行直到濾出物之導電度小於等於〇2 mS / cm，溶液再 濃縮至最終的6 L。所得產物進行凍乾法(iy〇phmzati〇n)後，當中的寡醣 磷酸鹽級分將以白色、具吸濕性之粉末呈現，約566克。產物經HpLC 檢測後純度約93%，足以在不須進一步純化的情況下用於本案醫藥組 成物之製備。' 前述之逆滲透步驟可將大量水解產物(每次水解約5〇〜6〇 L)快速地 減少至後續凍乾法可處理之程度，同時也是後續提高產量的關鍵步 驟。此外，逆滲透步驟還能移除級分中大量的無機鹽類，從而得到僅 含有少許無機鹽類的高純度終產物^ 最後’將前述由酵母菌户/c/wa NRRL Y-2448經培養、發酵、 水解及純化後所付之鱗酸甘露聚醣產物進行續化(sulf〇nati〇n)，以得到 本案醫藥組成物之活性成分。首先將前述純化所得之碳酸甘露聚醣產 物478克與10 L之二曱基甲醯胺(Dimethylformamide，DMF)混和，並 加入3.78公斤之三氧化硫-吡啶複合物（如版tri〇xide pyridine-complex) ’在25°C下攪拌混合三天，屆時產物將以厚實的油狀 (thick oil)物被分離出來。將二甲基甲醯胺抽乾，並以i [的乙醇清洗 殘餘物三次後將之溶於約3L水中。接著在該溶液中加入1M之Na〇H 溶液約5L以將酸鹼值調整至PH 9.5，以3L之二氣甲烧 (Dichloromethane)進行萃取三次以將釋出之吡啶移除。水相(aque〇us phase)部分以水進行稀釋並通過炭過渡(charcoal filter，CunoR53S)以脫 色。將溶液以水稀釋至20L並以八倍體積之1M NaCl溶液進行滲滤， 再以純水渗遽至滤出物之導電度小於0.2 mS / cm。最後將該溶液以逆 滲透法濃縮至6L，並以孔徑〇.2μιη濾膜進行過濾，經凍乾處理後得到 白色、具吸濕性 '無定形之固體物760克，為本案醫藥組成物之活性 成分° 201247208 本案醫藥組成物之確認 本案醫藥組成物之活性成分所含之組分係經由液相層析/電灑法-傅立葉轉換質譜儀(Liquid Chromatography / Electrospray Ionization -The aliquot (neutrai mound 3 out 〇 11) was flushed out with a 0.01 M NH4HC 〇 3 solution until the conductivity of the effluent was within the baseline of 0.2 mS / cm. Next, the oligosaccharide phosphate fraction was punched out using a 0.25 M NH4HC® solution, and the effluent was collected for high performance liquid chromatography (111 > 1 ) analysis. The appropriate fractions obtained by the above six flushing were combined and concentrated to 2 〇L by reverse osmosis to remove salts. The reverse osmosis system was continued in the percolation mode until the conductivity of the filtrate was less than or equal to 〇2 mS / cm, and the solution was concentrated again to the final 6 L. After the resulting product is subjected to lyophilization (iy〇phmzati〇n), the oligosaccharide phosphate fraction will be present in a white, hygroscopic powder, about 566 grams. The product was about 93% pure by HpLC and was sufficient for the preparation of the pharmaceutical composition of the present invention without further purification. The reverse osmosis step described above can rapidly reduce a large amount of hydrolyzed product (about 5 〇 to 6 〇 L per hydrolysis) to the extent that it can be treated by subsequent lyophilization, and is also a key step for subsequent increase in yield. In addition, the reverse osmosis step can also remove a large amount of inorganic salts in the fraction, thereby obtaining a high-purity end product containing only a small amount of inorganic salts. Finally, the above-mentioned yeast/c/wa NRRL Y-2448 is cultured. The phytate mannan product obtained after fermentation, hydrolysis and purification is continuously sulfonated to obtain the active ingredient of the pharmaceutical composition of the present invention. First, 478 g of the above-mentioned purified mannan carbonate product was mixed with 10 L of Dimethylformamide (DMF), and 3.78 kg of sulfur trioxide-pyridine complex (such as tri〇xide pyridine-) was added. Complex) 'Agitate and mix at 25 ° C for three days, at which time the product will be separated as a thick oil. The dimethylformamide was drained and the residue was washed three times with i [ethanol] and dissolved in about 3 L of water. Next, about 5 L of a 1 M Na〇H solution was added to the solution to adjust the pH to pH 9.5, and extraction was carried out three times with 3 L of dichloromethane to remove the released pyridine. The aque〇us phase portion was diluted with water and decolored by a charcoal filter (CunoR53S). The solution was diluted with water to 20 L and diafiltered with eight volumes of 1 M NaCl solution, and then permeated with pure water to a filtrate having a conductivity of less than 0.2 mS / cm. Finally, the solution was concentrated to 6 L by reverse osmosis method, and filtered with a pore size of 2.2 μιη filter, and lyophilized to obtain 760 g of a white, hygroscopic 'amorphous solid matter, which is a pharmaceutical composition of the present invention. Active ingredient ° 201247208 Confirmation of the pharmaceutical composition of the case The active ingredient of the pharmaceutical composition of the present invention is contained in a liquid chromatography/electrospray ionization-electrospray ionization mass spectrometer (Liquid Chromatography / Electrospray Ionization -
Fourier transform mass spectrometry，LC/ESI-FTMS)進行分析與確認， 其檢測分析條件描述如後。 高效能液相層析(high performance liquid chromatography，HPLC)部 分之條件設定：高效能液相層析系統為Shimadzu LC-20ATvp Shimadzu LC-20ADvp pumps，ShimadzuSIL-20AC 自動注射器(Autosampler)以及 Shimadzu SCL-20A System Controller;紫外線檢測器(UV Detector)為 Shimadzu SPD-20AV Detector 並設定於波長 280 nm ;資料系統(Data System)為Xcalibur 2.0.7 ;高效能液相層析管柱係使用ACE3, C18-AR， 4.6 X 150 mm ;保護管柱(guard column)係使用 ACE3, C18, 3·0μηι，3.2 X 10mm ;管柱溫度為周圍環境溫度；自動注射器溫度為4〇c ;移動相a (mobile phase A)為含有 5mM 二丁基醋酸敍(dibutyl ammonium acetate) 且由水·甲醇體積比8:2所混合配置之溶液；移動相b (mobile phase B) 為含有5mM二丁基醋酸敍(dibutyl ammonium acetate)且由水:甲醇體積 比1:9所混合配置之溶液。沖提梯度表則如下表丨所示： 色1、高效能液相層析所使用之沖提梯度表(Gradient Table)。 時間(分鐘） 流速(毫升/分鐘） 移動相A比例 移動相B比例 0 0.7 70% 30% 40 0.7 0 100% 45 0.7 0 100% 47 0.7 70% 30% 62 0.7 70% 30% 質J普儀部分之條件設定為:質譜儀係採用The腦LTQ 〇—叩 XL，資料系統(Data System)為 Xcalibur 2.0.7 ;離子化模式(IonizationFourier transform mass spectrometry, LC/ESI-FTMS) was used for analysis and confirmation, and the detection and analysis conditions are described as follows. High performance liquid chromatography (HPLC) part of the conditions: high performance liquid chromatography system is Shimadzu LC-20ATvp Shimadzu LC-20ADvp pumps, Shimadzu SIL-20AC autoinjector (Autosampler) and Shimadzu SCL-20A System Controller; UV Detector is Shimadzu SPD-20AV Detector and set at 280 nm; Data System is Xcalibur 2.0.7; High Performance LC Column is ACE3, C18-AR , 4.6 X 150 mm; guard column is ACE3, C18, 3·0μηι, 3.2 X 10mm; column temperature is ambient temperature; autoinjector temperature is 4〇c; mobile phase a (mobile phase A ) is a solution containing 5 mM dibutyl ammonium acetate and mixed by a water-methanol volume ratio of 8:2; mobile phase B is 5 mM dibutyl ammonium acetate And a solution prepared by mixing water: methanol volume ratio 1:9. The flushing gradient table is shown in the following table: Color 1. The Gradient Table used for high performance liquid chromatography. Time (minutes) Flow rate (ml/min) Mobile phase A proportional movement phase B ratio 0 0.7 70% 30% 40 0.7 0 100% 45 0.7 0 100% 47 0.7 70% 30% 62 0.7 70% 30% Some of the conditions are set as follows: The mass spectrometer uses The brain LTQ 〇-叩XL, the data system (Data System) is Xcalibur 2.0.7; Ionization mode (Ionization
Mode)為魏法之貞科赋；料賴電壓(lQn Spray v麵e)為 201247208 4.5kV ’ 毛細管溫度(Capillary Temperature)為 350°C，毛細管電壓 (Capillary Voltage)為-18V ;管透鏡(Tube Lens)為-100V，鞘流氣體流速 (Sheath Gas How)為 60 單位，輔助氣體流速(Auxiliary Gas Flow)為 30 單位’掃流氣體流速(Sweep Gas Flow)為5單位；碰撞氣體為氦氣 (Helium)。 經前述方法分析後’本案醫藥組成物之活性成分所含之組分呈現如 下表2。 表2、本案醫藥组成物之活性成分所含组分及其結構、分子式及分子量。Mode) is the method of Weifa; the material voltage (lQn Spray v surface e) is 201247208 4.5kV 'Capillary temperature is 350 ° C, capillary voltage is -18V; tube lens (Tube Lens) For -100V, the Sheath Gas How is 60 units, the Auxiliary Gas Flow is 30 units, the Sweep Gas Flow is 5 units, and the collision gas is Helium. . The components contained in the active ingredients of the pharmaceutical composition of the present invention after analysis by the foregoing method are as shown in Table 2 below. Table 2. The components contained in the active ingredients of the pharmaceutical composition of the present invention and their structures, molecular formulas and molecular weights.
如表2所示，本案醫藥組成物之活性成分在排除同分異構物的情況 下，包含組分1至組分8共八種組分。其基礎結構係為由二至五個甘 露酿(mannose)單元透過1—3及/或1—>2糖普鍵(glycosidicbond)所連接 而成的寡醣分子。進—步1•之，除與末麟單元係透過丨―2糖苦鍵連 接外’其餘醣單元㈣透過丨—3㈣鍵相連接^若對應表i中之結構 通式’當n=0時即代表雙醣、n=1時代表三酿、n=2時代表四聽而㈣ 時即代表五醣。 。一依據削述之基礎結構，本案醫藥組成物活性成分之各組分在首個醣 單元的6號碳(C6)位置上之經基(〇H gr0Up),其η被取代為p〇sH2或其 鹽類’其餘3n+7個經基中，則有3n+6或3n+7個經基其H被取 基(S〇3H)或其鹽類。若對應表2中之結構通式，其中組分丨、組分3二 201247208 、、且刀5及”且:7係、分別為有3n+6個經基其H被被取代為奶识或其鹽 類之雙醣、三酿、瞒及五酿，亦即除首個醣單元的6號碳㈣位置上 之輕基外’僅有-經基未被s〇3H或其鹽類所取代；而組分2、組分4、 、’且刀6及8係分別為有3n+7個經基其η被取代為s〇3H或其鹽類 之雙酿、三醣、四酿及五酿，亦即除首個醣單元的6號碳㈣位置上之 羥基外，所有羥基皆被取代為s〇3H或其鹽類。 第1圖係本案醫藥組成物之活性成分經^㈣不丁奶分析之總離 子層析圖(Total i〇n Chr0mat0gram ’ TIC)。如第i圖所示，本案醫藥組 成物之活性成分中，組分8及組分6係為主要之組分，亦即通式⑴化 合物t ’η為2或3对3n+7個R代表s〇3H者係為主要組分。此外， 第1圖亦顯示本案醫藥組成物之活性成分中，組分8之相對含量為最 高，亦即通式(I)化合物巾，n為3且有3n+7個R代表s〇3H 含量為最高。 由第1圖亦可得知，本案醫藥組成物之活性成分中，除組分丨至組 分8外，亦可能包含其他成分，如滞留時間丨86分鐘、9 33分鐘、75 分鐘及40.53分鐘所對應者。 依據前述LC/ESI-FTMS之總離子層析圖分析結果所得之各組分數 據’包括峰頂所對應之滞留時間(Apex Retention Time)、波峰面積、波 峰面積所佔比例、波峰高度及波峰高度所佔比例如下表3所示。/ 案醫藥组成物經LC/ESI-FTMS分析後各组分之相Μ务坤 名稱 峰頂所對應之 滯留時間(分鐘） •波峰面積 波峰面積 所佔比例 波峰高度 波峰高度 所佔比例 組分1 12.31 699406 0.09% 25711 0.1% 組分2 16.17 21279900 2.89% 422185 1.72% 組分3 20.55 10764686 1.46% 159934 0.65% 組分4 23.57 50971043 6.92% 942431 3.84% 組分5 25.62 30026265 4.08% 477234 1.95% 組分6 28.05 249954793 33.94% 9663862 39.39% 組分7 29.38 17374916 2.36% 532147 2.17% 組分8 31.36 355469535 48.26% 12308532 50.17% 由表3數據中各組分波峰面積所佔比例亦可得知，本案醫藥組成物 之活性成分中’組分8及組分6係為主要之組分，該二組分波峰面積 12 201247208 所佔比例總合為82.2%，亦即通式(i)化合物中η為2或3且有3n+7個 R代表S〇3H者係為主要組分β此外，表3亦顯示本案醫藥組成物之活 性成分中，組分8之相對含量為最高，其波峰面積所佔比例為48 26%， 亦即通式(I)化合物中η為3且有3η+7個R代表SChH者之相對含量為 最高。若以結構中羥基被取代為磺基(s〇3H)之情形加以區隔，由表3 之分析結果亦可得知，通式①化合物中，除首個醣單元的6號碳(C6) 位置上之羥基外，其餘3n+7個羥基皆完全被取代為磺基者，即組分2、 組分4、組分6及組分8 ,其波峰面積加總所佔之比例為92 〇1% :而通 式(I)化合物中，其3n+7個羥基中有3n+6個羥基被取代為磺基者，即 組分1、組分3、組分5及組分7，其波峰面積加總所佔之比例為7 99%， 足見本案醫藥組成物之活性成分中，係以3n+7個羥基皆完全被取代為 續基者為主要組分。 本案醫藥組成物之用途 本案之醫藥組成物目前係用於作為肝癌治療過程中之輔助療法，於 一較佳實施例中，本案之醫藥組成物係用於抑制肝癌復發、惡化或轉 移^再進一步言之，本案醫藥組成物之臨床功效係在於延長^癌患者 術後復發所需之時間，或降册癌患者之術後復發率。町係針對本 案醫藥組成物之適用病症、藥理作用、有效劑量、使用方法、藥理試 驗方法及結果進行實關，赠明本錄藥組成物之實際醫藥用 途。 、/、 *本案醫藥組成物之適用病症係為肝癌(LiverCancer)，其可與栓塞、 標f藥物治療、化學治療、放射練妍臟切除手術等轉方式中之 至少-種合併用於抑制肝癌之復發、惡化或轉移。於—較佳實施例中， 本案醫藥組成物係適用於接受肝臟切除手術(hepatect〇my)後之肝癌患 者°本案醫藥組成物之藥理侧係透過抑制類肝素酶及血管生長因子 ^活性’而抑制肝腫瘤轉移與肝腫瘤血管新生，以達琳卩制肝癌復發、 心化或轉移的效果。於—較佳實施例中，本案醫藥組成物之安全性、 初步有效性及有效劑量已透過人體臨床試驗而確認，且可有效延長肝 13 201247208 癌患者術後復發所^時間，或降低肝癌患者之碰㈣率。該人體 臨床:式驗方法係為_多試驗機構(—_、隨機分派(—冲 且平行試驗形式(parallel_gr0Up)之臨床試驗設計。 本案醫藥組成物於臨床使用時之給藥途徑係採注射方式，因其水溶 性高’因此可以水為溶劑。可使用的注射溶劑包括但不限於滅菌水、 以滅菌水為溶_生理食财萄糖水溶液等，此外亦不排除其他 油性溶劑。於-難實_巾，雜料_生理食鹽水(n_al沾丨㈣ 作為溶劑。 本，醫藥組成物係g&製為單位劑量之形式以便於細及劑量的均 一化。每單位劑量包含了定量的活性成分，該活性成分與-醫藥上可 接受之稀_、賦糊或載航合時，可提供麵的治療效果。 本案醫藥組成物之活性成分於水中之溶解度大於·^·，因此 160mg本案醫藥組成物之活性成分可完全溶解於〇4m丨水中。於一較 佳實施例中，每-劑藥物中係包含犯邮本案醫藥組成物之活性成分 及=1之生理食鹽水，混和溶解後之濃度為2〇〇爪咖丨，從中取〇 8池 進行注射’等同於本案冑藥組成物之活性成分含量為i6〇吨。 臨床試驗所使用之療效及安全分析群體(efficacy / safcty popular) 為意圖治療(Intent-t0-treat，ITT)分析群體，意圖治療分析群體之定義為 所有具試师格且賴齡派之钱者均取分析，_未服用任何 劑试驗用藥或在隨機分派後未具有任何紀錄者則被排除於分析之 外。 前述人體臨床試驗雜172名触過賴赚手術之肝癌患者隨 機分，三組’-組為未投藥之無臨床處置對照組(58人）…組為每日投 =劑罝為16Gmg本案醫藥組成紐性成分之試敝(57人），另一組為 每日投予継為25Gmg本案鶴組成物活性齡之試驗組(57人)’。試 驗組之受試者在接受賴嫌手織之4到6测始接受投藥，投藥 以4週為-個循環，在每個循環的前3週當中，每週連續4天以皮下 注射方式給藥，每個循環的第4週則不投藥。連續進行9個循環(址％ 週)後’接著12週的追蹤期_0评哪peri〇d)。兩組試驗组之受試者在 36週的投藥期間’每隔4週進行例行的檢查與追蹤，在後續12週的追 201247208 蹤期間則每隔6週檢查一次。對照組之受試者不投予對照藥物或安慰 劑，且對照組之受試者在總計48週的受試期間内，固定每隔6週進行 一次例行檢查與追蹤。 在每個循環開始前，受試者須進行醫病史的確認、理學檢查 (physical examination)、合併用藥(concomitant medication)情形確認，以 及包括α胎兒蛋白(α-fetoprotein，AFP)在内的例行實驗檢查。每月對所 有受試者進行腹部超音波檢測。在第4個循環及第7個循環的首日進 行腹部斷層掃瞄，另外若受試者被懷疑有肝癌復發的情況時亦須進行 腹部斷層掃瞄。胸部X光檢查、骨骼掃描及腦部斷層掃描亦於第4個 循環及第7個循環的首日進行以監測肝臟外的腫瘤復發情形 (extra-hepatic tumor recurrence) ° 血清中的α胎兒蛋白及腹部超音波檢查係例行地作為肝臟内腫瘤 復發之初步評估依據。當α胎兒蛋白濃度升高或腹部超音波檢查結果 顯示可能有新的肝腫瘤形成時，則須進行腹部斷層掃瞄檢查復發腫瘤 之血官結構。一旦該腫瘤在斷層掃晦顯影之動脈期(arterjal phase)顯示 出典型之多血管性(hypervascular)，且顯影劑(contraSt medium)於靜脈期 (venous phase)快速地被沖洗掉時，則該病灶即被定義為肝癌之復發 (HCC recurrence)。若受試者之病灶已接受過組織切片檢查或外科切除 手術’則肝癌之復發亦可透過組織學檢查來確認。 前述人體臨床試驗之主要療效試驗指標⑦加町efficacy endp〇int) 係為試驗終止時肝癌之未復發率(non_recurrence rate 〇f HCC)。而次要療 效試驗指標(secondary efficacy endpoint)為首次復發所需時間(time吣 first recurrence)，首次復發所需時間之計算係從隨機分派之日起，至透 過電腦斷層掃瞄或組織學檢查確認腫瘤復發之日止。 試驗結果之數據如下’龍組58人巾共58人為可納人資料分析 之思圖治療分析群體’其中26人於試驗結束時已復發肝癌，約佔45%， 其中29人於試驗絲時未復發職，約佔5()%，其巾3人於試驗中途 退出’約佔5%。投予劑量為每日16〇吨本案醫藥組成物活性 ^分^驗組57人中共56人為可納人資料分析之意圖治療分析群 體，其中16人於試驗結束時已復發肝癌，約佔29%，其中％人於試 201247208 的二〇。時未復發肝癌，約佔63%，其中5人於試驗中途退出(dr〇p〇ut)， S7人/〇〇投予劑量為每日25〇mg本案醫藥組成物活性成分之試驗組 λ 共54人為可納入資料分析之意圖治療分析群體，其中21人於 ^驗結束時已復發肝癌，約佔39%，其巾22人於試驗結束時未復發肝 ； 41 4其中11人於试驗中途退出(dropout),約佔20%。 第2圖係試驗終止時’對照組、投予劑量為每曰i60mg本案醫藥 2物活性成分之試驗組及投予劑量為每日25Gmg本案醫藥組成物活 =刀之試驗組之钱者群體巾’未復衡癌人數及巾賴出試驗人 比f °參閱第2圖’投予劑量為每日i6Gmg本案醫藥組成物 JL去^之試驗組’其未復發肝癌之比率為63%，而未投藥之對照組 其未復發肝癌之比率為5〇%，依統計方法判斷為具有差異(p = 〇 亦即本案醫藥組成物在活性成分劑量為每日⑽叫以皮下注射方式對 ΐίΪΓ臟姆手術之肝癌患者進行週雛投藥，能夠降低患者之肝 癌復發率。 t述試麟果賴^投㈣量躲日25Qmg本締齡成物活 = 驗組，其與未投藥之對照組相較之下，並無顯著降低患者 肝癌復發率之效果。 第3 @係比較制組與好絲日16()mg本案醫藥組成物活 性成分之3式驗、组’隨試驗之進行(〇至48週），未復發肝癌人數佔該組 比τ之變化情形’其中虛線係表示兩組受試者群體從試驗開 始至達到7G%受試者未復肝癌所需之時間。參閱第3目，對昭典 試者群體自試驗開始至_ 7G%受試者未復肝觸糾縣27週二 每日投予劑#為16Gmg本案«_物活性齡找敵受 體’其自試驗開始至_ 70%受試者未復肝癌所需時間為48週 結果顯不’在本實_巾’本錢藥組成物在活性成㈣量 160mg^皮财績接錢㈣姆手狀職患者進行週 投藥，旎夠顯著延長患者肝癌復發所需之時間。 此外，另有臨床實驗數據顯示，每日投予劑量為8〇呵本案 成物活性成分予受試者時，仍有抑麵_大的效果^而當每^ 劑量為315mg本案醫藥組成物活性成分予受試者時，可能產生嚴^血 201247208 小板低下(severe thrombocytopenia)之情形。 以上所提僅是本案的較佳實施例樣態，並不是用於限定本案的實施 範圍；任何在此領域具有通常知識者，在不脫離本案的精神與範圍下 所作的諸般變化與修飾，都不脫如附申請專利範圍所欲保護者。 【圖式簡單說明】 第1圖係本案醫藥組成物之活性成分經LC/ESI-FTMS分析之總離 子層析圖(Total ion chromatogram，TIC) 〇 第2圖係試驗終止時，對照組、投予劑量為每曰i6〇mg本案醫藥 組成物活性成分之試驗組及投予劑量為每日250mg本案醫藥組成物活 性成分之試驗組之受試者群體中，未復發肝癌人數及中途退出試驗人 數所佔之比率。 第3圖係比較對照組與投予劑量為每日i6〇mg本案醫藥組成物活 性成分之試驗組，隨試驗之進行(〇至48週），未復發肝癌人數佔該組受 試總人數比率之變化情形。 【主要元件符號說明】 益 17As shown in Table 2, the active ingredient of the pharmaceutical composition of the present invention contains eight components of Component 1 to Component 8 in the case of excluding the isomer. The basic structure is an oligosaccharide molecule which is formed by two to five mannose units connected by 1-3 and/or 1 -> 2 glycosidic bonds. In the first step, except for the connection with the end of the unit cell through the 丨2-saccharide bond, the remaining sugar units (4) are connected through the 丨-3 (four) bond ^ if the corresponding structural formula in the table i is 'when n=0 That is, it represents disaccharide, when n=1, it means three brewing, when n=2, it means four, and (4), it means five sugar. . According to the basic structure of the description, the components of the active constituents of the pharmaceutical composition of the present invention are substituted at the 6th carbon (C6) position of the first sugar unit (〇H gr0Up), and η is substituted with p〇sH2 or The salt of the remaining 3n+7 radicals has 3n+6 or 3n+7 groups based on its H group (S〇3H) or its salts. Corresponding to the structural formula in Table 2, wherein component 丨, component 3 2 201247208, and knife 5 and "and: 7 series, respectively, have 3n + 6 radicals, H is replaced by milk or The salt of the disaccharide, the third brewing, the glutinous rice and the five brewing, that is, except for the light base of the No. 6 carbon (4) position of the first sugar unit, the 'only-trans-base group is not replaced by s〇3H or its salts. And component 2, component 4, and 'and knives 6 and 8 are respectively 3n+7 bases, η is substituted with s〇3H or its salts, double brewed, trisaccharide, four brewed and five Brewing, that is, except for the hydroxyl group at the carbon (4) position of the first sugar unit, all the hydroxyl groups are substituted with s〇3H or its salts. Figure 1 shows the active ingredients of the pharmaceutical composition in this case. Total ion chromatogram of milk analysis (Total i〇n Chr0mat0gram ' TIC). As shown in Figure i, among the active ingredients of the pharmaceutical composition of this case, component 8 and component 6 are the main components, ie The compound t'η of the formula (1) is 2 or 3 pairs of 3n+7 R represents s〇3H as the main component. In addition, Fig. 1 also shows the relative content of the component 8 in the active ingredient of the pharmaceutical composition of the present invention. Highest (I) compound towel, n is 3 and 3n+7 R represents the highest content of s〇3H. It can also be seen from Fig. 1 that the active ingredient of the pharmaceutical composition of the present invention, except component 丨 to component 8 In addition, other components may be included, such as retention time 丨86 minutes, 913 minutes, 75 minutes, and 40.53 minutes. According to the results of the above LC/ESI-FTMS total ion chromatogram analysis, the data of each component' The proportion of the Apex Retention Time, the peak area, the proportion of the peak area, the height of the peak and the height of the peak including the peak top are shown in Table 3. The composition of the pharmaceutical composition was analyzed by LC/ESI-FTMS. The residence time of each component relative to the peak of the name of the Kun Kun (minutes) • The proportion of the peak area of the peak area. The peak height of the peak is the proportion of the component 1. 12.31 699406 0.09% 25711 0.1% Component 2 16.17 21279900 2.89 % 422185 1.72% Component 3 20.55 10764686 1.46% 159934 0.65% Component 4 23.57 50971043 6.92% 942431 3.84% Component 5 25.62 30026265 4.08% 477234 1.95% Component 6 28.05 249954793 33.94% 9663862 39.39% Component 7 29.38 17374916 2.36% 532147 2.17% Component 8 31.36 355469535 48.26% 12308532 50.17% It is also known from the proportion of the peak area of each component in the data in Table 3 that the active components of the pharmaceutical composition of this case are 'component 8 and component 6 As the main component, the ratio of the two-component peak area 12 201247208 is 82.2%, that is, the formula (i) has η of 2 or 3 and 3n+7 R represents S〇3H. In addition, Table 3 also shows that among the active ingredients of the pharmaceutical composition of the present case, the relative content of the component 8 is the highest, and the proportion of the peak area is 48 26%, that is, η in the compound of the general formula (I). The relative content of 3 and 3η+7 R represents SChH is the highest. If the hydroxy group in the structure is substituted with a sulfo group (s〇3H), it can be known from the analysis results in Table 3 that the compound of the formula 1 has the carbon No. 6 (C6) except the first sugar unit. In addition to the hydroxyl groups in the position, the remaining 3n+7 hydroxyl groups are completely substituted with sulfo groups, ie, component 2, component 4, component 6 and component 8, and the total peak area is 92 〇. 1% : and in the compound of the formula (I), 3n+6 of the 3n+7 hydroxyl groups are substituted with a sulfo group, ie, component 1, component 3, component 5 and component 7, The proportion of the peak area is 799%, which shows that the active ingredients of the pharmaceutical composition of this case are mainly composed of 3n+7 hydroxyl groups which are completely replaced by the continuation base. Use of the pharmaceutical composition of the present invention The pharmaceutical composition of the present invention is currently used as an adjuvant therapy in the treatment of liver cancer. In a preferred embodiment, the pharmaceutical composition of the present invention is for inhibiting recurrence, deterioration or metastasis of liver cancer. In other words, the clinical efficacy of the pharmaceutical composition of this case is to prolong the time required for postoperative recurrence of cancer patients, or the postoperative recurrence rate of patients with reduced cancer. The Department of Health conducts practical measures on the applicable conditions, pharmacological effects, effective doses, methods of use, pharmacological test methods and results of the pharmaceutical composition of the case, and presents the actual medical use of the recorded drug composition. , /, * The applicable disease of the pharmaceutical composition of this case is liver cancer (LiverCancer), which can be combined with at least one of embolization, drug treatment, chemotherapy, radiotherapy and sputum resection, etc. Recurrence, deterioration or metastasis. In a preferred embodiment, the pharmaceutical composition of the present invention is suitable for liver cancer patients after hepatectectomy (hepatect〇my). The pharmacological side of the pharmaceutical composition of the present invention inhibits heparanase and angiogenic factor activity by Inhibition of liver tumor metastasis and hepatic tumor angiogenesis, to achieve the effect of recurrence, cardiacization or metastasis of liver cancer. In the preferred embodiment, the safety, initial effectiveness and effective dose of the pharmaceutical composition of the present invention have been confirmed by human clinical trials, and can effectively prolong the time of recurrence of liver cancer 2012 2012208208 cancer patients, or reduce liver cancer patients Touch (four) rate. The human clinical: the test method is _ multi-test institutions (-_, random assignment (-rush and parallel test form (parallel_gr0Up) clinical trial design. The drug composition in this case is used in clinical use. Because of its high water solubility, it can be used as a solvent. The injectable solvents that can be used include, but are not limited to, sterilized water, sterilized water, _ physiological sugar, aqueous solution, etc., and other oily solvents are not excluded. _ towel, miscellaneous materials _ physiological saline (n_al 丨 丨 (4) as a solvent. The pharmaceutical composition g & is made in the form of a unit dose to facilitate the uniformity of the fine and the dose. Each unit dose contains a quantitative amount of active ingredients The active ingredient can provide a therapeutic effect on the surface when it is pharmaceutically acceptable, fused, or loaded. The solubility of the active ingredient of the pharmaceutical composition in water is greater than that of the solution, so 160 mg of the drug composition of the case The active ingredient of the substance can be completely dissolved in 〇4m 丨 water. In a preferred embodiment, each of the medicines comprises the active ingredient of the medicinal composition of the ruthenium case and the physiological saline solution of =1. The concentration after mixing and dissolving is 2 〇〇 claw curry, and 8 pools are taken for injection. The equivalent of the active ingredient content of the peony composition in this case is i6 ton. The efficacy and safety analysis group used in clinical trials (efficacy) / safcty popular) For the Intent-t0-treat (ITT) analysis group, the definition of the intended treatment analysis group is the analysis of all the testers and the money of the aging age, _ not taking any dose of the test medication Or if there is no record after random distribution, it is excluded from the analysis. The above-mentioned human clinical trials have 172 patients with liver cancer who have been exposed to Lai’s surgery, and the three groups are untreated. (58 persons)... The group is the daily dose = 16Gmg test for the drug composition of the case (57 persons), and the other group is the test group for the active age of the crane composition of 25Gmg per day. 57 people)'. The subjects in the experimental group received the drug for 4 to 6 tests, and the drug was administered for 4 weeks. In the first 3 weeks of each cycle, for 4 consecutive days per week. Subcutaneous injection, week 4 of each cycle Do not administer the drug. After 9 cycles (% of the week), 'following the 12-week follow-up period _0, which peri〇d.) The subjects in the two groups of trials were administered every 4 weeks during the 36-week administration period. Routine examinations and follow-ups were performed every 6 weeks during the following 12 weeks of follow-up 201247208. Subjects in the control group were not administered a control drug or placebo, and subjects in the control group were 48 weeks in total. During the test period, regular routine examinations and follow-ups were performed every 6 weeks. Before each cycle, subjects were required to confirm medical history, physical examination, and concomitant medication. Confirmation, as well as routine laboratory tests including alpha-fetoprotein (AFP). Abdominal ultrasound testing was performed on all subjects monthly. Abdominal tomography scans were performed on the first day of the fourth and seventh cycles, and abdominal tomography scans were also performed if the subject was suspected of having recurrence of liver cancer. Chest X-ray, bone scan and brain tomography were also performed on the first day of the 4th and 7th cycles to monitor extra-hepatic tumor recurrence ° serum alpha-fetoprotein and Abdominal ultrasonography is routinely used as a basis for the initial assessment of tumor recurrence in the liver. When the alpha fetal protein concentration is elevated or the abdominal ultrasound examination results indicate that a new liver tumor may form, a abdominal tomography scan is performed to examine the blood structure of the recurrent tumor. Once the tumor shows typical hypervascularity in the arterjal phase of the tomographic scan, and the developer (the contraSt medium) is rapidly washed away in the venous phase, the lesion is It is defined as the recurrence of liver cancer (HCC recurrence). If the subject's lesion has been subjected to a biopsy or surgical resection, the recurrence of liver cancer can also be confirmed by histological examination. The main efficacy test index of the aforementioned human clinical trials is the non-recurrence rate (〇f HCC) of the liver cancer at the end of the trial. The secondary efficacy endpoint is the time to first recurrence. The time required for the first relapse is calculated from the date of randomization to confirmation by computerized tomography or histological examination. The day of tumor recurrence. The data of the test results are as follows: 'Long group 58 people towel a total of 58 people for the analysis of the data analysis of the Canna people's data analysis' 26 of them have relapsed liver cancer at the end of the trial, accounting for about 45%, of which 29 were not tested Recurrence, accounting for about 5 ()%, the towel of 3 people in the middle of the trial exit 'about 5%. The dosage was 16 ton per day. The activity of the pharmaceutical composition of the case was divided into 57 groups of 57 people. The total of 56 people were the intentional treatment analysis group of the data analysis. Among them, 16 people had recurred liver cancer at the end of the trial, accounting for 29%. Among them, % of them tried the second of 201247208. There was no recurrence of liver cancer, accounting for about 63%, of which 5 were withdrawn from the trial (dr〇p〇ut), and S7 human/sputum was administered at a dose of 25 mg per day to the experimental group of the active ingredients of the pharmaceutical composition. 54 people were intentive treatment analysis groups that could be included in the data analysis. 21 of them had recurrence of liver cancer at the end of the test, accounting for 39%, and 22 of them had no recurrence of liver at the end of the trial; 41 4 of them were in the middle of the trial. Dropout, about 20%. Figure 2 is the test group at the end of the test, the dose of the active ingredient of 60 mg of the drug in each case, and the dose of 25 Gmg of the drug composition of the test group. 'The number of unreported cancers and the number of people who took out the test were compared with f °. See Figure 2'. The dose of the drug is the daily i6Gmg. The drug composition of the case JL to ^ the test group's rate of recurrence of liver cancer is 63%, but not The ratio of the non-recurring liver cancer in the control group was 5%, which was judged to be different according to the statistical method (p = 〇, that is, the pharmaceutical composition of the case was administered daily at the dose of the active ingredient (10) called subcutaneous injection. The liver cancer patients can be used to reduce the recurrence rate of liver cancer in patients with liver cancer. t The test results are based on the results of the test. The test results are compared with the control group, which is compared with the untreated control group. , did not significantly reduce the effect of recurrence rate of liver cancer in patients. The third @系 comparison group and the good silk day 16 () mg of the active ingredients of the pharmaceutical composition of the 3 test, the group 'with the test (〇 to 48 weeks) The number of non-recurrent liver cancers accounted for the change in the ratio τ of the group 'The dotted line indicates the time required for the two groups of subjects from the start of the trial to reach 7G% of the subjects without liver cancer. See item 3, starting from the trial group to _ 7G% of the subjects Fugan Touche County 27th Tuesday daily dose of agent #16Gmg this case «_ active age to find enemy receptors' from the beginning of the test to _ 70% of the subjects did not return to liver cancer, the time required for 48 weeks 'In this real _ towel' capital compound composition in the active (four) amount of 160mg ^ skin earned money (four) M hand patients for weekly dosing, sputum can significantly extend the time required for patients with liver cancer recurrence. In addition, there is another clinical The experimental data showed that when the daily dose was 8 〇 本 本 本 成 成 成 成 本 , , , , 本 本 本 本 本 本 本 本 本 本 本 本 本 本 而 而 而 而 而 而 而 而 而 而 而 而 而 而 而At the time, there may be a case where the blood is 201247208 small thrombocytopenia. The above is only a preferred embodiment of the present case, and is not intended to limit the scope of implementation of the case; any person having ordinary knowledge in this field Without departing from the spirit and scope of the case All kinds of changes and modifications are not subject to the scope of the patent application. [Simplified illustration] Figure 1 is the total ion chromatogram of the active constituents of the pharmaceutical composition of this case by LC/ESI-FTMS analysis ( Total ion chromatogram, TIC) 〇 Figure 2 At the end of the test, the control group, the dose of each dose of 〇6〇mg of the active ingredient of the pharmaceutical composition of the test group and the dose of 250mg daily active ingredients of the pharmaceutical composition The number of patients with no recurrence of liver cancer and the number of patients who withdrew from the trial in the test group. Figure 3 compares the control group with the dose of the active ingredient of the pharmaceutical composition of the daily dose of i6〇mg. According to the trial (〇 to 48 weeks), the number of patients without recurrence of liver cancer accounted for the change in the ratio of the total number of subjects in the group. [Main component symbol description] Benefit 17