200414903 玖、發明說明 本案請求美國臨時專利申請案60/2 26，94 8，申請日200 0 年8月22日、美國臨時專利申請案6〇/22 6,8 7 0，申請日 2 0 00年8月22日、以及美國臨時專利申請案6 0/22 6,8 7 1， 申請曰2000年8月22曰之權益，各案皆以引用方式倂入 此處。 【發明所屬之技術領域】 本發明所屬之技術領域爲疾病治療中特異性之改良。 【先前技術】 肝病特別是病毒性B型及C型肝炎仍然構成人類健康的 嚴重威脅，而且已經發展出多種治療之道。依據使用之藥 物而定，治療可分成直接抗病毒治療、間接抗病毒治療、 或直接與間接抗病毒治療的組合。 直接抗病毒治療係干擾病毒的複製及/或病毒的組裝。例 如可使用核脊類似物，經由抑制病毒反錄酶而減少病毒的 複製。但核苷類似物常見引發副作用，包括貧血及/或嗜中 性血球減少。此外，長期暴露於核苷類似物對某些病毒種 系容易出現抗藥性。爲了因應至少部分抗藥性相關問題， 可投予核苷類似物混合液。不幸核苷類似物混合液典型只 能延遲抗藥性出現的起始。此外，核苷類似物通常對病毒 複製以及有機體宿主複製時快速分裂的細胞不具有選擇 性，因此對宿主具有顯著細胞毒性。 另外，蛋白酶抑制劑可用來千擾病毒蛋白質的適當組 裝。蛋白酶抑制劑對病毒蛋白酶具有高度特異性，因此典200414903 发明 Description of the invention The present application requests US provisional patent application 60/2 26,94 8, application date August 22, 2000, US provisional patent application 60/22 6,8 70, application date 2000 August 22, 2010, and US provisional patent application 6 0/22 6, 8 71, the application for the rights and interests of August 22, 2000, each case is hereby incorporated by reference. [Technical Field to which the Invention belongs] The technical field to which the present invention pertains is the improvement of specificity in the treatment of diseases. [Previous Technology] Liver diseases, especially viral hepatitis B and C, still pose a serious threat to human health, and various treatments have been developed. Depending on the drug used, treatment can be divided into direct antiviral therapy, indirect antiviral therapy, or a combination of direct and indirect antiviral therapy. Direct antiviral therapy interferes with virus replication and / or virus assembly. For example, nuclear spine analogs can be used to reduce viral replication by inhibiting viral reverse-enzyme. However, nucleoside analogs often cause side effects, including anemia and / or decreased neutrophils. In addition, prolonged exposure to nucleoside analogs is prone to resistance to certain viral lines. To address at least some of the resistance-related issues, a mixture of nucleoside analogs may be administered. Unfortunately, a mixture of nucleoside analogs typically only delays the onset of resistance. In addition, nucleoside analogs are generally not selective for viral replication and cells that divide rapidly during host organism replication, and are therefore significantly cytotoxic to the host. In addition, protease inhibitors can be used to properly perturb viral proteins. Protease inhibitors are highly specific for viral proteases, so
6 312/發明說明書(補件)/92-04/92102500 200414903 型地可避免關於限制病毒複製與快速分裂中的宿主細胞複 製間之選擇性問題。但即使相對低劑量之蛋白酶抑制劑， 仍然容易出現包括噁心、腹瀉、糖尿病及腎結石等副作用。 此外某些蛋白酶抑制劑於水性溶液之溶解度不佳，因此減 低可輸送給病人之可能用量。此外於長期治療後顯示對某 些蛋白酶抑制劑產生病毒抗藥性。 間接病毒治療法係用來刺激免疫系統辨識病毒抗原，或 變更免疫系統之細胞分泌素平衡朝向第1型細胞分泌素反 應，第1型細胞分泌素反應相信有助於建立對抗病毒感染 細胞之細胞免疫力。例如IFN- α可用來治療慢性C型肝 炎。但多種接受IFN- α治療病人於停止治療後疾病復發， 某些病人於治療期間被病毒突破重圍。此外，IF Ν - α於較 低劑量時容易引發發燒、頭痛、無精打采、缺乏食慾、焦 慮及憂鬱；而於較高劑量時造成骨髓抑制以及血球數目降 低。 同時投予里巴維林（Rib a vi r in)及干擾素- α可達成直接 與間接抗病毒治療之一或二者，於許多病人顯示可顯著減 少發炎以及降低血淸ALT濃度。儘管里巴維林之治療效果 相對較佳，但使用里巴維林仍然有多種問題。例如特別使 用較高劑量時，里巴維林於紅血球之胞內磷酸化作用已知 會促成溶血性貧血。此外，磷酸化里巴維林傾向於積聚於 紅血球，因而顯著降低有效劑量。結果需要更高劑量才能 達成里巴維林於肝細胞之適當劑量。 雖然業界已知多種組成物及方法可用來鎖定肝病爲治 7 312/發明說明書(補件)/92-〇4/92102500 200414903 療目標’但全部或幾乎全部組成物及方法仍然有一或多項 缺點。因此仍然需要提供可鎖定疾病治療目標之經改良之 組成物及方法。 【發明內容】 本發明係有關提高藥物選擇性之方法及組成物。通常藥 物係藉封阻基共價改性。 特別於本發明主旨之一態樣，封阻基係透過封阻基之氮 原于而偶合至藥物。經過改性藥物之封阻基可減少藥物於 非目標細胞的代謝轉化，以及減少藥物的隔離（換言之積 聚），於目標細胞之封阻基藉酵素分解去除。 於本發明主旨之另一態樣，瞭解藥物之代謝轉化誘生對 目標細胞的損傷，進一步瞭解透過封阻基氮原子而附接至 藥物之封阻基可阻止代謝轉化，以及封阻基於目標細胞中 由藥物裂解。結果，預期使用封阻基改性藥物，將藥物投 予包含目標細胞以及非目標細胞系統可降低細胞毒性。 本發明主旨之又另一態樣，瞭解藥物於非目標細胞之代 謝轉化可降低藥物有效濃度，進一步瞭解透過封阻基氮原 子附接至藥物之封阻基可防止代謝轉化，以及封阻基於目 標細胞中由藥物裂解。結果，預期使用封阻基改性藥物可 降低劑量，且預期將藥物投予包含目標細胞以及非目標細 胞系統。 預期藥物包括羧醯胺基，特別預期藥物爲核糖 咲喃糖基· 1，2，4 -三π坐-3 -殘醯胺以及2 - /3 - D -核糖d夫喃糖基 -4-噻唑羧醯胺，其也可呈其個別之L-異構物形式。雖然封 8 312/發明說明書(補件)/92-〇4/92102500 200414903 * ， j * 阻基之化學性質並無特殊限制，但較佳封阻基包含氮原 子。特佳封阻基爲=ΝΗ。預期之目標細胞並未限於特定細 胞類型，預期之目標細胞可感染或未感染病毒，或可爲高 度增生細胞。但以受到病毒感染之或高度增生之肝細胞及 神經元爲特佳，非目標細胞包括紅血球。 多項本發明之目的、特色、態樣及優點由後文本發明之 較佳具體例之詳細說明連同附圖將顯然自明。 【實施方式】 用於此處「藥理功效」一詞表示任何變更細胞於含細胞 系統之代謝、複製、結構或壽命，該項藥理功效係由添加 至系統之分子所引起。例如抑制同化作用、異化作用、或 由酶催化之聚合酶類型反應被視爲藥理功效。同理，微管 蛋白藉Kinl激動素解聚合於此處定義範圍內也視爲藥理 效果。相反地，酶藉系統細胞內部產生的代謝產物進行異 位抑制不被視爲藥理效果，因該異位抑制劑並非由外部添 加至該系統。 進一步於此處使用「目標細胞」一詞表示一種細胞藥物 預期於該細胞具有藥理效果。例如病毒感染的肝細胞被視 爲藥物里巴維林之目標細胞。相反地，「非目標細胞」一詞 涵蓋含細胞系統中非屬目標細胞的全部細胞。 通常已知雖然某些藥物意圖對特定細胞（即目標細胞）具 有藥理功效，但非目標細胞可能以顯著速率代謝該等藥 物，經常導致非期望的非特異性副作用。發明人發現於非 目標細胞（亦即於細胞內或細胞表面）之此種非期望之代謝 9 312/發明說明書(補件)/92-04/92102500 200414903 轉化可藉由使用封阻基改性藥物予以避免，該封阻基於目 標細胞被選擇性去除，因此提高藥物藥理功效選擇性，同 時降低藥物之細胞毒性及劑量。 於本發明主旨之一態樣，藥物藥理功效之選擇性可藉由 一種方法提高，該方法中，於一步驟，藥物被識別爲對目 標細胞具有期望的藥理功效。於另一步驟，藥物以封阻基 改性，其中該封阻基係透過封阻基之氮原子而共價附接至 該藥物。封阻基進一步減少藥物積聚於非目標細胞，且於 目標細胞以酶裂解作用而自藥物中去除。用於此處，就藥 理功效而言，藥物之「選擇性」一詞表示相較於藥物對非 目標細胞產生藥理功效結果造成藥物於非目標細胞之非期 望副作用，該藥物傾向於對目標細胞朝向治療標的而發揮 藥理功效。 例如卜/3 -D-核糖呋喃糖基-1，2,4-三唑-3-羧醯胺（里巴 維林結構式1 )之藥理功效之選擇性可藉著=NH基共價偶合 至里巴維林形成羧戚基而升高。已知里巴維林於肝炎病毒 感染的肝細胞具有抗病毒性質（例如參考綜論[Marc ellin，P. and Benhamou J.; Treatment of chronic viral hepatitis, Baillieres Clin Gastroenterol 1 9 94 J υ n ; 8 ( 2 ) : 2 3 3 - 5 3 ] 〇 也 已知里巴維林易於紅血球內磷酸化而以顯著高比率轉變成 爲藥理活性形式，亦即里巴維林磷酸鹽（例如Ho mm a, M. et a 1. High-performance liquid chromatographic determination of Ribavirin in whole blood to assess disposition in erythrocytes; Antimicrob Agents Che mot her 10 312/發明說明書(補件)/92-04/92102500 200414903 1 9 9 9 Ν ο v ; 4 3 ( 1 1 ) ·· 2 7 1 6 - 9 )，其降低藥理效果之選擇性。出 乎意外地，發明人發現使用=NH基改性里巴維林（1- /3 -D-核糖呋喃糖基-1，2，4 -三唑-3 -羧，結構式2 )於紅血球未被 磷酸化或只能非顯著地被磷酸化，於1 - 0 - D -核糖呋喃糖 基-1，2,4-三唑-3-羧脉=NH基於肝細胞被特異性藉酶催化 去除而形成1- /3 -D-核糖呋喃糖基-1,2，4-三唑-3-羧醯胺。6 312 / Invention Specification (Supplement) / 92-04 / 92102500 200414903 Land type can avoid the problem of selectivity between restriction of virus replication and host cell replication during rapid division. However, even relatively low doses of protease inhibitors are still prone to side effects including nausea, diarrhea, diabetes, and kidney stones. In addition, certain protease inhibitors have poor solubility in aqueous solutions, thus reducing the possible amount that can be delivered to patients. In addition, it has been shown to develop viral resistance to certain protease inhibitors after prolonged treatment. Indirect virus therapy is used to stimulate the immune system to recognize viral antigens, or to change the secretory balance of cells in the immune system towards type 1 cytokine response, which is believed to help establish cells that fight virus infection Immunity. For example, IFN-α can be used to treat chronic hepatitis C. However, many patients receiving IFN-α treatment relapsed after stopping treatment, and some patients were surrounded by the virus during the treatment period. In addition, IF Ν-α tends to cause fever, headache, listlessness, lack of appetite, anxiety, and depression at lower doses, and bone marrow suppression and lower blood cell counts at higher doses. Simultaneous administration of Ribavirin and interferon-α can achieve one or both of direct and indirect antiviral therapy, which has been shown in many patients to significantly reduce inflammation and lower blood ALT levels. Although the therapeutic effect of ribavirin is relatively good, there are still many problems with using ribavirin. For example, at higher doses, intracellular phosphorylation of ribavirin in red blood cells is known to contribute to hemolytic anemia. In addition, phosphorylated ribavirin tends to accumulate in red blood cells, thereby significantly reducing the effective dose. As a result, higher doses are needed to achieve the proper dose of ribavirin to hepatocytes. Although various compositions and methods are known in the industry to target liver disease for treatment 7 312 / Explanation of the Invention (Supplement) / 92-〇4 / 92102500 200414903, all or almost all of the components and methods still have one or more disadvantages. Therefore, there is still a need to provide improved compositions and methods that can target disease treatment. [Summary of the Invention] The present invention relates to a method and a composition for improving drug selectivity. Drugs are usually covalently modified by blocking groups. In particular, in one aspect of the gist of the present invention, the blocking group is coupled to the drug through the nitrogen atom of the blocking group. Modified drug blocking groups can reduce the drug's metabolic transformation in non-target cells, as well as reduce drug isolation (in other words, accumulation). The blocking groups in target cells are removed by enzyme decomposition. In another aspect of the subject matter of the present invention, understanding the metabolic transformation of a drug induces damage to target cells, further understanding that a blocking group attached to a drug through a blocking nitrogen atom can prevent metabolic transformation, and blocking based on the target The cells are lysed by the drug. As a result, it is expected that the use of a blocking group-modified drug to reduce the cytotoxicity by administering the drug to a system containing target cells as well as non-target cells. Another aspect of the subject matter of the present invention is that understanding the metabolic transformation of a drug in non-target cells can reduce the effective concentration of the drug, further understanding that the blocking group attached to the drug through a blocking nitrogen atom can prevent metabolic transformation, and blocking is based on Target cells are lysed by the drug. As a result, the use of blocking group-modified drugs is expected to reduce the dose, and the drug is expected to be administered to a system containing target cells as well as non-target cells. Anticipated drugs include carboamido, and particularly expected drugs are riboglucosino, 1,2,4-triπ-sino-3 -residamine, and 2-/ 3 -D -ribose d-furanosyl-4- Thiazolamide may also be in the form of its individual L-isomer. Although the chemical properties of the blocking group 8 312 / Invention (Supplement) / 92-〇4 / 92102500 200414903 *, j * are not particularly limited, it is preferred that the blocking group include a nitrogen atom. A particularly good blocking group is = NΗ. The intended target cell is not limited to a specific cell type, and the intended target cell may be infected with or not infected with a virus, or may be a highly proliferative cell. However, virus-infected or highly proliferative hepatocytes and neurons are particularly preferred. Non-target cells include red blood cells. A number of objects, features, aspects, and advantages of the present invention will be apparent from the detailed description of the preferred embodiments of the invention in the following text, together with the accompanying drawings. [Embodiment] As used herein, the term "pharmacological effect" means any change in the metabolism, replication, structure, or longevity of cells in a cell-containing system, and the pharmacological effect is caused by molecules added to the system. For example, inhibition of assimilation, alienation, or polymerase-type reactions catalyzed by enzymes are considered pharmacological effects. In the same way, the tubulin depolymerized by Kinl kinetin within the scope defined here is also considered as a pharmacological effect. In contrast, ectopic inhibition of enzymes by metabolites produced inside the cells of the system is not considered a pharmacological effect because the ectopic inhibitor is not externally added to the system. The term "target cell" is further used herein to indicate that a cell drug is expected to have a pharmacological effect on the cell. For example, virus-infected liver cells are considered target cells for the drug ribavirin. In contrast, the term "non-target cells" encompasses all cells in a cell-containing system that are not target cells. It is generally known that although certain drugs are intended to have pharmacological effects on specific cells (ie, target cells), non-target cells may metabolize these drugs at a significant rate, often resulting in undesired non-specific side effects. The inventors found that such undesired metabolism in non-target cells (that is, in or on the cell surface) 9 312 / Explanation of the Invention (Supplement) / 92-04 / 92102500 200414903 Transformation can be modified by using blocking groups The drug is avoided. The blocking is based on the selective removal of target cells, thus increasing the selectivity of the pharmacological efficacy of the drug, while reducing the cytotoxicity and dose of the drug. In one aspect of the subject matter of the present invention, the selectivity of a pharmacological effect of a drug can be improved by a method in which the drug is identified in one step as having a desired pharmacological effect on a target cell. In another step, the drug is modified with a blocking group, wherein the blocking group is covalently attached to the drug through the nitrogen atom of the blocking group. The blocking group further reduces drug accumulation in non-target cells, and is removed from the drug by enzymatic lysis on the target cells. As used herein, in terms of pharmacological efficacy, the term "selectivity" of a drug means that the drug tends to cause undesired side effects of the drug on non-target cells compared to the drug's pharmacological effect on the non-target cells. Pharmacological effects towards the target of treatment. For example, the selectivity of the pharmacological effects of / 3 / 3-D-ribofuranosyl-1,2,4-triazol-3-carboxamide (ribavirin formula 1) can be covalently coupled by the = NH group. Ribavirin rises as carboxyl groups are formed. Ribavirin is known to have antiviral properties in hepatitis virus-infected hepatocytes (see, for example, [Marc ellin, P. and Benhamou J .; Treatment of chronic viral hepatitis, Baillieres Clin Gastroenterol 1 9 94 J υ n; 8 ( 2): 2 3 3-5 3] 〇 It is also known that ribavirin is prone to phosphorylation in red blood cells and changes to a pharmacologically active form at a significantly higher rate, that is, ribavirin phosphate (eg, Ho mma, M. et a 1. High-performance liquid chromatographic determination of Ribavirin in whole blood to assess disposition in erythrocytes; Antimicrob Agents Che mot her 10 312 / Invention Specification (Supplement) / 92-04 / 92102500 200414903 1 9 9 9 Ν ο v; 4 3 (1 1) ·· 2 7 1 6-9), which reduces the selectivity of pharmacological effects. Surprisingly, the inventors found that the use of = NH group modified ribavirin (1- / 3 -D-ribose Furanosyl-1,2,4-triazole-3-carboxy, structural formula 2) is not phosphorylated or can only be phosphorylated insignificantly in red blood cells, at 1-0-D-ribosefuranosyl-1 , 2,4-triazole-3-carboxamide = NH-based removal of liver cells by specific enzymes Form 1- / 3 -D- ribofuranosyl-1,2,4-triazol-3-2carboxamide.
結構式1 結構式2 改性里巴維林其選擇性積聚的減少顯示於圖1 A及 1 B。圖1 A中，紅血球（非目標細胞）1 〇 〇被提供以里巴維林 (R)。里巴維林進入紅血球，被磷酸化成爲藥理活性里巴維 林-磷酸鹽（R-P)，保留於紅血球內部。同理，肝細胞（目標 細胞）1 1 〇被提供以里巴維林（R)。里巴維林進入肝細胞， 被磷酸化成爲藥理活性里巴維林-磷酸鹽（R-P)，保留於肝 細胞。圖1 B中，紅血球（非目標細胞）1 0 1被提供以改性里 巴維林（R *，1 -冷-D -核糖呋喃糖基-1，2，4 -三唑-3 -羧棘）。改 性里巴維林進入紅血球，但未被磷酸化因此離開紅血球。 同理，肝細胞（目標細胞）1 1 1被提供以改性里巴維林（R*)。 改性里巴維林進入肝細胞且以酵素催化脫胺基成爲里巴維 11 312/發明說明書(補件)/92-04/92102500 200414903 林，隨後被磷酸化成爲具有藥理活性之磷酸化里巴維林 (R - P )而保有於肝細胞內部。 於本發明主旨之另一態樣，預期里巴維林以外有多種藥 物適合用於本發明之構想。通常適當藥物包括可於目標細 胞以外之細胞代謝、活化及/或去活化之藥物’特別預期之 藥物包括核苔、核苷酸、核苷類似物及核苷酸類似物。例 如堤亞左菲林（Tiaz〇furin)(2-冷-D-核糖呋喃糖基-4-噻唑-羧醯胺）爲一種帶有羧醯胺基之核苷類似物，其方便以=NH 基改性而獲得對應羧牌。另一例中，另一種藥物包含核苷 尿嘧啶或核苷類似物5’-氟尿嘧啶（5’-FU)。 於本發明主旨之又另一態樣，封阻基無需限於=NH基’ 可包括多種第一級胺及第二級胺，只要封阻基可透過氮原 子共價偶合至藥物即可。「封阻基」一詞用於此處表示共價 附接至藥物之化學基團，且當附接至藥物時，可封阻藥物 之至少一種代謝轉化作用。用於此處，藥物之「代謝轉化」 一詞表示藥物之任一種胞內及/或胞外化學變化係由細胞 或細胞系統之代謝所促成，特別包括酶分解（例如氧化、水 解裂解）以及酶改性（例如糖化、磷酸化）。 通常預期適當封阻基具有結構式Ν(Ι^)(Ι12)或=NR!，其 中R!及R2分別爲氫、直鏈或分支烷基、烯基、炔基、芳 院基、芳嫌基或芳炔基、芳基，其全部進一步包括氮、氧、 硫或鹵原子等雜原子。但特佳者爲其它封阻基可藉酶催化 作用而由藥物去除，並且特別預期酶包括肝特異性胺基水 解酶，包括去胺基酶（例如腺苷或胞嘧啶去胺基酶）、肝去 12 312/發明說明書(補件)/92-04/92102500 200414903 醯胺酶（例如菸鹼醯胺去醯胺酶）以及肝轉胺基酶（鍵胺酸_ 丙酮酸轉胺基酶）。 期望之封阻基可共價鍵結至藥物分子之多個位置，通常 較佳期望之樂物係於殘酸胺部分被改性，預期也可於竣醯 胺以外之多個位置被改性，特別羰基（例如羧酸以及酮類型 之羰基）。例如尿嘧啶或其類似物5，_FlJ之環部分之各個碳 基可藉封阻基改性。 雖然非囿限本發明之構想於此處所示，預期封阻基可將 藥物去活化，或一旦改性藥物呈現至非目標細胞時可防止 隨後的活化。例如若封阻基偶合至藥物之位置爲藥物與目 標分子（例如受體或酶基質結合位址）間之特異性交互作用 所必需的位置，則封阻基可將藥物去活化。它方面，封阻 基可偶合至藥物之防止代謝活化的位置。 依據藥物及/或封阻基之化學性質決定，預期封阻基可置 換官能基或取代基，或該封阻基萁附接至官能基或取代 基。例如若藥物爲里巴維林，而封阻基爲=NH基，則里巴 維林羧醯胺基之氧原子由=NH基所置換。它方面，若藥物 包含親核基團（例如-cr)，而封阻基包含帶有適當離去基之 第二級胺基，則第二級胺可附接至親核基團。 就藥物改性步驟而言，預期改性包含有機合成改性、酶 催化改性、或重新合成俾製造改性後的藥物。例如若藥物 包含活化羰基官能基，則羰基原子之醯胺化可於單一親核 交換反應達成。另外，特別當除了封阻基欲附接至基團之 外，藥物具有多個反應基時，改性藥物之重新合成於經濟 13 312/發明說明書(補件)/92·04/92102500 200414903 上更具有吸引力。特別也預期於採用藥物以及封阻基作爲 酶基質之反應中，經由將封阻基導入藥物而以酶催化方式 改性適當藥物。可能時，此種改性用之酶係衍生自目標細 胞（例如來自異種來源或同種異體來源，或來自於可表現酶 之重組編碼基因之來源）較佳。 須瞭解依據非目標細胞而定，以及依據藥物及/或封阻基 之化學性質而定，阻止藥物積聚於非目標細胞可藉多種機 轉中之至少一種達成，該等機轉包括減少經由藥物特異性 轉運因子的攝取，減少代謝轉化成爲將被保有的形式（例如 由於額外電荷或新電荷、疏水性變化、或由向外運送因子 辨識的改變），或由非目標細胞向外運送的增加（例如封阻 基之分泌信號等機轉），可達成防止藥物的堆積。例如預期 若藥物爲核苷類似物時，非目標細胞具有核苷轉運因子， 其可將不含親脂部分之核苷選擇性輸入細胞內部；將親脂 部分作爲封阻基加至藥物可防止藥物的積聚。另一例中， 預期紅血球之多個核苷之磷酸化（以及伴隨之積聚）可藉將 羧醯胺基轉成羧#基（參見上文）而予防止。 就封阻基於目標細胞之酶催化去除而言，依據目標細 胞、封阻基及藥物類別而定，酶催化去除有顯著變化。 酶催化去除包括各類型酶，包括水解酶、轉移酶、分解 酶以及氧化還原酶，特佳亞類型爲腺苷及胞苷去胺基酶、 精胺酸化酶、轉胺基酶以及芳基醯胺基酶。進一步須瞭解 用於酶催化去除封阻基之酶可排它地於目標細胞表現，但 於本發明主旨之其它態樣，適當酶也可以於目標細胞以外 14 312/發明說明書(補件)/92-04/92102500 200414903 的細胞表現。只要該酶並非到處表現於含細胞系統的全部 細胞即可。進一步須瞭解預期之酶於正常及/或病理情況下 於個別目標細胞可自然表現（換言之非重組）於個別目標細 胞。例如已知麩胺-丙酮酸轉胺基酶本質上係以相對高選擇 性於肝細胞表現，因此該種酶爲封阻基去除用之適當酶。 另外，已知胞苷去胺基酶以相對高數量於結腸癌細胞表 現，但於正常結腸細胞不表現或只微量表現。 本發明主旨之另一態樣，藥物對非目標細胞之細胞毒性 可藉一種方法降低，其中於一步驟，辨識非目標細胞藥物 之代謝轉化對非目標細胞誘生的損傷。「細胞毒性」一詞用 於此處表示對非目標細胞產生非期望的藥理效應，其中該 非期望的藥理效應特別包括抑制複製、能量代謝，或包括 細胞死亡。又一步驟中，藥物以封阻基改性，其中封阻基 係透過封阻基的氮原子而共價偶合至藥物，其中封阻基減 少藥物於非目標細胞代謝轉化，且於目標細胞藉酶催化而 由藥物裂解。又另一步驟中，藥物投予包含目標細胞及非 目標細胞的系統，其中封阻基係共價偶合至藥物。 就降低藥物對非目標細胞之細胞毒性之較佳態樣而 言，代謝轉化包括藥物於紅血球內磷酸化成爲對應藥物磷 酸鹽。例如業界眾所周知抗病毒藥物里巴維林於多種細胞 磷酸化，產生藥理活性里巴維林5’-單磷酸鹽（例如H omma， Μ. e t a 1. High-performance liquid chromatographic determination of Ribavirin in whole blood to assess disposition in erythrocytes; Antimicrob Agents C hem other 15 312/發明說明書(補件)/92-04/921 〇25〇〇 200414903 1 9 9 9 No V; 4 3 ( 1 1 ):2 7 1 6-9)，此種化合物爲可抑制肌苷一磷 酸鹽去氫酶（IMPDH)。不幸里巴維林-5 一磷酸鹽對紅血球 產生顯著細胞毒性（D e F 1· a n c e s c h i, e t a 1 _ ; H e m ο 1 y t i c anemia induced by ribavirin therapy in patients with chronic hepatitis C virus infection: role of membrane oxidative damage，Hepatology 2000 Apr; 3 1(4): 9 9 7 -10〇4)，結果得知預防或減少紅血球中形成里巴維林 -5 ’ - 一磷酸鹽將可顯著降低里巴維林的細胞毒性。 但須瞭解，除了磷酸化以外，藥物於非目標細胞之多種 代謝轉化作用預期也涵蓋於本發明之範圍，此等代謝轉化 包括氧化、還原、水解裂解藥物內部的共價鍵、加成或去 除旁懸基以及開環反應。例如預期若非目標細胞爲肝細 胞’則代謝轉化包括於肝臟已知可能出現之多種酶催化解 毒或溶解反應（例如糖化反應、胞色素P45G媒介氧化反應 等）。另一例中，代謝轉化包括磷酸酶或酯酶活性。 依據代謝轉化類別而定，轉化可能限於非目標細胞之單 一類型，但也可能出現多個細胞類型。例如若非目標細胞 有相對高的核酸合成速率，代謝轉化係由涉及核酸合成之 酶媒介，則多種快速生長類型當中的細胞將出現代謝轉 化。它方面，代謝轉化也可局限於藥物接近特定一組細胞 或器官。 須瞭解多種眾所周知之實驗程序可用來辨識及/或證實 封阻基共價偶合至藥物而減少於非目標細胞的代謝轉化。 舉例言之，當目標細胞於試管內培養時，預期非目標細胞 16 312/發明說明書(補件)/92-04/92102500 200414903 與·對應之放射性標記藥物共同培養，放射性標記藥物之代 謝產物可以多種檢定分析識別，包括免疫檢定分析、薄層 層析術或GC-MS。另外，當非目標細胞係局限於哺乳類， 則組織生檢將提供足夠檢體來分離及辨識投予藥物之代謝 產物。 進一步預期對非目標細胞損傷之類別可有實質變化，其 傷害係由非目標細胞之細胞代謝減慢至細胞死亡之範圍。 例如若代謝轉化產生糖解酶路徑上一種酶的抑制劑，則該 非目標細胞的能量至少部分須由救援路徑提供。同理，若 藥物代謝轉化成爲酶抑制劑係以相對緩慢速率進行，則抑 制劑影響酶表現的向上調節可幾乎完全補償活性位址數目 的減少。它方面，若代謝轉化產生自由基，則脂質過氧化 導致嚴重細胞膜受損，結果導致細胞死亡。 進一步預期藥物代謝轉化導致的損傷可直接或間接引 起。例如若代謝轉化產生酶抑制劑而封阻酶，則該種傷害 視爲直接傷害。它方面，若代謝轉化產生中間物，該中間 物於隨後之胞內或胞外改性之後進一步轉化成酶抑制劑， 則該傷害視爲間接傷害。 就藥物投予系統之步驟而言，預期適當藥物將以任一種 適當藥物調配物且以任一種適當方案投予。如此投藥可經 口、腸道外（包括皮下注射、靜脈肌肉、皮內注射或輸注技 術）、吸入噴霧、直腸、局部投藥等，以含有習知無毒醫藥 可接受性載劑、輔劑、及媒劑之單位劑量調配物投予。例 如預期適當藥物可呈藥理容許鹽形式口服投藥，或另外於 17 312/發明說明書(補件)/92-04/92102500 200414903 生理鹽溶液形式（例如緩衝至pH約7.2至7.5)經靜脈投 予。習知緩衝劑如磷酸鹽、碳酸氫鹽或檸檬酸鹽可用於此 項目的。此外，預期於業界人士之技巧範圔內，可修改特 定藥物之投藥途徑及用法用量俾控制藥物之藥動學而予病 人獲得最大裨益。 某些投藥形式預期使用藥物之前驅藥形式。熟諳技藝人 士瞭解如何方便改性預期的藥物成爲前驅藥形式，俾輔助 活性化合物輸送至宿主有機體或病人體內目標位址。熟諳 技藝人士也利用前驅藥形式（若有）之有利藥動學參數，輸 送化合物至宿主有機體或病人體內之目標位址俾獲得化合 物之最高期望效果。 進一步需瞭解期望藥物可單獨投予，或組合其它藥理活 性劑投予，其可分開或共同投藥，當分開投藥時可同時或 以任一種順序分開投藥。預期涵蓋之藥理活性劑包括抗病 毒劑如干擾素（例如干擾素α及7 );抗真菌劑如妥那飛 (tolnaftate)、芳吉鐘（Fungiz〇neTM)、羅崔明（LotriminTM)、 米斯雷克斯（MycelexTM)、尼史塔汀（Nystatin)及安佛特辛 (Amphoteracin);抗寄生蟲劑如明特左（MintezolTM)、尼可 拉席得（NicIocideTM)、微莫斯（Verm〇xTM)及富樂吉 (FlagylTM);腸道作用劑如瀉立停（Imm〇diumTM)、羅莫堤 (LomotilTM)及法茲米（PhazymeTM);抗腫瘤劑如干擾素α及 7、阿黴素（AdriamycinTM)、希妥山（CytoxanTM)、伊目朗 (ImuranTM)、美索崔謝（Methotrexate)、米斯拉辛 (MithracinTM)、堤左夫靈（TiazofurinTM)、紫杉醇 18 312/發明說明書(補件)/92-04/92102500 200414903 (TaxolTM);皮膚科用藥如阿羅微（AclovateTM)、西克羅科 (CyclocortTM)、迪諾樂斯（Denorex1M)、夫羅容 (F 1 〇 r ο n e τ M )、奧克索藍（Ο X s 〇 r a 1 e η τ M )、煤焦油及水楊酸； 偏頭痛製劑例如麥角胺化合物；前文未列舉之類固醇以及 免疫抑制劑包括環孢靈（c y c 1 〇 s ρ 〇 r i n s )、迪普松 (〇丨卩1*080 1^1^)、氫可體松（1：1)^1-0(：01*1；丨50 1^);福羅容 (F]oronTM)、麗得斯（LidexTM)、托琵可（Topicoft)及瓦利松 (Va] is one);以及代謝劑例如胰島素以及其它不屬於前述任 何類別之藥物，包括細胞分泌素如IL2、IL4、IL6、IL8、 IL10 及 IL12 。 關於預期藥物以及藥理活性劑劑量，治療有效量將隨接 受治療的病情、病情嚴重程度、採用的治療計畫、用藥之 藥動學以及接受治療的病人（動物或人類）而異。進一步預 期多種劑量皆適合，包括0.5毫克/千克至0.1毫克/千克及 以下之劑量，但也包括0.5至1 · 0毫克/千克及以上之劑 量。雖然通常較佳包含目標細胞及非目標細胞之系統爲哺 乳動物（最佳爲人類），但多種其它系統也適合，特別包括 體外試驗之細胞與組織培養。 關於藥物、封阻基、藥物改性步驟、目標細胞及非目標 細胞於預期降低藥物對非目標細胞之細胞毒性方法而言， 前文所述相同考量亦適用於此。 於本發明主旨之又另一態樣，藥物於系統之劑量可藉藥 物之提供方法予以降低，其中藥物於非目標細胞之代謝轉 化作用可降低藥物於包含非目標細胞以及目標細胞之系統 19 312/發明說明書(補件)/92-04/92102500 200414903 之濃度。又一步驟中，藥物使用封阻基改性，其中封阻基 係透過封阻基的氮原子而共價偶合至藥物，以及其中該封 阻基可降低藥物於非目標細胞的代謝轉化。隨後步驟中， 藥物投予系統，其中該封阻基共價偶合至藥物，以及其中 該封阻基係於目標細胞藉酶催化而自藥物中去除。 就降低藥物於系統劑量之較佳態樣而言，該藥物爲里巴 維林，該目標細胞爲感染病毒的肝細胞，以及非目標細胞 爲紅血球。業界眾所周知（參見上文）里巴維林被代謝轉化 成爲里巴維林磷酸鹽，以及里巴維林磷酸鹽被保留於紅血 球，因而顯著降低里巴維林濃度，里巴維林係經由附接=NH 封阻基至羧醯胺碳而被改性，藉此置換羧醯胺之羰基氧。 業已顯示（參見後文）里巴維林以=NH封阻基改性之代謝轉 化於紅血球顯著減低。進一步預期改性後里巴維林之較佳 劑量爲於人體單次口服50毫克- 300毫克。 已知里巴維林爲抗病毒藥，以至少約600毫克- 1 2 00毫 克的單一劑量口服投予人體。里巴維林於系統（例如人體） 之初濃度介於約1 # Μ至數百// Μ間，但因里巴維林於紅 血球被磷酸化，里巴維林之系統內濃度典型經由被隔離於 紅血球內部而在24小時內降至初濃度至約85%至50%。發 明人顯示將里巴維林改性成爲卜沒-D-核糖呋喃糖基-1,2, 4-三唑-3-羧勝，可顯著降低里巴維林之磷酸化數量（參見 後文）。因此預期全部或幾乎全部里巴維林之初濃度可用於 獲得目標細胞期望的藥理功效。結果，預期使用封阻基改 性里巴維林可降低里巴維林劑量達約5wt% ’較佳約 20 312/發明說明書(補件)/92-〇4/92102500 200414903 1 0 w t %，更佳 2 5 w t % 及最佳 5 0 W t %。 但須暸解也預期涵蓋600毫克-i 2 00毫克以外之多種劑 量，包括200毫克-600毫克劑量、20毫克- 200毫克劑量 及以下。例如若採用里巴維林作爲免疫調節藥，則約]〇 〇 毫克-3 0 0毫克之較低劑量即足。它方面，當需要相對高濃 度藥物時預期使用6 0 0毫克-1 8 0 〇毫克及以上之劑量。也 須瞭解依據特定代謝轉化而定，劑量的下降可有顯著變 化。例如若代謝轉化相當快，且於複數個非目標細胞進行， 則預期劑量減低2 5 w t %至8 0 w t %及以上。它方面，若代謝 轉化相當慢，則預期可減低劑量25 wt %至5 wt%及以下。 於預期降低藥物之方法中，有關藥物、封阻基、代謝轉 化、藥物改性步驟、系統、投藥步驟、目標細胞以及非目 標細胞，前文說明之考量也適用於此。 (實施例） (a)里巴維林之範例合成顯示於圖2，合成程序摘述如後。 1-(2,3,5 -二-0-乙醯基-/3 -D -核糖口夫喃糖基）-i，2,4 -三Π坐 -3-羧酸甲酯（3)以及1-(2,3,5-三-〇-乙醯基-/3-〇-核糖呋喃 糖基）-1,2，4-三唑-5-羧酸甲酯（4) 1，2,4-三唑-3-羧酸甲酯（25.4克，200毫莫耳）（1)1，2,3,5-四-〇-乙醯基-/3-D·核糖呋喃糖（ 6 3.6 6克，2 00毫莫耳）（2) 以及貳（對硝基苯基）磷酸酯（1克）之混合物置於圓底瓶 (5 0 0毫升）內。燒瓶置於1 6 5 · 1 7 5 °C之預熱油浴中，於水抽 吸器之真空下伴以攪拌經歷2 5分鐘時間。置換出之乙酸收 集於冰冷凝氣瓣，冰冷凝氣瓣係置於抽吸器與圓底瓶間。 21 312/發明說明書(補件)/92-04/½ 102500 200414903 燒瓶由油浴中移開讓其冷卻。當燒瓶溫度約達60- 7 0 °C 時，導入乙酸乙酯（ 3 00毫升）及飽和碳酸氫鈉（150毫升）， 於乙酸乙酯萃取。水層再度以乙酸乙酯（2 00毫升）萃取。 合倂乙酸乙酯萃取物以飽和碳酸氫鈉（3 00毫升）、水（200 毫升）及鹽水（1 5 0毫升）洗滌。有機萃取物以無水硫酸鈉脫 水，過濾，濾液蒸發至乾。殘餘物溶解於乙醇（1 0 0毫升）， 以甲醇（60毫升）稀釋，於0°C冷卻12小時獲得無色晶體。 固體經過濾，以最小量冷乙醇（20毫升）洗滌，於高度真空 於固體氫氧化鈉脫水獲得6 0克（7 8 %)。濾液蒸發至乾，使 用氯仿—乙酸乙酯（9:1)作爲洗提劑，於矽膠管柱純化。由 濾液分離兩種產物，快速移動的產物約8 . 5克（1 1 % )，緩慢 移動產物約5克（6.5 %)。緩慢移動產物匹配結晶產物。發 現快速移動產物爲4呈泡沬體。3組合產率爲65克（84%); 熔點 108-110°C ; 3 之1H-NMR (CDC13):(5 2.11 (s5 3H， COCH3)，2·12 (s，3H5 COCH3)，2.13 (s，3H，OCH3)，3.99 (s， 3H，COCH3)，4.22 (dd，1H)，4.46 (m，2H)，5.55 (t，1H， J = 6.0 Hz)，5.75 (m，1H)，6·05 (d，1H，C】’H J = 3.6 Hz)及 8.41 (s，1H，C5H)。分析（C15Hi9N309) C，H，N。4 之1H-NMR (CDC13):5 2.02 (s，3H，COCH3)，2.10 (s，3H，COCH3)，2.12 (s，3H，OCH3)，4.00 (s，3H，COCH3)，4.14 (m，1H)，4.42 (ni， 2H)，5·76 (t，1H)5 5.81 (m，1H)，6.94 (d，1H，CKH J = 2.1 Hz)，8.03 (s，1H，C5H)。分析（C15H19N309) C，H，N。 1-冷-D-核糖呋喃糖基-1，2,4-三唑-3-羧醯胺（5) 1-(2，3, 5 -三-0-乙醯基-/3 -D-核糖呋喃糖基）-1，2,4 -三唑 22 312/發明說明書(補件)/92-04/92102500 200414903 -3 -羧酸甲酯（6 2克，1 6 1毫莫耳）（3 )置於鋼彈內，使用新鮮 製備之甲醇系氨（350毫升，係將乾氨氣通入〇°C之無水甲 醇直到飽和製備）於0 t處理。鋼彈經封閉及於室溫攪拌1 8 小時。鋼彈冷卻至01，開啓，讓內容物蒸發至乾。殘餘 物以無水乙醇（1 00毫升）處理及蒸發至乾。所得殘餘物使 用丙酮濕磨獲得固體。固體經過濾及以丙酮洗滌。固體於 室溫脫水隔夜，溶解於熱乙醇（600毫升）及水（10毫升）混 合物。藉於熱板上加熱及攪拌而將乙醇溶液容積縮小至 1 5 0毫升。熱乙醇溶液於冷卻時獲得無色晶體，晶體經過 濾，以丙酮洗滌及真空脫水。濾液進一步濃縮獲得額外產 物。總產率：3 5 克（8 9 %);熔點 1 7 7 - 1 7 9 °C ; [ α ]2 0 D - 3 5 . 3 ( c， 10，H20); 】H-NMR (Me2SO-d6):5 3·46 (m，1H，C5，H)，3.60 (m，1H，C5’H)，3.94 (m，1H，C4，H), 4.12 (ηα，1H)，4.34 (m， 1H)，4.95 (t5 1H，C5’OH)，5.22 (d5 1H)，5.60 (d，1H)，5.80 (d，1H，J = 3.9 Hz，C】，H)，7.64 (bs，1H, NH2)，7.84 (bs, iH， NH2)，8.87 (s，1H，C5H).】3C NMR (Me2SO-d6)5 61·8，7〇·2, 74.4, 86.0, 91.6, 144·9, 157.4, 160.6。分析（C8H12N4〇5) C, H，N。 (b)以-NH基改性之里巴維林範例合成顯示於圖3，遵照後 文摘述之程序。 3-氰基-1-(2,3,5-三-〇-乙醯基-/3-1)-核糖呋喃糖基卜1,2， 三唑（7) 3-氰基-1，2,4-三唑（18·8 克，2〇〇 毫莫耳）（6)，12，]，^四 -0-乙醯基-冷·〇_核糖呋喃糖（6366克，2〇〇毫莫耳）及貳（對 312/發明說明書(補件)/92-04/92102500 23 200414903 硝基苯基）磷酸鹽（1克）之混合物置於圓底瓶（ 5 0 0毫升）。燒 瓶置於預熱之1 6 5 - 1 7 5 °C之油浴於水抽吸器之真空下伴以 攪拌放置2 5分鐘時間。被置換出之乙酸收集於冰冷凝器 瓣，凝器瓣係置於抽吸器與圓底瓶間。燒瓶由油浴中移開 讓其冷卻。當燒瓶溫度約降至60-70 °C時，導入乙酸乙酯 (3 0 0毫升）及飽和碳酸氫鈉（1 5 0毫升），萃取入乙酸乙酯。 水層再度以乙酸乙酯（200毫升）萃取。合倂乙酸乙酯萃取 物，以飽和碳酸氫鈉（3 00毫升）、水（200毫升）及鹽水（150 毫升）洗滌。有機萃取物以無水硫酸鈉脫水，過濾，濾液蒸 發至乾。殘餘物溶解於醚（1 0 0毫升），醚於0 t冷卻1 2小 時獲得無色晶體。固體經過濾，以最小量冷乙醇（20毫升） 洗滌，使用固體氫氧化鈉於高度真空脫水。產率：5 6.4克 (80%)。熔點 9 6- 97 °C 。】H-NMR (CDC13): δ 2.1 1 (s，3H， COCH3)，2.13 (s，3Η, COCH3)，2.14 (s，3Η，COCH3)，4·22 (dd? 1H)? 4.46 (m? 2H) 9 5.5 2 (t? 1H5 J = 6.〇 Hz); 5.70 (m; 1H)，6.01 (d，1H，C^’H J = 3.6 Hz)及 8.39 (s，1H，C5H)。 C]4H16N407 (3 5 2.3 0)之分析計算値：C，47.73; H，4.58; N， 15.90。實測値：C，4 7.7 0 ; H，4.6 3 ; N，1 6 · 0 卜 卜冷-D-核糖呋喃糖基-1，2,4-三唑-3-羧絲鹽酸鹽（8) 7(14.08克，40·0毫莫耳），氯化銨（2.14克，40.0毫莫 耳）及無水氨（1 5 0毫升）之混合物於鋼彈內於8 5它加熱1 8 小時。冷卻鋼彈，開啓鋼彈，內容物蒸發至乾。殘餘物由 乙腈-乙醇結晶獲得10.6克（95%) 8。熔點177] 79t：。 ]H-NMR (DMSO-d6):5 3.44-4.2 (m? 3H), 4.40 (m3 2H)? 5.04 24 312/發明說明書(補件)/92-04/92102500 200414903 (t，1H)，5.29 (m，1H)，5.74 (m，1H)，5.87 (d，1H，C】，H)， 8.96 (bs，3H)以及 9.17 (s，1H，C5H)。C8H14ClN5〇4 (279.68) 之分析計算値：C，34.35; H，5·05; N，25.04; Cl，12.69。實 測値：C, 34.39; Η，5·1〇; N，25.14; Cl，12.71。 始於里巴維林之另一種合成程序範例係如下進行： 2’，3’，5’-三-〇-乙醯基卜/3-0-核糖呋喃糖基-1，2,4-三唑-羧醯胺（9) 1- /3 核糖呋喃糖基-1，2,4-三唑-3-羧醯胺（28.4克， 11 6·4毫莫耳）（里巴維林）於乙酐（2 00毫升）及吡啶（50毫升） 之懸浮液於室溫攪拌隔夜。所得澄淸溶液經真空濃縮獲得 透明泡沬體（43.1克，定量產率）。泡沬體藉TLC證實爲均 質，未經純化直接用於次一步驟。小量泡沬體藉急速層析 術純化獲得分析樣本；]H-NMR (3 00MHz，D MS 0-d6) 5 2.01， 2.08, 2.09 (3 s，9H，COCH3)，4.10 (m，1 H)，3·52 (m，2H)， 5.58 (t? 1 H), 5.66 (m, 1 H); 6.33 (d? 1 H? J. = 3.0 Hz; C]H); 7.73, 7.92,（2 s，2 H，CONH2)，8.8 6 (s5 1 H，C5H 三唑）。 分析（C】〇H】8N408) C，H，N。 3_氰基_2’，3’，5’_三-〇-乙醯基-11 -D-核糖呋喃糖基 -1，2，4 -三唑（1 0 ) 於9(43·1克’ 116.4毫莫耳）於氯仿（5〇〇毫升）之溶液內 加入二乙基胺（2 4 4毫升），混合物於冰-鹽浴中冷卻至〇。 以攪拌緩慢逐滴加入憐醯氯（30.7毫升，330毫莫耳），讓 ί谷液iW熱至室？皿。“合物於室溫攪泮1小時後，T l c (己院 /丙酮3 : 1 ) ί曰不起物料完全消失。褐色反應混合物真空濃 312/發明說明書(補件)/92-04/92102500 25 200414903 縮至乾，殘餘物溶解於氯仿（5 0 0毫升），有機溶液以飽和 水性碳酸氫鈉（ 3 x 2 00毫升）洗滌，以無水硫酸鈉脫水及真 空濃縮。殘餘物於矽膠（急速層析術）使用20%丙酮於己烷 層析獲得3 3 . 1 4克（8 1 %得自里巴維林）純質1 0呈非晶形物 體。固體各方面皆與真實樣本完全相同：熔點1 0 1 -1 0 3 °C ; IR(溴化鉀）^ 22 5 0 (CN)，1 7 5 0 (C = 0)，cnTYH-NMR (300 MHz，CDC13)5 2.04，2.06, 2·07 (3 s，9H，，乙醯基甲基）， 4·15 (dd，1 H)，4.40 (m，1 Η)，5·47 (t，1 H)，5.63 (dd，1 H)， 5.95 (d，1 H，J = 3.2 Hz，C]H)，8.34 (s，1 H，C5H 三唑）。 1 -冷-D -核糖呋喃糖基-1，2，4 -三唑-3 -羧脒鹽酸鹽（8 ) 於10(4.0克，11·4毫莫耳）於甲醇（1〇〇毫升）之懸浮液內 加入甲醇系甲氧化鈉莫耳溶液（1 2毫升），混合物於室溫攪 拌隔夜。溶液使用經以甲醇洗滌之杜威（Dow ex) Η +樹脂酸 化至pH 4，樹脂經過濾去除，濾液經真空濃縮至乾。殘餘 物溶解於最小量甲醇（1 5毫升），移至加壓瓶內。加入氯化 銨（0 · 6 1克’ 1 1 · 4毫莫耳）以及於〇 °C使用無水氨氣飽和之 甲醇溶液（7 5毫升），加壓瓶密封，溶液於室溫攪拌隔夜。 溶液經真空濃縮至乾，結果所得殘餘物由乙腈/乙醇結晶獲 得8，呈結晶固體（2 · 9 5克，9 3 %)。此樣本各方面皆與真實 樣本相同。 又另一途徑中’卜/3 - D ·核糖呋喃糖基-1，2,4 -三唑-3 ·羧脒 鹽酸鹽（8 )可使用微生物培養、完好微生物細胞、或細胞萃 取物作爲酶來源（於微生物之非增生條件下）藉酶催化反應 製造。3-氰基-1-(2，3, 5-三-〇-乙醯基-/3 核糖呋喃糖 26 312/發明說明書(補件)/92-04/92102500 200414903 基）-1，2,4_三唑（7)可經由讓3-氰基-1，2，心三唑或其鹽與核 糖施體於基於微生物之酶來源存在下接觸製造。然後化合 物7經由使用液態氨溶液處理而轉換成爲（8)。另外’ 1,2,4-三唑-3-羧棘鹽酸鹽可與核糖施體於酶存在下反應而直接 製造（8)。 (〇改性里巴維林於肝臟脫胺基成爲里巴維林 於小鼠，重複口服投予3H-里巴維林及3H-( = NH)改性里 巴維林，每日劑量3 00毫克/千克連續8日後，里巴維林之 肝臟最小放射性濃度中間値Cmin比改性里巴維林之cmin 更低。特別須指出接受里巴維林處理小鼠，里巴維林占肝 放射性約90%，核糖呋喃糖基三作羧酸（RTCA)占肝放射性 約1 0 %。相反地，於使用改性里巴維林處理小鼠，改性里 巴維林占肝放射性約3 0%，而里巴維林占肝放射性約 7 0 %(也參考表1)。 3H-里巴維林 3H-( = NH)改性里巴 維林 ___ 總肝放射性 RTC A 里巴維林 改性里巴維林 1 8.4微克當量/克 約1.8微克當量/克 約1 6.6微克當量/ 克 未偵測 23.8微克當量/克 未偵測 約1 6.6微克當量/ 克 約7.2微克當量/克 表1 :小鼠肝臟放射性 (d)於紅血球（RBC)里巴維林及（ = NH)改性里巴維林之差異 放射性分佈 經顯示里巴維林可於紅血球磷酸化，進一步提示磷酸化 後的里巴維林爲人體長期或高劑量接受里巴維林後觀察得 溶血性貧血的起因。顯然（ = NH)改性里巴維林並未直接運 27 312/發明說明書(補件)/92-04/92102500 200414903 送入紅血球’如試管試驗硏究（資料未顯示於此處）可證， 隨後’預期改性里巴維林唯有於肝臟脫去胺基成爲里巴維 林且隨後被磷酸化成爲對應磷酸鹽之後才能積聚於紅血 球，如下表2所示。 小鼠以3 00毫克/千克一日劑量重複口服投予3H_里巴維 林及3 Η - ( = N Η )改性里巴維林連續8日後，改性里巴維林於 紅血球之最小放射性濃度中間値Cmin比里巴維林顯著更 低。由表1及2所示差異資料判定，改性里巴維林之治療 指數（亦即肝里巴維林濃度與紅血球里巴維林濃度之比）約 爲里巴維林之治療指數之三倍。 於肝門靜脈插管的獼猴，單次口服劑量3 0毫克/千克3 Η 里巴維林或（ = ΝΗ)改性3Η里巴維林後，24小時後達到紅血 球尖峰放射性濃度，且隨後維持穩定濃度。3Η里巴維林及 ( = ΝΗ)改性3Η里巴維林之尖峰放射性濃度具有半生期Τ1/2 分別約爲1 9 9 8小時及5 7 7小時。以3 0毫克/千克多重給藥 後，穩態放射性濃度預測里巴維林顯然比（ = ΝΗ)改性3Η里 巴維林更高（表2)。 里巴維林 3Η-( = ΝΗ)改性里 巴維林 紅血球放射性中 間値（小鼠） 紅血球放射性中 間値（猴-單劑） 紅血球放射性中 間値（猴·多劑） 1 .36微克當量/克 〜41微克當量/克 〜5 0 8 9微克當量/ 克 0.38微克當量/克 〜17微克當量/克 〜606微克當量/克 表2 :里巴維林及（ = ΝΗ)改性里巴維林於紅血球之差異放 射性分佈 28 312/發明說明書(補件)/92-04/92102500 200414903 表2所示資料也吻合毒性資料。恆河猴接受6 0毫克/千克 里巴維林，接著接受3 0毫克/千克連續每日1 〇日，出現溶 血性貧血以及紅血球顯著減少。相反地，接受相同劑量之 改性里巴維林的恆河猴的紅血球未見顯著變化。 基於肝門靜脈插管猴口服投予里巴維林或改性里巴糸隹 林後，肝門血漿與系統血漿間之差異，口服投予改性里巴 維林後，肝放射性濃度估計比里巴維林口服里巴，維林之肝 放射性濃度局約5 0 %。如此，改性里巴維林只需要里巴維 林之約6 6 %劑量即可達成與里巴維林相同的肝濃度。基於 改性里巴維林比里巴維林之紅血球放射性低（約1 2 %)以及 肝濃度較高（約5 0 % )，估計改性里巴維林之治療比約爲里 巴維林之治療比的1 2倍。因此，預期改性里巴維林可以里 巴維林之約65 %劑量投藥而達成約略與里巴維林的相等效 果，且實質不會出現溶血性貧血；或改性里巴維林可以里 巴維林之相等劑量投藥，而達成比里巴維林更高的功效， 但實質未出現溶血性貧血。進一步預期改性里巴維林可以 里巴維林劑量約5 % - 5 0 %，較佳2 0 % - 5 0 %，更佳1 〇 % -1 5 % 及最佳5 - 6 %投藥而達成里巴維林的相等治療效果。 (e )( = N Η )改性里巴維林於試管試驗脫去胺基成爲里巴維林 由小牛腸分離之腺苷脫胺基酶（A D A )係購自（百齡佳 曼翰（Bo ehringer Mannheim)公司。檢定分析係於室溫23 °C 於杜別克（Dulbecco)公司 pBS 緩衝液（Na2lip〇4，8mM; KH2P04，1.5 mM; KC1，2.7 mM; NaCl，138 mM; pH 7.2)進 行。獲得（ = NH)改性里巴維林及里巴維林（〇·2 mM)之紫外 29 312/發明說明書(補件)/92-04/92102500 . 200414903 線光譜，於2 4 0奈米之吸收差異用來追蹤（=Ν Η )改性里巴 維林水解脫去胺基成爲里巴維林。於無酶存在下，於緩衝 液（pH 7.2)觀察1.5小時時間未見（ = NH)改性里巴維林之自 動水解（資料未顯示於此處）’指示化合物極爲穩定。採用 紫外線檢定分析方法的極限’微拉米定（vir ami dine)出現自 動水解小於2.5 x 1 (Γ 5分鐘」。其它實驗顯示於緩衝液內添 加鋅離子不會升高自發水解速率（資料未顯示於此處）。Structural formula 1 Structural formula 2 Modified ribavirin The reduction in selective accumulation is shown in Figures 1A and 1B. In Figure 1A, red blood cells (non-target cells) 100 were provided with ribavirin (R). Ribavirin enters red blood cells and is phosphorylated into pharmacologically active ribavirin-phosphate (R-P), which remains inside the red blood cells. Similarly, hepatocytes (target cells) 110 were provided with ribavirin (R). Ribavirin enters liver cells and is phosphorylated into pharmacologically active ribavirin-phosphate (R-P), which remains in hepatocytes. In Figure 1B, red blood cells (non-target cells) 1 0 1 are provided with modified ribavirin (R *, 1-cold-D-ribofuranosyl-1,2,4-triazole-3-carboxyl spine). Modified ribavirin enters red blood cells but is not phosphorylated and therefore leaves red blood cells. Similarly, hepatocytes (target cells) 1 1 1 are provided to modify ribavirin (R *). The modified ribavirin enters the liver cells and is enzyme-catalyzed to deaminate to become ribavirin 11 312 / Inventory (Supplement) / 92-04 / 92102500 200414903 Lin, which is subsequently phosphorylated into a pharmacologically active phosphorylation Bavilin (R-P) is retained inside liver cells. In another aspect of the subject matter of the present invention, a variety of drugs other than ribavirin are expected to be suitable for use in the concept of the present invention. Generally suitable drugs include drugs that can be metabolized, activated, and / or deactivated by cells other than the target cell. Drugs that are specifically contemplated include ribs, nucleotides, nucleoside analogs, and nucleotide analogs. For example, TiazOfurin (2-cold-D-ribofuranosyl-4-thiazole-carboxamide) is a nucleoside analog with a carboxamido group, which is conveniently Modified to obtain the corresponding carboxy brand. In another example, another drug comprises a nucleoside uracil or a nucleoside analog 5'-fluorouracil (5'-FU). In yet another aspect of the subject matter of the present invention, the blocking group need not be limited to = NH group, and may include a variety of primary and secondary amines, as long as the blocking group can be covalently coupled to the drug through a nitrogen atom. The term "blocking group" is used herein to denote a chemical group covalently attached to a drug, and when attached to a drug, can block at least one metabolic conversion effect of the drug. As used herein, the term "metabolic transformation" of a drug means that any intracellular and / or extracellular chemical change of the drug is facilitated by the metabolism of a cell or cellular system, and specifically includes enzymatic breakdown (such as oxidation, hydrolytic lysis), and Enzymatic modification (eg saccharification, phosphorylation). A suitable blocking group is generally expected to have the structural formula N (Ι ^) (Ι12) or = NR !, where R! And R2 are hydrogen, straight-chain or branched alkyl, alkenyl, alkynyl, aryl, aryl, etc. Group, arylalkynyl, and aryl, all of which further include heteroatoms such as nitrogen, oxygen, sulfur, or halogen atoms. However, it is particularly preferred that other blocking groups can be removed by drugs through enzyme catalysis, and it is specifically expected that enzymes include liver-specific aminohydrolases, including deaminases (such as adenosine or cytosine deaminase), Liver de 12 312 / Inventory (Supplement) / 92-04 / 92102500 200414903 Pyramine (such as nicotine Pyramine deamidase) and liver transaminase (bonded amino acid_pyruvate transaminase) . The desired blocking group can be covalently bonded to multiple positions of the drug molecule. Generally, the desired fun is modified at the residual acid amine moiety, and it is expected that it can also be modified at multiple positions other than amine. , Especially carbonyl (such as carboxylic acid and ketone type carbonyl). For example, each carbon group in the ring portion of uracil or its analog 5, _FlJ can be modified by a blocking group. Although the concept of the present invention is not limited thereto, it is expected that the blocking group may deactivate the drug or prevent subsequent activation once the modified drug is presented to non-target cells. For example, if the blocking group is coupled to the drug at a position necessary for a specific interaction between the drug and the target molecule (such as a receptor or enzyme substrate binding site), the blocking group can deactivate the drug. In this regard, the blocking group can be coupled to the drug's site that prevents metabolic activation. Depending on the chemical properties of the drug and / or blocking group, it is expected that the blocking group may be replaced with a functional group or a substituent, or the blocking group may be attached to a functional group or a substituent. For example, if the drug is ribavirin and the blocking group is = NH group, the oxygen atom of the ribavirin carboxyamido group is replaced by the = NH group. In this regard, if the drug contains a nucleophilic group (e.g. -cr) and the blocking group contains a secondary amine group with a suitable leaving group, the secondary amine can be attached to the nucleophilic group. In the case of a drug modification step, the modification is expected to include organic synthesis modification, enzymatic modification, or re-synthesis to make a modified drug. For example, if the drug contains an activated carbonyl functional group, the amidation of the carbonyl atom can be achieved in a single nucleophilic exchange reaction. In addition, especially when the drug has multiple reactive groups in addition to the blocking group to be attached to the group, the modified drug is resynthesized on Economic 13 312 / Invention Specification (Supplement) / 92 · 04/92102500 200414903 More attractive. In particular, it is also expected that in reactions using drugs and blocking groups as enzyme substrates, appropriate drugs will be modified enzymatically by introducing blocking groups into the drug. When possible, the enzyme used for such modification is preferably derived from the target cell (for example, from a heterologous or allogeneic source, or from a source of a recombinantly encoded gene that can express the enzyme). It must be understood that depending on the non-target cells and the chemical properties of the drug and / or blocking group, preventing drug accumulation in non-target cells can be achieved by at least one of a variety of mechanisms, including the Uptake of specific transport factors, reducing metabolic conversion to a form that will be retained (for example, due to additional or new charges, changes in hydrophobicity, or changes recognized by outward transport factors), or increased outward transport by non-target cells (Such as the mechanism of the secretion signal of the blocking group) can prevent the accumulation of drugs. For example, if the drug is a nucleoside analog, non-target cells have a nucleoside transport factor, which can selectively introduce nucleosides without lipophilic moieties into the cell; adding a lipophilic moiety to the drug as a blocking group can prevent Accumulation of drugs. In another example, phosphorylation (and concomitant accumulation) of multiple nucleosides of red blood cells is expected to be prevented by converting a carboxyamido group to a carboxy # group (see above). In terms of blocking enzyme-catalyzed removal based on target cells, there are significant changes in enzyme-catalyzed removal based on target cells, blocking groups, and drug classes. Enzyme-catalyzed removal includes various types of enzymes, including hydrolases, transferases, degrading enzymes, and oxidoreductases. The most preferred subtypes are adenosine and cytidine deaminase, sperminase, transaminase, and aryl hydrazone Aminase. It is further understood that enzymes used for enzymatic removal of blocking groups can be expressed exclusively in target cells, but in other aspects of the subject matter of the present invention, appropriate enzymes can also be used outside of target cells 14 312 / Explanation of the Invention (Supplement) / 92-04 / 92102500 200414903. As long as the enzyme is not expressed everywhere in all cells of the cell-containing system. It is further necessary to understand that the expected enzymes will naturally (in other words, non-recombinant) behave in individual target cells in individual target cells under normal and / or pathological conditions. For example, it is known that glutamine-pyruvate transaminase essentially exhibits relatively high selectivity in hepatocytes, so this enzyme is a suitable enzyme for removing blocking groups. In addition, it is known that cytidine deaminase is expressed in colon cancer cells in a relatively high amount, but is not expressed in normal colon cells or only in a small amount. In another aspect of the subject matter of the present invention, the cytotoxicity of a drug to non-target cells can be reduced by a method in which, in one step, the damage induced by the non-target cell's metabolic transformation to non-target cells is identified. The term "cytotoxicity" is used herein to indicate an undesired pharmacological effect on non-target cells, where the undesired pharmacological effect specifically includes inhibition of replication, energy metabolism, or cell death. In a further step, the drug is modified with a blocking group, wherein the blocking group is covalently coupled to the drug through the nitrogen atom of the blocking group, wherein the blocking group reduces the metabolic transformation of the drug in non-target cells, and Enzymes catalyze cleavage by drugs. In yet another step, the drug is administered to a system comprising target cells and non-target cells, wherein the blocking group is covalently coupled to the drug. In the preferred aspect of reducing the cytotoxicity of the drug to non-target cells, metabolic transformation includes phosphorylation of the drug in the red blood cells to the corresponding drug phosphate. For example, it is well known in the industry that the antiviral drug ribavirin is phosphorylated on a variety of cells to produce pharmacologically active ribavirin 5'-monophosphate (eg Homma, M. e t a 1. High-performance liquid chromatographic determination of Ribavirin in whole blood to assess disposition in erythrocytes; Antimicrob Agents C hem other 15 312 / Description of Invention (Supplement) / 92-04 / 921 〇25〇〇200414903 1 9 9 9 No V; 4 3 (1 1): 2 7 1 6-9), this compound is an inhibitor of inosine monophosphate dehydrogenase (IMPDH). Unfortunately, ribavirin-5 monophosphate has significant cytotoxicity to red blood cells (D e F 1anceschi, eta 1 _; Hem ο 1 ytic anemia induced by ribavirin therapy in patients with chronic hepatitis C virus infection: role of membrane oxidative damage, Hepatology 2000 Apr; 3 1 (4): 9 9 7 -10〇4), the results show that preventing or reducing the formation of ribavirin-5'-monophosphate in red blood cells will significantly reduce ribavirin Cytotoxicity. However, it must be understood that in addition to phosphorylation, the various metabolic transformations of drugs in non-target cells are also expected to cover the scope of the present invention. Such metabolic transformations include oxidation, reduction, and hydrolysis to break the covalent bonds inside the drug, addition or removal. Pendant groups and ring-opening reactions. For example, it is expected that if the non-target cells are hepatocytes, metabolic transformation includes a variety of enzyme-catalyzed detoxification or lysis reactions (eg, glycation reaction, cytochrome P45G oxidative reaction, etc.) known to the liver. In another example, metabolic transformation includes phosphatase or esterase activity. Depending on the type of metabolic transformation, transformation may be limited to a single type of non-target cells, but multiple cell types may also occur. For example, if a non-target cell has a relatively high rate of nucleic acid synthesis and the metabolic transformation is by an enzyme vector involved in nucleic acid synthesis, the cells in a variety of fast-growing types will undergo metabolic transformation. In this regard, metabolic transformation can also be limited to drugs approaching a particular group of cells or organs. It is important to understand that a variety of well-known experimental procedures can be used to identify and / or confirm the covalent coupling of blocking groups to drugs to reduce metabolic transformation in non-target cells. For example, when target cells are cultured in a test tube, non-target cells are expected to be co-cultured with the corresponding radiolabeled drug 16 312 / Explanation of the Invention (Supplement) / 92-04 / 92102500 200414903. Identification of multiple assays, including immunoassay analysis, thin-layer chromatography or GC-MS. In addition, when non-target cell lines are restricted to mammals, a tissue biopsy will provide sufficient specimens to isolate and identify metabolites administered to the drug. It is further expected that the type of damage to non-target cells may be substantially changed, and the damage is from the slowdown of cell metabolism of non-target cells to the range of cell death. For example, if metabolic transformation produces an inhibitor of an enzyme on the glycolytic pathway, the energy of the non-target cell must be provided at least in part by the rescue pathway. In the same way, if the conversion of drug metabolism into enzyme inhibitors proceeds at a relatively slow rate, the upward regulation of inhibitors affecting enzyme performance can almost completely compensate for the decrease in the number of active sites. On the other hand, if free radicals are produced by metabolic transformation, lipid peroxidation will cause severe cell membrane damage, resulting in cell death. It is further expected that the damage caused by the metabolic transformation of drugs may be caused directly or indirectly. For example, if metabolic conversion produces an enzyme inhibitor and blocks the enzyme, the damage is considered direct damage. On the other hand, if the metabolic transformation produces an intermediate, which is further converted into an enzyme inhibitor after subsequent intracellular or extracellular modification, the injury is considered as indirect injury. With regard to the steps of the drug delivery system, it is expected that the appropriate drugs will be administered in any suitable drug formulation and in any appropriate protocol. Such administration can be performed orally, parenterally (including subcutaneous injection, intravenous muscle, intradermal injection or infusion technique), inhalation spray, rectum, topical administration, etc., to contain conventional non-toxic pharmaceutical acceptable carriers, adjuvants, and vehicles Dosage unit dose formulations are administered. For example, it is expected that an appropriate drug may be administered orally in the form of a pharmacologically acceptable salt, or otherwise in the form of a physiological salt solution (eg, buffered to a pH of about 7. 312 / Invention Specification (Supplement) / 92-04 / 92102500 200414903). 2 to 7. 5) Intravenous administration. Conventional buffers such as phosphate, bicarbonate or citrate can be used for this project. In addition, it is expected that within the skill range of the industry, the route of administration and usage of a particular drug can be modified and the pharmacokinetics of the drug controlled to maximize the benefit to the patient. Certain forms of administration are expected to expel the form before use. Skilled artisans will know how to easily modify the intended drug into a prodrug form and assist in the delivery of the active compound to a target site in the host organism or patient. The skilled artisan also utilizes the favorable pharmacokinetic parameters of the prodrug form (if any) to deliver the compound to the target location in the host organism or the patient's body to obtain the highest expected effect of the compound. It is further understood that the desired drugs may be administered alone or in combination with other pharmacologically active agents, which may be administered separately or together, and may be administered simultaneously or separately in any order when administered separately. Expected pharmacologically active agents include antiviral agents such as interferons (such as interferon alpha and 7); antifungal agents such as tolnaftate, FungizoneTM, LotriminTM, Mies MycelexTM, Nystatin, and Amphoteracin; antiparasitic agents such as MintezolTM, NicIocideTM, and Verm. xTM) and FlagylTM; intestinal agents such as ImmodiumTM, LomotilTM and PhazymeTM; antitumor agents such as interferon alpha and 7, Adriamycin (AdriamycinTM), CytoxanTM, ImuranTM, Methotrexate, MithracinTM, TiazofurinTM, Paclitaxel 18 312 / Invention Specification ( (Supplement) / 92-04 / 92102500 200414903 (TaxolTM); Dermatological drugs such as AclovateTM, CyclocortTM, Denorex1M, Frozon (F 1 〇r ο ne τ M), oxo blue (0 X s 〇ra 1 e η τ M), coal tar and salicylic acid; migraine preparations such as wheat Amine compounds; steroids and immunosuppressants not listed above include cyclosporine (cyc 1 〇s ρ 〇rins), diproxon (〇 丨 卩 1 * 080 1 ^ 1 ^), hydrocortisone (1: 1 ) ^ 1-0 (: 01 * 1; 丨 50 1 ^); Fororon (TM), Lidex (TM), Topicoft, and Vais is one); and Metabolic agents such as insulin and other drugs that do not fall into any of the foregoing categories, including cytosecretins such as IL2, IL4, IL6, IL8, IL10 and IL12. With regard to the expected drug and pharmacologically active agent dose, the therapeutically effective amount will vary depending on the condition being treated, the severity of the condition, the treatment plan used, the pharmacokinetics of the medication, and the patient (animal or human) being treated. It is further anticipated that a variety of doses will be suitable, including 0. 5 mg / kg to 0. Doses of 1 mg / kg and below, but also including 0. Doses of 5 to 1.0 mg / kg and above. Although it is generally preferred that the system containing target cells and non-target cells be mammals (most preferably humans), a variety of other systems are also suitable, including in particular cell and tissue cultures for in vitro testing. Regarding drugs, blocking groups, drug modification steps, target cells, and non-target cells, the same considerations described above apply to methods that are expected to reduce the cytotoxicity of drugs to non-target cells. In yet another aspect of the subject matter of the present invention, the dosage of a drug in a system can be reduced by a method of providing a drug, wherein the metabolic transformation of a drug in non-target cells can reduce the drug in a system containing non-target cells and target cells 19 312 / Invention Specification (Supplement) / 92-04 / 92102500 200414903. In a further step, the drug is modified with a blocking group, wherein the blocking group is covalently coupled to the drug through the nitrogen atom of the blocking group, and wherein the blocking group can reduce the metabolic transformation of the drug in non-target cells. In a subsequent step, the drug administration system, wherein the blocking group is covalently coupled to the drug, and wherein the blocking group is removed from the drug by the target cell by enzyme catalysis. In a preferred aspect of reducing the systemic dose of the drug, the drug is ribavirin, the target cells are liver cells infected with the virus, and the non-target cells are red blood cells. It is well known in the industry (see above) that Ribavirin is metabolized into Ribavirin phosphate, and Ribavirin phosphate is retained in red blood cells, thus significantly reducing the concentration of Ribavirin. It is modified by connecting the = NH blocking group to the carboxamide carbon, thereby replacing the carbonyl oxygen of the carboxamide. It has been shown (see below) that the conversion of ribavirin modified by = NH blocking group to red blood cells is significantly reduced. It is further expected that the optimal dose of modified ribavirin is 50 mg-300 mg in a single oral administration to the human body. Ribavirin is known as an antiviral drug and is administered orally to the human body in a single dose of at least about 600 mg-1200 mg. The initial concentration of ribavirin in the system (such as the human body) is between about 1 #M to several hundred // M, but because ribavirin is phosphorylated in red blood cells, the concentration of ribavirin in the system is typically Isolate inside the red blood cells and reduce the initial concentration to about 85% to 50% within 24 hours. The inventors show that modification of ribavirin to bufi-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine can significantly reduce the amount of phosphorylation of ribavirin (see later) ). It is therefore expected that all or almost all of the initial concentrations of ribavirin can be used to obtain the desired pharmacological efficacy of the target cells. As a result, it is expected that the use of blocking groups to modify ribavirin can reduce the dose of ribavirin by about 5 wt% 'preferably about 20 312 / Explanation of the Invention (Supplement) / 92-〇4 / 92102500 200414903 1 0 wt%, More preferably 2 5 wt% and optimal 50 W t%. However, it should be understood that it is also expected to cover a variety of doses other than 600 mg-i 200 mg, including 200 mg-600 mg doses, 20 mg-200 mg doses and below. For example, if ribavirin is used as an immunomodulatory drug, a lower dose of about 0.00 mg to 300 mg is sufficient. In this regard, when relatively high concentrations of the drug are required, dosages of 600 mg to 180 mg and above are expected. It is also important to understand that depending on the particular metabolic transformation, the reduction in dose can vary significantly. For example, if the metabolic transformation is relatively fast and is performed on a plurality of non-target cells, the expected dose reduction is 25 to 80% and more. On the other hand, if the metabolic conversion is quite slow, it is expected to reduce the dose by 25 wt% to 5 wt% and below. In the method of anticipating drug reduction, the considerations described above also apply to drugs, blocking groups, metabolic transformations, drug modification steps, systems, dosing steps, target cells, and non-target cells. (Example) (a) An example synthesis of ribavirin is shown in Fig. 2. The synthesis procedure is summarized below. 1- (2,3,5-di-0-ethenyl- / 3-D-ribose-glucosyl) -i, 2,4-tri-III-methyl-3-carboxylate (3) and 1- (2,3,5-tri-O-acetamido- / 3-O-ribofuranosyl) -1,2,4-triazole-5-carboxylic acid methyl ester (4) 1,2, 4-Triazole-3-carboxylic acid methyl ester (25. 4 g, 200 mmol) (1) 1,2,3,5-tetra-O-acetamido- / 3-D · ribosefuranose (63. A mixture of 66 grams, 200 millimoles) (2) and osmium (p-nitrophenyl) phosphate (1 grams) was placed in a round-bottomed bottle (500 ml). The flask was placed in a preheated oil bath at 16 5 · 17 5 ° C, and was stirred for 25 minutes under the vacuum of a water aspirator. The replaced acetic acid was collected in the ice condensation air flaps, which were placed between the aspirator and the round bottom bottle. 21 312 / Description of the Invention (Supplement) / 92-04 / ½ 102500 200414903 Remove the flask from the oil bath and allow it to cool. When the temperature of the flask reached about 60-70 ° C, ethyl acetate (300 ml) and saturated sodium bicarbonate (150 ml) were introduced and extracted with ethyl acetate. The aqueous layer was extracted again with ethyl acetate (200 ml). The ethyl acetate extract was washed with saturated sodium bicarbonate (300 ml), water (200 ml) and brine (150 ml). The organic extract was dehydrated with anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in ethanol (100 ml), diluted with methanol (60 ml), and cooled at 0 ° C for 12 hours to obtain colorless crystals. The solid was filtered, washed with a minimal amount of cold ethanol (20 ml), and dehydrated with solid sodium hydroxide under high vacuum to obtain 60 g (78%). The filtrate was evaporated to dryness and purified on a silica gel column using chloroform-ethyl acetate (9: 1) as the eluent. The two products were separated from the filtrate, and the rapidly moving product was about 8. 5 grams (11%), slowly moving the product about 5 grams (6. 5%). Slowly move the product to match the crystalline product. The fast-moving product was found to be vesicular carcasses. 3 combined yield is 65 g (84%); melting point 108-110 ° C; 1H-NMR (CDC13) of 3: (5 2. 11 (s5 3H, COCH3), 2.12 (s, 3H5 COCH3), 2. 13 (s, 3H, OCH3), 3. 99 (s, 3H, COCH3), 4. 22 (dd, 1H), 4. 46 (m, 2H), 5. 55 (t, 1H, J = 6. 0 Hz), 5. 75 (m, 1H), 6.05 (d, 1H, C) ’H J = 3. 6 Hz) and 8. 41 (s, 1H, C5H). (C15Hi9N309) C, H, N analysis. 1 of 1H-NMR (CDC13): 5 2. 02 (s, 3H, COCH3), 2. 10 (s, 3H, COCH3), 2. 12 (s, 3H, OCH3), 4. 00 (s, 3H, COCH3), 4. 14 (m, 1H), 4. 42 (ni, 2H), 5.76 (t, 1H) 5 5. 81 (m, 1H), 6. 94 (d, 1H, CKH J = 2. 1 Hz), 8. 03 (s, 1H, C5H). Analysis (C15H19N309) C, H, N. 1-Cold-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine (5) 1- (2,3, 5 -tri-0-acetamido- / 3 -D- Ribofuranosyl) 1,2,4-triazole 22 312 / Invention (Supplement) / 92-04 / 92102500 200414903 -3 -Carboxylic acid methyl ester (62 g, 16 1 mmol) 3) It is placed in a steel bomb and treated with freshly prepared methanol-based ammonia (350 ml, which is prepared by passing dry ammonia gas into anhydrous methanol at 0 ° C until saturation). The steel bomb was sealed and stirred at room temperature for 18 hours. The gundam cools to 01, turns on, and allows the contents to evaporate to dryness. The residue was treated with absolute ethanol (100 ml) and evaporated to dryness. The obtained residue was triturated with acetone to obtain a solid. The solid was filtered and washed with acetone. The solid was dehydrated at room temperature overnight and dissolved in a mixture of hot ethanol (600 ml) and water (10 ml). The volume of the ethanol solution was reduced to 150 ml by heating and stirring on a hot plate. Colorless crystals were obtained when the hot ethanol solution was cooled. The crystals were filtered, washed with acetone and dehydrated in vacuo. The filtrate was further concentrated to obtain additional product. Total yield: 35 g (89%); melting point 177-179 ° C; [α] 2 0 D-3 5. 3 (c, 10, H20);] H-NMR (Me2SO-d6): 5 3.46 (m, 1H, C5, H), 3. 60 (m, 1H, C5’H), 3. 94 (m, 1H, C4, H), 4. 12 (ηα, 1H), 4. 34 (m, 1H), 4. 95 (t5 1H, C5’OH), 5. 22 (d5 1H), 5. 60 (d, 1H), 5. 80 (d, 1H, J = 3. 9 Hz, C], H), 7. 64 (bs, 1H, NH2), 7. 84 (bs, iH, NH2), 8. 87 (s, 1H, C5H). 3C NMR (Me2SO-d6) 5 61 · 8, 70.2, 74. 4, 86. 0, 91. 6, 144 · 9, 157. 4, 160. 6. (C8H12N405) C, H, N analysis. (b) An example synthesis of Ribavirin modified with -NH group is shown in Figure 3, following the procedure outlined below. 3-cyano-1- (2,3,5-tri-O-ethylfluorenyl- / 3-1) -ribosefuranosyl group 1,2, triazole (7) 3-cyano-1,2 , 4-triazole (18.8 grams, 2000 millimoles) (6), 12,], ^ Tetra-0-acetamido-cold · 0-ribofuranose (6366 grams, 2000 millimoles) Mol) and plutonium (para. 312 / Invention Specification (Supplement) / 92-04 / 92102500 23 200414903 Nitrophenyl) phosphate (1 g) were placed in a round bottom bottle (500 ml). The flask was placed in a preheated oil bath at 165-175 ° C under vacuum of a water aspirator with stirring for 2 5 minutes. The replaced acetic acid was collected in the ice condenser flap, which was placed between the aspirator and the round bottom bottle. The flask was removed from the oil bath and allowed to cool. When the temperature of the flask dropped to about 60-70 ° C, ethyl acetate (300 ml) and saturated sodium bicarbonate (150 ml) were introduced, and extracted into ethyl acetate. The aqueous layer was extracted again with ethyl acetate (200 ml). The ethyl acetate extract was combined and washed with saturated sodium bicarbonate (300 ml), water (200 ml) and brine (150 ml). The organic extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness. The residue was dissolved in ether (100 ml), and the ether was cooled at 0 t for 12 hours to obtain colorless crystals. The solid was filtered, washed with a minimal amount of cold ethanol (20 ml), and dehydrated using solid sodium hydroxide under high vacuum. Yield: 5 6. 4 grams (80%). Melting point 9 6- 97 ° C. H-NMR (CDC13): δ 2. 1 1 (s, 3H, COCH3), 2. 13 (s, 3Η, COCH3), 2. 14 (s, 3Η, COCH3), 4.22 (dd? 1H)? 4. 46 (m? 2H) 9 5. 5 2 (t? 1H5 J = 6. 〇 Hz); 5. 70 (m; 1H), 6. 01 (d, 1H, C ^ ’H J = 3. 6 Hz) and 8. 39 (s, 1H, C5H). C] 4H16N407 (3 5 2. 3 0) Analysis and calculation 値: C, 47. 73; H, 4. 58; N, 15. 90. Found 値: C, 4 7. 7 0; H, 4. 6 3; N, 1 6 · 0 Fabry-D-ribosefuranosyl-1,2,4-triazole-3-carboxyline hydrochloride (8) 7 (14. 08 grams, 40.0 millimoles), ammonium chloride (2. 14 grams, 40. 0 millimolar) and anhydrous ammonia (150 ml) were heated in a steel bomb at 85 ° C for 18 hours. Cool the bullet, turn on the bullet, and evaporate the contents to dryness. The residue was crystallized from acetonitrile-ethanol to obtain 10. 6 grams (95%) 8. 177] 79t :. ] H-NMR (DMSO-d6): 53. 44-4. 2 (m? 3H), 4. 40 (m3 2H)? 5. 04 24 312 / Invention Specification (Supplement) / 92-04 / 92102500 200414903 (t, 1H), 5. 29 (m, 1H), 5. 74 (m, 1H), 5. 87 (d, 1H, C], H), 8. 96 (bs, 3H) and 9. 17 (s, 1H, C5H). C8H14ClN504 (279. 68) Analysis and calculation 値: C, 34. 35; H, 5.05; N, 25. 04; Cl, 12. 69. Measured radon: C, 34. 39; Η, 5.10; N, 25. 14; Cl, 12. 71. Another example of a synthetic procedure starting with ribavirin was performed as follows: 2 ', 3', 5'-tri-0-acetamib / 3-0-ribofuranosyl-1,2,4-tri Azole-carboxamide (9) 1- / 3 ribofuranosyl-1,2,4-triazole-3-carboxamide (28. A suspension of 4 g, 116.4 mmol (ribavirin) in acetic anhydride (200 ml) and pyridine (50 ml) was stirred overnight at room temperature. The obtained Chenghuang solution was concentrated in vacuo to obtain a transparent foamed carcass (43. 1 g, quantitative yield). The corpus callosum was confirmed to be homogeneous by TLC and used directly in the next step without purification. A small amount of corpus callosum was purified by flash chromatography;] H-NMR (300 MHz, D MS 0-d6) 5 2. 01, 2. 08, 2. 09 (3 s, 9H, COCH3), 4. 10 (m, 1 H), 3.52 (m, 2H), 5. 58 (t? 1 H), 5. 66 (m, 1 H); 6. 33 (d? 1 H? J. = 3. 0 Hz; C) H); 7. 73, 7. 92, (2 s, 2 H, CONH2), 8. 8 6 (s5 1 H, C5H triazole). (C] OH] 8N408) C, H, N. 3_cyano_2 ', 3', 5'_tri-o-ethylfluorenyl-11-D-ribosefuranosyl-1,2,4-triazole (1 0) in 9 (43.1 g '116. 4 mmol) was added to a solution of chloroform (500 ml) and diethylamine (244 ml) was added, and the mixture was cooled to 0 in an ice-salt bath. Slowly dropwise add dichloromethane (30. 7 ml, 330 millimoles), let gluten iW heat to the chamber? Dish. "After the mixture was stirred at room temperature for 1 hour, T lc (Keinin / Acetone 3: 1) could not completely disappear. The brown reaction mixture was concentrated under vacuum 312 / Invention Specification (Supplement) / 92-04 / 92102500 25 200414903 Shrinked to dryness, the residue was dissolved in chloroform (500 ml), the organic solution was washed with saturated aqueous sodium bicarbonate (3 x 2000 ml), dehydrated with anhydrous sodium sulfate and concentrated in vacuo. The residue was dried in silicone (rapid Chromatography) using 20% acetone in hexane chromatography to obtain 3 3. 14 grams (81% from ribavirin) of pure 10 were amorphous. All aspects of the solid are exactly the same as the real sample: melting point 1 0 1 -1 0 3 ° C; IR (potassium bromide) ^ 22 5 0 (CN), 1 7 5 0 (C = 0), cnTYH-NMR (300 MHz, CDC13) 5 2. 04, 2. 06, 2 · 07 (3 s, 9H ,, ethyl ethyl), 4.15 (dd, 1 H), 4. 40 (m, 1 Η), 5.47 (t, 1 H), 5. 63 (dd, 1 H), 5. 95 (d, 1 H, J = 3. 2 Hz, C] H), 8. 34 (s, 1 H, C5H triazole). 1-Cold-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (8) at 10 (4. 0 g, 11.4 mmol) was added to a suspension of methanol (100 ml), and a methanol-based sodium methoxide solution (12 ml) was added, and the mixture was stirred at room temperature overnight. The solution was acidified to pH 4 using Dow ex Η + resin washed with methanol, the resin was removed by filtration, and the filtrate was concentrated to dryness in vacuo. The residue was dissolved in a minimum amount of methanol (15 ml) and transferred to a pressure bottle. Ammonium chloride (0.61 g '11. 4 mmol) was added and a methanol solution (75 ml) saturated with anhydrous ammonia gas at 0 ° C was used. The bottle was sealed under pressure and the solution was stirred at room temperature overnight. The solution was concentrated to dryness in vacuo, and the resulting residue was crystallized from acetonitrile / ethanol to obtain 8 as a crystalline solid (2.95 g, 93%). This sample is identical in all respects to the real sample. In yet another approach, 'B / 3-D. ribofuranosyl-1,2,4-triazole-3. Carboxamidine hydrochloride (8) can be used as a microbial culture, intact microbial cells, or cell extracts. Enzyme sources (under non-proliferative conditions of microorganisms) are produced by enzyme-catalyzed reactions. 3-cyano-1- (2,3,5-tri-O-acetamido- / 3 ribofuranose 26 312 / Invention (Supplement) / 92-04 / 92102500 200414903 group) 1,2, 4-triazole (7) can be produced by contacting 3-cyano-1,2, carditriazole or a salt thereof with a ribose donor in the presence of a microorganism-based enzyme source. The compound 7 is then converted into (8) by treatment with a liquid ammonia solution. In addition, 1,2,4-triazole-3-carbachol hydrochloride can be directly produced by reacting with a ribose donor in the presence of an enzyme (8). (〇 Modified ribavirin in the liver deamination to become ribavirin in mice, repeated oral administration of 3H-ribavirin and 3H-(= NH) modified ribavirin, daily dose of 3 After 8 consecutive days of 00 mg / kg, the minimum radioactive concentration of Ribavirin in the liver is lower than the Cmin of modified Ribavirin. In particular, it must be noted that Ribavirin-treated mice account for liver Radioactivity is about 90%, ribofuranosyl tricarboxylic acid (RTCA) accounts for about 10% of liver radioactivity. Conversely, in mice treated with modified ribavirin, modified ribavirin accounts for about 3% of liver radioactivity 0%, while ribavirin accounts for about 70% of liver radioactivity (see also Table 1). 3H-ribavirin 3H-(= NH) modified ribavirin ___ total liver radioactivity RTC A ribavirin Forest modified ribavirin 1 8. 4 microgram equivalents / gram about 1. 8 μg equivalent / g approx. 16. 6 μg equivalent / g not detected 23. 8 μg equivalent / g undetected approx. 16. 6 μg equivalent / g approx. 7. 2 microgram equivalents / gram Table 1: Differential radioactive distribution of mouse liver (d) in red blood cell (RBC) ribavirin and (= NH) modified ribavirin. It has been shown that ribavirin can be phosphorylated in red blood cells. It further suggests that phosphorylated ribavirin is the cause of hemolytic anemia observed after long-term or high doses of ribavirin received by the human body. Apparently (= NH) modified ribavirin was not shipped directly 27 312 / Invention Note (Supplement) / 92-04 / 92102500 200414903 Sent into red blood cells' as tested in a test tube (data not shown here) can be proved Subsequently, it is expected that the modified ribavirin can accumulate in red blood cells only after the liver is deaminated to become ribavirin and subsequently phosphorylated to the corresponding phosphate, as shown in Table 2 below. Mice repeated oral administration of 3H_ribavirin and 3 Η-(= N 改性) modified ribavirin at a daily dose of 300 mg / kg for 8 consecutive days. The radioactive concentration of radon Cmin was significantly lower than that of ribavirin. Judging from the difference data shown in Tables 1 and 2, the therapeutic index of modified ribavirin (that is, the ratio of liver ribavirin concentration to red blood cell ribavirin concentration) is about three times the ribavirin therapeutic index. Times. After a single oral dose of 30 mg / kg 3 猕 ribavirin or (= ΝΗ) modified 3 Η ribavirin, the rhesus monkey intubated in the hepatic portal vein reached a peak red blood cell radioactivity concentration after 24 hours, and then remained stable. concentration. The peak radioactive concentrations of 3Ηribavirin and (= NΗ) modified 3Ηribavirin have half-life T1 / 2 of approximately 198 and 577, respectively. After multiple administration at 30 mg / kg, steady-state radioactive concentrations predicted that ribavirin was significantly higher than (= NΗ) modified ribavirin (Table 2). Ribavirin 3Η- (= ΝΗ) Modified Ribavirin Radioactive Intermediate Plutonium (Mouse) Red Blood Cell Intermediate Plutonium (Monkey-Single) Red Radioactive Intermediate Plutonium (Monkey · Multi-Dose) 1. 36 microgram equivalents / gram ~ 41 microgram equivalents / gram ~ 5 0 8 9 microgram equivalents / gram 0. 38 microgram equivalents / gram ~ 17 microgram equivalents / gram ~ 606 microgram equivalents / gram Table 2: Differential radioactive distribution of ribavirin and (= NΗ) modified ribavirin in red blood cells / 92-04 / 92102500 200414903 The data shown in Table 2 also agree with the toxicity data. Rhesus monkeys received 60 mg / kg of ribavirin, followed by 30 mg / kg for 10 consecutive days. Hemolytic anemia and marked reduction in red blood cells occurred. In contrast, rhesus monkeys receiving the same dose of modified ribavirin showed no significant changes. Based on the difference between hepatic portal plasma and systemic plasma after oral administration of hepatic portal vein intubated monkeys to ribavirin or modified ribavirin, hepatic radioactive concentrations were estimated after the oral administration of modified ribavirin. Bavirin is administered orally, and the liver radioactive concentration of vilin is about 50%. In this way, modified ribavirin requires only about 66% of the dose of ribavirin to achieve the same liver concentration as ribavirin. Based on the lower red blood cell radioactivity of modified ribavirin (about 12%) and higher liver concentration (about 50%), it is estimated that the therapeutic ratio of modified ribavirin is about ribavirin The treatment ratio is 12 times. Therefore, it is expected that modified ribavirin can be administered at a dose of about 65% of ribavirin to achieve approximately the same effect as ribavirin without substantial hemolytic anemia; or modified ribavirin can Bavirin was administered in equal doses and achieved higher efficacy than ribavirin, but hemolytic anemia did not occur in substance. It is further expected that modified ribavirin may be administered at a dose of ribavirin of about 5%-50%, preferably 20%-50%, more preferably 10%-15%, and optimally 5-6%. Achieve the same therapeutic effect of ribavirin. (e) (= NΗ) Modified ribavirin was deaminated in a test tube to become ribavirin. Adenosine deaminase (ADA) isolated from the calf intestine was purchased from (Bailingmanman Bo ehringer Mannheim). The assay was performed at room temperature at 23 ° C in Dulbecco's pBS buffer (Na2lip04, 8mM; KH2P04, 1. 5 mM; KC1, 2. 7 mM; NaCl, 138 mM; pH 7. 2) Go. Obtained (= NH) modified ribavirin and ribavirin (0.2 mM) UV 29 312 / Invention Specification (Supplement) / 92-04 / 92102500. 200414903 Line spectrum. The difference in absorption at 240 nm is used to track (= N Η) the modified ribavirin hydrolyzed to remove the amino group to become ribavirin. In the absence of enzyme, in buffer (pH 7. 2) Observe 1. No automatic hydrolysis of the modified ribavirin (= NH) within 5 hours (data not shown here) 'indicates that the compound is extremely stable. The limit of ‘vir ami dine’, which is the limit of the analytical method using ultraviolet detection, is less than 2. 5 x 1 (Γ 5 minutes ". Other experiments have shown that adding zinc ions to the buffer does not increase the rate of spontaneous hydrolysis (data not shown here).
於0.2 // M ADA存在下，（ = NH)改性里巴維林之脫胺基反應 加速。於目前檢定分析條件下，酶週轉數估計爲約2.5分 鐘―1。於0.2 m Μ ( = NH)改性里巴維林與〇·5 μ M ADA共同 培養隔夜後，酶反應產物之正交質譜分析，指示大於7 5 % ( = NH)改性里巴維林被轉成里巴維林。 如此，揭示於疾病治療中特異性改良之特定具體例以及 應用例。但熟請技藝人士顯然易知可未饽離此處本發明之 構想做出多種修改。因此，除了以隨附之申請專利範圍之 精髓限制外，本發明主旨絕非限制性。此外當解譯本說明 書及申請專利範圍時，全部術語須以符合本發明內文之最 廣義可能之方式加以解譯。特別「包含」一詞須解譯爲以 非排他方式敘述元件、組成元件或步驟，指示所述元件、 312/發明說明書(補件)/92-04/92102500 30 200414903 組成元件或步驟可存在、或利用或組合其它並未明白敘述 之元件、組成元件或步驟。 【圖式簡單說明】 圖1 A及1 B爲根據本發明主旨之範例藥物之攝取及留存 示意圖。 圖2爲里巴維林合成之範例合成圖。 圖3爲改性里巴維林合成之範例合成圖。 (元件符號說明） 100 紅血球 101 紅血球 110 肝細胞 111 肝細胞In the presence of 0.2 // M ADA, the deamination reaction of (= NH) modified ribavirin is accelerated. Under the current assay conditions, the enzyme turnover is estimated to be approximately 2.5 minutes -1. Orthogonal mass spectrometry analysis of the enzyme reaction product after co-cultivation of 0.2 m Μ (= NH) modified ribavirin with 0.5 μM ADA overnight, indicating greater than 75% (= NH) modified ribavirin Converted to Ribavirin. In this way, specific specific examples and application examples of specific improvement in disease treatment are disclosed. However, it will be apparent to those skilled in the art that various modifications can be made without departing from the concept of the invention herein. Therefore, the subject matter of the present invention is by no means restrictive, except that it is limited by the spirit of the scope of the accompanying patent application. In addition, when interpreting the scope of this specification and applying for a patent, all terms must be interpreted in the broadest possible manner consistent with the context of the present invention. In particular, the word "comprising" must be interpreted as a non-exclusive description of the element, constituent element or step, indicating that the element, 312 / Description of the Invention (Supplement) / 92-04 / 92102500 30 200414903 the constituent element or step may exist, Or use or combine other elements, constituent elements or steps that are not explicitly described. [Brief Description of the Drawings] Figures 1A and 1B are schematic diagrams of the uptake and retention of an exemplary drug according to the gist of the present invention. FIG. 2 is an exemplary synthesis diagram of ribavirin synthesis. FIG. 3 is an exemplary synthesis diagram of modified ribavirin synthesis. (Explanation of component symbols) 100 red blood cells 101 red blood cells 110 liver cells 111 liver cells
31 312/發明說明書(補件)/92-04/9210250031 312 / Invention Specification (Supplement) / 92-04 / 92102500