CN1325305A - Method of administering camptothecin compounds for the treatment of cancer with reduced side effects - Google Patents
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Abstract
Description
Translated fromChinese技术领域technical field
本发明涉及喜树碱化合物，具体地说，涉及盐酸伊立替康(irinotecanhydrochloride)组合物制剂，并涉及以低的副作用施用喜树碱化合物(例如盐酸伊立替康)治疗癌症和爱滋病的方法。The present invention relates to camptothecin compounds, in particular, to irinotecan hydrochloride (irinotecan hydrochloride) composition formulations, and to methods of administering camptothecin compounds (eg, irinotecan hydrochloride) to treat cancer and AIDS with low side effects.
背景技术Background technique
喜树碱是在中国喜树和亚洲nothapodytes树的树皮中发现的基于喹啉的生物碱。其与氨基喜树碱、CPT-11(伊立替康)、DX-8951F和topotecan具有密切的化学相关性。这些化合物在治疗乳腺癌、卵巢癌、结肠癌、恶性黑素瘤、小细胞肺癌、甲状腺癌、淋巴瘤和白血病上有用。这些化合物也用于爱滋病的治疗。Camptothecin is a quinoline-based alkaloid found in the bark of the Chinese camptotheca and Asian nothapodytes trees. It is closely chemically related to aminocamptothecin, CPT-11 (irinotecan), DX-8951F and topotecan. These compounds are useful in the treatment of breast cancer, ovarian cancer, colon cancer, malignant melanoma, small cell lung cancer, thyroid cancer, lymphoma and leukemia. These compounds are also used in the treatment of AIDS.
盐酸伊立替康(CPT-11)(4S)-4,11-二乙基-4-羟基-9-[(4-哌啶哌啶基)碳酰氧基]-1H-吡喃基[3′,4′:6,7]吲嗪基[1,2-b]喹啉-3,14(4h,12H)二酮盐酸盐具有新的抗肿瘤主动机理，即抑制DNA拓扑异构酶Ⅰ。拓扑异构酶是缠绕和不缠绕构成染色体的DNA的一些酶。染色体必须不被缠绕才能制造蛋白质，而喜树碱化合物保持染色体被紧密缠绕使得它们不能制造蛋白质。因为癌细胞以比正常细胞快得多的速率生长，它们比正常细胞更易受拓扑异构酶抑制作用的攻击。Irinotecan hydrochloride (CPT-11) (4S)-4,11-diethyl-4-hydroxy-9-[(4-piperidinylpiperidinyl)carbonyloxy]-1H-pyranyl[3 ',4':6,7]indolizinyl[1,2-b]quinoline-3,14(4h,12H)dione hydrochloride has a new active antitumor mechanism, namely inhibition of DNA topoisomerase I. Topoisomerases are enzymes that wind and unwind the DNA that makes up chromosomes. Chromosomes must be uncoiled to make proteins, and camptothecin compounds keep chromosomes so tightly wound that they cannot make proteins. Because cancer cells grow at a much faster rate than normal cells, they are more vulnerable to topoisomerase inhibition than normal cells.
CPT-11已经显示出有效的临床抗肿瘤活性(2,3)，并且，前不久CPT-11对存活的益处在结肠直肠癌上显示。然而，在临床实践中，其具有白细胞减少和腹泻这些严重的毒性。CPT-11以较高的剂量在临床上的使用与不希望的和高发的腹泻(4,6,7,12)有关，腹泻现在被认为是这一药物的受剂量影响的毒性(4-7)。虽然已经进行了许多药物分析(其已显示在不同的病人中很大的可变性)来预测腹泻的发生率，但是出现了一些有冲突的结果(8-11)。CPT-11 has shown potent clinical antitumor activity (2,3) and, more recently, a survival benefit of CPT-11 was shown in colorectal cancer. However, in clinical practice, it has severe toxicity of leukopenia and diarrhea. Clinical use of CPT-11 at higher doses has been associated with an undesired and high incidence of diarrhea (4,6,7,12), which is now recognized as a dose-dependent toxicity of this drug (4-7 ). Although many drug analyzes (which have shown great variability among different patients) have been performed to predict the incidence of diarrhea, some conflicting results have emerged (8-11).
CPT-11和其代谢物，SN-38以及SN-38-Glu，不仅在人血浆中而且在人胆汁中被检测到。在三种化合物中，SN-38具有强的细胞毒性，SN-38-Glu是从SN-38去活化的和葡萄糖醛酸化的，与SN-38相比，CPT-11具有低得多的细胞毒性。这些化合物具有α-羟基-3-内酯环，其以一定的速率进行可逆水解，所说的速率主要取决于pH值(15,16,17)。在高于生理pH值下，内酯形式不稳定，平衡有利于水解，打开内酯环，产生羧化物形式。在酸性条件下，形成内酯的逆向反应是有利的。CPT-11和SN-39-Glu也发生类似的反应。CPT-11 and its metabolites, SN-38 and SN-38-Glu, were detected not only in human plasma but also in human bile. Among the three compounds, SN-38 has strong cytotoxicity, SN-38-Glu is deactivated and glucuronidated from SN-38, and CPT-11 has much lower cytotoxicity compared with SN-38 toxicity. These compounds possess an α-hydroxy-3-lactone ring that undergoes reversible hydrolysis at a rate that is mainly dependent on pH (15, 16, 17). At pH values above physiologic, the lactone form is unstable and the equilibrium favors hydrolysis, which opens the lactone ring and produces the carboxylate form. Under acidic conditions, the reverse reaction to form lactones is favored. Similar reactions also occurred with CPT-11 and SN-39-Glu.
从几个报告可以认为，在人中的主要代谢途径如下：CPT-11通过主要为肝脏来源的羧基化酯酶水解为活性代谢物，7-乙基-10-羟基-喜树碱(SN-38)。一些SN-38尔后进行经由肝酶，UDP-葡萄糖醛酸基转移酶的与SN-38β-葡萄糖醛酸化物(SN-38-Glu)的缀合，与其它组分，CPT-11以及SN-38一同分泌进胆汁(13,14)。三种化合物被认为由肠细胞再吸收，进入肠肝循环。前不久已发现肝细胞色素P-4503A酶将CPT-11代谢为7-乙基-10-[4-N-(5-戊酸)-1-哌啶基]碳酰氧基喜树碱，其具有比SN-38弱500倍的抗肿瘤活性(Rivary等，1996b；Haaz等，1997)。CPT-11,SN-38和SN38-Glu具有α-羟基-3-内酯环，其以一定的速率进行可逆水解，所说的速率主要取决于pH值(Fassberg等，1992)。在生理pH值和高于该pH下，内酯形式不稳定，平衡有利于水解，打开内酯环，产生羧化物形式。在酸性条件下，内酯-羧化物内部转化移向内酯形式。CPT-11，SN-38和SN38-Glu被分泌进胆汁，并与其一同释放进小肠腔(Atsumi等，1991；Lokiec等，1995；Chu等，1997a,b)。此外，虽然次要(Atsumi等，1995)，但另外的途径牵涉CPT-11及其代谢物从血清跨越上皮细胞向腔室的直接转运。一旦在肠中，SN38-Glu经由细菌β-葡萄糖醛酸酶在盲囊和结肠中与SN-38去缀合(Takatsuna等，1996)。CPT-11,SN-38和SN38-Glu被认为由肠细胞某种程度的再吸收，进入肠肝循环。From several reports, it can be considered that the main metabolic pathway in humans is as follows: CPT-11 is hydrolyzed to the active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN- 38). Some SN-38 then underwent conjugation to SN-38β-glucuronidate (SN-38-Glu) via liver enzyme, UDP-glucuronosyltransferase, with other components, CPT-11 and SN- 38 are secreted together into bile (13,14). Three compounds are thought to be reabsorbed by enterocytes into the enterohepatic circulation. Not long ago, hepatic cytochrome P-4503A enzyme was found to metabolize CPT-11 to 7-ethyl-10-[4-N-(5-pentanoic acid)-1-piperidinyl]carbonyloxycamptothecin, It has 500-fold weaker antitumor activity than SN-38 (Rivary et al., 1996b; Haaz et al., 1997). CPT-11, SN-38 and SN38-Glu possess an α-hydroxy-3-lactone ring that undergoes reversible hydrolysis at a rate that is mainly dependent on pH (Fassberg et al., 1992). At physiological pH and above, the lactone form is unstable and the equilibrium favors hydrolysis, which opens the lactone ring and produces the carboxylate form. Under acidic conditions, the lactone-carboxylate internal conversion shifts to the lactone form. CPT-11, SN-38 and SN38-Glu are secreted into bile and released together with it into the lumen of the small intestine (Atsumi et al., 1991; Lokiec et al., 1995; Chu et al., 1997a,b). Furthermore, although secondary (Atsumi et al., 1995), an additional pathway involves the direct transport of CPT-11 and its metabolites from serum across epithelial cells to the compartment. Once in the intestine, SN38-Glu is deconjugated from SN-38 in the caecum and colon via bacterial β-glucuronidase (Takatsuna et al., 1996). CPT-11, SN-38 and SN38-Glu are thought to be reabsorbed to some extent by enterocytes into the enterohepatic circulation.
迄今为止，只有关于CPT-11和其衍生物的肠摄取和转运机理的很少信息。这一知识对CPT-11引起腹泻的机理的理解是关键的步骤。在本项研究中，CPT-11和SN-38由肠上皮细胞的摄取被估价，并与它们各自对细胞毒性的作用相关联。To date, there is little information on the mechanisms of intestinal uptake and transport of CPT-11 and its derivatives. This knowledge is a critical step towards the understanding of the mechanism by which CPT-11 causes diarrhea. In this study, the uptake of CPT-11 and SN-38 by intestinal epithelial cells was assessed and correlated with their respective effects on cytotoxicity.
几种喜树碱衍生物的结构是已知的。
此外，美国专利5,552,154公开了肌内或口头施用关闭的内酯环形式的喜树碱(CPT)和其衍生物。在这样的情况下，获得人类宽范围的癌症的总体上减轻而没有以前用CPT Na+观察到的毒性是可能的。所使用的CPT衍生物是9-氨基-20(S)-喜树碱(9AC)、9-硝基-20(S)-喜树碱(9NO2)。Furthermore, US Patent 5,552,154 discloses the intramuscular or oral administration of camptothecin (CPT) and its derivatives in the closed lactone ring form. Under such circumstances, it is possible to obtain overall remission of a wide range of cancers in humans without the toxicity previously observed with CPT Na+ . The CPT derivatives used were 9-amino-20(S)-camptothecin (9AC), 9-nitro-20(S)-camptothecin (9NO2 ).
美国专利5,468,754描述了CPT11和其它喜树碱衍生物进行E-环内酯的碱性、pH-依赖性水解。慢反应动力学使人们可以评估是否药物的内酯和非-内酯形式两者都稳定拓扑异构酶切割的DNA复合物。研究表明仅仅喜树的关闭的内酯形式帮助稳定可切割的复合物。因此，该专利推荐使用低于7的pH值水平，以使喜树碱的内酯形式占主导地位。该专利提出与药学上可接受的酸一起使用所说的化合物。US Patent 5,468,754 describes the alkaline, pH-dependent hydrolysis of E-cyclic lactones by CPT11 and other camptothecin derivatives. Slow reaction kinetics allow one to assess whether both lactone and non-lactone forms of the drug stabilize topoisomerase-cleaved DNA complexes. Studies have shown that only the closed lactone form of camptotheca helps stabilize the cleavable complex. Therefore, the patent recommends the use of pH levels below 7 so that the lactone form of camptothecin predominates. This patent proposes to use said compound together with a pharmaceutically acceptable acid.
美国专利5,447,936描述了药物的HECPT形式在酸性环境中比在具有高的胞内pH值水平的细胞中抑制拓扑异构酶-Ⅰ更有效。该专利描述了与酸(其是有机羧酸，例如柠檬酸)一起施用药物的方法。US Patent 5,447,936 describes that the HECPT form of the drug is more effective at inhibiting topoisomerase-I in acidic environments than in cells with high intracellular pH levels. This patent describes a method of administering the drug with an acid, which is an organic carboxylic acid, such as citric acid.
美国专利5,225,404描述了用水溶性化合物的水基溶剂(如生理盐水或磷酸盐缓冲的盐溶液)施用喜树碱化合物的方法。该专利指出阻止了腹泻和膀胱炎症状，并且没有出现任何全身性毒性。US Patent No. 5,225,404 describes methods of administering camptothecin compounds with water-based solvents for water-soluble compounds, such as physiological saline or phosphate-buffered saline. The patent states that diarrhea and cystitis symptoms were prevented without any systemic toxicity.
美国专利5,637,770描述了通过把水溶性环添加至喜树碱上获得环已基化合物(其具有喜树碱的很好的特性)的方法。美国专利5,633,016描述了组合癌症治疗方法，包括施用与顺式铂氨一起施用有效量的topotecan。US Patent 5,637,770 describes a method for obtaining cyclohexyl compounds (which have the very good properties of camptothecin) by adding a water soluble ring to camptothecin. US Patent 5,633,016 describes a method of combination cancer therapy comprising administering an effective amount of topotecan with cisplatin.
美国专利5,633,26021公开了7-11-取代的喜树碱衍生物。该专利也描述了在制剂中保持酸性pH值(3到4)对带有E-环-水解的羧化物的11,7-HECPT内脂的缓慢的转变(其在生理pH下发生)是重要的。这一专利规定了常规剂量以消除化合物的毒性。US Patent 5,633,26021 discloses 7-11-substituted camptothecin derivatives. The patent also describes that maintaining an acidic pH (3 to 4) in the formulation is important for the slow conversion of the 11,7-HECPT lactone bearing the E-ring-hydrolyzed carboxylate (which occurs at physiological pH) of. This patent prescribes conventional dosages to eliminate the toxicity of the compounds.
美国专利5,652,244描述了利用喜树碱衍生物治疗人癌症的方法。美国专利5,658,920描述了喜树碱的环己基化合物的衍生物。US Patent No. 5,652,244 describes methods of treating human cancers using camptothecin derivatives. US Patent 5,658,920 describes cyclohexyl derivatives of camptothecin.
美国专利5,597,829公开了CPT由肾排泄不变，虽然大比例的所施用的药物不能在尿中被计数，该专利提出CPT的羧化物形式的提高的肾排泄出现在暴露在pH值低于5时。因此，推荐通过施用具有有机羧酸的化合物施用所说的药物以保证酸性pH值。U.S. Patent 5,597,829 discloses that CPT is excreted unchanged by the kidneys, although a large proportion of the administered drug cannot be enumerated in the urine, the patent proposes that increased renal excretion of the carboxylate form of CPT occurs when exposed to pH values below 5 . Therefore, it is recommended to administer said drug by administering a compound having an organic carboxylic acid to ensure an acidic pH.
美国专利5,674,874描述了CPT11向HECPT的药理学转化。该专利描述了与药学上可接受的酸一起施用足够量的化合物，以保持制剂的pH值从约2到约6。US Patent 5,674,874 describes the pharmacological conversion of CPT11 to HECPT. This patent describes administering a sufficient amount of the compound with a pharmaceutically acceptable acid to maintain the pH of the formulation from about 2 to about 6.
癌症研究，第14卷增刊1第31号描述了使用伊立替康(CPT11)治疗结肠癌症和非小细胞肺癌。该出版物证实，在接受积极的洛哌丁胺治疗后，与使用CPT11相关的4级腹泻发生率从17％降至5％。Cancer Research, Vol. 14 Suppl. 1 No. 31 describes the use of irinotecan (CPT11) in the treatment of colon cancer and non-small cell lung cancer. The publication confirmed that the incidence of
用于进行性结肠癌的批准的伊立替康，药物科学通讯1996，第18卷12号指出，腹泻是施用伊立替康的一种普遍的副作用。Approval of Irinotecan for Progressive Colon Cancer, Pharmaceutical Science Letters 1996, Vol. 18, No. 12 states that diarrhea is a common side effect of irinotecan administration.
国家癌症研究所杂志，1996年9月4日，第88卷17号提出在盲囊中额外产生硫代粘蛋白可能是CPT-11-导致的腹泻的主要原因。The Journal of the National Cancer Institute, Sept. 4, 1996, Vol. 88, No. 17 suggested that additional thiomucin production in the caecum may be a major cause of CPT-11-induced diarrhea.
Camptosar患者管理指南提出通过施用洛哌丁胺和gatorade避免camptosar的腹泻副作用。Camptosar patient management guidelines suggest avoiding the diarrhea side effect of camptosar by administering loperamide and gatorade.
本发明克服了与施用喜树碱化合物，特别是盐酸伊立替康相联系的主要副作用之一，腹泻。其是在现有技术中施用盐酸伊立替康治疗肿瘤的主要缺陷之一。本发明克服了与施用盐酸伊立替康和其相关化合物有关的腹泻副作用。The present invention overcomes one of the major side effects associated with the administration of camptothecin compounds, particularly irinotecan hydrochloride, diarrhea. It is one of the main drawbacks of the prior art administration of irinotecan hydrochloride for the treatment of tumors. The present invention overcomes the diarrhea side effect associated with the administration of irinotecan hydrochloride and its related compounds.
发明概要Summary of the invention
本发明提供了施用喜树碱化合物(通过肝清除)，优选地是盐酸伊立替康，以及其衍生物的方法。The present invention provides methods of administering a camptothecin compound (cleared by the liver), preferably irinotecan hydrochloride, and derivatives thereof.
本发明提供了抑制由肝清除的喜树碱化合物(包括但不限于，irlnotecan盐酸盐(CPT-11),SN36-Clu，以及SN-38)的腹泻副作用的方法，该方法包括在保持肠腔碱性pH值的同时，施用盐酸伊立替康。The present invention provides methods of inhibiting the diarrheal side effects of hepatically cleared camptothecin compounds (including, but not limited to, irlnotecan hydrochloride (CPT-11), SN36-Clu, and SN-38) comprising maintaining intestinal Irinotecan hydrochloride was administered while the cavity was alkaline pH.
本发明也提供了治疗癌症的方法，该方法包括在保持肠腔碱性pH值的同时，施用喜树碱化合物如盐酸伊立替康。The present invention also provides a method of treating cancer comprising administering a camptothecin compound such as irinotecan hydrochloride while maintaining an alkaline pH of the intestinal lumen.
在一优选的实施方案中，所说的癌症选自但不限于乳腺癌、卵巢癌、结肠癌、恶性黑素瘤、小细胞肺癌、甲状腺癌、淋巴瘤和白血病。In a preferred embodiment, said cancer is selected from but not limited to breast cancer, ovarian cancer, colon cancer, malignant melanoma, small cell lung cancer, thyroid cancer, lymphoma and leukemia.
在另一个实施方案中，本发明提供了治疗艾滋病的方法，该方法包括在保持肠腔碱性pH值的同时，施用盐酸伊立替康。In another embodiment, the present invention provides a method of treating AIDS, the method comprising administering irinotecan hydrochloride while maintaining an alkaline pH of the intestinal lumen.
本发明有利地提供了静脉内施用喜树碱化合物如盐酸伊立替康(CPT-11)的方法，该方法包括在施用所说的喜树碱化合物之前或同时，口头施用碳酸氢盐与碱性H2O。The present invention advantageously provides a method of intravenously administering a camptothecin compound, such as irinotecan hydrochloride (CPT-11), comprising oral administration of bicarbonate and alkalineH2O .
本发明提供了静脉内施用喜树碱化合物如盐酸伊立替康(CPT-11)的方法，该方法包括在施用所说的喜树碱化合物之前或同时，口头施用包含硼酸(borbicacid)的组合物。The present invention provides a method of intravenously administering a camptothecin compound, such as irinotecan hydrochloride (CPT-11), comprising orally administering a composition comprising borbic acid prior to or simultaneously with said camptothecin compound .
本发明也提供了静脉内施用喜树碱化合物的方法，该方法包括在施用所说的喜树碱化合物之前或同时，口头施用包含乌索脱氧胆酸的组合物。The present invention also provides a method of intravenously administering a camptothecin compound comprising orally administering a composition comprising ursodeoxycholic acid prior to or simultaneously with said camptothecin compound.
在本说明书(其中以包括或包含特定组合物的形式描述了组合物，试剂盒，以及方法)中，发明人期望本发明的组合物也实质上包含或含有所叙述的组分。In this specification, where compositions, kits, and methods are described as comprising or comprising a particular composition, the inventors contemplate that compositions of the invention also substantially comprise or contain the recited components.
从下列详细描述与附图，相关领域的技术人员将易于理解本发明的以上和其他目的。在以下详细描述与附图中，仅简单地说明实施发明的最好方式，来显示和描述本发明的优选的实施方案。相关领域的技术人员容易认识到本发明可以被改进，但不偏离本发明的精神和范围。The above and other objects of the present invention will be readily understood by those skilled in the relevant art from the following detailed description and accompanying drawings. In the following detailed description and accompanying drawings, only the best mode of carrying out the invention is shown and described, showing and describing the preferred embodiment of the invention. Those skilled in the relevant art will readily recognize that the present invention can be modified without departing from the spirit and scope of the invention.
附图的简要说明Brief description of the drawings
图1显示CPT-11,SN-38和SN-38-葡萄糖醛酸化物(SN-38-GLU)的结构：CPT-11和SN-38的内酯形式是荷电的非离子，CPT-11和SN-38的羧化物形式是阴离子，不仅SN-38-Glu的羧化物形式，而且其内酯形成(在其葡糖苷酸组分中具有一个额外的羧基)都是阴离子。内酯和羧化物形式之间的可逆转变由pH值引起。Figure 1 shows the structures of CPT-11, SN-38 and SN-38-glucuronide (SN-38-GLU): the lactone forms of CPT-11 and SN-38 are charged nonionic, CPT-11 and the carboxylate form of SN-38 are anionic, not only the carboxylate form of SN-38-Glu, but also its lactone formation (with an extra carboxyl group in its glucuronide component) is anionic. The reversible transition between the lactone and carboxylate forms is induced by pH.
图2A和2B显示CPT-11和SN-38由分离的肠细胞摄取的时程：分别以内酯和羧化物形式的[14C]CPT-11(20μM)和[14C]SN-38(2μM)由分离的空肠肠细胞的摄取作为时间的函数测定。在时间0，各种试剂在持续振荡下添加至37℃保持的肠细胞悬液中。在15,30,45,60,90,120,240和480秒，除去细胞悬液的等分样，如材料和方法中的描述进行处理。结果以平均值±SE显示。Figures 2A and 2B show the time course of CPT-11 and SN-38 uptake by isolated enterocytes: [14 C]CPT-11 (20 μM) and [14 C]SN-38 (2 μM) in lactone and carboxylate forms, respectively. ) was determined by uptake by isolated jejunal enterocytes as a function of time. At
图3A,3B,3C和3D显示CPT-11的摄取初始的速率与其浓度之间的关系：在初始的90秒温育期间从细胞摄取的线型斜率测定初始摄取速率，数据用方程式V=(VmaxS)/(Km+S)+KdS经最小二乘法非线性回归分析处理，其中V代表摄取的初始速率，Vmax是摄取的最大速率，Km是表观米氏常数，Kd是扩散速率，S是CPT-11的浓度。Figure 3A, 3B, 3C and 3D show the relationship between the initial rate of uptake of CPT-11 and its concentration: during the initial 90 seconds of incubation, the initial uptake rate is determined from the linear slope of cell uptake, and the data use the equation V=( Vmax S)/(Km +S)+Kd S was processed by least squares nonlinear regression analysis, where V represents the initial rate of uptake, Vmax is the maximum rate of uptake, Km is the apparent Michaelis constant,Kd is the diffusion rate and S is the concentration of CPT-11.
图4A和4B显示SN-38的摄取初始的速率与其浓度之间的关系：初始摄取速率如图3的图例中与材料和方法中的描述测定，数据由最小二乘法线性回归处理。由于有限的溶解性，仅研究了达2μM浓度的SN-38。Figures 4A and 4B show the relationship between the initial rate of uptake of SN-38 and its concentration: the initial uptake rate was determined in the legend of Figure 3 and as described in Materials and Methods, and the data were processed by least squares linear regression. Due to limited solubility, only concentrations up to 2 μM of SN-38 were studied.
图5显示牛磺胆酸(TCA)分别对CPT-11和SN-38胶束形成的作用：[14C]CPT-11(20μM)和[14C]SN-38(2μM)在存在或不存在TCA(20mM)的情况下在Hank’s溶液中贮存一夜。如材料和方法所述经采用1000-分子量截断膜(YM1)超滤从胶束聚集体分离单体。数值是所示代谢物的单体形式，以超滤之前起始溶液浓度的百分比(％)表示。TCA和对照之间的比较由Mann-Whitney试验估价。(*)：在TCA存在下SN-38羧化物明显不同于其他试剂(Kruskal-Wallis试验：P=0.023,Student-Newman-Keuls法，P＜0.05)。使用的缩写：CPT内酯(CPTlact.)；CPT羧化物(CPT carb.)；SN-38内酯(SNlact.)；SN-38羧化物(SNcarb.)Figure 5 shows the effects of taurocholic acid (TCA) on the formation of micelles of CPT-11 and SN-38: [14 C]CPT-11 (20 μM) and [14 C]SN-38 (2 μM) in the presence or absence Store overnight in Hank's solution in the presence of TCA (20 mM). Monomers were isolated from micellar aggregates by ultrafiltration using a 1000-molecular weight cut-off membrane (YM1) as described in Materials and Methods. Values are the monomeric form of the indicated metabolites expressed as a percentage (%) of the starting solution concentration before ultrafiltration. Comparisons between TCA and controls were assessed by the Mann-Whitney test. (* ): SN-38 carboxylate was significantly different from other reagents in the presence of TCA (Kruskal-Wallis test: P=0.023, Student-Newman-Keuls method, P<0.05). Abbreviations used: CPT lactone (CPTlact.); CPT carboxylate (CPT carb.); SN-38 lactone (SNlact.); SN-38 carboxylate (SNcarb.)
图6A和6B显示pH值对CPT-11和SN-38的初始摄取速率的作用：[14C]CPT-11(20μM)和[14C]SN-38(2μM)溶解在pH6.2,6.8,7.4的PBS中，并且贮存一夜。通过添加药物至包含来源于整个小肠的肠细胞的Hank’s溶液中，完成摄取研究。由pH值引起的初始摄取速率的区别通过Kruskal-Wallis试验(对CPT-11和SN-38分别是p＜0.001,p＜0.001)和Student-Newman-Keuls法(*p＜0.05)分析。Figures 6A and 6B show the effect of pH on the initial uptake rate of CPT-11 and SN-38: [14 C]CPT-11 (20 μM) and [14 C]SN-38 (2 μM) were dissolved at pH 6.2, 6.8 , 7.4 in PBS, and store overnight. Uptake studies were done by adding drug to Hank's solution containing enterocytes derived from the whole small intestine. Differences in initial uptake rates caused by pH were analyzed by Kruskal-Wallis test (p<0.001, p<0.001 for CPT-11 and SN-38, respectively) and Student-Newman-Keuls method (* p<0.05).
图7显示pH值对HT29细胞初始摄取速率的作用。在pH6.2,6.8,7.4下，[14C]SN-38(2μM)溶解在PBS中一夜。通过添加化合物至包含HT29细胞的Hank’s溶液开始摄取研究。摄取的比较初始速率作为pH值的函数由Kruskal-Wallis试验(p＜0.001)和Dunn’s法(*p＜0.05)分析。Figure 7 shows the effect of pH on the initial uptake rate by HT29 cells. [14 C]SN-38 (2 μM) was dissolved in PBS overnight at pH 6.2, 6.8, 7.4. Uptake studies were initiated by adding compounds to Hank's solution containing HT29 cells. The comparative initial rates of uptake as a function of pH were analyzed by the Kruskal-Wallis test (p<0.001) and Dunn's method (* p<0.05).
图8显示SN-38的初始摄取速率和细胞毒性之间的关系。利用HT29细胞，生理pH值对2μM[14C]SN-38的初始摄取速率的作用如图3图例中的描述估价。在HT29细胞中，0.4μM SN-38-引起的细胞毒性由所描述的MTT测定法研究。SN-38的初始摄取速率和细胞毒性之间的关系由一简单的最小二乘法回归法图示。Figure 8 shows the relationship between initial uptake rate and cytotoxicity of SN-38. Using HT29 cells, the effect of physiological pH on the initial uptake rate of 2 μM [14 C]SN-38 was evaluated as described in the legend to FIG. 3 . In HT29 cells, 0.4 μM SN-38-induced cytotoxicity was studied by the described MTT assay. The relationship between the initial uptake rate of SN-38 and cytotoxicity is illustrated by a simple least squares regression.
发明详述Detailed description of the invention
喜树碱化合物如CPT-11和其代谢物由肠细胞转运的机理的知识对由喜树碱化合物，如CPT-11引起的腹泻的机理和在药代动力学中不同患者中的大的可变性的理解是关键的步骤。Knowledge of the mechanism of transport of camptothecin compounds such as CPT-11 and their metabolites by enterocytes to the mechanism of diarrhea induced by camptothecin compounds such as CPT-11 and the large variability among different patients in the pharmacokinetics Understanding is the key step.
本发明发明人研究了几种喜树碱化合物，CPT-11和SN-38由肠上皮细胞的摄取，结果提供了一种方法的新的设计，该方法是在用盐酸伊立替康和其相关化合物治疗癌症和肿瘤的临床实践上，阻止腹泻和在药代动力学中不同患者间的大的可变性的方法。The inventors of the present invention studied the uptake of several camptothecin compounds, CPT-11 and SN-38, by intestinal epithelial cells and provided a novel design of a method in which irinotecan hydrochloride and its related Clinical practice of compounds for the treatment of cancer and neoplasms, approach to prevent diarrhea and large interpatient variability in pharmacokinetics.
本发明提供了一种抑制喜树碱化合物(盐酸伊立替康(CPT-11)、SN-38-Glu、SN-38和其衍生物)的腹泻副作用的方法，该方法包括在保持胆汁和/或肠腔碱性pH值的同时，施用盐酸伊立替康。在一优选的实施方案中，通过施用碳酸氢盐与碱性H2O来保持肠腔处于碱性pH值。碳酸氢盐的量和碱性pH值适于降低喜树碱化合物的摄取量，并且由此降低包括腹泻副作用在内的细胞毒性副作用。喜树碱化合物或盐酸伊立替康可以通过静脉内，口头或饥内施用。本发明的方法阻止再吸收，并且降低肠对CPT-11和SN-38的内酯摄取，并且由此降低与喜树碱化合物如盐酸伊立替康相联系的腹泻副作用。The present invention provides a method for suppressing the side effect of diarrhea of camptothecin compounds (irinotecan hydrochloride (CPT-11), SN-38-Glu, SN-38 and derivatives thereof), which method comprises maintaining bile and/or Or the alkaline pH of the intestinal lumen while administering irinotecan hydrochloride. In a preferred embodiment, the intestinal lumen is maintained at an alkaline pH by administration of bicarbonate and alkalineH2O . The amount of bicarbonate and the alkaline pH are suitable to reduce the uptake of the camptothecin compound and thereby reduce cytotoxic side effects including diarrheal side effects. The camptothecin compound or irinotecan hydrochloride can be administered intravenously, orally or intragastrically. The methods of the invention prevent reabsorption and reduce intestinal lactone uptake of CPT-11 and SN-38, and thereby reduce the diarrhea side effect associated with camptothecin compounds such as irinotecan hydrochloride.
本发明也提供了一种治疗癌症的方法，该方法包括在保持肠腔处于碱性pH值状态的同时，施用盐酸伊立替康和其衍生物。在一优选的实施方案中，所说的癌症选自但不限于下组：乳腺癌、卵巢癌、结肠癌、恶性黑素瘤、小细胞肺癌、甲状腺癌、淋巴瘤和白血病。碱性pH值可以是从约7至约10。在一可替实施方案中，在保持肠腔处于碱性pH值状态的同时，通过施用选自7-羟甲基喜树碱，盐酸伊立替康，氨基喜树碱，DX-8951F,SN-3B,HAR4,HAR5,HAR6,HAR7,HAR8以及topotecan的化合物来治疗癌症。The present invention also provides a method for treating cancer, which comprises administering irinotecan hydrochloride and its derivatives while maintaining the intestinal lumen in an alkaline pH state. In a preferred embodiment, said cancer is selected from but not limited to the group consisting of breast cancer, ovarian cancer, colon cancer, malignant melanoma, small cell lung cancer, thyroid cancer, lymphoma and leukemia. Alkaline pH can be from about 7 to about 10. In an alternative embodiment, while maintaining the intestinal lumen in an alkaline pH state, by administering 7-hydroxymethylcamptothecin, irinotecan hydrochloride, aminocamptothecin, DX-8951F, SN- 3B, compounds of HAR4, HAR5, HAR6, HAR7, HAR8 and topotecan to treat cancer.
本发明有利地提供了一种治疗AIDS的方法，该方法包括在保持肠腔处于碱性pH值状态的同时，施用盐酸伊立替康和其衍生物。The present invention advantageously provides a method for treating AIDS, the method comprising administering irinotecan hydrochloride and its derivatives while maintaining the intestinal lumen in an alkaline pH state.
包含盐酸伊立替康(CPT-11)的药物组合物和试剂盒可以与碳酸氢盐结合施用，所说的结合的碳酸氢盐选自碳酸氢钠、碳酸氢镁和碳酸氢钾。另外盐酸伊立替康(CPT-11)可以与包含硼酸的组合物结合施用。这一化学品已经用于缓冲组合物(如Britton-Robinson缓冲液)，并且具有强的碱性缓冲作用。Pharmaceutical compositions and kits comprising irinotecan hydrochloride (CPT-11) may be administered in combination with a bicarbonate selected from the group consisting of sodium bicarbonate, magnesium bicarbonate and potassium bicarbonate. Additionally irinotecan hydrochloride (CPT-11) can be administered in combination with a composition comprising boric acid. This chemical has been used in buffer compositions (eg Britton-Robinson buffer) and has a strong alkaline buffering action.
本发明也提供了一种施用喜树碱化合物的方法，该方法包括在施用所说的喜树碱化合物之前或同时，口头施用包含乌索脱氧胆酸的组合物。这一组合物可以也可以不与碳酸氢盐一起施用。人们相信乌索脱氧胆酸刺激碳酸氢盐分泌进入胆汁。The present invention also provides a method of administering a camptothecin compound comprising orally administering a composition comprising ursodeoxycholic acid prior to or simultaneously with said camptothecin compound. This composition may or may not be administered with bicarbonate. It is believed that ursodeoxycholic acid stimulates the secretion of bicarbonate into bile.
下列实施例显示了依据本发明的方法，盐酸伊立替康化合物降低腹泻副作用的能力。The following examples demonstrate the ability of the irinotecan hydrochloride compound to reduce the side effect of diarrhea in accordance with the methods of the present invention.
实施例药物和动物Example Drugs and Animals
14C-标记的SN-38(3.68 MBg/mg)和14C-标记的CPT-11(1.47MBq/mg)由Daiichi药物有限公司(日本东京)友好赠送。非标记的CPT-11,SN-38和SN-38-Glu由Yakult Honsha有限公司(日本东京)供给。14C-标记的SN-38以2μM的终浓度溶解在DMSO中，这是因为其疏水性很强，并且在水中有差的可溶性。已证实在2％时，DMSO对标记的CPT-11和SN-38的起始摄取没有任何影响。其它药物溶解在蒸馏水中。通过分别在pH6或9的50mM磷酸缓冲液中溶解化合物一夜产生14C-标记的CPT-11和SN-38的内酯和羧化物形式。由谷胱甘肽和CDNB(1-氯-2,4-二硝基苯)化学制备DNP-SG。所有其它试剂是分析纯试剂。使用成年雄性金黄仓鼠(6-8周龄)，其模型代表类胆汁酸轮廓(28)，类似于在人中观察到的。肠细胞的制备14 C-labeled SN-38 (3.68 MBg/mg) and14 C-labeled CPT-11 (1.47 MBq/mg) were kind gifts from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan). Untagged CPT-11, SN-38 and SN-38-Glu were supplied by Yakult Honsha Co., Ltd. (Tokyo, Japan).14 C-labeled SN-38 was dissolved in DMSO at a final concentration of 2 μM because it is very hydrophobic and has poor solubility in water. At 2%, it was demonstrated that DMSO had no effect on the initial uptake of labeled CPT-11 and SN-38. Other drugs are dissolved in distilled water. The lactone and carboxylate forms of14 C-labeled CPT-11 and SN-38 were generated by dissolving the compounds overnight in 50 mM phosphate buffer,
如现有技术的描述分离肠细胞(28,29)。简言之，雄性仓鼠用戊巴比妥钠(Nembutol 70mg/kg体重)麻醉。移走整肠。肠腔管用37℃的Hank’s溶液洗涤。冲洗回肠囊(从盲肠12.5cm)和空肠(保留小肠)以及小肠肛门端的(从盲肠12.5cm)和口部端的(其它小肠)肠囊。以包含柠檬酸钠的氧化缓冲液(96mM NaCl,1.5mM KCl,5.6mM KH2PO4,27mM柠檬酸钠，pH7.3)冲洗囊，并于37℃在相同的缓冲液中温育10分钟。然后倒空囊，冲以包含乙二胺四乙酸的氧化缓冲液(140mM NaCl,16mM Na2HPO4,2mM乙二胺四乙酸，0.5mM二硫苏糖醇。pH7.3)，在37℃温育10分钟。然后将各囊放置在培养皿中，轻轻涡旋1分钟。用50mlHank’s溶液回收包含肠细胞的缓冲液，洗涤两次，并且用Hank’s介质(包含0.5％牛血清清蛋白的细胞贮存溶液，pH7.4)调节至106细胞/ml。Intestinal cells were isolated as described in the prior art (28,29). Briefly, male hamsters were anesthetized with sodium pentobarbital (Nembutol 70 mg/kg body weight). Remove the whole intestine. The intestinal lumen was washed with Hank's solution at 37°C. The ileal pouch (12.5 cm from the cecum) and jejunum (retaining the small intestine) and pouches at the anal (12.5 cm from the cecum) and oral (other small intestine) ends of the small bowel were flushed. Capsules were washed with an oxidation buffer containing sodium citrate (96 mM NaCl, 1.5 mM KCl, 5.6 mM KH2 PO4 , 27 mM sodium citrate, pH 7.3) and incubated in the same buffer for 10 minutes at 37°C. The capsule was then emptied, flushed with oxidation buffer containing EDTA (140 mM NaCl, 16 mM Na2 HPO4 , 2 mM EDTA, 0.5 mM dithiothreitol, pH 7.3), at 37° C. Incubate for 10 minutes. Each capsule was then placed in a Petri dish and vortexed gently for 1 minute. The enterocyte-containing buffer was recovered with 50 ml of Hank's solution, washed twice, and adjusted to 106 cells/ml with Hank's medium (cell stock solution containing 0.5% bovine serum albumin, pH 7.4).
分别测定14C-标记的CPT-11和SN-38的细胞摄取Cellular uptake of14 C-labeled CPT-11 and SN-38 were measured separately
14C-标记的CPT-11和SN-38的摄取由快速真空抽滤测定法(28,29)测定。将0.95ml细胞悬浮在37℃的水浴中，搅拌15分钟。通过在37℃添加0.05mlPBS(在pH3或9)开始摄取，所说的PBS含有标记的CPT-11和SN-38。在各时间间隔，100μL小份样品在4℃稀释进3毫升Hank’s介质，以终止摄取。真空下(20psi)用玻璃微型纤维滤膜(玻璃纤维滤膜圈G4,Fisherbrand,PA)过滤包含细胞的终止溶液。细胞以含0.5％牛血清白蛋白的Hank’s介质5ml洗涤一次(4℃)和)，用Hank’s溶液20ml洗涤一次(4℃)。将滤膜放置在包含4ml闪烁液的小瓶中，在β闪烁计数器(LS3801,Beckman,MD)中计数放射活性。Uptakeof14C -labeled CPT-11 and SN-38 was determined by a rapid vacuum filtration assay (28,29). Suspend 0.95ml of cells in a 37°C water bath and stir for 15 minutes. Uptake was initiated by adding 0.05 ml PBS (at
通过在14C-CPT-11和14C-SN-38(2μM)3分钟之前添加2,4-二硝基苯酚(1mM)至细胞中研究代谢抑制物2,4-二硝基苯酚(1mM)的作用。20mM牛磺胆酸(TCA)对CPT-11和SN-38的摄取的作用在pH7.4存在和不存在TCA下用Hank’s溶液过夜温育14C-CPT-11(20μM)和14C-SN-38(2μM)后进行。通过在14C-CPT-11(20μM)和14C-SN-38(2μM)7分钟之前添加DNP-SG或SN-38-Glu至细胞制剂中研究200μM DNP-SG或SN-38-Glu的作用。The
分别在pH6.2,6.8,7.4和8下用磷酸缓冲盐水过夜温育14C-CPT-11(20μM)和14C-SN-38(2μM)后，研究生理pH值对CPT-11和SN-38的初始肠摄取速率的作用。评估胶束形成After overnight incubation of14 C-CPT-11 (20 μM) and14 C-SN-38 (2 μM) with phosphate-buffered saline at pH 6.2, 6.8, 7.4 and 8, respectively, the effect of physiological pH on CPT-11 and SN The effect of the initial intestinal uptake rate of -38. Assessing micelle formation
为了评估CPT-11和SN-38是否形成胶束，用含有10mM TCA无钙和镁的Hank’s溶液pH4和9下温育这些药剂过夜。通过采用1000-分子量截断膜YM1(Diaflo,Amicon,MA)以0.04ml/分钟的稳定速度过滤各溶液。一旦停止过滤，就按照以前的描述测定初始溶液以及滤液和过滤后的保留的溶液中的放射活性。细胞毒性测定To assess whether CPT-11 and SN-38 form micelles, these agents were incubated overnight at
修改用于线粒体脱氢酶活性的快速比色测定法，并且用于评价SN-38的细胞毒性(Mosmann,1983)。简言之，将HT29细胞接种进12孔平板(Falcon-3043,Lincoln Park,NJ)，在48小时之后，在pH6.2,6.8,7.4和8.0下添加SN-38(0.4(M)。露置24小时，洗涤细胞两次，并且进行无药物温育24小时。然后，用0.5mg/ml 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-四溴化物(MTT)温育细胞4小时，通过添加在0.01N HCl中的n-十二烷基苯硫酸钠盐(SDS)和温育一夜溶解蓝色formazan晶体。蓝色formazan化合物的形成在560毫微米以分光光度法(Ultraspec 4050,LKB,Brornma,Sweden)测定。统计学分析A rapid colorimetric assay for mitochondrial dehydrogenase activity was adapted and used to evaluate the cytotoxicity of SN-38 (Mosmann, 1983). Briefly, HT29 cells were seeded into 12-well plates (Falcon-3043, Lincoln Park, NJ), and after 48 hours, SN-38 (0.4(M) was added at pH 6.2, 6.8, 7.4 and 8.0. Leave it for 24 hours, wash the cells twice, and carry out drug-free incubation for 24 hours. Then, use 0.5mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- Tetrabromide (MTT) incubated the cells for 4 hours, and dissolved the blue formazan crystals by adding n-dodecylbenzenesulfate sodium salt (SDS) in 0.01N HCl and incubating overnight. The formation of the blue formazan compound was at 560 nm was measured spectrophotometrically (Ultraspec 4050, LKB, Brornma, Sweden). Statistical analysis
CPT-11或SN-38的摄取的初始速率是从各自的回归线的线型回归分析中得出的，所说的回归线是从作为时间函数的摄取的曲线图获得的。将摄取的初始速率被对对应的浓度作图。数据用方程式V=(VmaxS)/(Km+S)+KdS经最小二乘法非线性回归分析(SigmaStat,Jandel scientific,CA)处理，其中V代表摄取的初始速率，Vmax是摄取的最大速率，Km是表观米氏常数，Kd是扩散速率，S是CPT-11或SN-38的浓度。The initial rates of uptake of CPT-11 or SN-38 were derived from linear regression analysis of the respective regression lines obtained from plots of uptake as a function of time. The initial rate of uptake was plotted against the corresponding concentration. Data were processed by least squares nonlinear regression analysis (SigmaStat, Jandel scientific, CA) using the equation V=(Vmax S)/(Km +S)+Kd S, where V represents the initial rate of uptake and Vmax is Maximum rate of uptake,Km is the apparent Michaelis constant,Kd is the diffusion rate, and S is the concentration of CPT-11 or SN-38.
两组之间的比较通过Mahn-whitney等级总数试验评价。两组以上的统计学差异显著性由Kruskal-Wallis单向等级方差分析确定，然后由Dunn’s法完成与对照组的多重比较。用简单的最小二乘法回归法将摄取的初始速率与SN-38的细胞毒性之间的关系作图。CPT-11和SN-38内酯和羧化物的分别由肠细胞的摄取Comparisons between two groups were assessed by the Mahn-Whitney rank sum test. The statistical significance of more than two groups was determined by Kruskal-Wallis one-way rank analysis of variance, and then multiple comparisons with the control group were completed by Dunn's method. The relationship between the initial rate of uptake and the cytotoxicity of SN-38 was plotted using simple least squares regression. Uptake of CPT-11 and SN-38 lactones and carboxylates, respectively, by enterocytes
20μM14C-CPT-11和2μM14C-SN-38的内酯和羧化物两种形式被分离的空肠细胞的时间-依赖性摄取在图2中显示。在时间0的摄取值的外推产生正的截距，表明非特异性结合，如对在细胞表面的标记的吸附。CPT-11和SN-38的内酯与羧化物两种形式各自的摄取在多达90秒内是线型的。因此，在初始时间期间，摄取的初始摄取速率由直线回归适配确定。各种药剂的内酯和羧化物形式之间的摄取速率的比较清楚地显示CPT-11和SN-38两者的内酯形式与羧化物形式比较被更迅速地摄取(图2)。The time-dependent uptake by isolated jejunal cells of both lactone and carboxylate forms of 20 μM14 C-CPT-11 and 2 μM14 C-SN-38 is shown in FIG. 2 . Extrapolation of the uptake value at
表1总结了20μM14C-CPT-11和2μM14C-SN-38被空肠和回肠细胞的初始摄取速率。在两种肠区的细胞中，CPT-11和SN-38内酯形式与它们的羧化物形式得到更迅速的摄取，但在空肠和回肠细胞之间没有显著性差异。CPT-11和SN-38内酯和羧化物的转运系统Table 1 summarizes the initial uptake rates of 20 μM14 CPT-11 and 2 μM14 C-SN-38 by jejunal and ileal cells. CPT-11 and SN-38 lactone forms and their carboxylate forms were taken up more rapidly in cells of both intestinal regions, but there was no significant difference between jejunum and ileal cells. Transport system for CPT-11 and SN-38 lactones and carboxylates
将CPT-11内酯和羧化物的各自的初始摄取速率作为浓度的函数作图，空肠以及回肠细胞中的数据用方程式V=(VmaxS)/(Km+S)+KdS经最小二乘法非线性回归分析处理(图3)，CPT-11内酯的摄取的优势组分是非饱和的。表明由被动扩散或液相胞吞作用摄取。CPT-11羧化物摄取的曲线分析也表明摄取过程的至少两个不同的组分。曲线的饱和组分的特征是147的最大摄取速率(Vmax)和157pmol·106细胞-1·分钟-1·μM-1，以及分别在空肠和回肠细胞中的51.3和50.5(M的米氏常数(Km)。次要的非饱和组分的特征是扩散速率(Kd)小于0.05pmol·106细胞-1·分钟-1·μM-1，并且代表在两个肠区的细胞中不到CPT-11内酯的二十分之一(表2)。此外，CPT-11内酯的Kd低于SN-38内酯的1.8-2.5倍。The respective initial uptake rates of CPT-11 lactone and carboxylate were plotted as a function of concentration, and data in jejunum and ileal cells were calculated using the equation V=(Vmax S)/(Km +S)+Kd S The least squares nonlinear regression analysis process (Figure 3), the dominant component of the uptake of CPT-11 lactone is unsaturated. Indicates uptake by passive diffusion or fluid phase endocytosis. Profile analysis of CPT-11 carboxylate uptake also indicated at least two distinct components of the uptake process. The saturated component of the curve is characterized by a maximum uptake rate (Vmax ) of 147 and 157 pmol·106 cell−1 ·min−1 ·μM−1 , and a meter of 51.3 and 50.5 (M in jejunum and ileum cells, respectively Constant (Km ). The minor unsaturated component is characterized by a diffusion rate (Kd ) of less than 0.05 pmol·106 cells-1 ·min-1 ·μM-1 and represents cells in both intestinal regions It is less than one-twentieth of CPT-11 lactone (Table 2). In addition, the Kd of CPT-11 lactone is lower than 1.8-2.5 times of SN-38 lactone.
将SN-38内酯和羧化物的初始摄取速率作为浓度的函数作图(图4)，用于这一研究的SN-38的最大浓度低于2μM，这是因为该化合物差的溶解性，由此使得测定摄取的饱和和不可饱和组分变得困难。在这一浓度水平上，SN-38内酯和羧化物的摄取是最不可饱和的(图4)The initial uptake rates of SN-38 lactone and carboxylate were plotted as a function of concentration (Figure 4). The maximum concentration of SN-38 used in this study was below 2 μM due to the poor solubility of this compound, This makes it difficult to determine the saturated and unsaturated components of the intake. Uptake of SN-38 lactone and carboxylate was least saturable at this concentration level (Figure 4)
载体介导的转运已知受代谢抑制剂抑制，如2,4-二硝基苯酚，其干扰细胞代谢，并且降低产生能量的反应(23)。因此，2,4-二硝基苯酚用于应用研究以分别确定CPT-11和SN-38内酯和羧化物的摄取的机理。这一研究的结果在表3中总结。虽然CPT-11和SN-38两者的内酯的摄取不明显地受添加2,4-二硝基苯酚的影响，但CPT-11以及SN-38羧化物的摄取速率分别被2,4-二硝基苯酚降低至22,6和30.85。表明对这两种化合物的主动转运。Carrier-mediated transport is known to be inhibited by metabolic inhibitors, such as 2,4-dinitrophenol, which interfere with cellular metabolism and reduce energy-producing reactions (23). Therefore, 2,4-dinitrophenol was used in applied studies to determine the mechanism of uptake of CPT-11 and SN-38 lactone and carboxylate, respectively. The results of this study are summarized in Table 3. Although the uptake of the lactones of both CPT-11 and SN-38 was not significantly affected by the addition of 2,4-dinitrophenol, the uptake rates of CPT-11 and SN-38 carboxylate were respectively influenced by 2,4- Dinitrophenol decreased to 22,6 and 30.85. Active transport for both compounds is indicated.
2,4-二硝基苯酚-S-谷胱甘肽(DNP-SG)已知在肝中是活性多特异性有机阴离子转运蛋白(cMOAT)的底物(24)。此外，由SN-38的UDP-葡糖醛酸基转移酶的缀合导致形成SN-38-Glu，其也是肝cMOAT的底物(12,17)。为了确定CPT-11羧化物和/或SN-38羧化物是否通过肠细胞的类cMOAT机理转运，存在或不存在DNP-SG和SN-38-Glu下研究CPT-11以及SN-38的摄取速率。结果在表3中总结。DNP-SG和SN-38-Glu明显抑制SN-38羧化物摄取的60％，而CPT-11羧化物的摄取保持不变，CPT-11和SN-38内酯形式的摄取速率不明显受到DNP-SG或SN38-Glu存在的影响。胶束形成和其对CPT-11和SN-38初始摄取速率的影响2,4-Dinitrophenol-S-glutathione (DNP-SG) is known to be a substrate of the active multispecific organic anion transporter (cMOAT) in the liver (24). Furthermore, conjugation by UDP-glucuronosyltransferase of SN-38 leads to the formation of SN-38-Glu, which is also a substrate of hepatic cMOAT (12,17). To determine whether CPT-11 carboxylate and/or SN-38 carboxylate is transported by a cMOAT-like mechanism in enterocytes, the uptake rates of CPT-11 and SN-38 were studied in the presence or absence of DNP-SG and SN-38-Glu . The results are summarized in Table 3. DNP-SG and SN-38-Glu significantly inhibited the uptake of SN-38 carboxylate by 60%, while the uptake of CPT-11 carboxylate remained unchanged, and the uptake rate of the lactone form of CPT-11 and SN-38 was not significantly affected by DNP - Effect of presence of SG or SN38-Glu. Micelle formation and its effect on the initial uptake rate of CPT-11 and SN-38
临界胶束浓度以上的牛磺胆酸(TCA)形成胶束(25)，与CPT-11和SN-38相反，其不能穿过1,000-分子量截断膜。我们利用这一性质来确定CPT-11和SN-38内酯和羧化物是否可以与TCA缔合成胶束。图5中报告的结果，表明TCA明显降低CPT-11内酯和羧化物以及SN-38内酯的％单体浓度。然而，SN-36羧化物不明显缔合成TCA胶束。Taurocholic acid (TCA) above the critical micelle concentration forms micelles (25), which, in contrast to CPT-11 and SN-38, cannot cross 1,000-molecular weight cutoff membranes. We exploited this property to determine whether CPT-11 and SN-38 lactones and carboxylates could associate with TCA to form micelles. The results, reported in Figure 5, show that TCA significantly reduces the % monomer concentration of CPT-11 lactone and carboxylate as well as SN-38 lactone. However, the SN-36 carboxylate did not significantly associate into TCA micelles.
下一个应用试验胶束形成对CPT-11和SN-38的细胞摄取的作用。在这一系列实验中，组合空肠与回肠细胞。在20mM TCA存在下，CPT-11和SN-38的初始摄取速率(平均值±SD)降低至没有TCA的对照的48.5(10.8和69.3±12.7％(N=5,Mann-Whitney试验，分别对CPT-11和SN-38 P=0.015和P=0.343)。pH值和碳酸氢盐对CPT-11和SN-38的初始摄取速率的影响The next application tested the effect of micelle formation on the cellular uptake of CPT-11 and SN-38. In this series of experiments, jejunal and ileal cells were combined. In the presence of 20 mM TCA, the initial uptake rates (mean ± SD) of CPT-11 and SN-38 were reduced to 48.5 (10.8 and 69.3 ± 12.7% of the control without TCA (N=5, Mann-Whitney test, respectively for CPT-11 and SN-38 P=0.015 and P=0.343).Effect of pH and bicarbonate on the initial uptake rate of CPT-11 and SN-38
CPT-11和SN-38各自的内酯和羧化物之间相互转化是可逆的和受pH值驱动的(11)。研究了生理pH值(pH6.2-8)对20μM14C-CPT-11，和2μM14C-SN-38的初始摄取速率的影响。结果在图6中总结，其显示在pH值大于6.8时，CPT-11和SN-38的摄取速率明显降低约65％。当在存在和不存在碳酸氢盐时测定CPT-11与SN-38的初始摄取速率时，也观察到摄取的改变。当Hank’s缓冲液的HEPES组分由碳酸氢钠代替，pH值调节至大于7时，CPT-11和SN-38的摄取降低。The interconversion between the respective lactone and carboxylate of CPT-11 and SN-38 is reversible and pH-driven (11). The effect of physiological pH (pH6.2-8) on the initial uptake rate of 20 μM14 C-CPT-11, and 2 μM14 C-SN-38 was studied. The results are summarized in Figure 6, which shows that the uptake rates of CPT-11 and SN-38 were significantly reduced by approximately 65% at pH values greater than 6.8. Changes in uptake were also observed when the initial uptake rates of CPT-11 and SN-38 were determined in the presence and absence of bicarbonate. When the HEPES component of Hank's buffer was replaced by sodium bicarbonate and the pH was adjusted to greater than 7, the uptake of CPT-11 and SN-38 decreased.
利用仓鼠肠细胞，本研究的结果显示CPT-11和SN-38两者的非离子内酯形式主要通过被动机理吸收，但是各自以几倍于其阴离子羧化物形式的速率(表1,2和3；图2,3和4)。在空肠和回肠细胞之间的转运机理以及动力学参数上存在明显差异(表2和3)。虽然没有显示出来，在采用盲肠和结肠细胞完成CPT-11和SN-38的摄取时，也观察到类似的结果(26)。Using hamster intestinal cells, the results of the present study showed that the nonionic lactone forms of both CPT-11 and SN-38 were absorbed mainly by passive mechanisms, but each at several times the rate of their anionic carboxylate forms (Tables 1, 2 and 3; Figures 2, 3 and 4). There were clear differences in the transport mechanism and kinetic parameters between jejunal and ileal cells (Tables 2 and 3). Although not shown, similar results were observed for CPT-11 and SN-38 uptake using cecal and colonic cells (26).
仓鼠肠细胞对估价SN-38的细胞毒性作用不是最好的模型，这是由于它们有限地存活约2小时(Gore等，1993)。因此，HT29细胞也用来研究生理pH值对2μM[14C]SN-38的初始摄取速率和0.4(MSN-38的细胞毒性的相对作用。SN-38的初始摄取速率在HT-29细胞中比在分离的仓鼠肠细胞中低(图3和4)。然而，与在分离的仓鼠肠细胞观察的一样，在HT29细胞中SN-38的摄取速率在pH6.2和6.8时明显大于在pH7.4和8.0时(Kruskal-Wallis试验：p=0.008,Dunn’s法：p＜0.05)(图7)。SN-38对HT29细胞的细胞毒性在pH6.2和6.8时明显大于在pH7.4和8.0时(Kruskal-Wallis试验：p=0.007；Dunn的方法：p＜0.05)。图5显示[14C]SN-38的初始摄取速率和SN-38的细胞毒性之间的相互关系，表明在降低的pH下，更高的摄取速率与更强的细胞毒性相关联。Hamster intestinal cells are not the best model for assessing the cytotoxic effects of SN-38 due to their limited survival of approximately 2 hours (Gore et al., 1993). Therefore, HT29 cells were also used to study the relative effect of physiological pH on the initial uptake rate of 2 μM [14 C]SN-38 and the cytotoxicity of 0.40 MSN-38. The initial uptake rate of SN-38 in HT-29 cells lower than in isolated hamster enterocytes (Figures 3 and 4). However, as observed in isolated hamster enterocytes, the uptake rate of SN-38 in HT29 cells was significantly greater at pH 6.2 and 6.8 than at
结果清楚地显示CPT-11和SN-38羧化物通过主动机理被肠细胞取(表2和3；图3)。前不久已经报道，在肝小管膜中表达的cMOAT作为主要主动转运系统转运几种类型的有机阴离子进入胆汁(24,27-29)。此外，已报道肝cMOAT对阴离子，SN-38羧化物，SN-3B-Glu内酯以及羧化物的胆汁分泌起作用(12,17)。报道阴离子CPT-11羧化物仅部分通过cMOAT消除(12,17)。发明人的工作显示，与CPT-11羧化物的不同，SN-38羧化物的初始摄取速率明显受DNP-SG和SN-38-Glu的抑制(表3)。这些结果与cho等(12,17)利用肝小管膜泡的结果一致。因此，这一工作界定cMOAT或类cMOAT转运蛋白牵涉到SN-38的空肠和回肠细胞摄取。The results clearly show that CPT-11 and SN-38 carboxylate is taken up by enterocytes by an active mechanism (Tables 2 and 3; Figure 3). It has been reported recently that cMOAT expressed in the hepatic tubular membrane serves as the main active transport system for transporting several types of organic anions into bile (24,27-29). In addition, hepatic cMOAT has been reported to contribute to the bile secretion of anion, SN-38 carboxylate, SN-3B-Glu lactone, and carboxylate (12,17). Anionic CPT-11 carboxylate was reported to be only partially eliminated by cMOAT (12,17). The inventors' work showed that, unlike that of CPT-11 carboxylate, the initial uptake rate of SN-38 carboxylate was significantly inhibited by DNP-SG and SN-38-Glu (Table 3). These results are consistent with those of Cho et al. (12,17) using hepatic tubular membrane vesicles. Thus, this work implicates cMOAT or cMOAT-like transporters in the jejunal and ileal cellular uptake of SN-38.
发明人也报道CPT-11内酯和羧化物，以及SN-38内酯，在存在高浓度的TCA时可以形成胶束(图5)。单体浓度的百分比在38至47％的范围内。这些浓度不同于长链脂肪酸的(即，油酸2.3％)和胆固醇的(3％)。此外，胶束形成抑制CPT-11摄取，不同于正向作用胆汁酸胶束形成，在长链脂肪酸和胆固醇的肠摄取中起作用。这些结果支持显示胶束形成抑制短链脂肪酸(如棕榈酸)摄取的数据。The inventors also reported that the CPT-11 lactone and carboxylate, as well as the SN-38 lactone, can form micelles in the presence of high concentrations of TCA (Figure 5). The percentage of monomer concentration ranged from 38 to 47%. These concentrations differed from those of long chain fatty acids (ie, oleic acid 2.3%) and cholesterol (3%). Furthermore, micelle formation inhibits CPT-11 uptake, unlike positively acting bile acid micelle formation, which plays a role in the intestinal uptake of long-chain fatty acids and cholesterol. These results support data showing that micelle formation inhibits the uptake of short chain fatty acids such as palmitic acid.
如图1中所描述的，从CPT-11和SN-18内酯转变至羧化物是受pH驱动的(11,12)。以前已经报道，在pH7.4时，SN-38和CPT-11分别有13％是其内酯形式的(30)。本研究显示CPT-11和SN-38的初始摄取速率在酸性pH值(pH6.2和6.8)几倍于在中性或碱性pH值时(pH7.4和8)(图6)。考虑以下事实：1)在酸性pH值，CPT-11和SN-38的非离子内酯形式被被动转运，2)在中性/碱性pH值，CPT-11和SN-38的阴离子羧化物形式主要是主动吸收，和3)CPT-11和SN-38两者的内酯的摄取速率比它们的羧化物形式的大几倍，CPT-11和SN38的肠细胞摄取机理密切类似短链脂肪酸。这一假说得到事实的支持，如同用短链脂肪酸，胶束形成降低CPT-11和SN-38的摄取，并且CPT-11和SN-38的摄取不限于小肠，而且也在盲囊与结肠中发生。As depicted in Figure 1, the transition from CPT-11 and SN-18 lactones to carboxylate is pH driven (11,12). It has been previously reported that SN-38 and CPT-11 were each 13% in their lactone form at pH 7.4 (30). This study showed that the initial uptake rates of CPT-11 and SN-38 were several times higher at acidic pH (pH 6.2 and 6.8) than at neutral or basic pH (pH 7.4 and 8) (Figure 6). Consider the fact that 1) at acidic pH, the nonionic lactone form of CPT-11 and SN-38 is passively transported, 2) at neutral/basic pH, the anionic carboxylate of CPT-11 and SN-38 The form is mainly actively absorbed, and 3) the uptake rate of the lactones of both CPT-11 and SN-38 is several times greater than that of their carboxylated forms, and the enterocyte uptake mechanism of CPT-11 and SN38 closely resembles short-chain fatty acids . This hypothesis is supported by the fact that, as with short-chain fatty acids, micelle formation reduces the uptake of CPT-11 and SN-38, and that the uptake of CPT-11 and SN-38 is not limited to the small intestine, but also in the caecum and colon occur.
因此，对短链脂肪酸而言，胆汁的碱化和肠腔含量降低CPT-11和SN-38的肠摄取。从两人(由顺式铂氨治疗，并且静脉内接收CPT-11)测定CPT-11和其代谢物的胆汁含量(9)。胆汁的主要组分是CPT-11(75.6-91.9％)，而SN-38和SN-38-Glu是次要组分，分别为0.9-3.3％和7.3-18.9％。此外，人胆汁的pH值已报道在6.5-8.0的范围内(31)。因此，可以认为，不仅CPT-11的羧化物而且内酯形式在药代动力学中扮演着重要作用，这是因为CPT-11内酯在肠上皮细胞中较大的吸收，导致在肠肝循环中增加CPT-11水平。Thus, alkalinization of bile and luminal content of short-chain fatty acids reduces intestinal uptake of CPT-11 and SN-38. The bile content of CPT-11 and its metabolites was determined from two persons (treated with cisplatin and receiving CPT-11 intravenously) (9). The major component of bile was CPT-11 (75.6-91.9%), while SN-38 and SN-38-Glu were minor components, 0.9-3.3% and 7.3-18.9%, respectively. Furthermore, the pH of human bile has been reported to be in the range of 6.5-8.0 (31). Therefore, it can be considered that not only the carboxylate but also the lactone form of CPT-11 plays an important role in the pharmacokinetics because of the greater absorption of the CPT-11 lactone in the enterocytes, resulting in a higher rate of activity in the enterohepatic circulation. in increasing CPT-11 levels.
SN-38主要以内酯形式为活性，而SN-38羧化物仅显示小的拓扑异构酶Ⅰ-抑制活性(32)。利用大鼠全身放射自显影，Ⅳ注射14C-SN-38 24小时后，在胃肠道中发现放射活性(33)。SN-38显示强的细胞毒性，SN38-Glu是SN-38的去活化葡萄糖醛酸化形式，与SN-38相比，CPT-11有较低的细胞毒性(Kavato等，1991)。SN-38在肠中的积累在大鼠中显示(Atsumi等，1995)，并且被认为是对在裸鼠中施用CPT-11引起的腹泻的反应(Araki等，1993)。在CPT-11施用之后的腹泻的小鼠和大鼠中观察到盲囊中肠上皮破裂(Takatsuna等，1996；Ikuno等，1995；Araki等，1993)。已报道在人中由CPT-11施用引起的腹泻为分泌腹泻(Bleiberg和Cvitkovic,1996)。然而，如在动物模型中报道的，在患者中我们观察到与CPT-11-诱导的副作用相关联的致死小肠损伤伤害(Kobayashi等，1998b)。SN-38 is mainly active in the lactone form, whereas the SN-38 carboxylate exhibits only minor topoisomerase I-inhibitory activity (32). Using whole-body autoradiography in rats, radioactivity was found in the gastrointestinal tract 24 hours after IV injection of14 C-SN-38 (33). SN-38 shows strong cytotoxicity, SN38-Glu is the deactivated glucuronidated form of SN-38, and CPT-11 has lower cytotoxicity compared with SN-38 (Kavato et al., 1991). Intestinal accumulation of SN-38 has been shown in rats (Atsumi et al., 1995) and is thought to be in response to diarrhea induced by administration of CPT-11 in nude mice (Araki et al., 1993). Disruption of the intestinal epithelium in the caeca was observed in diarrheal mice and rats following CPT-11 administration (Takatsuna et al., 1996; Ikuno et al., 1995; Araki et al., 1993). Diarrhea induced by CPT-11 administration has been reported as secretory diarrhea in humans (Bleiberg and Cvitkovic, 1996). However, in patients we observed lethal small bowel injury associated with CPT-11 -induced side effects as reported in animal models (Kobayashi et al., 1998b).
此外，SN-38的积累，放射活性仅在胃肠道中被发现(33)。SN-38在肠中的积累被证明是对裸鼠中CPT-11引起的腹泻的反应(34)。在盲囊中的肠上皮破裂被认为是对在大鼠中CPT-11-引起的腹泻的反应(35)。最后，从欧洲的临床评价来看，由CPT-1引起的腹泻被说成是分泌腹泻(36)，而在我们的研究中，应用者们经历了严重的小肠损伤(37)。Furthermore, accumulation of SN-38 radioactivity was only found in the gastrointestinal tract (33). Intestinal accumulation of SN-38 was shown to be in response to CPT-11-induced diarrhea in nude mice (34). Disruption of the intestinal epithelium in the caecum is thought to be in response to CPT-11-induced diarrhea in rats (35). Finally, from European clinical evaluations, diarrhea caused by CPT-1 was described as secretory diarrhea (36), whereas in our study users experienced severe small bowel damage (37).
尸体解剖揭示假膜空肠回肠炎的存在，在光学显微镜下，其特征是：肠上皮的破裂，表明损坏性腹泻可能在严重的情况下发生。CPT-11-引起的腹泻的机理被认为包括肠上皮的主要SN-38和CPT-11内酯的再吸收，导致肠上皮对这些代谢物的高暴露，所说的代谢物引起肠道的结构和功能损伤。Autopsy revealed the presence of pseudomembranous jejunoileitis, which, on light microscopy, was characterized by: rupture of the intestinal epithelium, suggesting damaging diarrhea that may occur in severe cases. The mechanism of CPT-11-induced diarrhea is thought to involve the reabsorption of major SN-38 and CPT-11 lactones by the intestinal epithelium, leading to high exposure of the intestinal epithelium to these metabolites, which cause structural changes in the gut and functional impairment.
如本研究所揭示的，胆汁碱化和/或肠腔含量降低肠上皮对CPT-11和SN-38内酯的摄取和暴露，肠的短链脂肪酸的吸收在过去十年中已有研究，并且曾有冲突的结果被报道。人们相信，降低pH值，导致降低摄取短链脂肪酸，如图6中报道的。这样，喜树碱和CPT-11-引起的腹泻的预防治疗集中于两个目标：1)肠腔的碱化，和2)从身体清除CPT-11和SN-38(即，大便控制)。在标准Ⅳ施用CPT-11之前和/或同时，给患者口头施用pH大于7的碳酸氢钠，氧化镁和水的组合物，腹泻的机会会降低。As revealed in this study, bile alkalinization and/or luminal content reduces the uptake and exposure of the intestinal epithelium to CPT-11 and SN-38 lactones, short-chain fatty acid absorption in the intestine that have been studied over the past decade, And conflicting results have been reported. It is believed that lowering the pH results in lower uptake of short chain fatty acids, as reported in FIG. 6 . Thus, preventive treatment of camptothecin and CPT-11-induced diarrhea focuses on two goals: 1) alkalinization of the intestinal lumen, and 2) clearance of CPT-11 and SN-38 from the body (ie, stool control). Oral administration of a composition of sodium bicarbonate, magnesium oxide and water at a pH greater than 7 to the patient prior to and/or concurrently with the administration of CPT-11 in Standard IV reduces the chance of diarrhea.
在本研究中也估价了SN-38的细胞摄取和与它相联系细胞毒性之间的关系。发现HT29细胞中，SN-38的细胞摄取和细胞毒性是pH-依赖性的，并且细胞毒性与初始摄取速率很好地相关(图8)。如以前的描述，我们认为，SN-38的占优势的形式在酸性pH值下是内酯。这将导致SN-38内酯较大的细胞摄取和胞内浓度。由于SN-38主要以内酯形式为活性，而SN-38羧化物仅显示小的拓扑异构酶Ⅰ-抑制活性(Kawato等，1991)，这应当与增加的细胞死亡相关。因此，有关CPT-11-引起的腹泻的一种可能的机理也许包括肠上皮对SN-38内酯的再吸收，导致肠道的结构和功能损伤。The relationship between cellular uptake of SN-38 and its associated cytotoxicity was also evaluated in this study. The cellular uptake and cytotoxicity of SN-38 were found to be pH-dependent in HT29 cells, and the cytotoxicity correlated well with the initial uptake rate (Figure 8). As previously described, we believe that the predominant form of SN-38 is a lactone at acidic pH. This will result in greater cellular uptake and intracellular concentration of SN-38 lactone. Since SN-38 is mainly active in the lactone form, whereas SN-38 carboxylate shows only a small topoisomerase I-inhibitory activity (Kawato et al., 1991), this should be associated with increased cell death. Therefore, a possible mechanism for CPT-11-induced diarrhea may involve the reabsorption of SN-38 lactone by the intestinal epithelium, resulting in structural and functional impairment of the intestinal tract.
总之，本研究首先估价CPT-11和SN-38被肠上皮细胞的摄取。CPT-11和SN-38内酯两者都被被动转运，而CPT-11和SN-38羧化物被主动吸收。CPT-11和SN-38内酯的摄取速率几倍于各自的羧化物形式。此外，在HT29细胞中，高的SN-38的摄取速率与增加的细胞毒性相联系。这些研究结果说明向羧化物转变将导致CPT-11和SN-38两者的细胞摄取。因而，这些研究结果对作为一种可能的机理，碱化肠腔以在临床实践中降低CPT-11和SN-38的再吸收提供了支持。可能的是，有限的肠再吸收反过来调节这一药物肠肝循环的生物利用率，并且降低SN-38对肠上皮的毒副作用。In summary, this study first assessed the uptake of CPT-11 and SN-38 by intestinal epithelial cells. Both CPT-11 and SN-38 lactones are passively transported, while CPT-11 and SN-38 carboxylates are actively absorbed. The uptake rates of CPT-11 and SN-38 lactones are several times that of the respective carboxylated forms. Furthermore, in HT29 cells, a high rate of SN-38 uptake was associated with increased cytotoxicity. These findings suggest that a shift to carboxylate will result in cellular uptake of both CPT-11 and SN-38. Thus, these findings provide support as a possible mechanism for alkalinizing the intestinal lumen to reduce the reabsorption of CPT-11 and SN-38 in clinical practice. It is possible that limited intestinal reabsorption in turn modulates the enterohepatic bioavailability of this drug and reduces the toxic side effects of SN-38 on the intestinal epithelium.
导致直接影响临床实践，以及喜树碱化合物的施用，，通过肝被清除，如盐酸伊立替康，以及其衍生物。发明者提供了喜树碱化合物的施用的口头的碱化通过肝被清除，包括CPT-11。Resulting in a direct impact on clinical practice, as well as the administration of camptothecin compounds, which are cleared by the liver, such as irinotecan hydrochloride, and its derivatives. The inventors provide that oral alkalinization by administration of camptothecin compounds is cleared by the liver, including CPT-11.
总而言之，发明人描述了喜树碱化合物(如CPT-11和SN-38)由肠上皮的摄取。尽管通过不同的转运机理，CPT-11和SN-38内酯两者由肠细胞被动转运。CPT-11和SN-38羧化物两者被主动吸收。TCA胶束的形成降低CPT-11和SN-38两者的摄取。CPT-11和SN-38内酯的摄取速率高于羧化物形式的几倍，而在碳酸氢盐的存在下和在增加的pH值条件下，摄取速率降低。这些有关CPT-11和SN-38的研究结果在临床实践中可能是有用的。In summary, the inventors describe the uptake of camptothecin compounds, such as CPT-11 and SN-38, by the intestinal epithelium. Both CPT-11 and SN-38 lactones are passively transported by enterocytes, albeit through different transport mechanisms. Both CPT-11 and SN-38 carboxylate are actively absorbed. Formation of TCA micelles reduces the uptake of both CPT-11 and SN-38. The uptake rate of CPT-11 and SN-38 lactones was several times higher than that of the carboxylate form, while in the presence of bicarbonate and under conditions of increasing pH, the uptake rate was reduced. These findings on CPT-11 and SN-38 may be useful in clinical practice.
表Ⅰ：肠细胞对CPT-11和SN-38的初始摄取速率 Table Ⅰ: Initial uptake rates of CPT-11 and SN-38 by enterocytes
空肠 回肠
比较了[14C]CPT-11(20μM)和[14C]SN-38(2μM)各自的内酯和羧化物的摄取初始速率。结果以pmol·106细胞-1·min-1表示，并且是10次实验的平均值±SE。Mann Whitney试验用于统计分析。The initial rates of uptake of lactone and carboxylate of [14 C]CPT-11 (20 μM) and [14 C]SN-38 (2 μM) respectively were compared. The results are expressed in pmol·106 cells−1 ·min−1 and are mean±SE of 10 experiments. Mann Whitney test was used for statistical analysis.
表Ⅱ CPT-11和SN-38由肠细胞摄取的动力学参数 Table Ⅱ Kinetic parameters of CPT-11 and SN-38 uptake by enterocytes
空肠 回肠jejunum ileum
Km Vmax Kd Km Vmax KdCPT-11 内酯 ND ND 0.95 ND ND 1.06Km Vmax Kd Km Vmax Kd CPT-11 lactone ND ND 0.95 ND ND 1.06
(0.15) (0.28)(0.15) (0.28)
羧化物 51.3 146.9 ＜0.05 50.5 157.3 ＜0.05 Carboxylate 51.3 146.9 ＜0.05 50.5 157.3 ＜0.05
(16.3) (41.3) (＜0.02) (13.0) (38.0) (＜0.02)SN-38(16.3) (41.3) (＜0.02) (13.0) (38.0) (＜0.02)SN-38
内酯* ND ND 2.38 ND ND 1.87Lactone* ND ND 2.38 ND ND 1.87
(0.26) (0.10)(0.26) (0.10)
羧化物* ND ND 0.44 ND ND 0.42Carboxylate* ND ND 0.44 ND ND 0.42
(0.17) (0.01)(0.17) (0.01)
(*)：由于有限的溶解性，仅研究了SN-38达2μM的浓度。(* ): Due to limited solubility, only concentrations of SN-38 up to 2 μΜ were studied.
(+)：由于从在二硝基苯酚的存在下SN-38羧化物摄取的评价中，SN-38羧化物被定为主动转运(表3)，这些值不被认为是生理学相关的。(+ ): Since the SN-38 carboxylate was assigned as active transport from the evaluation of SN-38 carboxylate uptake in the presence of dinitrophenol (Table 3), these values were not considered physiologically relevant.
注：数据用方程式V=(VmaxS)/(Km+S)+KdS经最小二乘法非线性回归分析处理，其中V代表摄取的初始速率，Vmax(pmol·106细胞-1·min-1)是摄取的最大速率，Km(μM)是表观米氏常数，Kd(pmol·106细胞-1·min-1·μM-1)是扩散速率，S(μm)是CPT-11或SN-38的浓度。值是平均值±SE。CPT-11内酯，SN-38内酯以及SN-38羧化物的摄取的主要组分分别是不可饱和的，因此，Km和Vmax值没有测定(ND)Note: The data were processed by least square nonlinear regression analysis using the equation V=(Vmax S)/(Km +S) +Kd S, where V represents the initial rate of uptake, Vmax (pmol·106 cells-1 ·min-1 ) is the maximum rate of uptake, Km (μM) is the apparent Michaelis constant, Kd (pmol·106 cells-1 ·min-1 ·μM-1 ) is the diffusion rate, S(μm ) is the concentration of CPT-11 or SN-38. Values are mean ± SE. The major components of the uptake of CPT-11 lactone, SN-38 lactone, and SN-38 carboxylate were not saturable, therefore, Km and Vmax values were not determined (ND)
表Ⅲ：二硝基苯酚，SN38-Glu和DNP-SG对 Table Ⅲ: Dinitrophenol, SN38-Glu and DNP-SG pairs
CPT-11和SN-38初始摄取速率影响Effect of CPT-11 and SN-38 on initial uptake rate
CPT-11羧化物 SN-38羧化物 CPT-11内酯 SN-38内酯 CPT-11 carboxylate SN-38 carboxylate CPT-11 lactone SN-38 lactone
空肠 回肠 空肠 回肠 空肠 回肠 空肠 回肠二硝基苯酚(1mM)jejunum ileum jejunum ileum jejunum ileum jejunum ileum dinitrophenol (1mM)
平均值 22.6 29.2 25.5 30.8 94.1 105.5 96.1 134.9(SE) (13.5) (9.2) (12.4) (13.1) (18.4) (15.6) (14.7) (19.0)P值1(n=5) 0.016 0.008 0.008 0.016 NS NS NS NSSN38-Glu(200μM)平均值 108.9 93.9 40.1* 28.9* 88.9 NE 54.3 NE(SE) (22.1) (14.3) (11.1) (11.2) (15.3) (20.6)DNP-SG(200μM)平均值 103.2 105.8 32.0* 28.5* 105.4 NE 78.8 NE(SE) (17.0) (36.3) (9.9) (11.9) (12.7) (24.4)P值2(n=5) NS NS 0.007 0.020 NS NSMean 22.6 29.2 25.5 30.8 94.1 105.5 96.1 134.9(SE) (13.5) (9.2) (12.4) (13.1) (18.4) (15.6) (14.7) (19.0)P-value1 (n=5) 0.016 0.008 0.008 NS 0.016 NS NS NSSN38-Glu (200μM) mean 108.9 93.9 40.1* 28.9* 88.9 NE 54.3 NE(SE) (22.1) (14.3) (11.1) (11.2) (15.3) (20.6) DNP-SG (200μM) mean 103.2 105.8 32.0* 28.5* 105.4 NE 78.8 NE(SE) (17.0) (36.3) (9.9) (11.9) (12.7) (24.4)P value2 (n=5) NS NS 0.007 0.020 NS NS
NE，未评估；NS，与对照无明显不同。NE, not assessed; NS, not significantly different from control.
注：在分别添加[14C]CPT-11(20μM)和[14C]SN-38(2μM)之前，将二硝基苯酚，SN-38葡糖苷酸(SN38-Glu)或2,4-二硝基苯基-S-谷胱甘肽(DNP-SG)添加到指示细胞悬液中(详细内容参见材料和方法)。在每种化合物的存在下CPT-11和SN-38的初始摄取速率以对照的百分比表示。二硝基苯酚和其对照之间的区别用1Mann-Whitney试验估价。SN-38Glu,DNP-SG以及它们的对照中间的区别用2Kriskal-Wallis试验估价，并且根据Durm’s法(*p＜0.05)分析与各自对照的显著性差异。盐酸伊立替康制剂Note: Dinitrophenol, SN-38 glucuronide(SN38 -Glu) or 2,4- Dinitrophenyl-S-glutathione (DNP-SG) was added to the indicator cell suspensions (see Materials and Methods for details). The initial uptake rates of CPT-11 and SN-38 in the presence of each compound are expressed as a percentage of control. The difference between dinitrophenol and its control was assessed bya Mann-Whitney test. Differences among SN-38Glu, DNP-SG and their controls were assessed with2 Kriskal-Wallis tests and significant differences from the respective controls were analyzed according to Durm's method (* p<0.05). Irinotecan hydrochloride preparation
在优选的实施方案中，碳酸氢钠，氧化镁和水以pH值高于约7，优选地8-10，最优选地8-9施用给用喜树碱化合物(如CPT-11和其衍生物)治疗的患者。In a preferred embodiment, sodium bicarbonate, magnesium oxide and water are administered with a camptothecin compound (such as CPT-11 and its derivatives) at a pH above about 7, preferably 8-10, most preferably 8-9 drug) treated patients.
此外，本发明的CPT-11化合物在对人类和动物的全身的施用的药物组合物中有用，其以单剂形式，如片剂，胶囊，丸剂，粉末，微粒，栓剂，无菌肠胃外溶液或悬液，无菌非肠胃外溶液或悬液，口服溶液或悬液，水包油或油包水乳剂等，它们包含合适量的有效成分。对于口服，固体或液体两种单剂都可以以化合物制备。所说的化合物在带有载体的活性成分的药物组合物中有用，活性成分约(wt％)1到20％，优选地约5到15％。Furthermore, the CPT-11 compounds of the present invention are useful in pharmaceutical compositions for systemic administration to humans and animals in single dosage forms such as tablets, capsules, pills, powders, granules, suppositories, sterile parenteral solutions or suspensions, sterile non-parenteral solutions or suspensions, oral solutions or suspensions, oil-in-water or water-in-oil emulsions, etc., which contain active ingredients in suitable amounts. For oral administration, both solid and liquid unit doses can be prepared with the compounds. Said compounds are useful in pharmaceutical compositions of active ingredient with a carrier, the active ingredient being about (wt%) 1 to 20%, preferably about 5 to 15%.
液体或固体单剂形式可以容易地制备供口服施用。例如，CPT-11可以与常规组分混合，如磷酸二钙，铝硅酸镁，硬脂酸镁，硫酸钙，淀粉，滑石，乳糖，阿拉伯胶，甲基纤维素，以及作为药物赋形剂和载体的功能性类似材料。可以也可以不施用持续释放制剂。可以通过将化合物与药物稀释剂混合来制备胶囊，将这种混合物插入到具有合适大小的硬明胶囊中。如果软胶囊是所需的，可以将化合物的浆状物与可接受的蔬菜，石油醚，或其它惰性油通过机器包裹少明胶囊中。Liquid or solid unit dosage forms are readily prepared for oral administration. For example, CPT-11 can be mixed with conventional ingredients such as dicalcium phosphate, magnesium aluminosilicate, magnesium stearate, calcium sulfate, starch, talc, lactose, acacia, methylcellulose, and as pharmaceutical excipients Functionally similar material to the carrier. Sustained release formulations may or may not be administered. Capsules can be prepared by mixing the compound with a pharmaceutical diluent and inserting this mixture into hard gelatin capsules of suitable size. If soft capsules are desired, a slurry of the compound and acceptable vegetable, petroleum ether, or other inert oils can be machine-coated into Shaoming capsules.
悬液，糖浆以及酏剂可以用于液体单剂形式的口服。包含油的液体制剂可以用于油溶性形式。蔬菜油(例如，玉米油，花生油或红花油)与调味剂，甜味剂以及任何防腐剂一道产生可接受的液体制剂。可以添加表面活性剂到水中以形成供液体单位给药的糖浆。可以使用具有可接受的甜味剂(如糖，糖精或生物增甜剂以及调味剂的酏剂形式的水-醇药物制剂。Suspensions, syrups and elixirs are available for oral administration in liquid single dose form. Liquid formulations containing oil may be used in oil soluble forms. Vegetable oil (eg, corn, peanut or safflower oil) together with flavoring, sweetening and any preservatives yields an acceptable liquid preparation. A surfactant may be added to water to form a syrup for liquid unit administration. Hydro-alcoholic pharmaceutical preparations may be used in the form of elixirs with acceptable sweetening agents such as sugar, saccharin or biological sweeteners and flavorings.
肠胃外和栓剂施用的药物组合物也可以利用本领域标准技术获得。Pharmaceutical compositions for parenteral and suppository administration can also be obtained using standard techniques in the art.
合适的药物载体包括无菌水；盐水，葡萄糖；在水或盐水中的葡萄糖；蓖麻油和环氧乙烷的缩合产品(每摩尔蓖麻油组合约30到约35摩尔的环氧乙烷)；液态酸；低级阿卡诺尔；油(如玉米油；花生油，芝麻油等)，具有乳化剂(如脂肪酸或磷脂的单或二甘油酯，如lecitbin等)；乙二醇；聚亚烷基二醇；有悬浮剂(如，羧甲基纤维素钠)存在的含水介质；褐藻酸钠；聚(聚乙烯吡咯烷酮)等；它们单独地或与合适的分散剂(如卵磷脂；聚氧化乙烯硬脂酸钠)等一起；载体也可以含有与本发明的渗透增强剂一起的佐剂，如保存稳定剂，润湿剂，乳化剂等。Suitable pharmaceutical carriers include sterile water; saline, dextrose; dextrose in water or saline; condensation products of castor oil and ethylene oxide (about 30 to about 35 moles of ethylene oxide per mole of castor oil combined); Acids in liquid form; lower alcanol; oils (such as corn oil; peanut oil, sesame oil, etc.), with emulsifiers (such as mono- or diglycerides of fatty acids or phospholipids, such as lecitbin, etc.); glycols; polyalkylene glycols ; aqueous media in the presence of suspending agents (e.g., sodium carboxymethylcellulose); sodium alginate; poly(vinylpyrrolidone), etc.; either alone or with suitable dispersants (e.g., lecithin; polyethylene oxide stearin Sodium acid) and the like; the carrier can also contain adjuvants together with the penetration enhancer of the present invention, such as storage stabilizers, wetting agents, emulsifiers and the like.
哺乳动物的有效剂量可以随一些因素变化，例如，年龄，体重水平，被治疗者的身体状况等。典型地，根据本发明的化合物的有效剂量在口头或直肠给药时，是从约10mg/m2到700mg/m2，每日1-3次。CPT-11可以优选地每周施用一次，用药1到5周。用于癌症和肿瘤治疗的CPT-11的施用次数和剂量是已知的。盐酸伊立替康的施用Effective doses for mammals may vary with factors such as age, body weight level, physical condition of the subject and the like. Typically, an effective dosage of a compound according to the invention is from about 10 mg/m2 to 700 mg/m2 , 1-3 times daily, when administered orally or rectally. CPT-11 can preferably be administered once a week for 1 to 5 weeks. The administration frequency and dose of CPT-11 for cancer and tumor therapy are known. Administration of irinotecan hydrochloride
盐酸伊立替康(Pharmacia-Upjohn)的平均最终排除的半衰期是约6小时。The mean terminal elimination half-life of irinotecan hydrochloride (Pharmacia-Upjohn) is about 6 hours.
喜树碱化合物可以单独施用，也可以与组合化疗药物结合起来施用，包括甲酰四氢叶酯，顺式铂氨，5-FU,oxiplatin以及其它已知化疗药物。在一可替实施方案中，喜树碱中化合物(如盐酸伊立替康)也可以与洛派丁胺一起施用。Camptothecin compounds can be administered alone or in combination with combination chemotherapy drugs, including leucovyl, cisplatin, 5-FU, oxiplatin and other known chemotherapy drugs. In an alternative embodiment, camptothecin compounds such as irinotecan hydrochloride may also be administered with loperamide.
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(31)胆汁pH(31) Bile pH
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(33)Atsumi R,Okazaki O,Hakusui H.SN-38[(+).(4S)-4,11-二乙基-4,9-二羟基-1H-吡喃[3’,4’:6,7]-吲嗪[1,2-b]喹啉-3,14(4H,12H)-二酮]，伊立替康的一种代谢物，在对大鼠单一静脉内使用14C-SN-38后的动力学。生物药学通讯。1995；18:1114-1119。(33)Atsumi R, Okazaki O, Hakusui H.SN-38[(+).(4S)-4,11-diethyl-4,9-dihydroxy-1H-pyran[3',4': 6,7]-Indolazine[1,2-b]quinoline-3,14(4H,12H)-dione], a metabolite of irinotecan, was administered in a single intravenous 14C-SN in rats Dynamics after -38. Biopharmaceutical Communications. 1995;18:1114-1119.
(34)Araki E,Ishikawa M,Ligo M,Koide T,Itabashi M,Hoshi A，在腹腔内施用CPT-11后，在无胸腺的小鼠的肠和血浆中，腹泻发生和SN-38,CPT-11的一种活性代谢物浓度之间的关系。日本癌症研究杂志。1991:51:4187-4191。(35)Takasuna K,Hagiwara T,Hirohashi M,Kato M,Nomura M,Nagai E，等。肠微生植系中β-葡萄糖醛酸酶牵涉到抗肿瘤物喜树碱衍生物盐酸伊立替康肠毒性。癌症研究。1996；56:3752-3757。(34) Araki E, Ishikawa M, Ligo M, Koide T, Itabashi M, Hoshi A, After intraperitoneal administration of CPT-11, diarrhea occurred in the intestine and plasma of athymic mice and SN-38, CPT The relationship between the concentration of an active metabolite of -11. Japanese Journal of Cancer Research. 1991:51:4187-4191. (35) Takasuna K, Hagiwara T, Hirohashi M, Kato M, Nomura M, Nagai E, et al. β-glucuronidase in intestinal microflora is involved in the enterotoxicity of the antineoplastic camptothecin derivative irinotecan hydrochloride. cancer research. 1996;56:3752-3757.
(36)Abigerges D,Armand JP,Chabot GG,DaCosta L,Fadel E,Cote C，等。采用增强的高剂量洛派丁胺高剂量增加伊立替康(CPT-11)，以控制腹泻。自然肿瘤研究杂志，1994:86:446-449。(36) Abigerges D, Armand JP, Chabot GG, DaCosta L, Fadel E, Cote C, et al. Diarrhea was controlled with boosted high-dose loperamide high-dose irinotecan (CPT-11). Journal of Nature Oncology Research, 1994:86:446-449.
(37)Kobayashi K,Shinbara A,Kamimura M,Takeda Y,Kudo K,Kabe J，等伊立替康(CPT-11)每周与cisplatin(CDDP)组合施用以治疗非小细胞肺癌。印刷的肿瘤化疗药理。(37) Kobayashi K, Shinbara A, Kamimura M, Takeda Y, Kudo K, Kabe J, et al. Irinotecan (CPT-11) administered weekly in combination with cisplatin (CDDP) in the treatment of non-small cell lung cancer. Printed Pharmacology of Cancer Chemotherapy.
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●TAKATSUNA,K.,HAGIWARA,T.,HIROHASHI,M.,KATO,M.,NOMURA,●TAKATSUNA, K., HAGIWARA, T., HIROHASHI, M., KATO, M., NOMURA,
●,NAGAI,E.,YOKOI,T.,KAMATAKI,T.,肠微生植系中β-葡萄糖醛酸酶牵涉到抗肿瘤物喜树碱衍生物盐酸伊立替康肠毒性。癌症研究。1996；56:3752-3757(1996)。●, NAGAI, E., YOKOI, T., KAMATAKI, T., Involvement of β-glucuronidase in intestinal microflora in enterotoxicity of antineoplastic camptothecin derivative irinotecan hydrochloride. cancer research. 1996;56:3752-3757 (1996).
进行以上描述和给出一些实施例的目的是为了说明本发明的实施方案，而没有任何限制本发明的意思。本领域技术人员清楚，对本发明的组合物和方法可以进行各种修改和变化而不偏离本发明的精神和范围。本文所引用的所有专利和出版物以其整体本文一并参考。The above description and some examples are given for the purpose of illustrating the embodiments of the present invention without any intention of limiting the present invention. It will be apparent to those skilled in the art that various modifications and changes can be made in the compositions and methods of the present invention without departing from the spirit and scope of the invention. All patents and publications cited herein are hereby incorporated by reference in their entirety.
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| CN107281462A (en)* | 2017-08-21 | 2017-10-24 | 滨州医学院 | Sha Kuilawei mitigates the new medical use of toxicity of irinotecan |
| US11090299B2 (en) | 2015-06-30 | 2021-08-17 | Hanmi Pharm. Co., Ltd. | Oral solid formulation containing irinotecan and method of preparing the same |
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| TWI581794B (en)* | 2014-10-21 | 2017-05-11 | 強普生技股份有限公司 | Methods and formulation for improving oral availability of cpt-11 while reducing cpt-11 induced gastronintestinal toxicity in cancer therapy |
| US11090299B2 (en) | 2015-06-30 | 2021-08-17 | Hanmi Pharm. Co., Ltd. | Oral solid formulation containing irinotecan and method of preparing the same |
| CN107281462A (en)* | 2017-08-21 | 2017-10-24 | 滨州医学院 | Sha Kuilawei mitigates the new medical use of toxicity of irinotecan |
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| JP2002518332A (en) | 2002-06-25 |
| EP1003516A1 (en) | 2000-05-31 |
| AU764370B2 (en) | 2003-08-14 |
| WO1999065493A1 (en) | 1999-12-23 |
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