Danks M K, Pawlik C A, Whipple D O, Wolverton J S
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Clin Cancer Res. 1997 Oct;3(10):1731-8.
Camptothecin analogues such as topotecan increase the number of covalent topoisomerase I-DNA complexes, which, in turn, have been proposed to initiate apoptosis. If induction of apoptosis by the camptothecins is, in fact, dependent on the formation of topoisomerase I-DNA complexes, then it would be of clinical relevance to identify schedules of exposure to the camptothecins that maximize the formation of these complexes but minimize the total amount of the drug administered. The time and dose dependence of topoisomerase I-DNA complex formation was determined by incubating Daoy pediatric medulloblastoma cells in vitro with topotecan at concentrations equivalent to those achievable in the plasma clinically (10, 50, or 200 nM) and measuring the number of complexes present in cells incubated for 15 min to 48 h with the drug. Regardless of the concentration of topotecan used, covalent topoisomerase I-DNA complexes were maximal within 15 min following addition of the lactone form of topotecan to the tissue culture medium. After 2 h of exposure to topotecan, complexes had decreased from maximum to approximately half of that value. Few, if any, complexes were detectable with topotecan incubations of 24-48 h. Growth inhibition studies showed that the IC50s of topotecan for the Daoy cell line (2.2 x 10(-9) M) and also for a second pediatric medulloblastoma cell line, SJ-Med3 (3.6 x 10(-9) M), exposed to topotecan 8 h daily for 5 days or continuous exposure were equivalent. The decrease in topoisomerase I-DNA complexes between 15 min and 1 h was consistent with a pH-dependent re-equilibration of topotecan to the less active hydroxyacid form of the drug. The decrease in complexes after a 2-48-h incubation with the drug was attributable neither to biological inactivation of topotecan as shown by sequential growth inhibition studies nor to a decrease in amount of topoisomerase I in the drug-treated cells. Indirect immunofluorescence labeling of topoisomerase I in Daoy cells incubated for 48 h with 10 nM topotecan showed a redistribution of nucleolar topoisomerase I. We are currently evaluating the antitumor effect of intermittent repetitive exposures to topotecan in mice bearing Daoy cells as a xenograft. The clinical utility of each effective schedule of exposure will depend on whether the therapeutic index of repetitive intermittent exposure to the drug is more or less favorable than the therapeutic index of continuous exposure.
喜树碱类似物如拓扑替康可增加共价拓扑异构酶I-DNA复合物的数量，而这些复合物反过来被认为可引发细胞凋亡。如果喜树碱诱导的细胞凋亡实际上依赖于拓扑异构酶I-DNA复合物的形成，那么确定喜树碱的给药方案就具有临床相关性，该方案应能使这些复合物的形成最大化，同时使给药的药物总量最小化。通过在体外将道氏小儿髓母细胞瘤细胞与拓扑替康以临床血浆中可达到的浓度（10、50或200 nM）孵育，并测量与药物孵育15分钟至48小时的细胞中存在的复合物数量，来确定拓扑异构酶I-DNA复合物形成的时间和剂量依赖性。无论使用的拓扑替康浓度如何，将内酯形式的拓扑替康添加到组织培养基中后15分钟内，共价拓扑异构酶I-DNA复合物的数量达到最大值。暴露于拓扑替康2小时后，复合物数量从最大值下降到该值的约一半。在24至48小时的拓扑替康孵育中，几乎检测不到复合物（如果有的话）。生长抑制研究表明，拓扑替康对道氏细胞系（2.2×10⁻⁹ M）以及另一个小儿髓母细胞瘤细胞系SJ-Med3（3.6×10⁻⁹ M）的IC50值在每天暴露8小时共5天或持续暴露的情况下是相当的。15分钟至1小时之间拓扑异构酶I-DNA复合物数量的减少与拓扑替康向活性较低的羟基酸形式的pH依赖性重新平衡一致。与药物孵育2至48小时后复合物数量的减少既不是由于拓扑替康的生物学失活（如连续生长抑制研究所显示），也不是由于药物处理细胞中拓扑异构酶I数量的减少。用10 nM拓扑替康孵育48小时的道氏细胞中拓扑异构酶I的间接免疫荧光标记显示核仁拓扑异构酶I发生了重新分布。我们目前正在评估间歇性重复暴露于拓扑替康对携带道氏细胞异种移植瘤的小鼠的抗肿瘤作用。每种有效的暴露方案的临床实用性将取决于重复间歇性暴露于该药物的治疗指数比连续暴露的治疗指数更有利还是更不利。
