Eliaz Rom E, Nir Shlomo, Marty Cornelia, Szoka Francis C
Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California, USA.
Cancer Res. 2004 Jan 15;64(2):711-8. doi: 10.1158/0008-5472.can-03-0654.
Various mathematical approaches have been devised to relate the cytotoxic effect of drugs in cell culture to the drug concentration added to the cell culture medium. Such approaches can satisfactorily account for drug response when the drugs are free in solution, but the approach becomes problematic when the drug is delivered in a drug delivery system, such as a liposome. To address this problem, we have developed a simple model that assumes that the cytotoxic potency of a drug is a function of the intracellular drug level in a critical compartment. Upon exposure to drug, cell death commences after a lag time, and the cell kill rate is dependent on the amount of drug in the critical intracellular compartment. The computed number of cells in culture, at any time after exposure to the drug, takes into account the cell proliferation rate, the cell kill rate, the average intracellular drug concentration, and a lag time for cell killing. We have applied this model to compare the cytotoxic effect of doxorubicin (DOX), or DOX encapsulated in a liposome that is targeted to CD44 on B16F10 melanoma cells in culture. CD44 is the surface receptor that binds to hyaluronan and is overexpressed on various cancer cells, including B16F10. We have shown previously that the drug encapsulated in hyaluronan-targeted liposomes was more potent than was the free drug. The model required the determination of the cell-associated DOX after the cells were incubated with various concentrations of the free or the encapsulated drug for 3 h, and the quantification of cell number at various times after exposure to the drug. The uptake of encapsulated drug was greater than that of the free drug, and the ratio of cell association of encapsulated:free drug was 1.3 at 0.5 micro g/ml and increased to 3.3 at 20 micro g/ml DOX. The results demonstrate that the enhanced potency of the encapsulated drug could stem from its enhanced uptake. However, in certain cases, where larger amounts of the free drug were added, such that the intracellular amounts of drug exceeded those obtained from the encapsulated drug, the numbers of viable cells were still significantly smaller for the encapsulated drug. This finding demonstrates that for given amounts of intracellular DOX, the encapsulated form was more efficient in killing B16F10 cells than the free drug. The outcome was expressed in the kinetic model as a 5-6-fold larger rate constant of cell killing potency for the encapsulated drug versus the free drug. The model provides a quantitative framework for comparing the cytotoxic effect in cultured cells when applying the drug in the free form or in a delivery system.
人们设计了各种数学方法，将细胞培养中药物的细胞毒性作用与添加到细胞培养基中的药物浓度联系起来。当药物在溶液中呈游离状态时，这些方法能够令人满意地解释药物反应，但当药物通过脂质体等药物递送系统给药时，该方法就会出现问题。为了解决这个问题，我们开发了一个简单的模型，该模型假设药物的细胞毒性效力是关键区室中细胞内药物水平的函数。细胞接触药物后，经过一段延迟时间开始死亡，细胞杀伤率取决于关键细胞内区室中的药物量。在接触药物后的任何时间，计算得出的培养细胞数量都考虑了细胞增殖率、细胞杀伤率、平均细胞内药物浓度以及细胞杀伤的延迟时间。我们应用这个模型来比较阿霉素（DOX）或包裹在靶向B16F10黑色素瘤细胞上CD44的脂质体中的DOX的细胞毒性作用。CD44是与透明质酸结合的表面受体，在包括B16F10在内的各种癌细胞上过度表达。我们之前已经表明，包裹在透明质酸靶向脂质体中的药物比游离药物更有效。该模型需要在细胞与不同浓度的游离或包裹药物孵育3小时后测定细胞相关的DOX，并在接触药物后的不同时间对细胞数量进行定量。包裹药物的摄取量大于游离药物，在0.5μg/ml时，包裹药物与游离药物的细胞结合比率为1.3，在20μg/ml DOX时增加到3.3。结果表明，包裹药物效力增强可能源于其摄取增加。然而，在某些情况下，添加大量游离药物使得细胞内药物量超过从包裹药物中获得的量时，包裹药物的活细胞数量仍然显著较少。这一发现表明，对于给定数量的细胞内DOX，包裹形式在杀死B16F10细胞方面比游离药物更有效。在动力学模型中，这一结果表现为包裹药物的细胞杀伤效力速率常数比游离药物大5至6倍。该模型为比较以游离形式或递送系统形式应用药物时培养细胞中的细胞毒性作用提供了一个定量框架。
