A novel assay reveals that weakly basic model compounds concentrate in lysosomes to an extent greater than pH-partitioning theory would predict
A novel assay reveals that weakly basic model compounds concentrate in lysosomes to an extent greater than pH-partitioning theory would predict
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DOI：
10.1021/mp050043s
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发表时间：
2005-11-01
影响因子：
4.9
通讯作者：
Krise, Jeffrey P.
中科院分区：
文献类型：
--
作者：
Duvvuri, Muralikrishna;Krise, Jeffrey P.
许多与细胞一起孵育的弱碱性药物已被证明可以在溶酶体中特异性积累。这种隔离的机制基础和底物特异性尚未经过严格评估；然而，条件有利于 pH 分配型积累。在某些情况下，这种区室化可能非常广泛，这可能会影响药物的治疗功效。尽管具有药学重要性，但之前尚未描述过对溶酶体中药物积累的直接定量评估。我们在此报告了一种新颖的磁性捕获技术，该技术可以从与模型化合物预孵育的培养 HL-60 细胞中快速有效地分离溶酶体。通过HPLC测定与分离的级分相关的化合物的量。分离的溶酶体的广泛生化和形态学特征以及 HPLC 数据可以对溶酶体中模型化合物的浓度进行估计。出于比较目的，还计算了基于 pH 分配理论的相应理论确定浓度值。有趣的是，实验确定的值比理论预测值大约高 3-15 倍。这一发现表明，除了 pH 分配之外，其他机制可能在药物在溶酶体中的积累中发挥重要作用。
Many weakly basic drugs incubated with cells have been shown to specifically accumulate in lysosomes. The mechanistic basis and substrate specificity for this sequestration have not been rigorously evaluated; however, conditions are favorable for a pH-partitioning type accumulation. In some circumstances, this compartmentalization can be very extensive, which can impact the therapeutic efficacy of a drug. Despite the pharmaceutical importance, direct quantitative assessments of drug accumulation in lysosomes have not been previously described. We report here a novel magnetic capture technique that allows for quick and efficient isolation of lysosomes from cultured HL-60 cells that have been preincubated with model compounds. The amount of compound associated with the isolated fraction is determined by HPLC. Extensive biochemical and morphological characterizations of isolated lysosomes, together with HPLC data, allowed for estimates to be made regarding the concentration of model compounds in lysosomes. The corresponding theoretically determined concentration values, based on pH-partitioning theory, were also calculated for comparison purposes. Interestingly, experimentally determined values were approximately 3-15 times higher than theoretically predicted values. This finding suggests that mechanisms, in addition to pH-partitioning, may play a significant role in the accumulation of drugs in lysosomes.