Bhandari Neetesh, Figueroa David J, Lawrence Jeffrey W, Gerhold David Lee
Merck Research Laboratories, Merck & Co., Inc., West Point, PA 19486-0004, USA.
Assay Drug Dev Technol. 2008 Jun;6(3):407-19. doi: 10.1089/adt.2007.119.
Phospholipidosis (PLD) is an accumulation of phospholipids in lysosome-derived multilamellar vesicles. More than 50 commercial drugs are known to cause PLD. In vitro screening assays were developed in HepG2 cells, rat primary hepatocytes, and rhesus monkey hepatocytes using the fluorescent-labeled phospholipid probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) or Nile Red lipid stain. The assays were qualified using amiodarone and fluoxetine as positive controls and precocene and valproic acid as negative controls. NBD-PE accumulation and Nile Red staining were first measured using fluorescence microscopy with morphometric analysis, and the throughput of the NBD-PE assay in HepG2 cells was increased by measuring fluorescence with a multiwell spectrofluorometer. The PLD potential values obtained for the tested compounds from the morphometric analysis were similar to the values obtained from the spectrofluorometer, suggesting the plate reader assay was effective at measuring the induction of NBD-PE accumulation. Fifteen commercial compounds were evaluated using the NBD-PE assay in HepG2 cells, rat primary hepatocytes, and rhesus monkey hepatocytes. The relative NBD-PE accumulation and PLD potentials of the evaluated compounds were similar and comparable to the values observed from other in vitro PLD assays referenced in the literature using different probes and cell lines. NBD-PE accumulations observed in rat hepatocytes after drug treatments were similar to those in HepG2 cells. NBD-PE accumulation potential observed in rhesus monkey hepatocytes after drug treatment was different for tamoxifen, perhexiline, clomipramine, and haloperidol. These agents caused potent NBD-PE accumulation in HepG2 cells, but minimal or no activity was observed in rhesus monkey hepatocytes. These data suggest that the NBD-PE spectrofluorometer assay in HepG2 cells has the speed and throughput to sensitively and quantitatively determine the PLD potential of various drug candidates. In addition, these data demonstrate the species differences in PLD potential between rat and monkey hepatocytes.
磷脂沉积症（PLD）是磷脂在溶酶体来源的多层囊泡中的蓄积。已知有50多种商业药物可导致PLD。使用荧光标记的磷脂探针N-(7-硝基苯并-2-恶唑-1,3-二氮杂萘-4-基)-1,2-二己酰基-sn-甘油-3-磷酸乙醇胺（NBD-PE）或尼罗红脂质染色剂，在HepG2细胞、大鼠原代肝细胞和恒河猴肝细胞中建立了体外筛选试验。使用胺碘酮和氟西汀作为阳性对照，早熟素和丙戊酸作为阴性对照对试验进行验证。首先使用荧光显微镜和形态计量分析测量NBD-PE蓄积和尼罗红染色，通过使用多孔荧光分光光度计测量荧光，提高了HepG2细胞中NBD-PE试验的通量。通过形态计量分析获得的受试化合物的PLD电位值与荧光分光光度计获得的值相似，表明酶标仪试验在测量NBD-PE蓄积诱导方面是有效的。使用HepG2细胞、大鼠原代肝细胞和恒河猴肝细胞中的NBD-PE试验对1５种商业化合物进行了评估。评估化合物的相对NBD-PE蓄积和PLD电位相似，与文献中使用不同探针和细胞系的其他体外PLD试验观察到的值相当。药物处理后在大鼠肝细胞中观察到的NBD-PE蓄积与在HepG2细胞中的相似。药物处理后在恒河猴肝细胞中观察到的NBD-PE蓄积潜力在他莫昔芬、哌克昔林、氯米帕明和氟哌啶醇方面有所不同。这些药物在HepG2细胞中引起强烈的NBD-PE蓄积，但在恒河猴肝细胞中观察到的活性最小或无活性。这些数据表明，HepG2细胞中的NBD-PE荧光分光光度计试验具有速度和通量，能够灵敏且定量地确定各种候选药物的PLD潜力。此外，这些数据证明了大鼠和猴肝细胞在PLD潜力方面的种属差异。
