Both eucaryotic and procaryotic cells are resistant to a large number of antibiotics because of the activities of export transporters. The most studied transporter in the mammalian ATP-binding cassette transporter superfamily, P glycoprotein (P-gp), ejects many structurally unrelated amphiphilic and lipophilic xenobiotics. Observed clinical interactions and some in vitro studies suggest that azole antifungals may interact with P-gp. Such an interaction could both affect the disposition and exposure to azole antifungal therapeutics and partially explain the clinical drug interactions observed with some antifungals. Using a whole-cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the abilities of the most widely prescribed orally administered azole antifungals to inhibit the function of this transporter. In a cell line presenting an overexpressed amount of the human P-gp transporter, itraconazole and ketoconazole inhibited P-gp function with 50% inhibitory concentrations (IC 50 s) of ϳ2 and ϳ6 M, respectively. Cyclosporin A was inhibitory with an IC 50 of 1.4 M in this system. Uniquely, fluconazole had no effect in this assay, a result consistent with known clinical interactions. The effects of these azole antifungals on ATP consumption by P-gp (representing transport activity) were also assessed, and the K m values were congruent with the IC 50 s. Therefore, exposure of tissue to the azole antifungals may be modulated by human P-gp, and the clinical interactions of azole antifungals with other drugs may be due, in part, to inhibition of P-gp transport.
Phospholipidosis (PLD) is characterized by the excessive intracellular accumulation of phospholipids. It is well established that a large number of cationic amphiphilic drugs have the potential to induce PLD. In the present study, we describe two facile in vitro methods to determine the PLD-inducing potential of a molecule. The first approach is based on a recent study by (Sawada et al., 2005, Toxicol. Sci. 83, 282-292) in which 17 genes were identified as potential biomarkers of PLD in HepG2 cells. To confirm the utility of this gene panel, we treated HepG2 cells with PLD-positive and -negative compounds and then analyzed gene expression using real-time PCR. Our initial analysis, which used a single dose of each drug, correctly identified five of eight positive compounds and four of four negative compounds. We then increased the doses of the three false negatives (amiodarone, tamoxifen, and loratadine) and found that the changes in gene expression became large enough to correctly identify them as PLD-inducing drugs. Our results suggest that a range of concentrations should be used to increase the accuracy of prediction in this assay. Our second approach utilized a fluorescently labeled phospholipid (LipidTox) which was added to the media of growing HepG2 cells along with compounds positive and negative for PLD. Phospholipid accumulation was determined using confocal microscopy and, more quantitatively, using a 96-well plate assay and a fluorescent plate reader. Using an expanded set of compounds, we show that this assay correctly identified 100% of PLD-positive and -negative compounds. Dose-dependent increases in intracellular fluorescent phospholipid accumulation were observed. We found that this assay was less time consuming, more sensitive, and higher throughput than gene expression analysis. To our knowledge, this study represents the first validation of the use of LipidTox in identifying drugs that can induce PLD.
Clinical studies indicate that the farnesyl protein transferase inhibitor SCH66336 (lonafarnib), an anticancer agent developed to antagonize oncogenic Ras, is generally well tolerated. Lonafarnib has also demonstrated therapeutic synergy with coadministered taxanes, vincristine, cisplatin, cyclophosphamide, 5-fluorouracil (5-FU) and Gleevec. Lonafarnib has recently been shown, in addition, to be a potent inhibitor of the transmembrane efflux transporter P-glycoprotein (P-gp), which confers cellular resistance to the substrates vincristine, taxol and paclitaxel. Treatment with lonafarnib would therefore be predicted to be synergistic with these coadministered cancer therapeutics that are substrates of P-gp. However, cisplatin, 5-FU and cyclophosphamide are not P-gp substrates, yet cisplatin, 5-FU and possibly cyclophosphamide are purported substrates for multidrug resistance proteins (MRPs) 1 and 2 (known to cause chemotherapy resistance). Lonafarnib is shown here to inhibit the function of MRP1 and MRP2 with a potency similar to that of cyclosporin A and may therefore cause the observed synergy with cisplatin and other agents by inhibiting these MRPs. Coadministration of lonafarnib could thus reduce chemotherapy dosage and hence produce lower exposure to normal cells and less undesired toxicity.
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