Validation of putative genomic biomarkers of nephrotoxicity in rats.

Drug-induced renal injury is a common finding in the early preclinical phase of drug development. But the specific genes responding to renal injury remain poorly defined. Identification of drug-induced gene changes is critical to provide insights into molecular mechanisms and detection of renal damage. To identify genes associated with the development of drug-induced nephrotoxicity, a literature survey was conducted and a panel of 48 genes was selected based on gene expression changes in multiple published studies. Male Sprague-Dawley rats were dosed daily for 1, 3 or 5 days to the known nephrotoxicants gentamicin, bacitracin, vancomycin and cisplatin, or the known hepatotoxicants ketoconazole, 1-naphthyl isothiocyanate and 4,4-diaminodiphenylmethane. Histopathological evaluation and clinical chemistry revealed renal proximal tubular necrosis in rats treated with the nephrotoxicants, but not from those treated with the hepatotoxicants. RNA was extracted from the kidney, and RT-PCR was performed to evaluate expression profiles of the selected genes. Among the genes examined, 24 genes are confirmed to be highly induced or repressed in rats treated with nephrotoxicants; further investigation identified that 5 of the 24 genes were also altered by hepatotoxicants. These data led to the identification of a set of genomic biomarker candidates whose expression in kidney is selectively regulated only by nephrotoxicants. Among those genes displaying the highest expression changes specifically in nephrotoxicant-treated rats were kidney injury molecule 1 (Kim1), lipocalin 2 (Lcn2), and osteopontin (Spp1). The establishment of such a genomic marker set offers a new tool in our ongoing quest to monitor nephrotoxicity.

Temporal evaluation of CYP mRNA in mice administered with prototypical P450 inducers: comparison with conventional protein/enzyme methods.

Assessment of cytochrome P450 (CYP) induction at the mRNA level in preclinical rodent studies has gained interest in recent years, but there are still concerns regarding correlations between the mRNA and the enzyme activity levels, especially in mice. The purpose of the present study was to systematically evaluate patterns of temporal changes of CYPs 1a1, 1a2, 2b10, 3a11, and 4a10 at mRNA, protein, and activity levels in order to determine to what extent mRNA levels could be used either qualitatively or quantitatively for the assessment of CYP enzyme induction. In this study, livers from male CD-1 mice treated daily with beta-naphthoflavone, phenobarbital, dexamethasone, clofibrate, and control vehicles were collected for RNA and microsomal analysis after 0.5, 1, 2, 4, and 8 days of daily dose. The results revealed a good correlation among mRNA, protein, and enzyme activity levels, with the best correlation at the time points between Days 2 and 8, suggesting that the appropriate time to monitor CYP mRNA may be beyond Day 2 of chemical treatments. Based on these results, we concluded that the mRNA approach is a useful tool to monitor CYP induction in mice, particularly when treatment duration is beyond 2 days.

In vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid based methodologies.

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.

Quantitative characterization of direct P-glycoprotein inhibition by St John's wort constituents hypericin and hyperforin.

The ATP-binding cassette transporter P-glycoprotein (P-gp) exerts a critical role in the systemic disposition of, and exposure to, lipophilic and amphipathic drugs, carcinogens, toxins and other xenobiotics. The ability of P-gp to transfer a wide variety of structurally unrelated compounds from the cell interior across the membrane bilayer remains intriguing. Since natural product chemicals in the widely consumed St John's wort appear to exert antidepressant effects by an unknown mechanism, the constituents are frequently studied for interactions with various biomacromolecules as well as cytotoxins or isolated cells. The drug interactions caused by this widely used herbal remedy are under-appreciated. Various clinical interactions have been observed upon the co-administration of St John's wort, and P-gp and CYP3A4 have been indicted as the cause. We characterized several St John's wort constituents for their interaction with P-gp and their specific effects on the P-gp export activity of several marker substrates. Two of these constituents, hyperforin and hypericin, inhibit the active efflux of the fluorescent markers daunorubicin (IC(50) approximately 30 microM) and calcein-AM. Herein, we show in-vitro results that can both explain the competing clinical observations of initial elevated exposure of P-gp substrate drugs (P-gp inhibition) followed by under-exposure (P-gp induction) when St John's wort is co-administered, and provide a further warning against unchecked co-administration of drugs with St John's wort.

Quantitative evaluation of isothiocyanates as substrates and inhibitors of P-glycoprotein.

The ATP-binding cassette transporter P-glycoprotein (P-gp) exerts a critical role in the systemic disposition of, and exposure to, lipophilic and amphipathic drugs, carcinogens, toxins and other xenobiotics. The ability of P-gp to transfer a wide variety of structurally unrelated compounds from the cell interior across the membrane bilayer remains intriguing. Since dietary chemicals in cruciferous and several other foods appear to exert anticarcinogenic effects by inducing phase II enzymes and inhibiting some phase I enzymes, the isothiocyanate constituents are frequently studied for interactions with various biomacromolecules as well as cytotoxins or isolated cells. Several prominent dietary isothiocyanates were characterized for their interaction with P-gp and their specific effects on the P-gp export activity of several marker substrates. Some of these compounds inhibit the active P-gp-mediated efflux of the fluorescent markers LDS-751 and daunorubicin with low potency, with the most potent among them, phenethyl isothiocyanate, inhibiting transport of the LDS-751 substrate with an IC(50) of approximately 240 microM. Overall, these isothiocyanates are unlikely to impede the xenobiotic defence function of P-gp even in the intestine where the concentrations are potentially high.

Co-induction of CYP3A12 and 3A26 in dog liver slices by xenobiotics: species difference between human and dog CYP3A induction.

The induction of dog CYP3A12 and CYP3A26 mRNA levels was evaluated in liver slices after treatment with 22 xenobiotics. Eleven of the 22 xenobiotics increased 3A12 mRNA by more than four-fold, while nine did the same for 3A26 mRNA. A four-fold increase in the mRNA level was used as the cut-off for indication of induction based on the noise level of the real time-PCR. A good correlation was found between the mRNA levels for 3A12 and 3A26 after treatment with compounds, suggesting that these two CYPs may be co-induced. Induction of CYP3A4 in human hepatocytes was evaluated after treatment with the same 22 compounds. Thirteen out of the 22 compounds increased the 3A4 mRNA levels by more than four-fold. When the mRNA levels of 3A4 and 3A12 were compared after treatment with compounds, no correlation was found. The regulation of CYP3A expression has been demonstrated to be controlled by pregnane X receptor (PXR). Upon examination of the sequence homology and the three-dimensional structures of human PXR and a dog PXR model, only two different amino acids (met323/val and arg410/lys) were found in the ligand-binding domain. This finding suggests that these two amino acids may play a role in the binding specificity of ligands.

The farnesyl protein transferase inhibitor lonafarnib (SCH66336) is an inhibitor of multidrug resistance proteins 1 and 2.

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.

Many P-glycoprotein substrates do not inhibit the transport process across cell membranes.

1. The critical role of P-glycoprotein (P-gp) in the clinical exposure of many pharmaceuticals and toxins has become widely appreciated. The P-gp-mediated influence can often be more significant than that of other well-known xenobiotic defence enzymes in both breadth and impact. The inhibition of P-gp, therefore, has often been examined by testing a compound for its influence on the P-gp-mediated transport of some marker substrate, often the compound is also evaluated for its active efflux mediated by P-gp. 2. Although a substrate for a xenobiotic defence enzyme is logically presumed to be an inhibitor of that enzyme toward an alternate substrate, that is not necessarily the case with a transmembrane active efflux transporter. A substrate that is ejected from the cytosolic side of the membrane bilayer that does not rapidly cross the membrane by passive diffusion back into the cell interior will not occlude the substrate binding site. Hence, some substrates may not significantly affect the overall P-gp function of causing a concentration gradient by efficient net transport. A wide variety of compounds that are documented as substrates of P-gp are characterized here as having no effect on the ability of P-gp to transport several conventional P-gp marker substrates. 3. Transbilayer passive diffusion apparently dictates the ability of a P-gp substrate to be an inhibitor, as described herein based on relative rates of transport (active efflux versus passive re-entry) and the interaction of amphipathic compounds with the cell membrane. 4. The portion of P-gp substrates whose disposition is dependent on P-gp function and which are not also inhibitors is striking. It is therefore important to characterize both the efflux rate parameters and those of inhibition. 5. This report affords a valuable list of known P-gp substrates that are non-inhibitors.

Elevation of P-glycoprotein function by a catechin in green tea.

The ABC transporter P-glycoprotein (P-gp) exerts a critical role in the systemic disposition of and exposure to lipophilic and amphipathic drugs, carcinogens, toxins, and other xenobiotics. The ability of P-gp to transfer a wide variety of structurally unrelated compounds from the cell interior across the membrane bilayer remains intriguing. Since dietary chemicals in green tea (and several other foods) appear to exert anticarcinogenic effects by an unknown mechanism, the constituents are frequently studied for interactions with various biomacromolecules as well as cytotoxins or isolated cells. We characterized several green tea catechins for their interaction with P-gp and their specific effects on P-gp export activity of several marker substrates. Some of these compounds inhibit the active efflux of the fluorescent markers LDS-751 (LDS) and rhodamine 123 (Rho) with low potency. Remarkably, others of these catechins facilitate the P-gp-mediated transport of LDS without affecting daunorubicin (DNR) transport or Rho. Moreover, (-)epicatechin, though an inhibitor of Rho transport, can significantly enhance the active net transport of another P-gp marker substrate, LDS. This result indicates that (-)epicatechin may bind to and activate an allosteric site that enhances P-gp overall function or efficiency. Such a mechanism of heterotropic allosteric enhancement of P-gp could serve as chemoprotective to many cells and contribute to the purported anticarcinogenic effect of green tea consumption.

Fluorescent substrates of sister-P-glycoprotein (BSEP) evaluated as markers of active transport and inhibition: evidence for contingent unequal binding sites.

PURPOSE: Although sister-P-glycoprotein (SPGP, BSEP) is closely related to P-glycoprotein, it is much more selective in distribution and substrate recognition. Moreover, because inhibition or lack of BSEP function has severe consequences including cholestasis, hepatotoxicity, exposure to toxic xenobiotics, and drug interactions, in vitro methods are necessary for quantifying and characterizing specific inhibition of BSEP. Therefore, the objective is to discern a method and quantitatively characterize several example BSEP inhibitors. METHODS: With fluorescent markers having been used successfully to evaluate and quantify inhibition of P-gp-mediated transport, this study evaluates several compounds for specific cell retention caused by BSEP inhibitors. In addition to the several compounds asserted to be BSEP inhibitors, the compounds suggested to be BSEP substrates might also inhibit BSEP competitively. Retained fluorescence of possible BSEP substrates was measured by a flow cell cytometer using transfected cells presenting the BSEP transporter specifically and abundantly. RESULTS: Several compounds were shown to inhibit BSEP active transport of the fluorescent substrates dihydrofluorescein and bodipy. The inhibition potency was quantified (i.e., cyclosporin A IC50 approximately 7 microM), revealing incongruent relative sensitivities among the substrate markers, with H2FDA generally the most sensitive of the series of substrate markers evaluated. CONCLUSIONS: The inconsistent sensitivities of the transport markers (H2FDA and bodipy) were reminiscent of the apparent multiple binding site behaviors observed for P-gp and could indicate opposing and unequal yet interacting binding sites akin to those of P-gp. Nonetheless, notable differences between P-gp and BSEP in marker substrate recognition/transport were apparent despite the observed overlap in xenobiotic recognition and transport. Thus far the most potent inhibitors seem to be cyclosporin, tamoxifen, and valinomycin. There are likely to be much more potent inhibitors, and other substrates also may be more sensitive to inhibition of transport.

Interaction of common azole antifungals with P glycoprotein.

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 approximately 2 and approximately 6 microM, respectively. Cyclosporin A was inhibitory with an IC(50) of 1.4 microM 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.

Garlic inhibits microsomal triglyceride transfer protein gene expression in human liver and intestinal cell lines and in rat intestine.

Epidemiologic studies have suggested that fresh garlic has lipid-lowering activity. Because the microsomal triglyceride transfer protein (MTP) plays a pivotal role in the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins, we evaluated the effect of garlic on the expression of the MTP gene in vitro in cell lines and in vivo in rats. Fresh garlic extract (FGE) reduced MTP mRNA levels in both the human hepatoma HepG2 and intestinal carcinoma Caco-2 cells in dose-dependent fashion; significant reductions were detected with 3 g/L FGE. Maximal 72 and 59% reductions, respectively, were observed with 6 g/L FGE. To evaluate the in vivo effect of garlic on MTP gene expression, rats were given a single oral dose of fresh garlic homogenate (FGH), with hepatic and intestinal MTP mRNA measured 3 h after dosing. Rats fed FGH had significantly (46% of the control) lower intestinal MTP mRNA levels compared with the control rats, whereas hepatic MTP mRNA levels were not affected. These results suggest a new mechanism for the hypolipidemic effect of fresh garlic. Long-term dietary supplementation of fresh garlic may exert a lipid-lowering effect partly through reducing intestinal MTP gene expression, thus suppressing the assembly and secretion of chylomicrons from intestine to the blood circulation.