Er-jia Wang

Interaction of Common Azole Antifungals with P Glycoprotein

ABSTRACT 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 (IC50s) of ∼2 and ∼6 μM, respectively. Cyclosporin A was inhibitory with an IC50 of 1.4 μM 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 Km values were congruent with the IC50s. 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.

HMG-CoA Reductase Inhibitors (Statins) Characterized as Direct Inhibitors of P-Glycoprotein

AbstractPurpose. HMG-CoA reductase inhibitors (statins) are commonly prescribed for lipid lowering to treat hypercholesterolemia. Although they are well tolerated, their pharmacokinetic interactions with other drugs can lead to some adverse clinical consequences. The avenue of interaction has been asserted to be CYP3A4 because most (or all) known interactions are with CYP3A4 inhibitors, and statin oxidative metabolism is mediated by CYP3A4 as well as other CYP enzymes. However, these same drugs that exert a clinical pharmacokinetic effect on statin disposition are generally also P-gp substrates/inhibitors; hence, this transporter may be, or may contribute to, the mechanism of interaction. Methods. This study shows directly, as well as quantifies, the inhibition of P-gp-mediated transport of a fluorescent marker substrate. Results. Lovastatin and simvastatin are very potent and effective inhibitors of P-gp transport with IC50s of 26 and 9 μM, respectively, for the human enzyme. Atorvastatin is also an effective P-gp inhibitor, but at higher concentrations. Uniquely, pravastatin, whose functional groups render it an inferior inhibitor of P-gp in the whole cell, had no effect in this assay. This result is consistent with known clinical interactions. The effect of these statins on ATP consumption by P-gp was also assessed, and the Km results were congruent with the IC50 observations. Conclusions. Therefore, the clinical interactions of statins with other drugs may be due, in part or all, to inhibition of P-gp transport.

Inhibition of P-Glycoprotein Transport Function by Grapefruit Juice Psoralen

AbstractPurpose. The grapefruit juice component bergamottin is known to inactivate cytochrome P450 3A4, with grapefruit juice consumption causing increased absorption and enhanced oral bioavailability of many cytochrome P450 3A4 substrates. Many of these substrates are also recognized by the efflux transporter P-glycoprotein. The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. Methods. Using a whole cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of grapefruit juice components to inhibit the function of this transporter. Results. In a cell line presenting an overexpressed amount of the human transporter, the enzyme exhibited a 40 μM IC50 for inhibition by bergamottin. Additionally, using the ATP-hydrolysis assay, we showed that bergamottin increases P-gp-mediated ATP hydrolysis by approximately 2.3 fold with a Km of 8 μM. The concentration for this interaction is similar to that for CYP3A4 inactivation. Conclusions. These results suggest that observed grapefruit juice - drug pharmacokinetic clinical interactions may be due to P-gp inhibition rather than or in addition to CYP3A4 inhibition. Inhibition of P-gp by citrus psoralens could present ways both to enhance bioavailability of therapies without increasing the dose and to diminish drug resistance in refractory cells.

Two transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependence.

The ATP-dependent transport enzyme known as P-glycoprotein (P-gp) confers multidrug resistance (MDR) against many unrelated drugs and xenobiotics. To understand better the broad substrate specificity of the enzyme as well as the mechanism of substrate transport out of the cell, it is critical to characterize the substrate binding sites. Since approximately 1 ATP is hydrolyzed per transport event, phosphate release rate provides a steady-state kinetics assay. Notably, the substrate H33342 causes a decrease in the baseline hydrolysis of ATP (probably due to competition for transport with an endogenous membrane lipid substrate) providing an excellent tool for a comprehensive graphical kinetic analysis of the interaction of substrate pairs at the transport site(s) allowing the determination of inhibition type and hence characterization of transport binding sites. The substrate H33342 interacted with quinidine, progesterone, and propranolol in a non-competitive manner, indicating that binding of H33342 precludes active transport of these other substrates at a distinct site. Compounds such as TPP+ and verapamil, and perhaps also nicardipine, interacted with H33342 as mixed-type inhibitors. This type of interaction results from a reduced affinity at the opposing active site by a factor of alpha and sometimes a partial activity of a fraction beta. Indeed, H33342 binding caused a roughly four-fold reduced affinity for TPP+. Using this definitive approach to inhibition kinetics, we were able to establish traits of a second transport site in P-gp. Therefore, the sites are unequal; however, the performance at one site is contingent on the other being unoccupied, and transport is also sometimes mitigated when the other site is occupied.

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.

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 Johns 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 Johns wort, and P‐gp and CYP3A4 have been indicted as the cause. We characterized several St Johns 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 (IC50 ˜ 30 μM) 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 Johns wort is co‐administered, and provide a further warning against unchecked co‐administration of drugs with St Johns wort.

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.

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

AbstractPurpose. 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 ∼ 7 μM), 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.

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.

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 IC50 of ˜240 μM. Overall, these isothiocyanates are unlikely to impede the xenobiotic defence function of P‐gp even in the intestine where the concentrations are potentially high.