Christopher N. Casciano

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.

Evaluation of loratadine as an inducer of liver microsomal cytochrome P450 in rats and mice

The non-sedating anti-histamine, loratadine [ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]-cyclohepta[1,2-b]pyridin- 11-ylidene-1-piperidinecarboxylate], was administered orally in the diet to mature male rats at dosages of 4, 10 and 25 mg/kg/day for 2 weeks. The effects of these treatments on liver microsomal cytochrome P450 were evaluated by immunochemical and biochemical techniques, and were compared with the effects of treating rats with three different inducers of cytochrome P450, namely phenobarbital, 3-methylcholanthrene and dexamethasone. Treatment of rats with loratadine caused a dose-dependent increase in the levels of P450 2B1 and 2B2, the major phenobarbital-inducible P450 enzymes, as determined by Western immunoblotting. At the highest dosage tested, loratadine was less effective than phenobarbital as an inducer of 2B1 and 2B2, although the induction of these proteins could be detected immunochemically even at the lowest dosage of loratadine tested. Consistent with these observations, treatment of rats with loratadine caused a dose-dependent increase in the rate of two reactions that are catalyzed predominantly by 2B1/2, namely testosterone 16 beta-hydroxylation and 7-pentoxyresorufin O-dealkylation. At the highest dosage tested, loratadine caused a 7.3- and 8.5-fold increase in the rate of testosterone 16 beta-hydroxylation and 7-pentoxyresorufin O-dealkylation, respectively, compared with a 22- and 45-fold increase caused by phenobarbital treatment. Treatment of rats with loratadine caused a 1.4- to 2.0-fold increase in the 2 beta-, 6 beta- and 15 beta-hydroxylation of testosterone, which was associated with a similar increase in the levels of immunoreactive P450 3A1 and/or 3A2. As an inducer of P450 3A1/2, loratadine was slightly less effective than phenobarbital, and was considerably less effective than dexamethasone, which caused a 10- to 33-fold increase in testosterone 2 beta-, 6 beta- and 15 beta-hydroxylase activity. At the dosages tested, loratadine did not increase the levels of P450 1A1, the major 3-methylcholanthrene-inducible P450 enzyme, as determined by Western immunoblotting. The rate of 7-ethoxyresorufin O-dealkylation, which is catalyzed predominantly by P450 1A1, increased 1.9-fold after loratidine treatment, but this increase was less than that caused by phenobarbital treatment (2.2-fold), and was considerably less than that caused by 3-methylcholanthrene treatment (33-fold). The effects of treating mature male mice with loratadine on liver microsomal cytochrome P450 resembled the effects observed in rats. These results indicate that loratadine is a phenobarbital-type inducer of liver microsomal cytochrome P450 in rats and mice.

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.