Quantitative distinctions of active site molecular recognition by P-glycoprotein and cytochrome P450 3A4.

The bulk of characterized xenobiotic defense and disposition is conferred by the abundant enzymes cytochrome P450 3A4 and P-glycoprotein. Although expressed in many tissues, these enzymes are most abundant in the liver and intestine and seem to share most substrates and inhibitors, with the apparent synergy between these two promiscuous enzymes asserted because of their extensive overlap of substrates and shared tissue location. Since the broad-spectrum tolerance to lipophilic compounds of various sizes naturally results in a similar pattern of substrate/inhibitor recognition, the cause or mechanism of many drug/drug and drug/herb interactions can be difficult to determine. These two seemingly indiscriminate enzymes, however, do not share some unique inhibitor selectivity. Particularly, we show various potent CYP3A4 inhibitors that do not affect P-gp active transport function. Remarkably, we have also identified several compounds-valinomycin, norverapamil, reserpine, nobiletin, emetine, gallopamil, fluphenazine-that uniquely inhibit P-gp function with affinities comparable to benchmark P-gp inhibitors despite a lack of effect on CYP3A4 function at physiologically relevant concentrations. Indeed, valinomycin inhibits P-gp with an IC(50) similar to cyclosporin A yet apparently does not affect CYP3A4 function, and emetine and nobiletin are also specific for interaction with P-gp. Additionally, norverapamil and reserpine have, respectively, a 60- and 40-fold preference for inhibition of P-gp over CYP3A4. Some striking structural analogies among these compounds are discussed. These distinguishing qualities of substrate recognition between CYP3A4 and P-gp should reveal nuances of active-site architecture unique to each and could serve as tools to probe for the specific discernment of P-gp-mediated drug/drug or drug/herb interactions. Learning more about binding distinctions and quantitative activity relationships of substrate/inhibitor interactions with these two enzymes and the differences between them may indicate how they recognize such a wide variety of molecules as substrates (and/or inhibitors). Moreover, identification of specific inhibitors will allow the determination of which enzyme is responsible for drug interactions and/or the extent of contribution in a multiple exposure situation.

Active transport of fluorescent P-glycoprotein substrates: evaluation as markers and interaction with inhibitors.

With P-glycoprotein (P-gp) continuing to have prominence among the ABC transporters for its ability to remove various xenobiotics from many cell types, accurate and robust methods for estimating the exposure of drug, carcinogen, toxicant, pesticide, and even some endobiotics to tissues and cells affected by P-gp are valuable. The inhibition of P-gp active transport of molecules, therefore, has often been quantified by concentration dependence of inhibitor effect on fluorescent substrate marker efflux mediated by this enzyme, with much evidence indicating two asymmetric yet interdependent substrate binding sites on P-gp. A uniqueness in the pair of binding sites could result in distinct effects of an inhibitor on the transport of certain substrates, thus leading to differences in fluorescent substrate responsiveness or sensitivity. Seven different fluorescent substrates of P-gp were quantitatively tested for their responsiveness to inhibition by a wide range of P-gp substrates/inhibitors. Interesting differences were observed in the IC(50) values caused by each of the inhibitors employed, in part exemplified by DNR and LDS being generally more sensitive to inhibition effects than any other fluorescent marker. However, no clear trend emerged to designate any fluorochrome marker as the most or least responsive to inhibition. Furthermore, LDS is more sensitive to some P-gp inhibitors than the substrate marker DNR, generally the most responsive. These results support the assertion of two unequal substrate binding sites that are allosterically dependent on each other. Therefore, an inhibitor that favors binding to the site opposite from that favored by a particular marker may have significant transduced effects through the protein between the two binding sites. Nevertheless, although either DNR or LDS is generally the fluorescent substrate most responsive to inhibition, there may be other substrates yet even more sensitive.

The farnesyl protein transferase inhibitor SCH66336 is a potent inhibitor of MDR1 product P-glycoprotein.

P-glycoprotein (Pgp)-mediated drug efflux is a major factor contributing to the variance of absorption and distribution of many drugs, particularly cancer chemotherapeutics. Multidrug resistance (MDR) is caused largely by the efflux of therapeutics out of the tumor cell by Pgp, resulting in reduced efficacy of chemotherapy. SCH66336, a farnesyl transferase inhibitor in development for cancer therapy, was examined in the present study for its ability to inhibit Pgp. In a test system consisting of a NIH-G185 cell line presenting an overexpressed amount of the human transporter Pgp, known Pgp inhibitors, such as cyclosporin A, paclitaxel, verapamil, tamoxifen, and vinblastine, were demonstrated to inhibit the Pgp-mediated efflux of daunorubicin. SCH66336 significantly inhibited daunorubicin transport with an IC50 of about 3 microM and similarly affected the transport of rhodamine 123 with a potency similar to cyclosporin A. Additionally, by an ATP-hydrolysis assay, SCH66336 was shown to decrease Pgp-mediated ATP hydrolysis by >70% with a Km of 3 microM. This observation indicates that SCH66336 directly interacts with the substrate binding site of Pgp, a quality unique to SCH66336 and its analogues, although not inherent to farnesyl transferase inhibitors in general. Moreover, low concentrations of SCH66336 exhibit synergy with the Pgp substrate/inhibitors paclitaxel, tamoxifen, and vinblastine respectively by significantly potentiating their inhibition of Pgp. Treatment with SCH66336 would be predicted to be synergistic with coadministered cancer therapeutics that are substrates of Pgp. A further benefit of coadministration of SCH66336 could be reduced chemotherapy dosage, hence, lower exposure to normal cells and, therefore, less undesired toxicity.

In vitro characterization of the inhibition profile of loratadine, desloratadine, and 3-OH-desloratadine for five human cytochrome P-450 enzymes.

The purpose of this study was to evaluate loratadine, desloratadine, and 3-OH-desloratadine as inhibitors of certain human liver cytochrome P-450 enzymes. Pooled human liver microsomes were used to determine whether loratadine, desloratadine, and 3-OH-desloratadine were inhibitors of cytochrome P-450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4. Loratadine did not inhibit CYP1A2 or CYP3A4 at concentrations up to 3829 ng/ml, which is approximately 815-fold greater than the expected maximal human plasma concentration (4.7 +/- 2.7 ng/ml) following the recommended dose of 10 mg/day. Loratadine inhibited CYP2C19 and CYP2D6 with IC(50) values of approximately 0.76 microM [291 ng/ml; K(i) congruent with 0.61 microM (234 ng/ml)] and 8.1 microM [3100 ng/ml; K(i) congruent with 2.7 microM (1034 ng/ml)], respectively, which are approximately 62 and 660 times the expected loratadine therapeutic exposure concentration. Neither desloratadine nor 3-OH-desloratadine inhibited CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 greater than 25% at concentrations of 3108 or 3278 ng/ml, respectively. These results suggest that loratadine and the active metabolites desloratadine and 3-OH-desloratadine are unlikely to affect the pharmacokinetics of coadministered drugs which are metabolized by these five cytochrome P-450 enzymes.

Inhibition of P-glycoprotein transport function by grapefruit juice psoralen.

PURPOSE: 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 ceil 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 microM 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 microM. 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.

Evaluation of the interaction of loratadine and desloratadine with P-glycoprotein.

The absorption of many drugs is affected by their interaction with ATP-binding cassette (ABC) transporters. The most extensively studied of these ABC transporters is the proein product of MDR1 (multidrug resistance) that encodes a 170-kDa integral plasma membrane phosphorylated glycoprotein known as P-glycoprotein (P-gp). The purpose of this study was to determine, using two different methods, whether the nonsedating antihistamine loratadine (L) and its active metabolite desloratadine (DL) interact with P-gp. MDR cells presenting human P-gp were incubated with the fluorescent P-gp substrate daunorubicin with or without L, DL, and several positive controls. The IC(50) of loratadine (approximately 11 microM) was approximately 160 times the maximum observed plasma concentration (C(max)) following a dose of 10 mg. The IC(50) of desloratadine (approximately 43 microM) was approximately 880 times the C(max) following a dose of 5 mg. The positive control, cyclosporin A, had an IC(50) of approximately 1 microM. ATP hydrolysis activity was measured in the membrane fraction prepared from MDR cells presenting P-gp, which were exposed to various concentrations of test compounds. Known substrates of P-gp demonstrated clear, repeatable, concentration-dependent increases in ATP hydrolysis activity. L caused an increase in ATPase activity above basal levels. L had a V(max) about 200% basal activity and K(m) of approximately 3 microM for P-gp. In contrast, DL had no significant effect on baseline ATP hydrolysis. L inhibited human P-gp much less than verapamil or cyclosporin A. DL inhibited human P-gp significantly less than L (4 times). DL therefore is not a significant inhibitor of P-gp and should not cause clinical drug interactions with agents that are P-gp substrates.

HMG-CoA reductase inhibitors (statins) characterized as direct inhibitors of P-glycoprotein.

PURPOSE: 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 IC50's of 26 and 9 microM, 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.

Mechanism-based inactivation of CYP2D6 by 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine.

SCH 66712 [5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine] caused a time- and NADPH-dependent loss of CYP2D6 activity. The inactivation of human liver (HL) microsomal dextromethorphan O-demethylase activity, a prototype marker for CYP2D6, was characterized by a K(I) of 4.8 microM and a maximal rate constant of inactivation (k(inact)) of 0.14 min(-1). The inactivation of the recombinant CYP2D6 in Supersomes (r-CYP2D6) was characterized by a K(I) of 0.55 microM and a k(inact) of 0.32 min(-1). Extensive dialysis of the SCH 66712-inhibited enzyme failed to restore the activity to control levels (dialyzed reaction mixture lacking SCH 66712) for both HL microsomes and r-CYP2D6. Addition of glutathione, superoxide dismutase, or mannitol to the reaction mixture failed to protect CYP2D6 against SCH 66712-NADPH-catalyzed inactivation. Addition of quinidine, a reversible inhibitor of CYP2D6, to a preincubation mixture consisting of SCH 66712, HL microsomes, or Supersomes and NADPH partially protected CYP2D6 from inactivation. SCH 66712 also inhibited HL microsomal CYP3A4, CYP2C9, and CYP2C19; however, the concentrations required to inhibit those isoforms were 5- to 10-fold higher than those required to inhibit CYP2D6. These results demonstrate that SCH 66712 is a potent and fairly selective mechanism-based inhibitor of CYP2D6.

Cooperativity in the inhibition of P-glycoprotein-mediated daunorubicin transport: evidence for half-of-the-sites reactivity.

P-Glycoprotein (Pgp) is an important transport enzyme composed of two homologous domains and transports a wide range of structurally diverse xenobiotics from the cell. Recent studies have indicated that allosteric interactions occur between the nucleotide binding domains and between the substrate binding domains of the two halves, but the extent of this interaction as well as the means by which the enzyme can transport such a wide variety of substrates has not been elucidated. Herein, the Pgp-mediated transport of a marker substrate, daunorubicin (DNR), out of viable cells was examined in the presence of a variety of other known substrates of Pgp. For most of the typical Pgp substrates examined, the relationship between inhibition of DNR efflux and competing substrate concentration was sigmoidal and therefore not a simple mutually exclusive competitive inhibition of transport. The Hill coefficient ranged from about 3 to 5 for the inhibition of transport of DNR. This negative cooperativity in combination with recent evidence, including several examples of noncompetitive inhibition between the homologous halves of Pgp, indicates a "half-of-the-sites" reactivity. Our data support the mechanistic proposal that substrate binding at one putative transport binding site precludes activity at another unequal site; many of the substrates examined exert a negative allosteric effect on the other transport site (and vice versa). A half-of-the-sites reactivity model would account for many of these observations and may be critical to the efficiency of Pgp substrate transport of a broad spectrum of compounds.

Cholesterol interaction with the daunorubicin binding site of P-glycoprotein.

The inherent complexities of cholesterol disposition and metabolism preclude a single transmembrane active transport avenue for this steroid-precursor, cell-membrane constituent. Yet the ABC (ATP binding cassette) transporters are inextricably linked to elements of cholesterol disposition. Recent observations have suggested that, under certain settings, the ABC transporter P-glycoprotein (P-gp) performs a direct role in cholesterol disposition. The gene product of MDR1 (multidrug resistance transporter), P-glycoprotein also confers protection against xenobiotics. Using a whole cell assay in which the retention of a marker substrate is evaluated and quantified, we studied the ability of cholesterol to inhibit directly the function of this transporter. In a NIH-G185 cell line presenting an overexpressed amount of the human transporter P-gp, cholesterol caused dramatic inhibition of daunorubicin transport with an IC(50) of about 8 microM yet had no effect on the parent cell line nor rhodamine 123 transport. Additionally, using the ATP-hydrolysis assay, we showed that cholesterol increases P-gp-mediated ATP hydrolysis by approximately 1.6-fold with a K(s) of 5 microM. Suggesting that cholesterol directly interacts with the substrate binding site of P-gp, these results are consistent with cholesterol being transported by MDR1 P-gp.