Evaluation of Human P-Glycoprotein (MDR1/ABCB1) ATPase Activity Assay Method by Comparing with in Vitro Transport Measurements: Michaelis-Menten Kinetic Analysis to Estimate the Affinity of P-Glycoprotein to Drugs

Shirasaka, Yoshiyuki; Onishi, Yuko; Sakurai, Aki; Nakagawa, Hiroshi; Ishikawa, Toshihisa; Yamashita, Shinji

doi:10.1248/bpb.29.2465

Cited by 27 publications

(17 citation statements)

References 26 publications

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“…By using the series of same cell lines, Katoh et al (2006) examined the species differences in maximal activity (V max / K m ) of P-gp to transport its substrate drugs and clarified the fact that maximal activity of rat MDR1b correlated well with that of human MDR1. In addition, the apparent K m value of quinidine to rat MDR1a was reported to be approximately 5 M (Müller et al, 1994) and corresponded well with that of human MDR1 in other reports [5.42 M by Adachi et al (2001) and 7.88 M by Shirasaka et al (2006b)]. On the basis of these facts, it is possible to validate our method by simulating the concentration-dependent permeability of three P-gp substrate drugs to rat small intestine and then by comparing the results of simulation with the experimentally measured permeability of those drugs.…”

Section: Discussionsupporting

confidence: 87%

“…This finding might be explained by its high permeability (high P app, PD ), high affinity to P-gp (low K m ), and/or low V max compared with other drugs, as shown in Fig. 2B (Borgnia et al, 1996;Adachi et al, 2001;Troutman and Thakker, 2003;Shirasaka et al, 2006b). Ogihara et al (2006) investigated the influence of P-gp on the intestinal absorption of verapamil in each intestinal segment in wild-type and mdr1a/1b gene-deficient mice, whereas no significant effects of P-gp on the absorption of verapamil were observed in all intestinal segments.…”

Section: Downloaded Frommentioning

confidence: 97%

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Scaling of in Vitro Membrane Permeability to Predict P-glycoprotein-Mediated Drug Absorption in Vivo

Shirasaka¹,

Masaoka²,

Kataoka³

et al. 2008

Drug Metab Dispos

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ABSTRACT:In a previous study, the concentration-dependent permeability of P-glycoprotein (P-gp) substrate drugs, quinidine, verapamil, and vinblastine, in several cell monolayers with different levels of P-gp expression was analyzed kinetically to obtain fundamental parameters for P-gp-mediated transport, V max and K m(app) values. Both V max and K m(app) values of each drug were found to show linear correlations with the expression level of P-gp. These findings imply the possibility of estimating the V max and K m(app) values of P-gp substrate drugs in the in vivo intestinal membrane on the basis of the P-gp expression level. In the present study, concentrationdependent drug permeability to the rat small intestines (upper jejunum and ileum) was simulated on the basis of V max and K m(app) values of each drug estimated from the P-gp expression level in the rat small intestines. To validate the predictability of these procedures, drug permeability in the rat small intestines was measured by the in situ single-pass perfusion method. It was confirmed that simulated permeability of each drug in the rat jejunum and ileum corresponded well with permeability measured by the in situ single-pass perfusion method. This study clearly demonstrated the potential to estimate the permeability of P-gp substrate drugs in the human intestine from its P-gp expression level and thus the possibility to predict the oral absorption of those drugs.P-glycoprotein (P-gp), one of the most important efflux transporters in the field of biopharmacy, is an ATP-binding cassette (ABC) transporter encoded by the multidrug resistance 1 gene (MDR1/ABCB1). P-gp is expressed not only in cancer cells but also in many normal tissues (Loo and Clarke, 1999). So far, many reports have described the effects of P-gp on the pharmacokinetic profiles of clinically important drugs, by denaturing absorption, distribution, and excretion. For example, P-gp located in the apical domain of the enterocytes of the gastrointestinal tract limits the uptake and absorption of its substrate drugs after oral administration (Hunter and Hirst, 1997;Meijer et al., 1997;Jonker et al., 1999). Because of the great impact of P-gp on the pharmacokinetic profiles of a variety of drugs, various in vitro experimental techniques are now used to identify the compounds subjected to P-gp-mediated efflux (Kim et al., 1998;Polli et al., 1999Polli et al., , 2001Susanto and Benet, 2002; Shirasaka et al., 2006a,b). However, it is still difficult to quantitatively predict the effect of P-gp on in vivo intestinal absorption of such drugs.In a previous study, we measured the apical (AP) to basal (BL) absorptive permeability of P-gp substrate drugs in several cultured cell monolayers with different expression levels of P-gp (Shirasaka et al., 2008). In all cell monolayers, AP to BL permeability of P-gp substrate drugs showed a sigmoid-type relation to their donor (AP) concentration and reached a maximal value at the higher concentration range because of the saturation of P-gp-mediated efflux. Con...

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Section: Discussionsupporting

confidence: 87%

Section: Downloaded Frommentioning

confidence: 97%

Scaling of in Vitro Membrane Permeability to Predict P-glycoprotein-Mediated Drug Absorption in Vivo

Shirasaka¹,

Masaoka²,

Kataoka³

et al. 2008

Drug Metab Dispos

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“…A "true" P-gp inhibitor is defined as one which inhibits P-gp without being effluxed by P-gp itself and simultaneously does not activate ATPase 16 . In the P-gp-Glo assay system, a significant decrease in residual ATP content was produced by VER (a P-gp substrate and competitive inhibitor) while a significant increase in inhibited it, consistent with previous results 17 . We then found that trantinterol produced an increasing tendency in residual ATP content especially at concentrations of 10 µM and 100 µM (P<0.01) (Figure 4).…”

Section: P-gp Expression Validationsupporting

confidence: 91%

Trantinterol, a Novel β₂-Adrenoceptor Agonist, Noncompetitively Inhibits P-Glycoprotein Functionin Vitroandin Vivo

Wang

Sun

et al. 2014

Mol. Pharmaceutics

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P-glycoprotein (P-gp)-mediated drug-drug interactions are important factors causing adverse effects of drugs in clinical use. The aim of this study was to determine whether trantinterol (also known as SPFF), a novel β2-adrenoceptor agonist, was a P-gp inhibitor or substrate. The results showed that trantinterol was not a substrate of P-gp but increased rhodamine 123 (Rho 123) uptake by MDCK-MDR1 cells and decreased the efflux transport of both Rho 123 and cyclosporine A (CsA) in bidirectional transport studies across MDCK-MDR1 cell monolayers. This suggested that trantinterol was a P-gp inhibitor but not a P-gp substrate. The mechanism of inhibition was investigated in the P-gp-Glo assay system, where it was found that trantinterol inhibited P-gp ATPase activity in a dose-dependent manner. A subsequent study using the antibody binding assay with the conformation-sensitive P-gp-specific antibody UIC2 confirmed that trantinterol decreased UIC2 binding at 10 μM in contrast to the competitive inhibitor, verapamil. This suggested that trantinterol was a noncompetitive inhibitor of P-gp. Finally, a pharmacokinetic study in rat showed that trantinterol significantly increased the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of digoxin and paclitaxel (PAC), and the Cmax of cyclosporine A (CsA). In summary, trantinterol is a potent noncompetitive P-gp inhibitor which may increase the bioavailability of other P-gp substrate drugs coadministered with it.

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“…Also, its expression has been described in lymphoid cell populations from human bone marrow and peripheral blood (5). Specifically, it has been shown on the membrane of pluripotent stem cells, monocytes, dendritic cells, CD4 + and CD8 + T lymphocytes, NK cells, and B lymphocytes, suggesting that Pgp may influence cell-mediated immune responses (6)(7)(8)(9).…”

Section: Introductionmentioning

confidence: 99%

Impact of Small Molecules Immunosuppressants on P-Glycoprotein Activity and T-cell Function

et al. 2012

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-Purpose. P-glycoprotein (Pgp) is a member of the ABC-transporter family that transports substances across cellular membranes acting as an efflux pump extruding drugs out of the cells. Pgp plays a key role on the pharmacokinetics of several drugs. Herein, we have studied the effects of immunosuppressants on Pgp function, assessing rhodamine-123 (Rho123) uptake and efflux in different Tcell subsets. Methods. Different immunosuppressants such as Cyclosporine (CsA), Rapamycin (Rapa) and Tacrolimus (Tac) were used to assess the in vitro effect on Pgp function of main T-cell subsets among healthy volunteers. We measured Rho123 uptake, efflux and kinetic of extrusion in CD4 + and CD8 + subsets by flow cytometry. Antigen-specific memory T-cell responses were assessed by measuring T-cell proliferation and cytokine secretion using an allogeneic mixed lymphocyte reaction. Results. Rho123 uptake in groups treated with CsA and CsA+Rapa was significantly decreased compared to non-treated group and the other immunosupressants in both T cells subsets. Pgp activity was also reduced in CsA and CsA+Rapa compared to the other immunosupressants but it was only significant in the CsA group for CD8 + subset. Kinetic extrusion of Rho123 by Pgp in all groups was faster in CD8 + T cells. All immunosuppressants and the specific Pgp inhibitor PSC833 diminished antigen-primed T-cell proliferation, especially CD8 + T-cell subset. Conclusions. Our data indicate that small molecules immunosuppressants, especially CsA, inhibit Pgp activity and T-cell function being the CD8 + T cells more susceptible to this effect. These findings support the importance of Pgp when designing combined immunosuppressive regimens.

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Evaluation of Human P-Glycoprotein (MDR1/ABCB1) ATPase Activity Assay Method by Comparing with in Vitro Transport Measurements: Michaelis-Menten Kinetic Analysis to Estimate the Affinity of P-Glycoprotein to Drugs

Cited by 27 publications

References 26 publications

Scaling of in Vitro Membrane Permeability to Predict P-glycoprotein-Mediated Drug Absorption in Vivo

Scaling of in Vitro Membrane Permeability to Predict P-glycoprotein-Mediated Drug Absorption in Vivo

Trantinterol, a Novel β₂-Adrenoceptor Agonist, Noncompetitively Inhibits P-Glycoprotein Functionin Vitroandin Vivo

Impact of Small Molecules Immunosuppressants on P-Glycoprotein Activity and T-cell Function

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Evaluation of Human P-Glycoprotein (MDR1/ABCB1) ATPase Activity Assay Method by Comparing with in Vitro Transport Measurements: Michaelis-Menten Kinetic Analysis to Estimate the Affinity of P-Glycoprotein to Drugs

Cited by 27 publications

References 26 publications

Scaling of in Vitro Membrane Permeability to Predict P-glycoprotein-Mediated Drug Absorption in Vivo

Scaling of in Vitro Membrane Permeability to Predict P-glycoprotein-Mediated Drug Absorption in Vivo

Trantinterol, a Novel β2-Adrenoceptor Agonist, Noncompetitively Inhibits P-Glycoprotein Functionin Vitroandin Vivo

Impact of Small Molecules Immunosuppressants on P-Glycoprotein Activity and T-cell Function

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Product

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Trantinterol, a Novel β₂-Adrenoceptor Agonist, Noncompetitively Inhibits P-Glycoprotein Functionin Vitroandin Vivo