Abstract
Purpose. Using rhodamine123 (RH123) cell exclusion, 17 clinically used compounds were screened for their inhibitory effect on P-glycoprotein (P-gp), which was compared with the drugs' inhibitory activity against CYP3A4. The same assay was used to study induction of P-gp activity.
Methods. P-gp inhibition was assessed using RH123 accumulation into LS180V cells as well as Rh123 transport across Caco-2 monolayers. Inhibition of CYP3A4 was determined in human liver microsomes using triazolam-4-hydroxylation. Induction of P-gp expression and activity was measured using western blot analysis and RH123 accumulation into LS180V cells, respectively.
Results. The observed inhibition of RH123 cell exclusion ranged from little or no effect (digoxin, indinavir, fexofenadine) up to a nearly 10-fold increase in RH123 accumulation (ivermectin, terfenadine). No correlation between P-gp and CYP3A4 inhibition was observed. The rank order in P-gp inhibitory potency for terfenadine, verapamil, ritonavir, and indomethacin was identical in both LS180V and Caco-2 models. Ritonavir and St. John's wort extract showed a concentration-dependent P-gp induction, with good correlation between western blot analysis and RH123 accumulation.
Conclusions. The RH123 accumulation assay in LS180V cells can be used as a valuable screening tool to study both inhibition and induction of P-gp activity and expression. This assay has the potential to predict P-gp-mediated alterations in intestinal absorption of drugs.
Similar content being viewed by others
REFERENCES
I. Sugawara, I. Kataoka, Y. Morishita, H. Hamada, T. Tsuruo, S. Itoyama, and S. Mori. Tissue distribution of P-glycoprotein encoded by a multidrug-resistant gene as revealed by a monoclonal antibody, MRK 16. Cancer Res. 48:1926-1929 (1988).
C. Cordon-Cardo, J. P. O'Brien, D. Casals, L. Rittman-Grauer, J. L. Biedler, M. R. Melamed, and J. R. Bertino. Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc. Natl. Acad. Sci. USA 86:695-698 (1989).
V. Ling, N. Kartner, T. Sudo, L. Siminovitch, and J. R. Riordan. Multidrug-resistance phenotype in Chinese hamster ovary cells. Cancer Treat. Rep. 67:869-874 (1983).
M. D. Perloff, L. L. von Moltke, J. E. Marchand, and D. J. Greenblatt. Ritonavir induces P-glycoprotein expression, multidrug resistance-associated protein (MRP1) expression, and drug transporter-mediated activity in a human intestinal cell line. J. Pharm. Sci. 90:1829-1837 (2001).
A. R. Quesada, M. M. Barbacid, E. Mira, M. Aracil, and G. Marquez. Chemosensitization and drug accumulation assays as complementary methods for the screening of multidrug resistance reversal agents. Cancer Lett. 99:109-114 (1996).
J. G. Sarver, W. A. Klis, J. P. Byers, and P. W. Erhardt. Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein. J. Biomol. Screen. 7:29-34 (2002).
J. S. Lee, K. Paull, M. Alvarez, C. Hose, A. Monks, M. Grever, A. T. Fojo, and S. E. Bates. Rhodamine efflux patterns predict P-glycoprotein substrates in the National Cancer Institute drug screen. Mol. Pharmacol. 46:627-638 (1994).
C. E. Herzog, M. Tsokos, S. E. Bates, and A. T. Fojo. Increased mdr-1/P-glycoprotein expression after treatment of human colon carcinoma cells with P-glycoprotein antagonists. J. Biol. Chem. 268:2946-2952 (1993).
E. StÖrmer, L. L. von Moltke, M. D. Perloff, and D. J. Greenblatt. Differential modulation of P-glycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture. Pharm. Res. 19:1038-1045 (2002).
M. D. Perloff, L. L. von Moltke, M. H. Court, T. Kotegawa, R. I. Shader, and D. J. Greenblatt. Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative contribution of CYP3A and CYP2C isoforms. J. Pharmacol. Exp. Ther. 292:618-628 (2000).
P. Artursson, K. Palm, and K. Luthman. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev. 22:67-84 (1996).
E. J. Wang, C. N. Casciano, R. P. Clement, and W. W. Johnson. In vitro flow cytometry method to quantitatively assess inhibitors of P-glycoprotein. Drug Metab. Dispos. 28:522-528 (2000).
L. Profit, V. A. Eagling, and D. J. Back. Modulation of P-glycoprotein function in human lymphocytes and Caco-2 cell monolayers by HIV-1 protease inhibitors. AIDS 13:1623-1627 (1999).
R. B. Kim, C. Wandel, B. Leake, M. Cvetkovic, M. F. Fromm, P. J. Dempsey, M. M. Roden, F. Belas, A. K. Chaudhary, D. M. Roden, A. J. Wood, and G. R. Wilkinson. Interrelationship between substrates and inhibitors of human CYP3A and P-glycoprotein. Pharm. Res. 16:408-414 (1999).
P. B. Watkins. The barrier function of CYP3A4 and P-glycoprotein in the small bowel. Adv. Drug Deliv. Rev. 27:161-170 (1997).
L. L. von Moltke, D. J. Greenblatt, J. M. Grassi, B. W. Granda, S. X. Duan, S. M. Fogelman, J. P. Daily, J. S. Harmatz, and R. I. Shader. Protease inhibitors as inhibitors of human cytochromes P450: high risk associated with ritonavir. J. Clin. Pharmacol. 38:106-111 (1998).
C. Wandel, R. B. Kim, S. Kajiji, P. Guengerich, G. R. Wilkinson, and A. J. Wood. P-glycoprotein and cytochrome P-450 3A inhibition: dissociation of inhibitory potencies. Cancer Res. 59:3944-3948 (1999).
M. D. Perloff, L. L. von Moltke, E. StÖrmer, R. I. Shader, and D. J. Greenblatt. Saint John's wort: An in vitro analysis of P-glycoprotein induction due to extended exposure. Br. J. Pharmacol. 134:1601-1608 (2001).
E. G. Schuetz, W. T. Beck, and J. D. Schuetz. Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-#x00AEulate these proteins in human colon carcinoma cells. Mol. Pharmacol. 49:311-318 (1996).
E. StÖrmer, L. L. von Moltke, M. D. Perloff, and D. J. Greenblatt. P-glycoprotein interactions of nefazodone and trazodone in cell culture. J. Clin. Pharmacol. 41:708-714 (2001).
D. Durr, B. Stieger, G. A. Kullak-Ublick, K. M. Rentsch, H. C. Steinert, P. J. Meier, and K. Fattinger. St John's Wort induces intestinal P-glycoprotein/MDR1 and intestinal and hepatic CYP3A4. Clin. Pharmacol. Ther. 68:598-604 (2000).
B. Greiner, M. Eichelbaum, P. Fritz, H. P. Kreichgauer, O. von Richter, J. Zundler, and H. K. Kroemer. The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J. Clin. Invest. 104:147-153 (1999).
K. Westphal, A. Weinbrenner, M. Zschiesche, G. Franke, M. Knoke, R. Oertel, P. Fritz, O. von Richter, R. Warzok, T. Hachenberg, H. M. Kauffmann, D. Schrenk, B. Terhaag, H. K. Kroemer, and W. Siegmund. Induction of P-glycoprotein by rifampin increases intestinal secretion of talinolol in human beings: a new type of drug/drug interaction. Clin. Pharmacol. Ther. 68:345-355 (2000).
M. Pinto, S. Robine-Leon, M. Appay, M. Kedinger, N. Triadou, E. Dussaulx, B. Lacroix, P. Simon-Assmann, K. Haffen, J. Fogh, and A. Zweibaum. Enterocyte-like differentiation and polarisation of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47:323-330 (1983).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Perloff, M.D., Störmer, E., von Moltke, L.L. et al. Rapid Assessment of P-Glycoprotein Inhibition and Induction in Vitro . Pharm Res 20, 1177–1183 (2003). https://doi.org/10.1023/A:1025092829696
Issue Date:
DOI: https://doi.org/10.1023/A:1025092829696