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Novel Experimental Parameters to Quantify the Modulation of Absorptive and Secretory Transport of Compounds by P-Glycoprotein in Cell Culture Models of Intestinal Epithelium

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Abstract

Purpose. The purpose of this work was to elucidate the asymmetric effect of P-gp on modulation of absorptive and secretory transport of compounds across polarized epithelium, to develop experimental parameters to quantify P-gp-mediated modulation of absorptive and secretory transport, and to elucidate how P-gp-mediated modulation of transport is affected by passive diffusion properties, interaction of the substrate with P-gp, and P-gp expression.

Methods. The permeability of a set of P-gp substrates was determined in absorptive and secretory directions in Madine-Darby Canine kidney (MDCK), Caco-2, and MDR-MDCK monolayers. The transport was also determined in the presence of GW918, a non-competitive P-gp inhibitor, to quantify the permeability without the influence of P-gp. From these two experimental permeability values in each direction, two new parameters, absorptive quotient (AQ) and the secretory quotient (SQ), were defined to express the functional activity of P-gp during absorptive and secretory transport, respectively. Western blot analysis was used to quantify P-gp expression in these monolayers and in normal human intestinal.

Results. P-gp expression in Caco-2 and MDR-MDCK monolayers was comparable to that in normal intestine, and much less in MDCK cells. For all models, the substrates encompassed a wide range of apparent permeability due to passive diffusion (P PD). The parameters AQ and SQ, calculated for all compounds, assessed the attenuation in absorptive and enhancement of secretory transport, respectively, normalized to the permeability due to passive diffusion. Analysis of these parameters showed that 1) P-gp affected absorptive and secretory transport differentially and 2) compounds could be stratified into distinct groups with respect to the modulation of their absorptive and secretory transport by P-gp. Compounds could be identified whose absorptive transport was either strongly affected or poorly affected by changes in P-gp expression. For certain compounds, AQ values showed parabolic relationship with respect to passive diffusivity, and for others AQ was unaffected by changes in passive diffusivity.

Conclusions. The relationship between attenuation of absorptive transport and enhancement of secretory transport of compounds by P-gp is asymmetric, and different for different sets of compounds. The relationship between attenuation of absorption by P-gp and passive diffusivity of compounds, their interaction potential with P-gp, and levels of P-gp expression is complex; however, compounds can be classified into sets based on these relationships. A classification system that describes the functional activity of P-gp with respect to modulation of absorptive and secretory transport was developed from these results.

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REFERENCES

  1. F. Thiebaut, T. Tsuruo, H. Hamada, M. M. Gottesman, I. Pastan, and M. C. Willingham. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc. Natl. Acad. Sci. USA 84:7735-7738 (1987).

    Google Scholar 

  2. J. Hunter, B. H. Hirst, and N. L. Simmons. Drug absorption limited by P-glycoprotein-mediated secretory drug transport in human intestinal epithelial Caco-2 cell layers. Pharm. Res. 10:743-749 (1993).

    Google Scholar 

  3. P. F. Augustijns, T. P. Bradshaw, L. S. Gan, R. W. Hendren, and D. R. Thakker. Evidence for a polarized efflux system in CACO-2 cells capable of modulating cyclosporin A transport. Biochem. Biophys. Res. Commun. 197:360-365 (1993).

    Google Scholar 

  4. T. Gramatte, R. Oertel, B. Terhaag, and W. Kirch. Direct demonstration of small intestinal secretion and site-dependent absorption of the #x0392-blocker talinolol in humans. Clin. Pharmacol. Ther. 59:541-549 (1996).

    Google Scholar 

  5. M. F. Hebert. Contributions of hepatic and intestinal metabolism and P-glycoprotein to cyclosporine and tacrolimus oral drug delivery. Adv. Drug Deliv. Rev. 27:201-214 (1997).

    Google Scholar 

  6. W. M. Kan, Y. T. Liu, C. L. Hsiao, C. Y. Shieh, J. H. Kuo, J. D. Huang, and S. F. Su. Effect of hydroxyzine on the transport of etoposide in rat small intestine. Anticancer Drugs 12:267-273 (2001).

    Google Scholar 

  7. M. Sababi, O. Borga, and U. Hultkvist-Bengtsson. The role of P-glycoprotein in limiting intestinal #x00AEional absorption of digoxin in rats. Eur. J. Pharm. Sci. 14:21-27 (2001).

    Google Scholar 

  8. H. Saitoh and B. J. Aungst. Possible involvement of multiple P-glycoprotein-mediated efflux systems in the transport of verapamil and other organic cations across rat intestine. Pharm. Res. 12:1304-1310 (1995).

    Google Scholar 

  9. H. Saitoh, M. Hatakeyama, O. Eguchi, M. Oda, and M. Takada. Involvement of intestinal P-glycoprotein in the restricted absorption of methylprednisolone from rat small intestine. J. Pharm. Sci. 87:73-75 (1998).

    Google Scholar 

  10. H. Spahn-Langguth, G. Baktir, A. Radschuweit, A. Okyar, B. Terhaag, P. Ader, A. Hanafy, and P. Langguth. P-glycoprotein transporters and the gastrointestinal tract: evaluation of the potential in vivo relevance of in vitro data employing talinolol as model compound. Int. J. Clin. Pharmacol. Ther. 36:16-24 (1998).

    Google Scholar 

  11. A. Sparreboom, J. van Asperen, U. Mayer, A. H. Schinkel, J. W. Smit, D. K. Meijer, P. Borst, W. J. Nooijen, J. H. Beijnen, and O. van Tellingen. Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine. Proc. Natl. Acad. Sci. USA 94:2031-2035 (1997).

    Google Scholar 

  12. R. B. Kim, M. F. Fromm, C. Wandel, B. Leake, A. J. Wood, D. M. Roden, and G. R. Wilkinson. The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J. Clin. Invest. 101:289-294 (1998).

    Google Scholar 

  13. 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).

    Google Scholar 

  14. W. L. Chiou, S. M. Chung, and T. C. Wu. Commentary: Potential role of P-glycoprotein in affecting hepatic metabolism of drugs. Pharm. Res. 17:901-903 (2000).

    Google Scholar 

  15. K. Arimori and M. Nakano. Drug exsorption from blood into the gastrointestinal tract. Pharm. Res. 15:371-376 (1998).

    Google Scholar 

  16. U. Mayer, E. Wagenaar, J. H. Beijnen, J. W. Smit, D. K. Meijer, J. van Asperen, P. Borst, and A. H. Schinkel. Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdr 1a P-glycoprotein. Br. J. Pharmacol. 119:1038-1044 (1996).

    Google Scholar 

  17. T. Gramatte and R. Oertel. Intestinal secretion of intravenous talinolol is inhibited by luminal R-verapamil. Clin. Pharmacol. Ther. 66:239-245 (1999).

    Google Scholar 

  18. J. van Asperen, A. H. Schinkel, J. H. Beijnen, W. J. Nooijen, P. Borst, and O. van Tellingen. Altered pharmacokinetics of vinblastine in Mdr1a P-glycoprotein-deficient Mice. J. Natl. Cancer Inst. 88:994-999 (1996).

    Google Scholar 

  19. J. van Asperen, O. van Tellingen, and J. H. Beijnen. The role of mdr1a P-glycoprotein in the biliary and intestinal secretion of doxorubicin and vinblastine in mice. Drug Metab. Dispos. 28:264-267 (2000).

    Google Scholar 

  20. U. Wetterich, H. Spahn-Langguth, E. Mutschler, B. Terhaag, W. Rosch, and P. Langguth. Evidence for intestinal secretion as an additional clearance pathway of talinolol enantiomers: concentration-and dose-dependent absorption in vitro and in vivo. Pharm. Res. 13:514-522 (1996).

    Google Scholar 

  21. S. Ito, C. Woodland, P. A. Harper, and G. Koren. P-glycoprotein-mediated renal tubular secretion of digoxin: the toxicological significance of the urine-blood barrier model. Life Sci. 53:L25-L31 (1993).

    Google Scholar 

  22. A. Johne, J. Brockmoller, S. Bauer, A. Maurer, M. Langheinrich, and I. Roots. Pharmacokinetic interaction of digoxin with an herbal extract from St John's wort (Hypericum perforatum). Clin. Pharmacol. Ther. 66:338-345 (1999).

    Google Scholar 

  23. 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).

    Google Scholar 

  24. J. G. Theis, H. S. Chan, M. L. Greenberg, D. Malkin, V. Karaskov, I. Moncica, and G. Koren. and J. Doyle. Increased systemic toxicity of sarcoma chemotherapy due to combination with the P-glycoprotein inhibitor cyclosporin. Int. J. Clin. Pharmacol. Ther. 36:61-64 (1998).

    Google Scholar 

  25. M. F. Paine, L. Y. Leung, H. K. Lim, K. Liao, A. Oganesian, M. Y. Zhang, K. E. Thummel, and P. B. Watkins. Identification of a novel route of extraction of sirolimus in human small intestine: roles of metabolism and secretion. J. Pharmacol. Exp. Ther. 301:174-186 (2002).

    Google Scholar 

  26. L. Z. Benet, S. Oie, and J. B. Schwartz. Design and Optimization of Dosage Regimens; Pharmacokinetic Data. In J. G. Hardman, L. E. Limbird, P. B. Molinoff, R. W. Ruddon and G.G. A. (eds), Goodman and Gilman's The Pharmacological Basis of Therapeutics (J. G. Hardman L. E. Limbird P. B. Molinoff R. W. Ruddonand G. G. A., eds), McGraw-Hill, New York, 1990, pp. 1707-1792.

    Google Scholar 

  27. J. W. Polli, S. A. Wring, J. E. Humphreys, L. Huang, J. B. Morgan, L. O. Webster, and C. S. Serabjit-Singh. Rational use of in vitro P-glycoprotein assays in drug discovery. J. Pharmacol. Exp. Ther. 299:620-628 (2001).

    Google Scholar 

  28. A. H. Schinkel. Pharmacological insights from P-glycoprotein knockout mice. Int. J. Clin. Pharmacol. Ther. 36:9-13 (1998).

    Google Scholar 

  29. T. Terao, E. Hisanaga, Y. Sai, I. Tamai, and A. Tsuji. Active secretion of drugs from the small intestinal epithelium in rats by P-glycoprotein functioning as an absorption barrier. J. Pharm. Pharmacol. 48:1083-1089 (1996).

    Google Scholar 

  30. M. D. Troutman and D. R. Thakker. Rhodamine 123 Requires Carrier-Mediated Influx for Its Activity as a P-glycoprotein Substrate in Caco-2 Cells. Pharm. Res. 20:1192-1199 (2003).

    Google Scholar 

  31. M. D. Troutman and D. R. Thakker. The Efflux Ratio Cannot Assess P-glycoprotein-Mediated Attenuation of Absorptive Transport-Asymmetric Effect of P-glycoprotein on Absorptive and Secretory Transport Across Caco-2 Cell Monolayers. Pharm Res. 20:1200-1209 (2003).

    Google Scholar 

  32. P. Schmiedlin-Ren, K. E. Thummel, J. M. Fisher, M. F. Paine, K. S. Lown, and P. B. Watkins. Expression of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix-coated permeable supports in the presence of 1alpha,25-dihydroxyvitamin D3. Mol. Pharmacol. 51:741-754 (1997).

    Google Scholar 

  33. U. A. Germann, M. C. Willingham, I. Pastan, and M. M. Gottesman. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus. Biochemistry 29:2295-2303 (1990).

    Google Scholar 

  34. K. Lee and D. R. Thakker. Saturable transport of H2-antagonists ranitidine and famotidine across Caco-2 cell monolayers. J. Pharm. Sci. 88:680-687 (1999).

    Google Scholar 

  35. M. J. Cho, D. P. Thompson, C. T. Cramer, T. J. Vidmar, and J. F. Scieszka. The Madin Darby canine kidney (MDCK) epithelial cell monolayer as a model cellular transport barrier. Pharm. Res. 6:71-77 (1989).

    Google Scholar 

  36. O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275 (1951).

    Google Scholar 

  37. F. Hyafil, C. Vergely, P. Du Vignaud, and T. Grand-Perret. In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative. Cancer Res. 53:4595-4602 (1993).

    Google Scholar 

  38. F. Ingels, S. Deferme, E. Destexhe, M. Oth, G. Van den Mooter, and P. Augustijns. Simulated intestinal fluid as transport medium in the Caco-2 cell culture model. Int. J. Pharm. 232:183-192 (2002).

    Google Scholar 

  39. J. Gao, O. Murase, R. L. Schowen, J. Aube, and R. T. Borchardt. A functional assay for quantitation of the apparent affinities of ligands of P-glycoprotein in Caco-2 cells. Pharm. Res. 18:171-176 (2001).

    Google Scholar 

  40. S. Doppenschmitt, H. Spahn-Langguth, C. G. Regardh, and P. Langguth. Role of P-glycoprotein-mediated secretion in absorptive drug permeability: An approach using passive membrane permeability and affinity to P-glycoprotein. J. Pharm. Sci. 88:1067-1072 (1999).

    Google Scholar 

  41. N. F. Ho, P. S. Burton, R. A. Conradi, and C. L. Barsuhn. A biophysical model of passive and polarized active transport processes in Caco-2 cells: approaches to uncoupling apical and basolateral membrane events in the intact cell. J. Pharm. Sci. 84:21-27 (1995).

    Google Scholar 

  42. S. H. Jang, M. G. Wientjes, and J. L. Au. Kinetics of P-glycoprotein-mediated efflux of paclitaxel. J. Pharmacol. Exp. Ther. 298:1236-1242 (2001).

    Google Scholar 

  43. K. A. Lentz, J. W. Polli, S. A. Wring, J. E. Humphreys, and J. E. Polli. Influence of passive permeability on apparent P-glycoprotein kinetics. Pharm. Res. 17:1456-1460 (2000).

    Google Scholar 

  44. C. Martin, G. Berridge, C. F. Higgins, P. Mistry, P. Charlton, and R. Callaghan. Communication between multiple drug binding sites on P-glycoprotein. Mol. Pharmacol. 58:624-632 (2000).

    Google Scholar 

  45. M. Horio, K. V. Chin, S. J. Currier, S. Goldenberg, C. Williams, I. Pastan, and M. M. Gottesman. and J. Handler. Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia. J. Biol. Chem. 264:14880-14884 (1989).

    Google Scholar 

  46. Y. Zhang and L. Z. Benet. Characterization of P-glycoprotein mediated transport of K02, a novel vinylsulfone peptidomimetic cysteine protease inhibitor, across MDR1-MDCK and Caco-2 cell monolayers. Pharm. Res. 15:1520-1524 (1998).

    Google Scholar 

  47. S. Ito, C. Woodland, B. Sarkadi, G. Hockmann, S. E. Walker, and G. Koren. Modeling of P-glycoprotein-involved epithelial drug transport in MDCK cells. Am. J. Physiol. 277:F84-F96 (1999).

    Google Scholar 

  48. C. J. Matheny, M. W. Lamb, K. R. Brouwer, and G. M. Pollack. Pharmacokinetic and pharmacodynamic implications of P-glycoprotein modulation. Pharmacotherapy 21:778-796 (2001).

    Google Scholar 

  49. W. D. Stein. Kinetics of the P-glycoprotein, the multidrug transporter. Exp. Physiol. 83:221-232 (1998).

    Google Scholar 

  50. D. R. Ferry, P. J. Malkhandi, M. A. Russell, and D. J. Kerr. Allosteric #x00AEulation of [3H]vinblastine binding to P-glycoprotein of MCF-7 ADR cells by dexniguldipine. Biochem. Pharmacol. 49:1851-1861 (1995).

    Google Scholar 

  51. R. Liu and F. J. Sharom. Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains. Biochemistry 35:11865-11873 (1996).

    Google Scholar 

  52. C. Martin, G. Berridge, C. F. Higgins, and R. Callaghan. The multi-drug resistance reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by an allosteric interaction. Br. J. Pharmacol. 122:765-771 (1997).

    Google Scholar 

  53. C. Martin, G. Berridge, P. Mistry, C. Higgins, P. Charlton, and R. Callaghan. Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis. Biochemistry 39:11901-11906 (2000).

    Google Scholar 

  54. K. Simons and S. D. Fuller. Cell surface polarity in epithelia. Annu. Rev. Cell Biol. 1:243-288 (1985).

    Google Scholar 

  55. K. Simons and G. van Meer. Lipid sorting in epithelial cells. Biochemistry 27:6197-6202 (1988).

    Google Scholar 

  56. I. Chantret, A. Barbat, E. Dussaulx, M. G. Brattain, and A. Zweibaum. Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Res. 48:1936-1942 (1988).

    Google Scholar 

  57. C. Le Grimellec, G. Friedlander, E. H. el Yandouzi, P. Zlatkine, and M. C. Giocondi. Membrane fluidity and transport properties in epithelia. Kidney Int. 42:825-836 (1992).

    Google Scholar 

  58. C. Le Grimellec, G. Friedlander, and M. C. Giocondi. Asymmetry of plasma membrane lipid order in Madin-Darby Canine Kidney cells. Am. J. Physiol. 255:F22-F32 (1988).

    Google Scholar 

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  1. Division of Drug Delivery and Disposition, School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599

    Matthew D. Troutman & Dhiren R. Thakker

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Troutman, M.D., Thakker, D.R. Novel Experimental Parameters to Quantify the Modulation of Absorptive and Secretory Transport of Compounds by P-Glycoprotein in Cell Culture Models of Intestinal Epithelium. Pharm Res 20, 1210–1224 (2003). https://doi.org/10.1023/A:1025001131513

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