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Characterization of the Regional Intestinal Kinetics of Drug Efflux in Rat and Human Intestine and in Caco-2 Cells

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Abstract

Purpose. The aim of the present study was to investigate the transport kinetics of intestinal secretory processes in the jejunum, ileum and colon of rats and humans and in Caco-2 cells, in vitro.

Methods. Etoposide, vinblastine sulphate and verapamil hydrochloride were chosen as model substrates since they have been reported to undergo efflux in various other tissues. The concentration dependence, inhibition, directionality, temperature dependence, proton/sodium dependence, and ATP dependence of efflux were studied using side-by-side diffusion chambers and brush border membrane vesicles (BBMVs). Intestinal tissue from rats and humans and Caco-2 cells (passage no. 26) were used. Directional steady state effective permeabilities were calculated from drug appearance in the apical (AP) or basolateral (BL) chambers. Kinetic studies were carried out by investigating substrate efflux at concentrations ranging from 0.2 μM to 1000 μM. Since substrate efflux may be a result of more than one transporter, the hybrid efflux Km (Michaelis-constant), Pc (carrier-mediated permeability), and Pm (passive permeability) were determined as a function of intestinal region. Inhibitor studies were performed using quinidine (0.2 mM), a mixed inhibitor of P-glycoprotein (Pgp) and Multidrug Resistance-Associated Protein (MRP), and Leukotriene C4(100 nM), an inhibitor of MRP and the canalicular multispecific organic anion transporter (cMOAT). Temperature dependent efflux was determined by investigating the BL to AP transport at temperatures ranging from 3°C to 37°C. Energies of activation (Ea) were determined from an Arrhenius analysis. Sodium, proton, and ATP dependence were determined using BBMVs. Immunoquantitation of Pgp, MRP and Lung Resistance Protein (LRP) in Caco-2 cells were carried out using Western blot analysis.

Results. Active efflux of all substrates was observed in all regions of rat and human intestine and in Caco-2 cells. Directionality was observed with BL to AP transport exceeding AP to BL transport. The BL to AP/AP to BL permeability ratio, the efflux ratio, ranged from 1.4 to 19.8. Heal efflux was significantly higher (p < 0.001) than in other regions. Kinetic studies revealed that hybrid efflux Km values ranged from 4 to 350 μM. In some cases, efflux was not saturable due to the solubility limits of the compounds utilized in this study. In presence of inhibitors, efflux ratios approached 1. BL to AP transport was temperature dependent in rat ileum for all substrates. Ea of intestinal efflux was found to be 11.6, 8.3, and 15.8 kcal/mole for etoposide, vinblastine and verapamil, respectively, suggesting an active, energy-dependent efflux mechanism. Substrate efflux was not sodium or proton dependent but was dependent on ATP. Using Western blot analysis the presence of Pgp, MRP, and LRP was demonstrated in Caco-2 cells and the amount of each transport protein varied as a function of passage number.

Conclusions. Using multiple putative efflux substrates, the current results demonstrate that intestinal efflux was regionally dependent, mediated by multiple efflux transporters, the Km’s were in the micro-molar range, and involved an energy dependent mechanism(s).

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REFERENCES

  1. G. D. Kruh, A. Chan, K. Myers, K. Gaughan, T. Miki, and S. A. Aaronson. Expression complementary DNA library transfer establishes mrp as a multidrug resistance gene. Cancer Res. 54:1649-1652 (1994).

    PubMed  Google Scholar 

  2. S. P. C. Cole, K. E. Sparks, K. Fraser, D. W. Loe, C. E. Grant, G. M. Wilson, and R. G. Deeley. Pharmacological characterization of multidrug resistant MRP-transfected human tumor cells. Cancer Res. 54:5902-5910 (1994).

    PubMed  Google Scholar 

  3. D. Schadendorf, A. Makki, C. Stahr, A. van Dyck, R. Wanner, G. L. Scheffer, M. J. Flens, R. Scheper and B. M. Henz. Membrane transport proteins associated with drug resistance expressed in human melanoma. Am. J. Pathol. 147:1545-52 (1995).

    PubMed  Google Scholar 

  4. Z. C. Gatmaitan and I. M. Arias. Structure and function of P-glycoprotein in normal liver and small intestine. Advances in Pharmacology 24:77-91 (1993).

    PubMed  Google Scholar 

  5. I. Sagawara, S. Akiyama, R. J. Scheper, and S. Itoyama. Lung resistance protein (LRP) expression in human normal tissues in comparison with that of MDR1 and MRP. Cancer Lett. 112:23-31 (1997).

    PubMed  Google Scholar 

  6. K. Ito, H. Suzuki, T. Hirohashi, K. Kume, T. Shimizu, and Y. Sugiyama. Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR Am. J Physiol. 272:G16-22 (1997).

    PubMed  Google Scholar 

  7. D. Keppler, J. Konig, and M. Buchler. The canalicular multidrug resistance protein' cMRP/MRP2' a novel conjugate export pump expressed in the apical membrane of hepatocytes. Adv. Enzyme Regul. 37:321-33 (1997).

    PubMed  Google Scholar 

  8. M. A. Izquierdo, G. L. Scheffer, M. J. Flens, G. Giaccone, H. J. Broxterman, C. J. Meijer, and P. van der Valk. Broad distribution of the multidrug resistance-related vault lung resistance protein in normal human tissues and tumors. Am. J. Pathol. 148:877-87 (1996).

    PubMed  Google Scholar 

  9. L. Z. Benet, C.-Y. Wu, M. F. Hebert, and V. J. Wacher. Intestinal drug metabolism and antitransport processes: A potential paradigm shift in oral drug delivery. J Contr. Rel. 39:139-143 (1996).

    Google Scholar 

  10. L. C. Floren, I. Bekersky, L. Z. Benet, Q. Mekki, D. Dressler, J. W. Lee, J. P. Roberts, and M. F. Hebert. Tacrolimus oral bioavailability doubles with coadministration of ketoconazole. Clin. Pharmacol. Ther. 62:41-9 (1997).

    PubMed  Google Scholar 

  11. J. Alsenz, H. Steffen, and R. Alex. Active apical secretory efflux of the HIV protease inhibitors saquinavir and ritonavir in Caco-2 cells monolayers. Pharm. Res. 15:423-(1998).

    PubMed  Google Scholar 

  12. J. Hunter and B. H. Hirst. Intestinal secretion of drugs. The role of P-glycoprotein and related drug efflux systems in limiting oral drug absorption. Adv. Drug Delivery Rev. 25:129-157 (1997).

    Google Scholar 

  13. B. L. Leu and J. D. Huang. Inhibition of intestinal P-glycoprotein and effects on etoposide absorption. Cancer Chemother. Pharmacol. 35:432-436(1995).

    PubMed  Google Scholar 

  14. J. Hunter, M. A. Jepson, T. Tsuruo, N. L. Simmons, and B. H. Hirst. Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine secretion and interaction with modulators. J. Biol. Chem. 268:14991-14997 (1993).

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  16. R. B. Kim, M. F. Fromm, C. Wandel, B. Leake, A. J. 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).

    PubMed  Google Scholar 

  17. D. W. Loe, K. C. Almquist, R. G. Deeley, and S. P. C. Cole. Multidrug resistance protein (MRP)-mediated transport of Leukotriene C4and chemotherapeutic agents in membrane vesicles. J. Biol. Chem. 271:9675-9682 (1996).

    PubMed  Google Scholar 

  18. I. Leier, G. Jedlitschky, U. Buchholz, S. P. C. Cole, R. G. Deeley, and D. Keppler. The MRP gene encodes an ATP-dependent export pump for Leukotriene C4and structurally related conjugates. J. Biol. Chem. 269:27807-27810 (1994).

    PubMed  Google Scholar 

  19. A. P. Waclawski and P. J. Sinko. Oral Absorption of Anti-AIDS Nucleoside Analogues. 2. Carrier-Mediated Absorption of Stavudine in Rat and Rabbit Preparations. J. Pharm. Sci. 85:478-485 (1996).

    PubMed  Google Scholar 

  20. M. Donowitz, E. Emmer, J. McCullen, L. Reinlib, M. E. Cohen, R. P. Rood, J. Madara, G. W. G. Sharp, H. Muer, and K. Malmstrom. Freeze-thaw and high voltage discharge allow macromolecule uptake into ileal brush-border vesicles. Am. J. Physiol. 252:G273-G735 (1987).

    Google Scholar 

  21. S. Hsing, Z. Gatmaitan, and I. M. Arias. The Function of Gp170, the Multidrug-Resistance Gene Product, in the Brush Border of Rat Intestinal Mucosa. Gastroenterology 102:879-885 (1992).

    PubMed  Google Scholar 

  22. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227:680-685 (1970).

    Google Scholar 

  23. Flens, M. J., Izquierdo, M. A., Scheffer, G. L., Fritz, J. M., Meijer, C. J. L. M., Scheper, R. J., and Zaman, G. J. R. Immunochemical detection of the multidrug resistance-associated protein MRP in human multidrug-resistant tumor cells by monoclonal antibodies. Cancer Res. 54:4557-4563 (1994).

    PubMed  Google Scholar 

  24. M. Muller, H. Roelofsen, and P. L. Jansen. Secretion of organic anions by hepatocytes: involvement of homologues of the multidrug resistance protein. Semin Liver. Dis. 16:211-20 (1996).

    PubMed  Google Scholar 

  25. T. Terao, E. Hisabaga, 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).

    PubMed  Google Scholar 

  26. H. Saitoh, C. Gerard, and B. J. Aungst. The secretory intestinal transport of some beta-lactam antibiotics and anionic compounds. J. Pharmacol. Exp. Ther. 278:205-211 (1996).

    PubMed  Google Scholar 

  27. P. A. Augustijns, T. Timothy, P. Bradshaw, L-S. L. 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).

    PubMed  Google Scholar 

  28. J. Karlsson, S. M. Kuo, J. Ziemniak, and P. Artursson. Transport of celiprolol across human intestinal epithelial (Caco-2) cells: mediation of secretion by multiple transporters including P-glycoprotein. Br. J. Pharmacol. 110:1009-1016 (1993). See also: S. M. Kuo, B. R. Whitby, P. Artursson, and J. A. Ziemniak. The contribution of intestinal secretion to the dose-dependent absorption of celiprolol. Pharm. Res. 11:648–653 (1994).

    PubMed  Google Scholar 

  29. P. S. Burton, R. A. Conradi, A. R. Hilgers, and N. H. Ho. Evidence for a polarized efflux system for peptides in the apical membrane of Caco-2 cells. Biochem. Biophys. Res. Commun. 190:760-766 (1993).

    PubMed  Google Scholar 

  30. P. Wils, V. Phung-Ba, A. Warnery, D. Lechardeur, S. Raeissi, I. J. Hidalgo, and D. Scherman. Polarized transport of docetaxel and vinblastine mediated by P-glycoprotein in human intestinal epithelial cell monolayers. Biochem. Pharmacol. 48:1528-1530 (1994).

    PubMed  Google Scholar 

  31. M. E. Cavet, M. West, and N. L. Simmons. Transport and epithelial secretion of the cardiac glycoside, digoxin by human intestinal epithelial (Caco-2) cells. Br. J. Pharmacol. 118:1389-1396 (1996).

    PubMed  Google Scholar 

  32. S. Paul, L. M. Breuninger, K. D. Tew, H. Shen, and G. D. Kruh. ATP-dependent uptake of natural product cytotoxic drugs by membrane vesicles establishes MRP as a broad specificity transporter. Proc. Natl. Acad. Sci.USA 93:6929-6934 (1996).

    PubMed  Google Scholar 

  33. I. J. Hidalgo and R. T. Borchardt. Transport of a large neutral amino acid (phenylalanine) in a human intestinal epithelial cell line: Caco-2. Biochim. Biophys. Acta. 1028:25-30 (1990).

    PubMed  Google Scholar 

  34. K. Goda, L. Balkay, T. Marian, L. Tron, A. Aszalos, and G. Szabo Jr. Intracellular pH does not affect drug extrusion by P-glycoprotein. J. Photochem. Photobiol. B. 34:177-82 (1996).

    PubMed  Google Scholar 

  35. Ken-ichi, Hosoya, K.-J. Kim, and V. H. L. Lee. Age-dependent expression of P-glycoprotein gp170 in Caco-2 cell monolayers. Pharm. Res. 13:885-890 (1996).

    PubMed  Google Scholar 

  36. S. F. Su, J. D. Huang. Inhibition of the intestinal digoxin absorption and exsorption by quinidine. Drug Metab. Dispos. 24:142-7 (1996).

    PubMed  Google Scholar 

  37. A. H. Schinkel, J. J. Smit, O. van Tellingen, J. H. Beijnen, E. Wagenaar, L. van Deemter et. al. Disruption of the mouse mdr la P-glycoprtoein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell. 77:491-502 (1994).

    Google Scholar 

  38. A. H. Schinkel, E. Wagenaar, C. A. Mol and L. van Deemter. P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J. Clin. Invest. 97:2517-2524 (1996).

    PubMed  Google Scholar 

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  1. College of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey, 08854

    Vijaya D. Makhey, Ailan Guo, Daniel A. Norris, Peidi Hu, Jiansheng Yan & Patrick J. Sinko

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  1. Vijaya D. Makhey
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Makhey, V.D., Guo, A., Norris, D.A. et al. Characterization of the Regional Intestinal Kinetics of Drug Efflux in Rat and Human Intestine and in Caco-2 Cells. Pharm Res 15, 1160–1167 (1998). https://doi.org/10.1023/A:1011971303880

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