Skip to main content
SpringerLink
Log in

The Steady-State Michaelis–Menten Analysis of P-Glycoprotein Mediated Transport Through a Confluent Cell Monolayer Cannot Predict the Correct Michaelis Constant Km

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Purpose

Typically, the kinetics of membrane transport is analyzed using the steady-state Michaelis–Menten (or Eadie–Hofstee or Hanes) equations. This approach has been successful when the substrate is picked up from the aqueous phase, like a water-soluble enzyme, for which the Michaelis–Menten steady-state analysis was developed. For membrane transporters whose substrate resides in the lipid bilayer of the plasma membrane, like P-glycoprotein (P-gp), there has been no validation of the accuracy of the steady-state analysis because the elementary rate constants for transport were not known.

Methods

Recently, we fitted the mass action elementary kinetic rate constants of P-gp transport of three different drugs through a confluent monolayer of MDCKII-hMDR1 cells. With these elementary rate constants in hand, we use computer simulations to assess the accuracy of the steady-state Michaelis–Menten parameters. This limits the simulation to parameter ranges known to be physiologically relevant.

Results

Using over 2,300 different vectors of initial elementary parameters spanning the space bounded by the three drugs, which defines 2,300 “virtual substrates”, the concentrations of substrate transported were calculated and fitted to Eadie–Hofstee plots. Acceptable plots were obtained for 1,338 cases.

Conclusion

The fitted steady-state Vmax values from the analysis correlated to within a factor of 2–3 with the values predicted from the elementary parameters. However, the fitted Km value could be generated by a wide range of underlying “molecular” Km values. This is because of the convolution of the drug passive permeability kinetics into the fitted Km. This implies that Km values measured in simpler systems, e.g., microsomes or proteoliposomes, even if accurate, would not predict the Km values for the confluent monolayer system or, by logical extension, in vivo. Reliable in vitro–in vivo extrapolation seems to require using the elementary rate constants rather than the Michaelis–Menten steady-state parameters.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

Abbreviations

A > B (or B > A):

transport across the confluent cell monolayer when the donor chamber is apical (or basolateral) and the receiver chamber is basolateral (or apical)

P-gp:

the P-glycoprotein product of the hMDR1 gene

References

  1. K. S. Lown R. R. Mayo A. B. Leichtman H. L. Hsiao D. K. Turgeon P. Schmiedlin-Ren M. B. Brown W. Guo S. J. Rossi L. Z. Benet P. B. Watkins (1997) ArticleTitleRole of intestinal P-glycoprotein (mdr1) in interpatient variation in the oral bioavailability of cyclosporine Clin. Pharmacol. & Therap. 62 IssueID3 248–260

    Google Scholar 

  2. A. H. Schinkel (1998) ArticleTitlePharmacological insights from P-glycoprotein knockout mice Int. J. Clin. Pharmacol. & Therap. 36 IssueID1 9–13

    Google Scholar 

  3. L. B. Goh K. J. Spears D. Yao A. Ayrton P. Morgan W. C. Roland T. Friedberg (2002) ArticleTitleEndogenous drug transporters in in vitro and in vivo models for the prediction of drug disposition in man Biochem. Pharmacol. 64 1569–1578 Occurrence Handle10.1016/S0006-2952(02)01355-2 Occurrence Handle12429346

    Article  PubMed  Google Scholar 

  4. P. Borst R. O. Elferink (2002) ArticleTitleMammalian ABC transporters in health and disease Ann. Rev. Biochem. 71 537–592 Occurrence Handle10.1146/annurev.biochem.71.102301.093055 Occurrence Handle12045106

    Article  PubMed  Google Scholar 

  5. M. M. Gottesman (2002) ArticleTitleMechanisms of cancer drug resistance Annu. Rev. Med. 53 615–627 Occurrence Handle10.1146/annurev.med.53.082901.103929 Occurrence Handle11818492

    Article  PubMed  Google Scholar 

  6. S. V. Ambudkar C. Kimchi-Sarfaty Z. E. Sauna M. M. Gottesman (2003) ArticleTitleP-glycoprotein: from genomics to mechanism Oncogene 22 7468–7485 Occurrence Handle10.1038/sj.onc.1206948 Occurrence Handle14576852

    Article  PubMed  Google Scholar 

  7. A. E. Senior M. K. Al-Shawi I. L. Urbatsch (1995) ArticleTitleThe catalytic cycle of P-glycoprotein FEBS Lett. 377 IssueID3 285–289 Occurrence Handle10.1016/0014-5793(95)01345-8 Occurrence Handle8549739

    Article  PubMed  Google Scholar 

  8. H. W. Veen Particlevan A. Margolles M. Muller C. F. Higgins W. N. Konings (2000) ArticleTitleThe homodimeric ATP-binding cassette transporter LmrA mediates multidrug transport by an alternating two-site (two-cylinder engine) mechanism EMBO J. 19 2503–2514 Occurrence Handle10.1093/emboj/19.11.2503 Occurrence Handle10835349

    Article  PubMed  Google Scholar 

  9. C. Martin G. Berridge C. F. Higgens P. Mistry P. Charlton R. Callaghan (2000) ArticleTitleCommunication between Multiple Drug binding sites on P-glycoprotein Mol. Pharmacol. 58 624–632 Occurrence Handle10953057

    PubMed  Google Scholar 

  10. C. Martin C. F. Higgins R. Callaghan (2001) ArticleTitleThe vinblastine binding site adopts high- and low-affinity conformations during a transport cycle of P-glycoprotein Biochemistry 40 15733–15742 Occurrence Handle10.1021/bi011211z Occurrence Handle11747450

    Article  PubMed  Google Scholar 

  11. Q. Qu J. W. Chu F. J. Sharom (2003) ArticleTitleTransition state P-glycoprotein binds drugs and modulators with unchanged affinity, suggesting a concerted transport mechanism Biochemistry 42 1345–1353 Occurrence Handle10.1021/bi0267745 Occurrence Handle12564938

    Article  PubMed  Google Scholar 

  12. M. K. Al-Shawi M. K. Polar H. Omote R. A. Figler (2003) ArticleTitleTransition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein J. Biol. Chem. 278 52629–52640 Occurrence Handle10.1074/jbc.M308175200 Occurrence Handle14551217

    Article  PubMed  Google Scholar 

  13. S. H. Jang M. G. Wientjes J. L.-S. Au (2003) ArticleTitleInterdependent effect of P-glycoprotein-mediated drug efflux and intracellular drug binding on intracellular paclitaxel pharmacokinetics: Application of computational modeling J. Pharmacol. Exp. Therap. 304 773–780 Occurrence Handle10.1124/jpet.102.044172

    Article  Google Scholar 

  14. T. W. Loo M. C. Bartlett D. M. Clarke (2003) ArticleTitleSubstrate-induced conformational changes in the transmembrane segments of human P-glycoprotein. Direct evidence for the substrate-induced fit mechanism for drug binding J. Biol. Chem. 278 IssueID16 13603–13606 Occurrence Handle10.1074/jbc.C300073200 Occurrence Handle12609990

    Article  PubMed  Google Scholar 

  15. I. L. Urbatsch G. A. Tyndall G. Tombline A. E. Senior (2003) ArticleTitleP-glycoprotein catalytic mechanism: Studies of the ADP-vanadate inhibited state J. Biol. Chem. 278 IssueID25 23171–23179 Occurrence Handle10.1074/jbc.M301957200 Occurrence Handle12670938

    Article  PubMed  Google Scholar 

  16. F. Tang K. Horie R. T. Borchardt (2002) ArticleTitleAre MDCK cells transfected with the human MRP2 gene a good model of the human intestinal mucosa? Pharm. Res. 19 765–772 Occurrence Handle10.1023/A:1016140429238 Occurrence Handle12134945

    Article  PubMed  Google Scholar 

  17. F. Tang K. Horie R. T. Borchardt (2002) ArticleTitleAre MDCK cells transfected with the human MDR1 gene a good model of the human intestinal mucosa? Pharm. Res. 19 773–779 Occurrence Handle10.1023/A:1016192413308 Occurrence Handle12134946

    Article  PubMed  Google Scholar 

  18. M. D. Troutman D. R. Thakker (2003) ArticleTitleNovel experimental parameters to quantify the modulation of absorptive and secretory transport of substrates by P-glycoprotein in cell culture models of intestinal epithelium Pharm. Res. 220 1210–1224 Occurrence Handle10.1023/A:1025001131513

    Article  Google Scholar 

  19. M. D. Troutman D. R. Thakker (2003) ArticleTitleEfflux 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 Occurrence Handle10.1023/A:1025049014674 Occurrence Handle12948018

    Article  PubMed  Google Scholar 

  20. A. Seelig E. Gatlik-Landwojtowicz (2005) ArticleTitleBiophysical characterization of inhibitors of multidrug efflux transporters: Their membrane and protein interactions Med. Chem. (Minirev.) 5 IssueID2 135–151

    Google Scholar 

  21. H. Omote R. A. Figler M. K. Polar M. K. Al-Shawi (2004) ArticleTitleImproved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation Biochemistry 43 3917–3928 Occurrence Handle10.1021/bi035365l Occurrence Handle15049699

    Article  PubMed  Google Scholar 

  22. G. Tombline G. Lori A. Bartholomew G. A. Tyndall K. Gimi I. L. Urbatsch A. E. Senior (2004) ArticleTitleProperties of P-glycoprotein with mutations in the “catalytic carboxylate” glutamate residues J. Biol. Chem. 279 46518–46526 Occurrence Handle10.1074/jbc.M408052200 Occurrence Handle15326176

    Article  PubMed  Google Scholar 

  23. T. T. Tran A. Mittal T. Gales B. Maleeff T. Aldinger J. W. Polli A. Ayrton H. Ellens J. Bentz (2004) ArticleTitleAn exact kinetic analysis of passive transport across a polarized confluent MDCK cell monolayer modeled as a single barrier J. Pharm. Sci. 93 2108–2123 Occurrence Handle10.1002/jps.20105 Occurrence Handle15236458

    Article  PubMed  Google Scholar 

  24. T. T. Tran A. Mittal T. Aldinger J. W. Polli A. Ayrton H. Ellens J. Bentz (2005) ArticleTitleThe elementary mass action rate constants of P-gp transport for a confluent monolayer of MDCKII-hMDR1 cells Biophys. J. 88 715–738 Occurrence Handle10.1529/biophysj.104.045633 Occurrence Handle15501934

    Article  PubMed  Google Scholar 

  25. W. D. Stein (1997) ArticleTitleKinetics of the multidrug transporter (P-glycoprotein) and its reversal Physiol. Rev. 77 IssueID2 545–590 Occurrence Handle9114823

    PubMed  Google Scholar 

  26. N. F. H. Ho P. S. Burton R. A. Conradi C. L. Barsuhn (1995) ArticleTitleA 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 IssueID1 21–27 Occurrence Handle7714738

    PubMed  Google Scholar 

  27. N. F. H. Ho T. J. Raub P. S. Burton C. L. Bausuhn A. Adson K. L. Audus R. Borchardt (2000) Quantitative approaches to delineate and passive transport mechanisms in cell culture monolayers G. L. Amidon P. I. Lee (Eds) Transport Processes in Pharmaceutical Systems Marcel Dekker New York 219–316

    Google Scholar 

  28. S. Doppenschmitt H. Spahn-Langguth C. G. Regardh P. Langguth (1999) ArticleTitleRole 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 Occurrence Handle10.1021/js980378j Occurrence Handle10514357

    Article  PubMed  Google Scholar 

  29. S. Ito C. Woodland B. Sarkadi G. Hockmann S. E. Walker G. Koren (1999) ArticleTitleModeling of P-glycoprotein-involved epithelial drug transport in MDCK cells Am. J. Physiol.-Renal Fluid Electrolyte Physiol. 46 F84–F96

    Google Scholar 

  30. H. J. Kuh S. H. Jang M. G. Wientjes J. L. Au (2000) ArticleTitleComputational model of intracellular pharmacokinetics of paclitaxel J. Pharmacol. Exp. Ther. 293 761–770 Occurrence Handle10869374

    PubMed  Google Scholar 

  31. A. Ruth W. D. Stein E. Rose I. B. Roninson (2001) ArticleTitleCoordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein Biochemistry 40 4332–4339 Occurrence Handle10.1021/bi001373f Occurrence Handle11284689

    Article  PubMed  Google Scholar 

  32. M. Sasaki H. Suzuki K. Ito T. Abe Y. Sugiyama (2002) ArticleTitleTranscellular transport of organic anions across a double transfected Madin–Darby Canine Kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and multidrug resistance-associated protein 2 (MRP2/ABCC2) J. Biol. Chem. 277 6497–6503 Occurrence Handle10.1074/jbc.M109081200 Occurrence Handle11748225

    Article  PubMed  Google Scholar 

  33. F. J. Meyer-Almes M. Auer (2000) ArticleTitleEnzyme inhibition assays using fluorescence correlation spectroscopy: A new algorithm for the derivation of kcat/KM and Ki values at substrate concentrations much lower than the Michaelis constant Biochemistry 39 IssueID43 13261–13268 Occurrence Handle10.1021/bi000057y Occurrence Handle11052679

    Article  PubMed  Google Scholar 

  34. F. J. Sharom P. D. Eckford (2003) ArticleTitleReconstitution of membrane transporters Methods Mol. Biol. 227 129–154 Occurrence Handle12824648

    PubMed  Google Scholar 

  35. M. R. Lugo F. J. Sharom (2005) ArticleTitleInteraction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site Biochemistry 44 643–655 Occurrence Handle10.1021/bi0485326 Occurrence Handle15641790

    Article  PubMed  Google Scholar 

  36. J. W. Polli S. A. Wring J. E. Humphreys L. Huang J. B. Morgan L. O. Webster C. S. Serabjit-Singh (2001) ArticleTitleRational use of in vitro P-glycoprotein assays in drug discovery J. Pharmacol. & Exp. Ther. 299 IssueID2 620–628

    Google Scholar 

  37. R. Evers M. Kool A. J. Smith L. Deemter Particlevan M. Haas Particlede P. Borst (2000) ArticleTitleInhibitory effect of the reversal agents V-104, GF120918 and Pluronic L61 on MDR1 Pgp-MRP1- and MRP2-mediated transport Br. J. Cancer 83 366–374 Occurrence Handle10.1054/bjoc.2000.1260 Occurrence Handle10917553

    Article  PubMed  Google Scholar 

  38. F. Hyafil C. Vergely P. Vignaud ParticleDu T. Grand-Perret (1993) ArticleTitleIn vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative Cancer Res. 53 4595–4602 Occurrence Handle8402633

    PubMed  Google Scholar 

  39. C. Butor J. Davoust (1992) ArticleTitleApical to basolateral surface area ratio and polarity of MDCK cells grown on different supports Exp. Cell Res. 203 115–127 Occurrence Handle10.1016/0014-4827(92)90046-B Occurrence Handle1426034

    Article  PubMed  Google Scholar 

  40. S. Modok C. Heyward R. Callaghan (2004) ArticleTitleP-glycoprotein retains function when reconstituted into a sphingolipid and cholesterol rich environment J. Lipid Res. 45 1910–1918 Occurrence Handle10.1194/jlr.M400220-JLR200 Occurrence Handle15258203

    Article  PubMed  Google Scholar 

  41. J. Troost H. Lindenmaier W. E. Haefeli J. Weiss (2004) ArticleTitleModulation of cellular cholesterol alters P-glycoprotein activity in multidrug-resistant cells Mol. Pharmacol. 66 1332–1339 Occurrence Handle10.1124/mol.104.002329 Occurrence Handle15308763

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania, 19104, USA

    Joe Bentz & Thuy Thanh Tran

  2. Preclinical Drug Metabolism and Pharmacokinetics, GlaxoSmith- Kline, King of Prussia, Pennsylvania, 19406, USA

    Thuy Thanh Tran & Harma Ellens

  3. Preclinical Drug Metabolism and Pharmacokinetics, GlaxoSmith- Kline, Research Triangle Park, North Carolina, 27709, USA

    Joseph W. Polli

  4. Preclinical Drug Metabolism and Pharmacokinetics, GlaxoSmith- Kline, Welwyn, UK

    Andrew Ayrton

Authors
  1. Joe Bentz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thuy Thanh Tran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joseph W. Polli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrew Ayrton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Harma Ellens
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joe Bentz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bentz, J., Tran, T.T., Polli, J.W. et al. The Steady-State Michaelis–Menten Analysis of P-Glycoprotein Mediated Transport Through a Confluent Cell Monolayer Cannot Predict the Correct Michaelis Constant Km. Pharm Res 22, 1667–1677 (2005). https://doi.org/10.1007/s11095-005-6627-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-005-6627-z

Key Words

Search

Navigation