Advertisement

The AAPS Journal

, Volume 19, Issue 3, pp 787–796 | Cite as

Reliable Rate Measurements for Active and Passive Hepatic Uptake Using Plated Human Hepatocytes

  • Yi-an Bi
  • Renato J. Scialis
  • Sarah Lazzaro
  • Sumathy Mathialagan
  • Emi Kimoto
  • Julie Keefer
  • Hui Zhang
  • Anna M. Vildhede
  • Chester Costales
  • A. David Rodrigues
  • Larry M. Tremaine
  • Manthena V. S. VarmaEmail author
Research Article

Abstract

Transporter-mediated hepatic uptake is proven to be the rate-determining step in the systemic clearance of several drugs. Therefore, accurate measurement of active and passive uptake clearances in vitro is critical to facilitate pharmacokinetics and drug-drug interaction predictions. Here, we evaluated the plated human hepatocytes (PHH) and studied the effect of incubation temperature and inhibitor concentration on uptake measurements, in order to reliably estimate hepatic uptake components. Uptake rates measured using PHH, at 37°C without and with rifamycin SV, were comparable with those obtained from suspension hepatocytes and sandwich-cultured hepatocytes for a set of 10–13 compounds. Apparent permeability across monolayers of low-efflux Madin-Darby canine kidney cells was measured at 4, 10, and 37°C. Of the 23 compounds evaluated, 13 compounds showed >2-fold reduction in passive permeability at 4°C compared to 37°C, inferring that low-temperature incubations may underestimate passive uptake. Inhibition studies using transporter-transfected cells suggested that ∼20 μM rifamycin SV completely inhibited organic anion-transporting polypeptides (OATPs), while no significant inhibition was noted for other hepatic uptake transporters. On the basis of inhibition profiles, the contribution of active versus passive and OATP versus non-OATP transport to the PHH uptake was discerned for various endogenous substrates and statins. With the exception of fluvastatin, the statins studied were predominantly transported by OATPs in PHH and the non-OATP transporters, such as Na+-taurocholate co-transporting polypeptide, played a minimal role. In conclusion, PHH is useful for uptake measurements, and rifamycin SV employed at different concentrations can reliably estimate active and passive uptake and characterize OATP-dependent active uptake.

KEY WORDS

drug transporters hepatic uptake organic anion-transporting polypeptide pharmacokinetics plated human hepatocytes 

Supplementary material

12248_2017_51_MOESM1_ESM.pdf (273 kb)
ESM 1 (PDF 272 kb)

References

  1. 1.
    Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KL, Chu X, et al. Membrane transporters in drug development. Nat Rev Drug Discov. 2010;9(3):215–36.CrossRefPubMedGoogle Scholar
  2. 2.
    Shitara Y, Horie T, Sugiyama Y. Transporters as a determinant of drug clearance and tissue distribution. Eur J Pharm Sci. 2006;27(5):425–46.CrossRefPubMedGoogle Scholar
  3. 3.
    Niemi M, Pasanen MK, Neuvonen PJ. Organic anion transporting polypeptide 1B1: a genetically polymorphic transporter of major importance for hepatic drug uptake. Pharmacol Rev. 2011;63(1):157–81.CrossRefPubMedGoogle Scholar
  4. 4.
    Treiber A, Schneiter R, Häusler S, Stieger B. Bosentan is a substrate of human OATP1B1 and OATP1B3: inhibition of hepatic uptake as the common mechanism of its interactions with cyclosporin A, rifampicin, and sildenafil. Drug Metab Dispos. 2007;35(8):1400–7.CrossRefPubMedGoogle Scholar
  5. 5.
    Yamashiro W, Maeda K, Hirouchi M, Adachi Y, Hu Z, Sugiyama Y. Involvement of transporters in the hepatic uptake and biliary excretion of valsartan, a selective antagonist of the angiotensin II AT1-receptor, in humans. Drug Metab Dispos: Biologic Fate Chem. 2006;34(7):1247–54.CrossRefGoogle Scholar
  6. 6.
    Varma MV, Steyn SJ, Allerton C, El-Kattan AF. Predicting clearance mechanism in drug discovery: Extended Clearance Classification System (ECCS). Pharm Res. 2015;32(12):3785–802.CrossRefPubMedGoogle Scholar
  7. 7.
    Bi YA, Kazolias D, Duignan DB. Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab Dispos: Biologic Fate Chem. 2006;34(9):1658–65.CrossRefGoogle Scholar
  8. 8.
    Brouwer KL, Keppler D, Hoffmaster KA, Bow DA, Cheng Y, Lai Y, et al. In vitro methods to support transporter evaluation in drug discovery and development. Clin Pharmacol Ther. 2013;94(1):95–112.CrossRefPubMedGoogle Scholar
  9. 9.
    Zamek-Gliszczynski MJ, Lee CA, Poirier A, Bentz J, Chu X, Ellens H, et al. ITC recommendations for transporter kinetic parameter estimation and translational modeling of transport-mediated PK and DDIs in humans. Clin Pharmacol Ther. 2013;94(1):64–79.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Watanabe T, Kusuhara H, Maeda K, Kanamaru H, Saito Y, Hu Z, et al. Investigation of the rate-determining process in the hepatic elimination of HMG-CoA reductase inhibitors in rats and humans. Drug Metab Dispos: Biologic Fate Chem. 2010;38(2):215–22.CrossRefGoogle Scholar
  11. 11.
    Varma MV, Bi Y-a, Kimoto E, Lin J. Quantitative prediction of transporter-and enzyme-mediated clinical drug-drug interactions of organic anion-transporting polypeptide 1B1 substrates using a mechanistic net-effect model. J Pharmacol Exp Ther. 2014;351(1):214–23.CrossRefPubMedGoogle Scholar
  12. 12.
    Menochet K, Kenworthy KE, Houston JB, Galetin A. Use of mechanistic modeling to assess interindividual variability and interspecies differences in active uptake in human and rat hepatocytes. Drug Metab Dispos: Biologic Fate Chem. 2012;40(9):1744–56.CrossRefGoogle Scholar
  13. 13.
    Camenisch G, Umehara K. Predicting human hepatic clearance from in vitro drug metabolism and transport data: a scientific and pharmaceutical perspective for assessing drug-drug interactions. Biopharm Drug Dispos. 2012;33(4):179–94.CrossRefPubMedGoogle Scholar
  14. 14.
    Paine SW, Parker AJ, Gardiner P, Webborn PJ, Riley RJ. Prediction of the pharmacokinetics of atorvastatin, cerivastatin, and indomethacin using kinetic models applied to isolated rat hepatocytes. Drug Metab Dispos: Biologic Fate Chem. 2008;36(7):1365–74.CrossRefGoogle Scholar
  15. 15.
    Poirier A, Cascais AC, Funk C, Lave T. Prediction of pharmacokinetic profile of valsartan in human based on in vitro uptake transport data. J Pharmacokinet Pharmacodyn. 2009;36(6):585–611.CrossRefPubMedGoogle Scholar
  16. 16.
    Watanabe T, Kusuhara H, Maeda K, Shitara Y, Sugiyama Y. Physiologically based pharmacokinetic modeling to predict transporter-mediated clearance and distribution of pravastatin in humans. J Pharmacol Exp Ther. 2009;328(2):652–62.CrossRefPubMedGoogle Scholar
  17. 17.
    Jones HM, Barton HA, Lai Y, Bi YA, Kimoto E, Kempshall S, et al. Mechanistic pharmacokinetic modeling for the prediction of transporter-mediated disposition in humans from sandwich culture human hepatocyte data. Drug Metab Dispos: Biologic Fate Chem. 2012;40(5):1007–17.CrossRefGoogle Scholar
  18. 18.
    Varma MV, Lai Y, Kimoto E, Goosen TC, El-Kattan AF, Kumar V. Mechanistic modeling to predict the transporter- and enzyme-mediated drug-drug interactions of repaglinide. Pharm Res. 2013;30(4):1188–99.CrossRefPubMedGoogle Scholar
  19. 19.
    Cheng Y, Vapurcuyan A, Shahidullah M, Aleksunes LM, Pelis RM. Expression of organic anion transporter 2 in the human kidney and its potential role in the tubular secretion of guanine-containing antiviral drugs. Drug Metab Dispos: Biologic Fate Chem. 2012;40(3):617–24.CrossRefGoogle Scholar
  20. 20.
    Bi YA, Qiu X, Rotter CJ, Kimoto E, Piotrowski M, Varma MV, et al. Quantitative assessment of the contribution of sodium-dependent taurocholate co-transporting polypeptide (NTCP) to the hepatic uptake of rosuvastatin, pitavastatin and fluvastatin. Biopharm Drug Dispos. 2013;34(8):452–61.CrossRefPubMedGoogle Scholar
  21. 21.
    Varma MV, Gardner I, Steyn SJ, Nkansah P, Rotter CJ, Whitney-Pickett C, et al. pH-dependent solubility and permeability criteria for provisional biopharmaceutics classification (BCS and BDDCS) in early drug discovery. Mol Pharm. 2012;9(5):1199–212.CrossRefPubMedGoogle Scholar
  22. 22.
    Di L, Whitney‐Pickett C, Umland JP, Zhang H, Zhang X, Gebhard DF, et al. Development of a new permeability assay using low‐efflux MDCKII cells. J Pharm Sci. 2011;100(11):4974–85.CrossRefPubMedGoogle Scholar
  23. 23.
    Eng H, Scialis RJ, Rotter CJ, Lin J, Lazzaro S, Varma MV, et al. The antimicrobial agent fusidic acid inhibits organic anion transporting polypeptide-mediated hepatic clearance and may potentiate statin-induced myopathy. Drug Metab Dispos: Biologic Fate Chem. 2016;44(5):692–9.CrossRefGoogle Scholar
  24. 24.
    Kalgutkar AS, Chen D, Varma MV, Feng B, Terra SG, Scialis RJ, et al. Elucidation of the biochemical basis for a clinical drug-drug interaction between atorvastatin and 5-(N-(4-((4-ethylbenzyl)thio)phenyl)sulfamoyl)-2-methyl benzoic acid (CP-778875), a subtype selective agonist of the peroxisome proliferator-activated receptor alpha. Xenobiotica; Fate Foreign Compounds Biologic Syst. 2013;43(11):963–72.CrossRefGoogle Scholar
  25. 25.
    Li R, Barton HA, Varma MV. Prediction of pharmacokinetics and drug-drug interactions when hepatic transporters are involved. Clin Pharmacokinet. 2014;53(8):659–78.CrossRefPubMedGoogle Scholar
  26. 26.
    Ulvestad M, Bjorquist P, Molden E, Asberg A, Andersson TB. OATP1B1/1B3 activity in plated primary human hepatocytes over time in culture. Biochem Pharmacol. 2011;82(9):1219–26.CrossRefPubMedGoogle Scholar
  27. 27.
    Poirier A, Lavé T, Portmann R, Brun M-E, Senner F, Kansy M, et al. Design, data analysis, and simulation of in vitro drug transport kinetic experiments using a mechanistic in vitro model. Drug Metab Dispos: Biologic Fate Chem. 2008;36(12):2434–44.CrossRefGoogle Scholar
  28. 28.
    Nordell P, Winiwarter S, Hilgendorf C. Resolving the distribution-metabolism interplay of eight OATP substrates in the standard clearance assay with suspended human cryopreserved hepatocytes. Mol Pharm. 2013;10(12):4443–51.CrossRefPubMedGoogle Scholar
  29. 29.
    Papahadjopoulos D, Nir S, Ohki S. Permeability properties of phospholipid membranes: effect of cholesterol and temperature. Biochim Biophys Acta -Biomembranes. 1972;266(3):561–83.CrossRefGoogle Scholar
  30. 30.
    Lipowsky R. The conformation of membranes. Nature. 1991;349(6309):475–81.CrossRefPubMedGoogle Scholar
  31. 31.
    Izumi S, Nozaki Y, Komori T, Maeda K, Takenaka O, Kusano K, et al. Substrate-dependent inhibition of organic anion transporting polypeptide 1B1: comparative analysis with prototypical probe substrates estradiol-17β-glucuronide, estrone-3-sulfate, and sulfobromophthalein. Drug Metab Dispos: Biologic Fate Chem. 2013;41(10):1859–66.CrossRefGoogle Scholar
  32. 32.
    Shitara Y, Maeda K, Ikejiri K, Yoshida K, Horie T, Sugiyama Y. Clinical significance of organic anion transporting polypeptides (OATPs) in drug disposition: their roles in hepatic clearance and intestinal absorption. Biopharm Drug Dispos. 2013;34(1):45–78.CrossRefPubMedGoogle Scholar
  33. 33.
    Benet LZ, Hosey CM, Ursu O, Oprea TI. BDDCS, the rule of 5 and drugability. Adv Drug Del Rev. 2016;101:89–98.CrossRefGoogle Scholar
  34. 34.
    Niemi M, Pasanen MK, Neuvonen PJ. SLCO1B1 polymorphism and sex affect the pharmacokinetics of pravastatin but not fluvastatin. Clin Pharmacol Ther. 2006;80(4):356–66.CrossRefPubMedGoogle Scholar
  35. 35.
    Park JW, Siekmeier R, Lattke P, Merz M, Mix C, Schuler S, et al. Pharmacokinetics and pharmacodynamics of fluvastatin in heart transplant recipients taking cyclosporine A. J Cardiovasc Pharmacol Ther. 2001;6(4):351–61.CrossRefPubMedGoogle Scholar
  36. 36.
    Holdaas H, Hagen E, Asberg A, Lund K, Hartman A, Vaidyanathan S, et al. Evaluation of the pharmacokinetic interaction between fluvastatin XL and cyclosporine in renal transplant recipients. Int J Clin Pharmacol Ther. 2006;44(4):163–71.CrossRefPubMedGoogle Scholar
  37. 37.
    Ho RH, Tirona RG, Leake BF, Glaeser H, Lee W, Lemke CJ, et al. Drug and bile acid transporters in rosuvastatin hepatic uptake: function, expression, and pharmacogenetics. Gastroenterology. 2006;130(6):1793–806.CrossRefPubMedGoogle Scholar
  38. 38.
    McRae MP, Lowe CM, Tian X, Bourdet DL, Ho RH, Leake BF, et al. Ritonavir, saquinavir, and efavirenz, but not nevirapine, inhibit bile acid transport in human and rat hepatocytes. J Pharmacol Exp Ther. 2006;318(3):1068–75.CrossRefPubMedGoogle Scholar
  39. 39.
    Martinez-Becerra P, Briz O, Romero MR, Macias RI, Perez MJ, Sancho-Mateo C, et al. Further characterization of the electrogenicity and pH sensitivity of the human organic anion-transporting polypeptides OATP1B1 and OATP1B3. Mol Pharmacol. 2011;79(3):596–607.CrossRefPubMedGoogle Scholar
  40. 40.
    Jacquemin E, Hagenbuch B, Stieger B, Wolkoff AW, Meier PJ. Expression cloning of a rat liver Na(+)-independent organic anion transporter. Proc Natl Acad Sci U S A. 1994;91(1):133–7.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Sai Y, Kaneko Y, Ito S, Mitsuoka K, Kato Y, Tamai I, et al. Predominant contribution of organic anion transporting polypeptide OATP-B (OATP2B1) to apical uptake of estrone-3-sulfate by human intestinal Caco-2 cells. Drug Metab Dispos: Biologic Fate Chem. 2006;34(8):1423–31.CrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2017

Authors and Affiliations

  • Yi-an Bi
    • 1
  • Renato J. Scialis
    • 1
    • 2
  • Sarah Lazzaro
    • 1
  • Sumathy Mathialagan
    • 1
  • Emi Kimoto
    • 1
  • Julie Keefer
    • 1
  • Hui Zhang
    • 1
  • Anna M. Vildhede
    • 1
  • Chester Costales
    • 1
  • A. David Rodrigues
    • 1
  • Larry M. Tremaine
    • 1
  • Manthena V. S. Varma
    • 1
    Email author
  1. 1.Pharmacokinetics Dynamics and Metabolism, Pfizer Global Research and DevelopmentPfizer Inc.GrotonUSA
  2. 2.Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Research and DevelopmentPrincetonUSA

Personalised recommendations