Skip to main content

Advertisement

SpringerLink
Log in

Inter-Subject Variability in OCT1 Activity in 27 Batches of Cryopreserved Human Hepatocytes and Association with OCT1 mRNA Expression and Genotype

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

Abstract

Purpose

OCT1/3 (Organic Cation Transporter-1 and -3; SLC22A1/3) are transmembrane proteins localized at the basolateral membrane of hepatocytes. They mediate the uptake of cationic endogenous compounds and/or xenobiotics. The present study was set up to verify whether the previously observed variability in OCT activity in hepatocytes may be explained by inter-individual differences in OCT1/3 mRNA levels or OCT1 genotype.

Methods

Twenty-seven batches of cryopreserved human hepatocytes (male and female, age 24–88 y) were characterized for OCT activity, normalized OCT1/3 mRNA expression, and OCT1 genetic mutation. ASP+ (4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide) was used as probe substrate.

Results

ASP+ uptake ranged between 75 ± 61 and 2531 ± 202 pmol/(min × million cells). The relative OCT1 and OCT3 mRNA expression ranged between 0.007–0.46 and 0.0002–0.005, respectively. The presence of one or two nonfunctional SLC22A1 alleles was observed in 13 batches and these exhibited significant (p = 0.04) association with OCT1 and OCT3 mRNA expression. However, direct association between genotype and OCT activity could not be established.

Conclusion

mRNA levels and genotype of OCT only partially explain inter-individual variability in OCT-mediated transport. Our findings illustrate the necessity of in vitro transporter activity profiling for better understanding of inter-individual drug disposition behavior.

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

Similar content being viewed by others

Abbreviations

ASP+ :

4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide

CGamF:

Cholyglycylamidofluoresein

DDIs:

Drug-drug interactions

FBS:

Fetal bovine serum

FLX:

Fluoxetine

HEPES:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

MAF:

Minor allele frequency

NaFluo:

Sodium fluorescein

NTCP/Ntcp:

Na+-taurocholate co-transporting peptide (Human/Rat)

OATP/Oatp:

Organic anion transporting polypeptide (Human/Rat)

OCT/Oct:

Organic cation transporter (Human/Rat)

PBS:

Phosphate buffered saline

SLC:

SoLute Carrier

Tauro-nor-THCA-24-DBD:

(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3 benzoxadiazole)] amino-3α,7α,12α-trihydroxy-27-nor-5βcholestan-26-oyl)-2′-aminoethanesulfonate

References

  1. Hagenbuch B. Drug uptake systems in liver and kidney: a historic perspective. Clin Pharmacol Ther. 2010;87(1):39–47.

    Article  CAS  PubMed  Google Scholar 

  2. Zolk O, Fromm MF. Transporter-mediated drug uptake and efflux: important determinants of adverse drug reactions. Clin Pharmacol Ther. 2011;89(6):798–805.

    Article  CAS  PubMed  Google Scholar 

  3. Koepsell H, Lips K, Volk C. Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharm Res. 2007;24(7):1227–51.

    Article  CAS  PubMed  Google Scholar 

  4. Ahlin G, Karlsson J, Pedersen JM, Gustavsson L, Larsson R, Matsson P, et al. Structural requirements for drug inhibition of the liver specific human organic cation transport protein 1. J Med Chem. 2008;51(19):5932–42.

    Article  CAS  PubMed  Google Scholar 

  5. Minuesa G, Volk C, Molina-Arcas M, Gorboulev V, Erkizia I, Arndt P, et al. Transport of lamivudine (3TC) and high-affinity interaction of nucleoside reverse transcriptase inhibitors with human organic cation transporters 1, 2, and 3. J Pharmacol Exp Ther. 2009;392(1):252–61.

    Article  Google Scholar 

  6. Sogame Y, Kitamura A, Yabuki M, Komuro S. A comparison of uptake of metformin and phenformin mediated by hOCT1 in human hepatocytes. Biopharm Drug Dispos. 2009;30(8):476–84.

    Article  CAS  PubMed  Google Scholar 

  7. Sogame Y, Kitamura A, Yabuki M, Komuro S. Liver uptake of biguanides in rats. Biomed Pharmacother Bioméd Pharmacothérapie. 2011;65(6):451–5.

    Article  CAS  Google Scholar 

  8. Tzvetkov MV, Saadatmand AR, Lötsch J, Tegeder I, Stingl JC, Brockmöller J. Genetically polymorphic OCT1: another piece in the puzzle of the variable pharmacokinetics and pharmacodynamics of the opioidergic drug tramadol. Clin Pharmacol Ther. 2011;90(1):143–50.

    Article  CAS  PubMed  Google Scholar 

  9. Tzvetkov MV, dos Santos Pereira JN, Meineke I, Saadatmand AR, Stingl JC, Brockmöller J. Morphine is a substrate of the organic cation transporter OCT1 and polymorphisms in OCT1 gene affect morphine pharmacokinetics after codeine administration. Biochem Pharmacol. 2013;86(5):666–78.

    Article  CAS  PubMed  Google Scholar 

  10. Koepsell H. The SLC22 family with transporters of organic cations, anions and zwitterions. Mol Asp Med. 2013;34(2–3):413–35.

    Article  CAS  Google Scholar 

  11. Nies AT, Koepsell H, Damme K, Schwab M. Organic cation transporters (OCTs, MATEs), in vitro and in vivo evidence for the importance in drug therapy. In: Fromm MF, Kim RB, editors. Drug transporters [Internet]. Springer Berlin Heidelberg; 2011 [cited 2015 Jun 9]. p. 105–67. (Handbook of Experimental Pharmacology). Available from: http://link.springer.com/chapter/10.1007/978-3-642-14541-4_3.

  12. Shu Y, Sheardown SA, Brown C, Owen RP, Zhang S, Castro RA, et al. Effect of genetic variation in the organic cation transporter 1 (OCT1) on metformin action. J Clin Invest. 2007;117(5):1422–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Becker ML, Visser LE, van Schaik RHN, Hofman A, Uitterlinden AG, Stricker BHC. Genetic variation in the organic cation transporter 1 is associated with metformin response in patients with diabetes mellitus. Pharmacogenomics J. 2009;9(4):242–7.

    Article  CAS  PubMed  Google Scholar 

  14. Tzvetkov MV, Vormfelde SV, Balen D, Meineke I, Schmidt T, Sehrt D, et al. The effects of genetic polymorphisms in the organic cation transporters OCT1, OCT2, and OCT3 on the renal clearance of metformin. Clin Pharmacol Ther. 2009;86(3):299–306.

    Article  CAS  PubMed  Google Scholar 

  15. Chen L, Takizawa M, Chen E, Schlessinger A, Segenthelar J, Choi JH, et al. Genetic polymorphisms in organic cation transporter 1 (OCT1) in Chinese and Japanese populations exhibit altered function. J Pharmacol Exp Ther. 2010;335(1):42–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Matic M, de Wildt SN, Elens L, de Hoon JN, Annaert P, Tibboel D, et al. SLC22A1/OCT1 genotype affects O-desmethyltramadol exposure in newborn infants. Ther Drug Monit. 2016;38(4):487–92.

    Article  CAS  PubMed  Google Scholar 

  17. Shikata E, Yamamoto R, Takane H, Shigemasa C, Ikeda T, Otsubo K, et al. Human organic cation transporter (OCT1 and OCT2) gene polymorphisms and therapeutic effects of metformin. J Hum Genet. 2007;52(2):117–22.

    Article  CAS  PubMed  Google Scholar 

  18. Umehara K-I, Iwatsubo T, Noguchi K, Kamimura H. Functional involvement of organic cation transporter1 (OCT1/Oct1) in the hepatic uptake of organic cations in humans and rats. Xenobiotica Fate Foreign Compd Biol Syst. 2007;37(8):818–31.

    Article  CAS  Google Scholar 

  19. Roth M, Obaidat A, Hagenbuch B. OATPs, OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. Br J Pharmacol. 2012;165(5):1260–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Li L, Tu M, Yang X, Sun S, Wu X, Zhou H, et al. The contribution of human OCT1, OCT3, and CYP3A4 to nitidine chloride-induced hepatocellular toxicity. Drug Metab Dispos Biol Fate Chem. 2014;42(7):1227–34.

    Article  PubMed  Google Scholar 

  21. Zamek-Gliszczynski MJ, Xiong H, Patel NJ, Turncliff RZ, Pollack GM, Brouwer KLR. Pharmacokinetics of 5 (and 6)-carboxy-2′,7′-dichlorofluorescein and its diacetate promoiety in the liver. J Pharmacol Exp Ther. 2003;304(2):801–9.

    Article  CAS  PubMed  Google Scholar 

  22. Ye Z-W, Augustijns P, Annaert P. Cellular accumulation of cholyl-glycylamido-fluorescein in sandwich-cultured rat hepatocytes: kinetic characterization, transport mechanisms, and effect of human immunodeficiency virus protease inhibitors. Drug Metab Dispos Biol Fate Chem. 2008;36(7):1315–21.

    Article  CAS  PubMed  Google Scholar 

  23. de Waart DR, Häusler S, Vlaming MLH, Kunne C, Hänggi E, Gruss H-J, et al. Hepatic transport mechanisms of cholyl-L-lysyl-fluorescein. J Pharmacol Exp Ther. 2010;334(1):78–86.

    Article  CAS  PubMed  Google Scholar 

  24. Gui C, Obaidat A, Chaguturu R, Hagenbuch B. Development of a cell-based high-throughput assay to screen for inhibitors of organic anion transporting polypeptides 1B1 and 1B3. Curr Chem Genomics. 2010;4:1–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yamaguchi K, Murai T, Yabuuchi H, Hui S-P, Kurosawa T. Measurement of bile salt export pump transport activities using a fluorescent bile acid derivative. Drug Metab Pharmacokinet. 2010;25(2):214–9.

    Article  CAS  PubMed  Google Scholar 

  26. De Bruyn T, Fattah S, Stieger B, Augustijns P, Annaert P. Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3. J Pharm Sci. 2011;100(11):5018–30.

    Article  CAS  PubMed  Google Scholar 

  27. Mehrens T, Lelleck S, Cetinkaya I, Knollmann M, Hohage H, Gorboulev V, et al. The affinity of the organic cation transporter rOCT1 is increased by protein kinase C-dependent phosphorylation. J Am Soc Nephrol JASN. 2000;11(7):1216–24.

    CAS  PubMed  Google Scholar 

  28. Schlatter E, Mönnich V, Cetinkaya I, Mehrens T, Ciarimboli G, Hirsch JR, et al. The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP. J Membr Biol. 2002;189(3):237–44.

    Article  CAS  PubMed  Google Scholar 

  29. De Bruyn T, Ye Z-W, Peeters A, Sahi J, Baes M, Augustijns PF, et al. Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes. Eur J Pharm Sci Off J Eur Fed Pharm Sci. 2011;43(4):297–307.

    CAS  Google Scholar 

  30. Fattah S, Augustijns P, Annaert P. Age-dependent activity of the uptake transporters Ntcp and Oatp1b2 in male rat hepatocytes: from birth till adulthood. Drug Metab Dispos. 2015;43(1):1–8.

    Article  PubMed  Google Scholar 

  31. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods San Diego Calif. 2001;25(4):402–8.

    Article  CAS  Google Scholar 

  32. Seitz T, Stalmann R, Dalila N, Chen J, Pojar S, Dos Santos Pereira JN, et al. Global genetic analyses reveal strong inter-ethnic variability in the loss of activity of the organic cation transporter OCT1. Genome Med. 2015;7:56.

    Article  PubMed  PubMed Central  Google Scholar 

  33. De Bruyn T, Sempels W, Snoeys J, Holmstock N, Chatterjee S, Stieger B, et al. Confocal imaging with a fluorescent bile acid analogue closely mimicking hepatic taurocholate disposition. J Pharm Sci. 2014;103(6):1872–81.

    Article  CAS  PubMed  Google Scholar 

  34. Yasujima T, Ohta K, Inoue K, Ishimaru M, Yuasa H. Evaluation of 4′,6-diamidino-2-phenylindole as a fluorescent probe substrate for rapid assays of the functionality of human multidrug and toxin extrusion proteins. Drug Metab Dispos Biol Fate Chem. 2010;38(4):715–21.

    Article  CAS  PubMed  Google Scholar 

  35. Soars MG, McGinnity DF, Grime K, Riley RJ. The pivotal role of hepatocytes in drug discovery. Chem Biol Interact. 2007;168(1):2–15.

    Article  CAS  PubMed  Google Scholar 

  36. Maeda K, Sugiyama Y. The use of hepatocytes to investigate drug uptake transporters. Methods Mol Biol Clifton NJ. 2010;640:327–53.

    Article  CAS  Google Scholar 

  37. Zhang D, Surapaneni S. ADME-enabling technologies in drug design and development. John Wiley & Sons; 2012. p. 625.

  38. Ebner T, Ishiguro N, Taub ME. The use of transporter probe drug cocktails for the assessment of transporter-based drug–drug interactions in a Clinical setting—proposal of a four component transporter cocktail. J Pharm Sci. 2015;104(9):3220–8.

    Article  CAS  PubMed  Google Scholar 

  39. Jigorel E, Vee ML, Boursier-Neyret C, Bertrand M, Fardel O. Functional expression of sinusoidal drug transporters in primary human and rat hepatocytes. Drug Metab Dispos. 2005;33(10):1418–22.

    Article  CAS  PubMed  Google Scholar 

  40. Badolo L, Rasmussen LM, Hansen HR, Sveigaard C. Screening of OATP1B1/3 and OCT1 inhibitors in cryopreserved hepatocytes in suspension. Eur J Pharm Sci. 2010;40(4):282–8.

    Article  CAS  PubMed  Google Scholar 

  41. Haenisch B, Drescher E, Thiemer L, Xin H, Giros B, Gautron S, et al. Interaction of antidepressant and antipsychotic drugs with the human organic cation transporters hOCT1, hOCT2 and hOCT3. Naunyn Schmiedeberg’s Arch Pharmacol. 2012;385(10):1017–23.

    Article  CAS  Google Scholar 

  42. Hilgendorf C, Ahlin G, Seithel A, Artursson P, Ungell A-L, Karlsson J. Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines. Drug Metab Dispos Biol Fate Chem. 2007;35(8):1333–40.

    Article  CAS  PubMed  Google Scholar 

  43. Vildhede A, Wiśniewski JR, Norén A, Karlgren M, Artursson P. Comparative proteomic analysis of human liver tissue and isolated hepatocytes with a focus on proteins determining drug exposure. J Proteome Res. 2015;14(8):3305–14.

    Article  CAS  PubMed  Google Scholar 

  44. Artursson P, Matsson P, Karlgren M. In vitro characterization of interactions with drug transporting proteins. In: Sugiyama Y, Steffansen B, editors. Transporters in drug development [Internet]. Springer New York; 2013 [cited 2015 Mar 2]. p. 37–65. (AAPS Advances in the Pharmaceutical Sciences Series). Available from: http://link.springer.com/chapter/10.1007/978-1-4614-8229-1_3.

  45. Lundquist P, Lööf J, Sohlenius-Sternbeck A-K, Floby E, Johansson J, Bylund J, et al. The impact of solute carrier (SLC) drug uptake transporter loss in human and rat cryopreserved hepatocytes on clearance predictions. Drug Metab Dispos Biol Fate Chem. 2014;42(3):469–80.

    Article  PubMed  Google Scholar 

  46. Nies AT, Koepsell H, Winter S, Burk O, Klein K, Kerb R, et al. Expression of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) is affected by genetic factors and cholestasis in human liver. Hepatology. 2009;50(4):1227–40.

    Article  CAS  PubMed  Google Scholar 

  47. Choi M-K, Song I-S. Organic cation transporters and their pharmacokinetic and Pharmacodynamic consequences. Drug Metab Pharmacokinet. 2008;23(4):243–53.

    Article  PubMed  Google Scholar 

  48. Shu Y, Brown C, Castro R, Shi R, Lin E, Owen R, et al. Effect of genetic variation in the organic cation transporter 1, OCT1, on metformin pharmacokinetics. Clin Pharmacol Ther. 2008;83(2):273–80.

    Article  CAS  PubMed  Google Scholar 

  49. Ahlin G, Chen L, Lazorova L, Chen Y, Ianculescu AG, Davis RL, et al. Genotype-dependent effects of inhibitors of the organic cation transporter, OCT1: predictions of metformin interactions. Pharmacogenomics J. 2011;11(6):400–11.

    Article  CAS  PubMed  Google Scholar 

  50. Leschziner GD, Andrew T, Pirmohamed M, Johnson MR. ABCB1 genotype and PGP expression, function and therapeutic drug response: a critical review and recommendations for future research. Pharmacogenomics J. 2007;7(3):154–79.

    Article  CAS  PubMed  Google Scholar 

  51. Kim M-H, Shin HJ, Lim SJ, Park J-S, Lee S-S, Song I-S, et al. Inter-individual variability in OCT1 expression and its relationship with OCT1 genotype in liver samples from a Korean population. Drug Metab Pharmacokinet. 2012;27(5):530–5.

    Article  CAS  PubMed  Google Scholar 

  52. Nies AT, Niemi M, Burk O, Winter S, Zanger UM, Stieger B, et al. Genetics is a major determinant of expression of the human hepatic uptake transporter OATP1B1, but not of OATP1B3 and OATP2B1. Genome Med. 2013;5(1):1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Zhou T, Hu M, Cost M, Poloyac S, Rohan L. Short communication: expression of transporters and metabolizing enzymes in the female lower genital tract: implications for microbicide research. AIDS Res Hum Retrovir. 2013;29(11):1496–503.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kuhlmann JB, Wensing G, Kuhlmann J. Correlation of genotype, phenotype, and mRNA expression of CYP2D6 and CYP2C19 in peripheral blood leukocytes (PBLs). Int J Clin Pharmacol Ther. 2014;52(2):143–50.

    Article  CAS  PubMed  Google Scholar 

  55. Prasad B, Lai Y, Lin Y, Unadkat JD. Interindividual variability in the hepatic expression of the human breast cancer resistance protein (BCRP/ABCG2): effect of age, sex, and genotype. J Pharm Sci. 2013;102(3):787–93.

    Article  CAS  PubMed  Google Scholar 

  56. Niemi M, Arnold KA, Backman JT, Pasanen MK, Gödtel-Armbrust U, Wojnowski L, et al. Association of genetic polymorphism in ABCC2 with hepatic multidrug resistance-associated protein 2 expression and pravastatin pharmacokinetics. Pharmacogenet Genomics. 2006;16(11):801–8.

    Article  CAS  PubMed  Google Scholar 

  57. Urban TJ, Sebro R, Hurowitz EH, Leabman MK, Badagnani I, Lagpacan LL, et al. Functional genomics of membrane transporters in human populations. Genome Res. 2006;16(2):223–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments and Disclosures

The authors would like to acknowledge professor Per Artursson for his scientific input and critical review of this manuscript. This work is partly supported by internal funds of the KU Leuven Drug Delivery and Disposition laboratory. The authors declare that there is no conflict of interest.

Author information

Authors and Affiliations

  1. Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Campus Gasthuisberg O&N II Herestraat 49 Box 921, 3000, Leuven, Belgium

    Sarinj Fattah, Patrick Augustijns & Pieter Annaert

  2. Laboratory of Cell Metabolism, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium

    Abhijit Babaji Shinde & Myriam Baes

  3. Department Clinical Chemistry, Erasmus University Medical Centre, Rotterdam, Netherlands

    Maja Matic & Ron H. N. van Schaik

  4. Intensive Care and Department of Surgery, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands

    Maja Matic & Karel Allegaert

  5. Department of Development and Regeneration, KU Leuven, Leuven, Belgium

    Karel Allegaert

  6. KaLy-Cell, Plobsheim, France

    Celine Parmentier & Lysiane Richert

  7. Université de Franche-Comté, 4267, Besançon, EA, France

    Lysiane Richert

Authors
  1. Sarinj Fattah
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abhijit Babaji Shinde
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Maja Matic
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Myriam Baes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ron H. N. van Schaik
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Karel Allegaert
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Celine Parmentier
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lysiane Richert
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Patrick Augustijns
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Pieter Annaert
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pieter Annaert.

Electronic supplementary material

ESM 1

(DOCX 123 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fattah, S., Shinde, A.B., Matic, M. et al. Inter-Subject Variability in OCT1 Activity in 27 Batches of Cryopreserved Human Hepatocytes and Association with OCT1 mRNA Expression and Genotype. Pharm Res 34, 1309–1319 (2017). https://doi.org/10.1007/s11095-017-2148-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-017-2148-9

KEY WORDS

Search

Navigation