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Establishment and Characterization of the Transformants Stably-Expressing MDR1 Derived from Various Animal Species in LLC-PK1

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Purpose

Stable transformants expressing human multidrug resistance 1 (MDR1), monkey MDR1, canine MDR1, rat MDR1a, rat MDR1b, mouse mdr1a, and mouse mdr1b in LLC-PK1 were established to investigate species differences in P-glycoprotein (P-gp, ABCB1) mediated efflux activity.

Methods

The seven cDNAs of MDR1 from five animals were cloned, and their transformants stably expressing the series of MDR1 in LLC-PK1 were established. Transport studies of clarithromycin, daunorubicin, digoxin, erythromycin, etoposide, paclitaxel, propranolol, quinidine, ritonavir, saquinavir, verapamil, and vinblastine were performed by using these cells, and efflux activity was compared among the species.

Results

Except for propranolol, all compounds showed efflux activity in all transformants, and were judged to be substrates of P-gp. There were slight interspecies and interisoforms differences in the substrate recognition. However, the efflux ratio among the series of the MDR1 stably expressing cells showed good correlation as represented between human and monkey MDR1, and poor correlation as represented between human MDR1 and mouse mdr1a, and human and canine MDR1.

Conclusions

Results in the present study indicate that all MDR1 stably expressing cells have efflux activity for various P-gp substrates, and that interspecies differences and similarities of the P-gp substrate efflux activity may exist.

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Abbreviations

A:

apical

B:

basolateral

BA:

bioavailability

BSA:

bovine serum albumin

calcein AM:

calcein acetoxymethyl ester

cDNA:

complementary DNA

FBS:

fetal bovine serum

MDR:

multidrug resistance

PBS:

phosphate buffered saline

P-gp:

P-glycoprotein

RT-PCR:

reverse transcriptase polymerase chain reaction

References

  1. R. L. Juliano V. Ling (1976) ArticleTitleA surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants Biochim. Biophys. Acta 455 152–162 Occurrence Handle990323 Occurrence Handle1:CAS:528:DyaE28XlvV2qs70%3D Occurrence Handle10.1016/0005-2736(76)90160-7

    Article  PubMed  CAS  Google Scholar 

  2. F. Thiebaut T. Tsuruo H. Hamada M. M. Gottesman I. Pastan M. C. Willingham (1987) ArticleTitleCellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues Proc. Natl. Acad. Sci. USA 84 7735–7738 Occurrence Handle2444983 Occurrence Handle1:CAS:528:DyaL2sXmtlels7k%3D Occurrence Handle10.1073/pnas.84.21.7735

    Article  PubMed  CAS  Google Scholar 

  3. C. J. Matheny M. W. Lamb K. R. Brouwer G. M. Pollack (2001) ArticleTitlePharmacokinetic and pharmacodynamic implications of P-glycoprotein modulation Pharmacotherapy 21 778–796 Occurrence Handle11444575 Occurrence Handle1:CAS:528:DC%2BD3MXlsFSqtrY%3D Occurrence Handle10.1592/phco.21.9.778.34558

    Article  PubMed  CAS  Google Scholar 

  4. A. Sparreboom J. Asperen Particlevan U. Mayer A. H. Schinkel J. W. Smit D. K. Meijer P. Borst W. J. Nooijen J. H. Beijnen O. Tellingen Particlevan (1997) ArticleTitleLimited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine Proc. Natl. Acad. Sci. USA 94 2031–2035 Occurrence Handle9050899 Occurrence Handle1:CAS:528:DyaK2sXhslCjs7o%3D Occurrence Handle10.1073/pnas.94.5.2031

    Article  PubMed  CAS  Google Scholar 

  5. M. Sababi O. Borga U. Hultkvist-Bengtsson (2001) ArticleTitleThe role of P-glycoprotein in limiting intestinal regional absorption of digoxin in rats Eur. J. Pharm. Sci. 14 21–27 Occurrence Handle11457646 Occurrence Handle1:CAS:528:DC%2BD3MXltF2jt7g%3D Occurrence Handle10.1016/S0928-0987(01)00161-0

    Article  PubMed  CAS  Google Scholar 

  6. L. M. Chan S. Lowes B. H. Hirst (2004) ArticleTitleThe ABCs of drug transport in intestine and liver: efflux proteins limiting drug absorption and bioavailability Eur. J. Pharm. Sci. 21 25–51 Occurrence Handle14706810 Occurrence Handle1:CAS:528:DC%2BD2cXovFSr Occurrence Handle10.1016/j.ejps.2003.07.003

    Article  PubMed  CAS  Google Scholar 

  7. M. F. Fromm R. B. Kim C. M. Stein G. R. Wilkinson D. M. Roden (1999) ArticleTitleInhibition of P-glycoprotein-mediated drug transport: a unifying mechanism to explain the interaction between digoxin and quinidine Circulation 99 552–557 Occurrence Handle9927403 Occurrence Handle1:CAS:528:DyaK1MXht1Gnsr4%3D

    PubMed  CAS  Google Scholar 

  8. B. Greiner M. Eichelbaum P. Fritz H. P. Kreichgauer O. Richter ParticleVon J. Zundler H. K. Kroemer (1999) ArticleTitleThe role of intestinal P-glycoprotein in the interaction of digoxin and rifampin J. Clin. Invest. 104 147–153 Occurrence Handle10411543 Occurrence Handle1:CAS:528:DyaK1MXkslSmsb8%3D Occurrence Handle10.1172/JCI6663

    Article  PubMed  CAS  Google Scholar 

  9. A. Harrison A. Betts K. Fenner K. Beaumont A. Edgington S. Roffey J. Davis P. Comby P. Morgan (2004) ArticleTitleNonlinear oral pharmacokinetics of the α-antagonist 4-amino-5-(4-fluorophenyl)-6,7-dimethoxy-2-[4-(morpholinocarbonyl)-perhydro-1,4-diazepin-1-yl]quinoline in humans: use of preclinical data to rationalize clinical observations Drug Metab. Dispos. 32 197–204 Occurrence Handle14744941 Occurrence Handle1:CAS:528:DC%2BD2cXhsVWisbk%3D Occurrence Handle10.1124/dmd.32.2.197

    Article  PubMed  CAS  Google Scholar 

  10. A. H. Schinkel J. J. Smit O. Tellingen Particlevan J. H. Beijnen E. Wagenaar L. Deemter Particlevan C. A. Mol M. A. Valk Particlevan der E. C. Robanus-Maandag H. P. Rielete A. J. M. Berns P. Borst (1994) ArticleTitleDisruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood–brain barrier and to increased sensitivity to drugs Cell 77 491–502 Occurrence Handle7910522 Occurrence Handle1:CAS:528:DyaK2cXkt1Knsbc%3D Occurrence Handle10.1016/0092-8674(94)90212-7

    Article  PubMed  CAS  Google Scholar 

  11. A. H. Schinkel U. Mayer E. Wagenaar C. A. Mol L. Deemter Particlevan J. J. Smit M. A. Valk Particlevan der A. C. Voordouw H. Spits O. Tellingen Particlevan J. M. Zijlmans W. E. Fibbe P. Borst (1997) ArticleTitleNormal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins Proc. Natl. Acad. Sci. USA 94 4028–4033 Occurrence Handle9108099 Occurrence Handle1:CAS:528:DyaK2sXis1akurk%3D Occurrence Handle10.1073/pnas.94.8.4028

    Article  PubMed  CAS  Google Scholar 

  12. M. M. Gottesman I. Pastan (1993) ArticleTitleBiochemistry of multidrug resistance mediated by the multidrug transporter Annu. Rev. Biochem. 62 385–427 Occurrence Handle8102521 Occurrence Handle1:CAS:528:DyaK3sXlsFWnsbY%3D Occurrence Handle10.1146/annurev.bi.62.070193.002125

    Article  PubMed  CAS  Google Scholar 

  13. D. F. Tang-Wai S. Kajiji F. DiCapua D. Graaf Particlede I. B. Roninson P. Gros (1995) ArticleTitleHuman (MDR1) and mouse (mdr1, mdr3) P-glycoproteins can be distinguished by their respective drug resistance profiles and sensitivity to modulators Biochemistry 34 32–39 Occurrence Handle7819214 Occurrence Handle1:CAS:528:DyaK2MXivVelsLs%3D Occurrence Handle10.1021/bi00001a005

    Article  PubMed  CAS  Google Scholar 

  14. M. Yamazaki W. E. Neway T. Ohe I. Chen J. F. Rowe J. H. Hochman M. Chiba J. H. Lin (2001) ArticleTitle In vitro substrate identification studies for P-glycoprotein-mediated transport: species difference and predictability of in vivo results J. Pharmacol. Exp. Ther. 296 723–735 Occurrence Handle11181899 Occurrence Handle1:CAS:528:DC%2BD3MXhtlOlurg%3D

    PubMed  CAS  Google Scholar 

  15. S. Kumar G. Y. Kwei G. K. Poon S. A. Iliff Y. Wang Q. Chen R. B. Franklin V. Didolkar R. W. Wang M. Yamazaki S. H. Chiu J. H. Lin P. G. Pearson T. A. Baillie (2003) ArticleTitlePharmacokinetics and interactions of a novel antagonist of chemokine receptor 5 (CCR5) with ritonavir in rats and monkeys: role of CYP3A and P-glycoprotein J. Pharmacol. Exp. Ther. 304 1161–1171 Occurrence Handle12604693 Occurrence Handle1:CAS:528:DC%2BD3sXit1eis70%3D Occurrence Handle10.1124/jpet.102.045096

    Article  PubMed  CAS  Google Scholar 

  16. D. K. Walker S. Abel P. Comby G. J. Muirhead A. N. R. Nedderman D. A. Smith (2005) ArticleTitleSpecies differences in the disposition of the CCR5 antagonist, UK-427,857, a new potential treatment for HIV Drug Metab. Dispos. 33 587–595 Occurrence Handle15650075 Occurrence Handle1:CAS:528:DC%2BD2MXjtFCjs78%3D Occurrence Handle10.1124/dmd.104.002626

    Article  PubMed  CAS  Google Scholar 

  17. D. K. Yu (1999) ArticleTitleThe contribution of P-glycoprotein to pharmacokinetic drug–drug interactions J. Clin. Pharmacol. 39 1203–1211 Occurrence Handle10586385 Occurrence Handle1:CAS:528:DC%2BD3cXisVaktA%3D%3D Occurrence Handle10.1177/00912709922012006

    Article  PubMed  CAS  Google Scholar 

  18. A. Tsuji (2002) ArticleTitleTransporter-mediated drug interactions Drug Metab. Pharmacokinet. 17 253–274 Occurrence Handle15618677 Occurrence Handle1:CAS:528:DC%2BD38XpsVamt74%3D Occurrence Handle10.2133/dmpk.17.253

    Article  PubMed  CAS  Google Scholar 

  19. Y. Tanigawara N. Okamura M. Hirai M. Yasuhara K. Ueda N. Kioka T. Komano R. Hori (1992) ArticleTitleTransport of digoxin by human P-glycoprotein expressed in a porcine kidney epithelial cell line (LLC-PK1) J. Pharmacol. Exp. Ther. 263 840–845 Occurrence Handle1359120 Occurrence Handle1:CAS:528:DyaK3sXitlA%3D

    PubMed  CAS  Google Scholar 

  20. K. Ueda N. Okamura M. Hirai Y. Tanigawara T. Saeki N. Kioka T. Komano R. Hori (1992) ArticleTitleHuman P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone J. Biol. Chem. 267 24248–24252 Occurrence Handle1360010 Occurrence Handle1:CAS:528:DyaK38XmtVGgu78%3D

    PubMed  CAS  Google Scholar 

  21. M. Katoh M. Nakajima H. Yamazaki T. Yokoi (2000) ArticleTitleInhibitory potencies of 1,4-dihydropyridine calcium antagonists to P-glycoprotein-mediated transport: comparison with the effects on CYP3A4 Pharm. Res. 17 1189–1197 Occurrence Handle11145223 Occurrence Handle1:CAS:528:DC%2BD3MXit1Wg Occurrence Handle10.1023/A:1007568811691

    Article  PubMed  CAS  Google Scholar 

  22. Y. Adachi H. Suzuki Y. Sugiyama (2001) ArticleTitleComparative studies on in vitro methods for evaluating in vivo function of MDR1 P-glycoprotein Pharm. Res. 18 1660–1668 Occurrence Handle11785684 Occurrence Handle1:CAS:528:DC%2BD38XhvVagtw%3D%3D Occurrence Handle10.1023/A:1013358126640

    Article  PubMed  CAS  Google Scholar 

  23. D. S. Misfeldt M. J. Sanders (1981) ArticleTitleTransepithelial transport in cell culture: d-glucose transport by a pig kidney cell line (LLC-PK1) J. Membr. Biol. 59 13–18 Occurrence Handle7241572 Occurrence Handle1:CAS:528:DyaL3MXhvV2hsLc%3D Occurrence Handle10.1007/BF01870816

    Article  PubMed  CAS  Google Scholar 

  24. C. Fauth B. Rossier F. Roch-Ramel (1988) ArticleTitleTransport of tetraethylammonium by a kidney epithelial cell line (LLC-PK1) Am. J. Physiol. 254 F351–F357 Occurrence Handle3279818 Occurrence Handle1:CAS:528:DyaL1cXmtVyktbo%3D

    PubMed  CAS  Google Scholar 

  25. A. K. Fouda C. Fauth F. Roch-Ramel (1990) ArticleTitleTransport of organic cations by kidney epithelial cell line LLC-PK1 J. Pharmacol. Exp. Ther. 252 286–292 Occurrence Handle2137175 Occurrence Handle1:CAS:528:DyaK3cXhtFKhtbc%3D

    PubMed  CAS  Google Scholar 

  26. A. Takayama Y. Okazaki K. Fukuda M. Takano K. Inui R. Hori (1991) ArticleTitleTransport of cyclosporin A in kidney epithelial cell line (LLC-PK1) J. Pharmacol. Exp. Ther. 257 200–204 Occurrence Handle2019985 Occurrence Handle1:CAS:528:DyaK3MXkt1WmtLo%3D

    PubMed  CAS  Google Scholar 

  27. L. B. Goh K. J. Spears D. Yao A. Ayrton P. Morgan C. R. Wolf 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 Handle12429346 Occurrence Handle1:CAS:528:DC%2BD38XosFKls78%3D Occurrence Handle10.1016/S0006-2952(02)01355-2

    Article  PubMed  CAS  Google Scholar 

  28. C. Chen X. Liu B. J. Smith (2003) ArticleTitleUtility of mdr1-gene deficient mice in assessing the impact of P-glycoprotein on pharmacokinetics and pharmacodynamics in drug discovery and development Curr. Drug. Metab. 4 272–291 Occurrence Handle12871045 Occurrence Handle1:CAS:528:DC%2BD3sXmt1GhsrY%3D Occurrence Handle10.2174/1389200033489415

    Article  PubMed  CAS  Google Scholar 

  29. U. Wenzel D. Diehl M. Herget H. Daniel (1998) ArticleTitleEndogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells Am. J. Physiol. 275 C1573–C1579 Occurrence Handle9843719 Occurrence Handle1:CAS:528:DyaK1MXhsFGltg%3D%3D

    PubMed  CAS  Google Scholar 

  30. H. Saito K. Inui R. Hori (1986) ArticleTitleMechanisms of gentamicin transport in kidney epithelial cell line (LLC-PK1) J. Pharmacol. Exp. Ther. 238 1071–1076 Occurrence Handle3091808 Occurrence Handle1:CAS:528:DyaL28Xls1Cnt7c%3D

    PubMed  CAS  Google Scholar 

  31. A. Collett J. Tanianis-Hughes G. Warhurst (2004) ArticleTitleRapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro: a possible source of transporter mediated drug interactions? Biochem. Pharmacol. 68 783–790 Occurrence Handle15276086 Occurrence Handle1:CAS:528:DC%2BD2cXmtVWhs70%3D Occurrence Handle10.1016/j.bcp.2004.05.006

    Article  PubMed  CAS  Google Scholar 

  32. 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 Handle12948018 Occurrence Handle1:CAS:528:DC%2BD3sXmtVGrtLY%3D Occurrence Handle10.1023/A:1025049014674

    Article  PubMed  CAS  Google Scholar 

  33. J. H. Lin M. Yamazaki (2003) ArticleTitleRole of P-glycoprotein in pharmacokinetics Clin. Pharmacokinet. 42 59–98 Occurrence Handle12489979 Occurrence Handle1:CAS:528:DC%2BD3sXptVymuw%3D%3D Occurrence Handle10.2165/00003088-200342010-00003

    Article  PubMed  CAS  Google Scholar 

  34. W. L. Chiou P. W. Buehler (2002) ArticleTitleComparison of oral absorption and bioavailability of drugs between monkey and human Pharm. Res. 19 868–874 Occurrence Handle12134959 Occurrence Handle1:CAS:528:DC%2BD38XltVSgtro%3D Occurrence Handle10.1023/A:1016169202830

    Article  PubMed  CAS  Google Scholar 

  35. C. P. Siegers S. Denker B. Steffen W. Jelkmann (1987) ArticleTitleBiotransformation enzymes in two renal epithelial cell lines (LLC-PK1 and RK-L) Mol. Toxicol. 1 335–339 Occurrence Handle3509689 Occurrence Handle1:CAS:528:DyaL1MXlvV2jtbY%3D

    PubMed  CAS  Google Scholar 

  36. J. J. Gribar M. Ramachandra C. A. Hrycyna S. Dey S. V. Ambudkar (2000) ArticleTitleFunctional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system J. Membr. Biol. 173 203–214 Occurrence Handle10667916 Occurrence Handle1:CAS:528:DC%2BD3cXht1Gnt7c%3D Occurrence Handle10.1007/s002320001020

    Article  PubMed  CAS  Google Scholar 

  37. A. H. Schinkel S. Kemp M. Dolle G. Rudenko E. Wagenaar (1993) ArticleTitle N-Glycosylation and deletion mutants of the human MDR1 P-glycoprotein J. Biol. Chem. 268 7474–7481 Occurrence Handle8096511 Occurrence Handle1:CAS:528:DyaK3sXit1ylsb8%3D

    PubMed  CAS  Google Scholar 

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Acknowledgments

We are deeply thankful to Professor Dr. Yusuke Tanigawara, Keio University, and Professor Dr. Kazumitsu Ueda, Kyoto University, for supplying LLC-GA5-COL300 cells as a positive control to establish the MDR1 stably expressing cells.

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Authors and Affiliations

  1. Development Research Center, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-17-85 Juso-Honmachi, Yodogawa-ku, Osaka, 532-8686, Japan

    Toshiyuki Takeuchi, Tomoaki Higuchi, Takuya Ebihara & Satoru Asahi

  2. Discovery Research Center, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-17-85 Juso-Honmachi, Yodogawa-ku, Osaka, 532-8686, Japan

    Sumie Yoshitomi, Keiko Ikemoto & Shin-Ichi Niwa

  3. Department of Graduate School of Medical Science, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan

    Miki Katoh & Tsuyoshi Yokoi

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  1. Toshiyuki Takeuchi
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  2. Sumie Yoshitomi
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  3. Tomoaki Higuchi
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  4. Keiko Ikemoto
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Correspondence to Toshiyuki Takeuchi.

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Takeuchi, T., Yoshitomi, S., Higuchi, T. et al. Establishment and Characterization of the Transformants Stably-Expressing MDR1 Derived from Various Animal Species in LLC-PK1 . Pharm Res 23, 1460–1472 (2006). https://doi.org/10.1007/s11095-006-0285-7

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