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Characterization of Aripiprazole Uptake Transporter in the Blood-Brain Barrier Model hCMEC/D3 Cells by Targeted siRNA Screening

Abstract

Aim

Identification of blood-brain barrier (BBB) uptake transporters is a major challenge in the research and development of central nervous system (CNS) drugs. However, conventional methods that consider known drug uptake characteristics have failed at identifying the responsible transporter molecule. The present study aimed at identifying aripiprazole uptake transporters in BBB model hCMEC/D3 cells using a knockdown screening study targeting various transporters, including uncharacterized ones.

Methods

We evaluated the effect of 214 types of siRNA targeting transporters on the uptake of aripiprazole, an atypical antipsychotic drug, in hCMEC/D3 cells. Aripiprazole uptake was determined using Xenopus oocytes expressing the candidate genes extracted from the siRNA screening assay.

Results

The estimated unbound brain to plasma concentration ratio (Kp,uu,brain) of aripiprazole was estimated as 0.67 in wild-type mice and 1.94 in abcb1a/1b/abcg2 knockout mice, suggesting the involvement of both uptake and efflux transporters in BBB permeation. According to siRNA knockdown screening studies, organic cation/carnitine transporter 2 (OCTN2) and long-chain fatty acid transporter 1 (FATP1) were identified as candidate genes. The uptake of aripiprazole by hCMEC/D3 cells was decreased by OCTN2 inhibitors, but not by FATP1 inhibitors. A partially increased uptake of aripiprazole was observed in OCTN2-expressing Xenopus oocytes. Finally, to evaluate transporter-mediated BBB permeation of drugs, the reported and estimated Kp,uu,brain values were summarized.

Conclusions

A knockdown screening study in combination with Kp,uu,brain values showed that aripiprazole was a potential substrate of OCTN2. The technique described in this study can be applied to identifying novel BBB transporters for CNS drugs.

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References

  1. Tsuji A, Tamai I. Carrier-mediated or specialized transport of drugs across the blood-brain barrier. Adv Drug Deliv Rev. 1999;36(2-3):277–90.

    CAS  PubMed  Article  Google Scholar 

  2. Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis. 2010;37(1):13–25.

    CAS  PubMed  Article  Google Scholar 

  3. Mueckler M. Facilitative glucose transporters. Eur J Biochem. 1994;219(3):713–25.

    CAS  PubMed  Article  Google Scholar 

  4. Kageyama T, Nakamura M, Matsuo A, Yamasaki Y, Takakura Y, Hashida M, et al. The 4F2hc/LAT1 complex transports L-DOPA across the blood-brain barrier. Brain Res. 2000;879(1-2):115–21.

    CAS  PubMed  Article  Google Scholar 

  5. Kido Y, Tamai I, Uchino H, Suzuki F, Sai Y, Tsuji A. Molecular and functional identification of large neutral amino acid transporters LAT1 and LAT2 and their pharmacological relevance at the blood-brain barrier. J Pharm Pharmacol. 2001;53(4):497–503.

    CAS  PubMed  Article  Google Scholar 

  6. Anraku Y, Kuwahara H, Fukusato Y, Mizoguchi A, Ishii T, Nitta K, et al. Glycaemic control boosts glucosylated nanocarrier crossing the BBB into the brain. Nat Commun. 2017;8(1):1001.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. Mochizuki T, Mizuno T, Kurosawa T, Yamaguchi T, Higuchi K, Tega Y, et al. Functional investigation of solute carrier family 35, member F2, in three cellular models of the primate blood-brain barrier. Drug Metab Dispos. 2021;49(1):3–11.

    CAS  PubMed  Article  Google Scholar 

  8. Okura T, Hattori A, Takano Y, Sato T, Hammarlund-Udenaes M, Terasaki T, et al. Involvement of the pyrilamine transporter, a putative organic cation transporter, in blood-brain barrier transport of oxycodone. Drug Metab Dispos. 2008;36(10):2005–13.

    CAS  PubMed  Article  Google Scholar 

  9. Sadiq MW, Borgs A, Okura T, Shimomura K, Kato S, Deguchi Y, et al. Diphenhydramine active uptake at the blood-brain barrier and its interaction with oxycodone in vitro and in vivo. J Pharm Sci. 2011;100(9):3912–23.

    CAS  PubMed  Article  Google Scholar 

  10. Shimomura K, Okura T, Kato S, Couraud PO, Schermann JM, Terasaki T, et al. Functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood-brain barrier model. Fluids Barriers CNS. 2013;10(1):8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Yamazaki M, Terasaki T, Yoshioka K, Nagata O, Kato H, Ito Y, et al. Carrier-mediated transport of H1-antagonist at the blood-brain barrier: mepyramine uptake into bovine brain capillary endothelial cells in primary monolayer cultures. Pharm Res. 1994;11(7):975–8.

    CAS  PubMed  Article  Google Scholar 

  12. Ishiguro N, Nozawa T, Tsujihata A, Saito A, Kishimoto W, Yokoyama K, et al. Influx and efflux transport of H1-antagonist epinastine across the blood-brain barrier. Drug Metab Dispos. 2004;32(5):519–24.

    CAS  PubMed  Article  Google Scholar 

  13. Hiranaka S, Tega Y, Higuchi K, Kurosawa T, Deguchi Y, Arata M, et al. Design, synthesis, and blood-brain barrier transport study of pyrilamine derivatives as histone deacetylase inhibitors. ACS Med Chem Lett. 2018;9(9):884–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. Geier EG, Chen EC, Webb A, Papp AC, Yee SW, Sadee W, et al. Profiling solute carrier transporters in the human blood-brain barrier. Clin Pharmacol Ther. 2013;94(6):636–9.

    CAS  PubMed  Article  Google Scholar 

  15. Ohtsuki S, Ikeda C, Uchida Y, Sakamoto Y, Miller F, Glacial F, et al. Quantitative targeted absolute proteomic analysis of transporters, receptors and junction proteins for validation of human cerebral microvascular endothelial cell line hCMEC/D3 as a human blood-brain barrier model. Mol Pharm. 2013;10(1):289–96.

    CAS  PubMed  Article  Google Scholar 

  16. Uchida Y, Ohtsuki S, Katsukura Y, Ikeda C, Suzuki T, Kamiie J, et al. Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. J Neurochem. 2011;117(2):333–45.

    CAS  PubMed  Article  Google Scholar 

  17. Andre P, Debray M, Scherrmann JM, Cisternino S. Clonidine transport at the mouse blood-brain barrier by a new H+ antiporter that interacts with addictive drugs. J Cereb Blood Flow Metab. 2009;29(7):1293–304.

    CAS  PubMed  Article  Google Scholar 

  18. Okura T, Ito R, Ishiguro N, Tamai I, Deguchi Y. Blood-brain barrier transport of pramipexole, a dopamine D2 agonist. Life Sci. 2007;80(17):1564–71.

    CAS  PubMed  Article  Google Scholar 

  19. Okura T, Higuchi K, Kitamura A, Deguchi Y. Proton-coupled organic cation antiporter-mediated uptake of apomorphine enantiomers in human brain capillary endothelial cell line hCMEC/D3. Biol Pharm Bull. 2014;37(2):286–91.

    CAS  PubMed  Article  Google Scholar 

  20. Higuchi K, Kitamura A, Okura T, Deguchi Y. Memantine transport by a proton-coupled organic cation antiporter in hCMEC/D3 cells, an in vitro human blood-brain barrier model. Drug Metab Pharmacokinet. 2015;30(2):182–7.

    CAS  PubMed  Article  Google Scholar 

  21. Kurosawa T, Higuchi K, Okura T, Kobayashi K, Kusuhara H, Deguchi Y. Involvement of proton-coupled organic cation antiporter in Varenicline transport at blood-brain barrier of rats and in human brain capillary endothelial cells. J Pharm Sci. 2017;106(9):2576–82.

    CAS  PubMed  Article  Google Scholar 

  22. Liu X, Chen C, Smith BJ. Progress in brain penetration evaluation in drug discovery and development. J Pharmacol Exp Ther. 2008;325(2):349–56.

    CAS  PubMed  Article  Google Scholar 

  23. Tamai I, Tsuji A. Transporter-mediated permeation of drugs across the blood-brain barrier. J Pharm Sci. 2000;89(11):1371–88.

    CAS  PubMed  Article  Google Scholar 

  24. Friden M, Gupta A, Antonsson M, Bredberg U, Hammarlund-Udenaes M. In vitro methods for estimating unbound drug concentrations in the brain interstitial and intracellular fluids. Drug Metab Dispos. 2007;35(9):1711–9.

    CAS  PubMed  Article  Google Scholar 

  25. Raub TJ, Wishart GN, Kulanthaivel P, Staton BA, Ajamie RT, Sawada GA, et al. Brain exposure of two selective dual CDK4 and CDK6 inhibitors and the antitumor activity of CDK4 and CDK6 inhibition in combination with Temozolomide in an intracranial glioblastoma xenograft. Drug Metab Dispos. 2015;43(9):1360–71.

    CAS  PubMed  Article  Google Scholar 

  26. Summerfield SG, Zhang Y, Liu H. Examining the uptake of central nervous system drugs and candidates across the blood-brain barrier. J Pharmacol Exp Ther. 2016;358(2):294–305.

    CAS  PubMed  Article  Google Scholar 

  27. Nagaya Y, Nozaki Y, Kobayashi K, Takenaka O, Nakatani Y, Kusano K, et al. Utility of cerebrospinal fluid drug concentration as a surrogate for unbound brain concentration in nonhuman primates. Drug Metab Pharmacokinet. 2014;29(5):419–26.

    PubMed  Article  CAS  Google Scholar 

  28. Bundgaard C, Jensen CJ, Garmer M. Species comparison of in vivo P-glycoprotein-mediated brain efflux using mdr1a-deficient rats and mice. Drug Metab Dispos. 2012;40(3):461–6.

    CAS  PubMed  Article  Google Scholar 

  29. Nagasaka Y, Sano T, Oda K, Kawamura A, Usui T. Impact of genetic deficiencies of P-glycoprotein and breast cancer resistance protein on pharmacokinetics of aripiprazole and dehydroaripiprazole. Xenobiotica. 2014;44(10):926–32.

    CAS  PubMed  Article  Google Scholar 

  30. Liu H, Chen Y, Huang L, Sun X, Fu T, Wu S, et al. Drug distribution into peripheral nerve. J Pharmacol Exp Ther. 2018;365(2):336–45.

    CAS  PubMed  Article  Google Scholar 

  31. Choo EF, Ly J, Chan J, Shahidi-Latham SK, Messick K, Plise E, et al. Role of P-glycoprotein on the brain penetration and brain pharmacodynamic activity of the MEK inhibitor cobimetinib. Mol Pharm. 2014;11(11):4199–207.

    CAS  PubMed  Article  Google Scholar 

  32. Choo EF, Belvin M, Boggs J, Deng Y, Hoeflich KP, Ly J, et al. Preclinical disposition of GDC-0973 and prospective and retrospective analysis of human dose and efficacy predictions. Drug Metab Dispos. 2012;40(5):919–27.

    CAS  PubMed  Article  Google Scholar 

  33. Huang L, Li X, Roberts J, Janosky B, Lin MH. Differential role of P-glycoprotein and breast cancer resistance protein in drug distribution into brain, CSF and peripheral nerve tissues in rats. Xenobiotica. 2015;45(6):547–55.

    CAS  PubMed  Article  Google Scholar 

  34. Kalvass JC, Maurer TS, Pollack GM. Use of plasma and brain unbound fractions to assess the extent of brain distribution of 34 drugs: comparison of unbound concentration ratios to in vivo p-glycoprotein efflux ratios. Drug Metab Dispos. 2007;35(4):660–6.

    CAS  PubMed  Article  Google Scholar 

  35. Uchida Y, Ohtsuki S, Kamiie J, Terasaki T. Blood-brain barrier (BBB) pharmacoproteomics: reconstruction of in vivo brain distribution of 11 P-glycoprotein substrates based on the BBB transporter protein concentration, in vitro intrinsic transport activity, and unbound fraction in plasma and brain in mice. J Pharmacol Exp Ther. 2011;339(2):579–88.

    CAS  PubMed  Article  Google Scholar 

  36. Maurer TS, Debartolo DB, Tess DA, Scott DO. Relationship between exposure and nonspecific binding of thirty-three central nervous system drugs in mice. Drug Metab Dispos. 2005;33(1):175–81.

    CAS  PubMed  Article  Google Scholar 

  37. Doran A, Obach RS, Smith BJ, Hosea NA, Becker S, Callegari E, et al. The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1A/1B knockout mouse model. Drug Metab Dispos. 2005;33(1):165–74.

    CAS  PubMed  Article  Google Scholar 

  38. Kitamura A, Okura T, Higuchi K, Deguchi Y. Cocktail-dosing microdialysis study to simultaneously assess delivery of multiple organic-cationic drugs to the brain. J Pharm Sci. 2016;105(2):935–40.

    CAS  PubMed  Article  Google Scholar 

  39. Sadiq MW, Uchida Y, Hoshi Y, Tachikawa M, Terasaki T, Hammarlund-Udenaes M. Validation of a P-glycoprotein (P-gp) humanized mouse model by integrating selective absolute quantification of human MDR1, mouse Mdr1a and Mdr1b protein expressions with in vivo functional analysis for blood-brain barrier transport. PLoS One. 2015;10(5):e0118638.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  40. Kim RB, Fromm MF, Wandel C, Leake B, Wood AJ, Roden DM, et al. The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J Clin Invest. 1998;101(2):289–94.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. Chen C, Hanson E, Watson JW, Lee JS. P-glycoprotein limits the brain penetration of nonsedating but not sedating H1-antagonists. Drug Metab Dispos. 2003;31(3):312–8.

    CAS  PubMed  Article  Google Scholar 

  42. Kemper EM, Cleypool C, Boogerd W, Beijnen JH, van Tellingen O. The influence of the P-glycoprotein inhibitor zosuquidar trihydrochloride (LY335979) on the brain penetration of paclitaxel in mice. Cancer Chemother Pharmacol. 2004;53(2):173–8.

    CAS  PubMed  Article  Google Scholar 

  43. Laramy JK, Kim M, Parrish KE, Sarkaria JN, Elmquist WF. Pharmacokinetic assessment of cooperative efflux of the multitargeted kinase inhibitor Ponatinib across the blood-brain barrier. J Pharmacol Exp Ther. 2018;365(2):249–61.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. Hendrikse NH, Schinkel AH, de Vries EG, Fluks E, Van der Graaf WT, Willemsen AT, Vaalburg W, Franssen EJ. Complete in vivo reversal of P-glycoprotein pump function in the blood-brain barrier visualized with positron emission tomography. Br J Pharmacol. 1998;124(7):1413–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Tamai I, Ohashi R, Nezu J, Yabuuchi H, Oku A, Shimane M, et al. Molecular and functional identification of sodium ion-dependent, high affinity human carnitine transporter OCTN2. J Biol Chem. 1998;273(32):20378–82.

    CAS  PubMed  Article  Google Scholar 

  46. Arakawa H, Amezawa N, Kawakatsu Y, Tamai I. Renal Reabsorptive transport of uric acid precursor xanthine by URAT1 and GLUT9. Biol Pharm Bull. 2020;43(11):1792–8.

    CAS  PubMed  Article  Google Scholar 

  47. Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, Leveque M, et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J. 2005;19(13):1872–4.

    CAS  PubMed  Article  Google Scholar 

  48. Shirasaka Y, Sakane T, Yamashita S. Effect of P-glycoprotein expression levels on the concentration-dependent permeability of drugs to the cell membrane. J Pharm Sci. 2008;97(1):553–65.

    CAS  PubMed  Article  Google Scholar 

  49. Ohashi R, Tamai I, Yabuuchi H, Nezu JI, Oku A, Sai Y, et al. Na(+)-dependent carnitine transport by organic cation transporter (OCTN2): its pharmacological and toxicological relevance. J Pharmacol Exp Ther. 1999;291(2):778–84.

    CAS  PubMed  Google Scholar 

  50. Wagner CA, Lukewille U, Kaltenbach S, Moschen I, Broer A, Risler T, et al. Functional and pharmacological characterization of human Na(+)-carnitine cotransporter hOCTN2. Am J Physiol Renal Physiol. 2000;279(3):F584–91.

    CAS  PubMed  Article  Google Scholar 

  51. Ohashi R, Tamai I, Nezu JI, Nikaido H, Hashimoto N, Oku A, et al. Molecular and physiological evidence for multifunctionality of carnitine/organic cation transporter OCTN2. Mol Pharmacol. 2001;59(2):358–66.

    CAS  PubMed  Article  Google Scholar 

  52. Ohashi R, Tamai I, Inano A, Katsura M, Sai Y, Nezu J, et al. Studies on functional sites of organic cation/carnitine transporter OCTN2 (SLC22A5) using a Ser467Cys mutant protein. J Pharmacol Exp Ther. 2002;302(3):1286–94.

    CAS  PubMed  Article  Google Scholar 

  53. Ochiai Y, Uchida Y, Ohtsuki S, Tachikawa M, Aizawa S, Terasaki T. The blood-brain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport. J Neurochem. 2017;141(3):400–12.

    CAS  PubMed  Article  Google Scholar 

  54. Kido Y, Tamai I, Ohnari A, Sai Y, Kagami T, Nezu J, et al. Functional relevance of carnitine transporter OCTN2 to brain distribution of L-carnitine and acetyl-L-carnitine across the blood-brain barrier. J Neurochem. 2001;79(5):959–69.

    CAS  PubMed  Article  Google Scholar 

  55. Miecz D, Januszewicz E, Czeredys M, Hinton BT, Berezowski V, Cecchelli R, et al. Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood-brain barrier. J Neurochem. 2008;104(1):113–23.

    CAS  PubMed  Google Scholar 

  56. Inano A, Sai Y, Nikaido H, Hasimoto N, Asano M, Tsuji A, et al. Acetyl-L-carnitine permeability across the blood-brain barrier and involvement of carnitine transporter OCTN2. Biopharm Drug Dispos. 2003;24(8):357–65.

    CAS  PubMed  Article  Google Scholar 

  57. Kano T, Kato Y, Ito K, Ogihara T, Kubo Y, Tsuji A. Carnitine/organic cation transporter OCTN2 (Slc22a5) is responsible for renal secretion of cephaloridine in mice. Drug Metab Dispos. 2009;37(5):1009–16.

    CAS  PubMed  Article  Google Scholar 

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors declare no conflicts of interest.

Funding

This study was supported by a Grant-in-Aid for Early-Career Scientists (Grant number: 20 K16039), Grant-in-Aid for Transformative Research Areas (Grant number: 20H05745), Kanazawa University SAKIGAKE project 2020, The Uehara Memorial Foundation, and Takeda Science Foundation.

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Correspondence to Ikumi Tamai.

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Kadoguchi, M., Arakawa, H., Honda, R. et al. Characterization of Aripiprazole Uptake Transporter in the Blood-Brain Barrier Model hCMEC/D3 Cells by Targeted siRNA Screening. Pharm Res 39, 1549–1559 (2022). https://doi.org/10.1007/s11095-022-03223-z

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  • DOI: https://doi.org/10.1007/s11095-022-03223-z

KEY WORDS

  • aripiprazole
  • blood-brain barrier
  • distribution
  • screening
  • transporter