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Drug Metabolism and Bioanalysis Letters

Author(s): Elin Eneberg*, Christopher R. Jones, Thomas Jensen, Kristine Langthaler and Christoffer Bundgaard

DOI: 10.2174/1872312815666220222091032

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Practical Application of Rodent Transporter Knockout Models to Assess Brain Penetration in Drug Discovery

Page: [12 - 21] Pages: 10

  • * (Excluding Mailing and Handling)

Abstract

Background and Objective: Compound X is a drug candidate for the treatment of neurodegenerative diseases. Its brain distribution was evaluated as part of the lead identification and optimization activities undertaken in early drug discovery.

Methods: The brain distribution of compound X was studied in genetic transporter knockout rodent models, in vivo models with a chemical inhibitor, and in vitro transporter cell systems.

Results: Compound X was found to be a substrate for human Breast Cancer-Resistance Protein (BCRP) in vitro (efflux ratio 8.1) and rodent Bcrp in vivo (Kp, uuKO/Kp, uuWT = 0.15/0.057 = 2.7, p< 0.05) but not a substrate for human P-glycoprotein (P-gp) in vitro (efflux ratio 1.0) nor rodent P-gp in vivo (Kp, uuKO/Kp, uuWT = 0.056/ 0.051 = 1.1, p> 0.05). When both transporters were knocked out in vivo, Kp, uu increased to 0.51±0.02. A similar pattern observed across compounds with related chemistry corroborating the structure-activity relationship.

Conclusion: While in vitro assays showed compound X to be a substrate for human BCRP and not P-gp, in vivo studies indicated a synergistic effect between rodent efflux transporters. However, this only accounted for ~50% of restricted BBB-transport, suggesting involvement of other efflux transporters. Considering Kp, uu as a key criterion for assessing the technical quality of CNS candidates before progression into clinical development, it is important to identify relevant screening assays for a better understanding of low Kp, uu and brain distribution in pre-clinical models for translation to humans.

Keywords: CNS, blood-brain-barrier, ABC transporters, P-gp, BCRP, Kp, uu, drug discovery.

Graphical Abstract

[1]
McGonigle, P. Animal models of CNS disorders. Biochem. Pharmacol., 2014, 87(1), 140-149.
[http://dx.doi.org/10.1016/j.bcp.2013.06.016] [PMID: 23811310]
[2]
Hammarlund-Udenaes, M.; de Lange, E.C.M.; Thorne, R.G. Drug delivery to the brain: Physiological concepts, methodologies and ap-proaches.AAPS Advances in the Pharmaceutical Sciences Series; Springer New York: New York, NY, 2014, p. 10.
[http://dx.doi.org/10.1007/978-1-4614-9105-7]
[3]
Abbott, N.J.; Rönnbäck, L.; Hansson, E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat. Rev. Neurosci., 2006, 7(1), 41-53.
[http://dx.doi.org/10.1038/nrn1824] [PMID: 16371949]
[4]
Hammarlund-Udenaes, M.; Bredberg, U.; Fridén, M. Methodologies to assess brain drug delivery in lead optimization. Curr. Top. Med. Chem., 2009, 9(2), 148-162.
[http://dx.doi.org/10.2174/156802609787521607] [PMID: 19200002]
[5]
Jeffrey, P.; Summerfield, S. Assessment of the blood-brain barrier in CNS drug discovery. Neurobiol. Dis., 2010, 37(1), 33-37.
[http://dx.doi.org/10.1016/j.nbd.2009.07.033] [PMID: 19664709]
[6]
Huang, L.; Wells, M.C.; Zhao, Z. A practical perspective on the evaluation of small molecule CNS penetration in drug discovery. Drug Metab. Lett., 2019, 13(2), 78-94.
[http://dx.doi.org/10.2174/1872312813666190311125652] [PMID: 30854983]
[7]
Urquhart, B.L.; Kim, R.B. Blood-brain barrier transporters and response to CNS-active drugs. Eur. J. Clin. Pharmacol., 2009, 65(11), 1063-1070.
[http://dx.doi.org/10.1007/s00228-009-0714-8] [PMID: 19727692]
[8]
Stouch, T.R.; Gudmundsson, O. Progress in understanding the structure-activity relationships of P-glycoprotein. Adv. Drug Deliv. Rev., 2002, 54(3), 315-328.
[http://dx.doi.org/10.1016/S0169-409X(02)00006-6] [PMID: 11922950]
[9]
Estrada, E.; Molina, E.; Nodarse, D.; Uriarte, E. Structural contributions of substrates to their binding to P-Glycoprotein. A TOPS-MODE approach. Curr. Pharm. Des., 2010, 16(24), 2676-2709.
[http://dx.doi.org/10.2174/138161210792389243] [PMID: 20642431]
[10]
Hodgetts, K.J. Case studies of CNS drug optimization-medicinal chemistry and CNS biology perspectives. In: Blood-Brain Barrier in Drug Discovery; Di, L.; Kerns, E.H., Eds.; John Wiley & Sons, Inc.: Hoboken, NJ, 2015; pp. 425-445.
[http://dx.doi.org/10.1002/9781118788523.ch19]
[11]
Di, L.; Rong, H.; Feng, B. Demystifying brain penetration in central nervous system drug discovery. Miniperspective. J. Med. Chem., 2013, 56(1), 2-12.
[http://dx.doi.org/10.1021/jm301297f] [PMID: 23075026]
[12]
Uchida, Y.; Ohtsuki, S.; Katsukura, Y.; Ikeda, C.; Suzuki, T.; Kamiie, J.; Terasaki, T. Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. J. Neurochem., 2011, 117(2), 333-345.
[http://dx.doi.org/10.1111/j.1471-4159.2011.07208.x] [PMID: 21291474]
[13]
Hoshi, Y.; Uchida, Y.; Tachikawa, M.; Inoue, T.; Ohtsuki, S.; Terasaki, T. Quantitative atlas of blood-brain barrier transporters, receptors, and tight junction proteins in rats and common marmoset. J. Pharm. Sci., 2013, 102(9), 3343-3355.
[http://dx.doi.org/10.1002/jps.23575] [PMID: 23650139]
[14]
Tillement, J.P.; Urien, S.; Chaumet-Riffaud, P.; Riant, P.; Bree, F.; Morin, D.; Albengres, E.; Barre, J. Blood binding and tissue uptake of drugs. Recent advances and perspectives. Fundam. Clin. Pharmacol., 1988, 2(3), 223-238.
[http://dx.doi.org/10.1111/j.1472-8206.1988.tb00634.x] [PMID: 3042568]
[15]
Urien, S.; Pinquier, J.L.; Paquette, B.; Chaumet-Riffaud, P.; Kiechel, J.R.; Tillement, J.P. Effect of the binding of isradipine and darodipine to different plasma proteins on their transfer through the rat blood-brain barrier. Drug binding to lipoproteins does not limit the transfer of drug. J. Pharmacol. Exp. Ther., 1987, 242(1), 349-353.
[PMID: 2956411]
[16]
de Lange, E.C. The mastermind approach to CNS drug therapy: translational prediction of human brain distribution, target site kinetics, and therapeutic effects. Fluids Barriers CNS, 2013, 10(1), 12.
[http://dx.doi.org/10.1186/2045-8118-10-12] [PMID: 23432852]
[17]
Hammarlund-Udenaes, M.; Fridén, M.; Syvänen, S.; Gupta, A. On the rate and extent of drug delivery to the brain. Pharm. Res., 2008, 25(8), 1737-1750.
[http://dx.doi.org/10.1007/s11095-007-9502-2] [PMID: 18058202]
[18]
Syvänen, S.; Lindhe, O.; Palner, M.; Kornum, B.R.; Rahman, O.; Långström, B.; Knudsen, G.M.; Hammarlund-Udenaes, M. Species dif-ferences in blood-brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport. Drug Metab. Dispos., 2009, 37(3), 635-643.
[http://dx.doi.org/10.1124/dmd.108.024745] [PMID: 19047468]
[19]
Bauer, M.; Zeitlinger, M.; Karch, R.; Matzneller, P.; Stanek, J.; Jäger, W.; Böhmdorfer, M.; Wadsak, W.; Mitterhauser, M.; Bankstahl, J.P.; Löscher, W.; Koepp, M.; Kuntner, C.; Müller, M.; Langer, O. Pgp-mediated interaction between (R)-[11C]verapamil and tariquidar at the human blood-brain barrier: a comparison with rat data. Clin. Pharmacol. Ther., 2012, 91(2), 227-233.
[http://dx.doi.org/10.1038/clpt.2011.217] [PMID: 22166851]
[20]
Liu, X.; Van Natta, K.; Yeo, H.; Vilenski, O.; Weller, P.E.; Worboys, P.D.; Monshouwer, M. Unbound drug concentration in brain homog-enate and cerebral spinal fluid at steady state as a surrogate for unbound concentration in brain interstitial fluid. Drug Metab. Dispos., 2009, 37(4), 787-793.
[http://dx.doi.org/10.1124/dmd.108.024125] [PMID: 19116265]
[21]
Bundgaard, C.; Eneberg, E.; Sánchez, C. P-glycoprotein differentially affects escitalopram, levomilnacipran, vilazodone and vortioxetine transport at the mouse blood-brain barrier in vivo. Neuropharmacology, 2016, 103, 104-111.
[http://dx.doi.org/10.1016/j.neuropharm.2015.12.009] [PMID: 26700248]
[22]
Culot, M.; Fabulas-da Costa, A.; Sevin, E.; Szorath, E.; Martinsson, S.; Renftel, M.; Hongmei, Y.; Cecchelli, R.; Lundquist, S. A simple method for assessing free brain/free plasma ratios using an in vitro model of the blood brain barrier. PLoS One, 2013, 8(12), e80634.
[http://dx.doi.org/10.1371/journal.pone.0080634] [PMID: 24312489]
[23]
Wager, T.T.; Hou, X.; Verhoest, P.R.; Villalobos, A. Moving beyond rules: The development of a central nervous system multiparameter optimization (CNS MPO) approach to enable alignment of druglike properties. ACS Chem. Neurosci., 2010, 1(6), 435-449.
[http://dx.doi.org/10.1021/cn100008c] [PMID: 22778837]
[24]
Wager, T.T.; Hou, X.; Verhoest, P.R.; Villalobos, A. Central nervous system multiparameter optimization desirability: Application in drug discovery. ACS Chem. Neurosci., 2016, 7(6), 767-775.
[http://dx.doi.org/10.1021/acschemneuro.6b00029] [PMID: 26991242]
[25]
Di, L.; Kerns, E.H.; Carter, G.T. Strategies to assess blood-brain barrier penetration. Expert Opin. Drug Discov., 2008, 3(6), 677-687.
[http://dx.doi.org/10.1517/17460441.3.6.677] [PMID: 23506148]
[26]
Borst, P.; Schinkel, A.H. P-glycoprotein ABCB1: A major player in drug handling by mammals. J. Clin. Invest., 2013, 123(10), 4131-4133.
[http://dx.doi.org/10.1172/JCI70430] [PMID: 24084745]
[27]
Risgaard, R.; Ettrup, A.; Balle, T.; Dyssegaard, A.; Hansen, H.D.; Lehel, S.; Madsen, J.; Pedersen, H.; Püschl, A.; Badolo, L.; Bang-Andersen, B.; Knudsen, G.M.; Kristensen, J.L. Radiolabelling and PET brain imaging of the α1-adrenoceptor antagonist Lu AE43936. Nucl. Med. Biol., 2013, 40(1), 135-140.
[http://dx.doi.org/10.1016/j.nucmedbio.2012.09.010] [PMID: 23165140]
[28]
Redrobe, J.P.; Jørgensen, M.; Christoffersen, C.T.; Montezinho, L.P.; Bastlund, J.F.; Carnerup, M.; Bundgaard, C.; Lerdrup, L.; Plath, N. In vitro and in vivo characterisation of Lu AF64280, a novel, brain penetrant phosphodiesterase (PDE) 2A inhibitor: potential relevance to cognitive deficits in schizophrenia. Psychopharmacology (Berl.), 2014, 231(16), 3151-3167.
[http://dx.doi.org/10.1007/s00213-014-3492-7] [PMID: 24577516]
[29]
Schinkel, A.H.; Wagenaar, E.; van Deemter, L.; Mol, C.A.; Borst, P. Absence of the mdr1a P-Glycoprotein in mice affects tissue distribu-tion and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J. Clin. Invest., 1995, 96(4), 1698-1705.
[http://dx.doi.org/10.1172/JCI118214] [PMID: 7560060]
[30]
Polli, J.W.; Olson, K.L.; Chism, J.P.; St. John-Williams, L.; Yeager, R.L.; Woodard, S.M.; Otto, V.; Castellino, S.; Demby, V.E. An unex-pected synergist role of P-glycoprotein and breast cancer resistance protein on the central nervous system penetration of the tyrosine ki-nase inhibitor lapatinib (N- -6-[5-(Methyl)-2-Furyl]-4-quinazolinamine; gw572016). Drug Metab. Dispos., 2009, 37(2), 439-442.
[http://dx.doi.org/10.1124/dmd.108.024646] [PMID: 19056914]
[31]
Hyafil, F.; Vergely, C.; Du Vignaud, P.; Grand-Perret, T. in vitro and in vivo reversal of multidrug resistance by GF120918, an acridone-carboxamide derivative. Cancer Res., 1993, 53(19), 4595-4602.
[PMID: 8402633]
[32]
Allen, J.D.; Brinkhuis, R.F.; Wijnholds, J.; Schinkel, A.H. The mouse Bcrp1/Mxr/Abcp gene: amplification and overexpression in cell lines selected for resistance to topotecan, mitoxantrone, or doxorubicin. Cancer Res., 1999, 59(17), 4237-4241.
[PMID: 10485464]
[33]
Lai, Y. Transporter Study Methodologies. In: Transporters in Drug Discovery and Development; Elsevier, 2013; pp. 675-718.
[http://dx.doi.org/10.1533/9781908818287.675]
[34]
Lagas, J.S.; van Waterschoot, R.A.B.; van Tilburg, V.A.C.J.; Hillebrand, M.J.; Lankheet, N.; Rosing, H.; Beijnen, J.H.; Schinkel, A.H. Brain accumulation of dasatinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by elacridar treatment. Clin. Cancer Res., 2009, 15(7), 2344-2351.
[http://dx.doi.org/10.1158/1078-0432.CCR-08-2253] [PMID: 19276246]
[35]
Benjamini, Y.; Krieger, A.M.; Yekutieli, D. Adaptive linear step-up procedures that control the false discovery rate. Biometrika, 2006, 93(3), 491-507.
[http://dx.doi.org/10.1093/biomet/93.3.491]
[36]
Kalvass, J.C.; Polli, J.W.; Bourdet, D.L.; Feng, B.; Huang, S-M.; Liu, X.; Smith, Q.R.; Zhang, L.K.; Zamek-Gliszczynski, M.J. International Transporter Consortium. Why clinical modulation of efflux transport at the human blood-brain barrier is unlikely: the ITC evidence-based position. Clin. Pharmacol. Ther., 2013, 94(1), 80-94.
[http://dx.doi.org/10.1038/clpt.2013.34] [PMID: 23588303]
[37]
Jetté, L.; Pouliot, J.F.; Murphy, G.F.; Béliveau, R.; Isoform, I. Isoform I (mdr3) is the major form of P-glycoprotein expressed in mouse brain capillaries. Evidence for cross-reactivity of antibody C219 with an unrelated protein. Biochem. J., 1995, 305(Pt 3), 761-766.
[http://dx.doi.org/10.1042/bj3050761] [PMID: 7848274]
[38]
Regina, A.; Koman, A.; Piciotti, M.; El Hafny, B.; Center, M.S.; Bergmann, R.; Couraud, P-O.; Roux, F. Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells. J. Neurochem., 1998, 71(2), 705-715.
[http://dx.doi.org/10.1046/j.1471-4159.1998.71020705.x] [PMID: 9681461]
[39]
Wang, Q.; Zuo, Z. Impact of transporters and enzymes from blood-cerebrospinal fluid barrier and brain parenchyma on CNS drug uptake. Expert Opin. Drug Metab. Toxicol., 2018, 14(9), 961-972.
[http://dx.doi.org/10.1080/17425255.2018.1513493] [PMID: 30118608]
[40]
Flores, K.; Manautou, J.E.; Renfro, J.L. Gender-specific expression of ATP-binding cassette (Abc) transporters and cytoprotective genes in mouse choroid plexus. Toxicology, 2017, 386, 84-92.
[http://dx.doi.org/10.1016/j.tox.2017.05.019] [PMID: 28587784]
[41]
Leggas, M.; Adachi, M.; Scheffer, G.L.; Sun, D.; Wielinga, P.; Du, G.; Mercer, K.E.; Zhuang, Y.; Panetta, J.C.; Johnston, B.; Scheper, R.J.; Stewart, C.F.; Schuetz, J.D. Mrp4 confers resistance to topotecan and protects the brain from chemotherapy. Mol. Cell. Biol., 2004, 24(17), 7612-7621.
[http://dx.doi.org/10.1128/MCB.24.17.7612-7621.2004] [PMID: 15314169]
[42]
Ito, K.; Uchida, Y.; Ohtsuki, S.; Aizawa, S.; Kawakami, H.; Katsukura, Y.; Kamiie, J.; Terasaki, T. Quantitative membrane protein expres-sion at the blood-brain barrier of adult and younger cynomolgus monkeys. J. Pharm. Sci., 2011, 100(9), 3939-3950.
[http://dx.doi.org/10.1002/jps.22487] [PMID: 21254069]
[43]
Kodaira, H.; Kusuhara, H.; Ushiki, J.; Fuse, E.; Sugiyama, Y. Kinetic analysis of the cooperation of P-glycoprotein (P-gp/Abcb1) and breast cancer resistance protein (Bcrp/Abcg2) in limiting the brain and testis penetration of erlotinib, flavopiridol, and mitoxantrone. J. Pharmacol. Exp. Ther., 2010, 333(3), 788-796.
[http://dx.doi.org/10.1124/jpet.109.162321] [PMID: 20304939]
[44]
de Lange, E.C.M. Utility of CSF in translational neuroscience. J. Pharmacokinet. Pharmacodyn., 2013, 40(3), 315-326.
[http://dx.doi.org/10.1007/s10928-013-9301-9] [PMID: 23400635]
[45]
Doran, A.; Obach, R.S.; Smith, B.J.; Hosea, N.A.; Becker, S.; Callegari, E.; Chen, C.; Chen, X.; Choo, E.; Cianfrogna, J.; Cox, L.M.; Gibbs, J.P.; Gibbs, M.A.; Hatch, H.; Hop, C.E.; Kasman, I.N.; Laperle, J.; Liu, J.; Liu, X.; Logman, M.; Maclin, D.; Nedza, F.M.; Nelson, F.; Ol-son, E.; Rahematpura, S.; Raunig, D.; Rogers, S.; Schmidt, K.; Spracklin, D.K.; Szewc, M.; Troutman, M.; Tseng, E.; Tu, M.; Van Deusen, J.W.; Venkatakrishnan, K.; Walens, G.; Wang, E.Q.; Wong, D.; Yasgar, A.S.; Zhang, C. 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), 165-174.
[http://dx.doi.org/10.1124/dmd.104.001230] [PMID: 15502009]