P-Glycoprotein Contributes to the Blood-Brain, but Not Blood-Cerebrospinal Fluid, Barrier in a Spontaneous Canine P-Glycoprotein Knockout Model □ S Katrina L. Mealey, Stephen Greene, Rodney Bagley, John Gay, Russ Tucker, Patrick Gavin, Kari Schmidt, and Frederick Nelson Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington (K.L.M., S.G., R.B., J.G., R.T., P.G.); and Department of Pharmacokinetics, Dynamics, and Drug Metabolism, Pfizer Global Research and Development, Groton Laboratories, Groton, Connecticut (K.S., F.N.) Received September 25, 2007; accepted March 7, 2008 ABSTRACT: P-glycoprotein is considered to be a major factor impeding effec- tive drug therapy for many diseases of the central nervous system (CNS). Thus, efforts are being made to gain a better understanding of P-glycoprotein’s role in drug distribution to brain parenchyma and cerebrospinal fluid (CSF). The goal of this study was to validate and introduce a novel P-glycoprotein–deficient (ABCB1-1) canine model for studying P-glycoprotein–mediated effects of drug distri- bution to brain tissue and CSF. CSF concentrations of drug are often used to correlate efficacy of CNS drug therapy as a surrogate for determining drug concentration in brain tissue. A secondary goal of this study was to investigate the validity of using CSF concentrations of P-glycoprotein substrates to predict brain tissue concentrations. Loperamide, an opioid that is excluded from the brain by P-glycoprotein, was used to confirm a P-glycoprotein–null phenotype in the dog model. ABCB1-1 dogs experienced CNS depression following loperamide administration, whereas ABCB1 wild-type dogs experienced no CNS depression. In summary, we have validated a novel P-glycoprotein–deficient canine model and have used the model to investigate transport of the P-glycoprotein substrate 99m Tc-sestamibi at the blood-brain barrier and blood- CSF barrier. The effectiveness of many pharmacological agents, including hu- man immunodeficiency virus 1 protease inhibitors, antineoplastic agents, and antiepileptic drugs, is limited by their ability to cross the blood-brain barrier (BBB) (Lo ¨ scher and Potschka, 2002; McGee et al., 2006; Peak and Abrey, 2006). In addition to factors such as the drug’s molecular weight, hydrophobicity, degree of ionization, and plasma protein and tissue binding (Golden and Pollack, 2003), whether the drug is a substrate for P-glycoprotein greatly influences its ability to cross the BBB. P-glycoprotein is a membrane transporter belonging to the ATP binding cassette superfamily encoded by the ABCB1 gene (formerly known as MDR1) (Lin and Yamazaki, 2003). P-glycopro- tein is expressed on the lumenal surface of brain capillary endothelial cells, where it is believed to contribute to the BBB (Sugawara et al., 1990; Bendayan et al., 2002), and on the apical surface of choroid plexus epithelial cells, where it is believed to contribute to the blood– cerebrospinal fluid barrier (CSF) barrier (Rao et al., 1999). Delineation of P-glycoprotein’s role in the BBB and blood-CSF barrier is necessary to improve treatment strategies for patients with disorders of the central nervous system (CNS) such as AIDS demen- tia, brain tumors, and epilepsy. However, a quantitative, mechanistic understanding of the role of P-glycoprotein in the BBB and blood- CSF barrier is hindered by current models [cell culture and abcb1ab(-/-) knockout murine models]. When studying drug pen- etration of the BBB, particularly for P-glycoprotein substrate drugs, many investigators have resorted to using CSF drug concentrations as a surrogate for brain parenchymal concentrations in both rodent and human studies (Christensen et al., 2001; Antinori et al., 2005; Cap- parelli et al., 2005). The results of the study presented here, in which a spontaneous canine P-glycoprotein knockout model is used, ques- tion the validity of using CSF drug concentrations to predict the concentrations of P-glycoprotein substrate drugs in brain parenchyma. We recently identified a functional polymorphism of the ABCB1 gene in collies (Mealey et al., 2001) and other herding breed dogs (Neff et al., 2004). Affected dogs (ABCB1-1) harbor a 4-base pair deletion mutation at the 5' end of the canine ABCB1 open reading frame. The frame shift generates several premature stop codons oc- curring within the first 10% of the coding region, resulting in prema- ture termination of P-glycoprotein synthesis. Required elements for P-glycoprotein’s drug-efflux function (ATP binding sites, substrate binding sites, phosphorylation sites, and multiple membrane-spanning motifs) (Yoshimura et al., 1989; Skach, 1998) are absent, resulting in This work was supported by Pfizer, Inc., and by National Institutes of Health Grant MH074956. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.107.018978. □ S The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. ABBREVIATIONS: BBB, blood-brain barrier; CSF, cerebrospinal fluid; CNS, central nervous system; MRI, magnetic resonance imaging; BIS, bispectral index system. 0090-9556/08/3606-1073–1079$20.00 DRUG METABOLISM AND DISPOSITION Vol. 36, No. 6 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 18978/3339574 DMD 36:1073–1079, 2008 Printed in U.S.A. 1073 http://dmd.aspetjournals.org/content/suppl/2008/03/10/dmd.107.018978.DC1 Supplemental material to this article can be found at: at ASPET Journals on July 19, 2018 dmd.aspetjournals.org Downloaded from