Use of the Cassette-Dosing Approach to Assess Brain Penetration in Drug Discovery Xingrong Liu, Xiao Ding, Gauri Deshmukh, Bianca M. Liederer, and Cornelis E. C. A. Hop Genentech, Inc., South San Francisco, California Received December 23, 2011; accepted February 10, 2012 ABSTRACT: The objective of the present study was to examine the cassette dosing method in determination of brain-to-plasma concentration ratio (area under the concentration-time profiles for plasma/area un- der the concentration-time profiles for brain, K p ). Eleven model com- pounds, amprenavir, citalopram, digoxin, elacridar, imatinib, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methyl- propyl)-1,4-dioxopyrazino[1,2:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester (Ko143), loperamide, prazosin, quinidine, sulfasalazine, and verapamil, were selected to compare their K p de- termined from discrete dosing in wild-type mice and their K p from cassette dosing in wild-type, Mdr1a/1b(/), Bcrp1(/), and Mdr1a/1b(/)/Bcrp1(/) mice at 1 to 3 mg/kg. The mice brain and plasma were collected at 0.25, 1, and 3 h and were analyzed using high-performance liquid chromatography-tandem mass spectrome- try methods. The K p determined from discrete dosing versus cassette dosing in the wild-type mice were within 2-fold for all the compounds except sulfasalazine and Ko143. The brain concentrations of sul- fasalazine and Ko143 and the plasma concentrations of Ko143 were below the lower limit of quantitation. In addition, the K p values esti- mated by mass spectrometry responses, namely the ratio of com- pound peak area to internal standard peak area, were within 2-fold of the K p observed from the actual concentrations. Furthermore, the ratios of K p in Mdr1a/1b(/), Bcrp1(/), and Mdr1a/1b(/)/ Bcrp1(/) mice versus the K p in the wild-type mice from cassette dosing were consistent with the ones reported in the literature where the compounds were dosed discretely. These results demonstrate that drug-drug interactions at the blood-brain barrier are un- likely at a subcutaneous dose of 1 to 3 mg/kg and support the use of the cassette dosing approach to assess brain penetration in drug discovery. Introduction The blood-brain barrier (BBB) consists of a continuous layer of en- dothelial cells joined by tight junctions at the cerebral vasculature. It represents a physical, enzymatic, and transporter barrier to restrict and regulate the penetration of compounds into and out of the brain (Davson and Segal, 1995). The main mechanisms limiting the delivery of drugs from blood into the brain are that the BBB exhibits very low paracellular permeability and expresses multiple drug transporters. Two efflux drug transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are the main efflux transporters expressed at the luminal side of the BBB, and their functional importance in limiting brain penetration of various compounds has been demonstrated (Schinkel et al., 1994; Chen et al., 2003; Breedveld et al., 2005; Enokizono et al., 2007; Polli et al., 2009; Zhou et al., 2009; Agarwal et al., 2011). In mice, P-gp is the product of Mdr1a (Abcb1a) and Mdr1b (Abcb1b) genes, and Bcrp is the product of Bcrp1 (Abcg2) gene (Schinkel, 1999; Scherrmann, 2005). A useful parameter to assess the efficiency of a drug to cross the BBB is the ratio of unbound brain concentration to unbound plasma concentration (K p,uu ) (Liu et al., 2008; Hammarlund-Udenaes et al., 2009). The common method to estimate K p,uu is to determine in vivo plasma and brain concentrations (K p ) and in vitro unbound fraction in plasma and brain tissue (Maurer et al., 2005). K p can be determined at steady state after intravenous infusion or from the area under the curve (AUC) of brain and plasma concentrations after a single dose. These experimental approaches are low throughput and resource-intensive. In the present study, we evaluated the cassette dosing approach to increase the throughput and reduce resource consumption in determi- nation of K p . Traditionally pharmacokinetic parameters were generated by dos- ing compounds discretely. To increase the throughput of pharmaco- kinetic studies, cassette dosing (also called N-in-1 dosing) has been used in animal pharmacokinetic studies in drug discovery screening (Manitpisitkul and White, 2004). Extensive research work has been published to assess the cassette dosing approach for screening sys- temic pharmacokinetics in drug discovery. However, much less orig- inal research work has been published to evaluate cassette dosing to study brain penetration. Frick et al. (1998) briefly described in a review for the cassette dosing method to study a series of compounds in mice and observed good agreement between the Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. http://dx.doi.org/10.1124/dmd.111.044420. ABBREVIATIONS: BBB, blood-brain barrier; P-gp, P-glycoprotein; Bcrp, breast cancer resistance protein; K p , brain-to-plasma concentration ratio; K p,uu , unbound brain-to-plasma concentration ratio; AUC, area under the curve; Ko143, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9- methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester; LLOQ, low limit of quanti- tation; AUC p,R , area under the curve values calculated by using the mass spectrometer response for plasma; AUC b,R , area under the curve values calculated by using the mass spectrometer response for brain; KO/WT ratio, K p in knockout mice versus the K p from wild-type mice; PF-407288, 2-(4-(2-(2-(4-methoxyphenyl)-5-methyloxazol-4-yl)ethoxy)benzyl)-tetrahydrofuran-2-carboxylic acid. 1521-009X/12/4005-963–969$25.00 DRUG METABOLISM AND DISPOSITION Vol. 40, No. 5 Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics 44420/3764268 DMD 40:963–969, 2012 963 at ASPET Journals on June 26, 2017 dmd.aspetjournals.org Downloaded from