Supporting Information

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1 Supporting Information Table S1. Animal data, brain sample and choroid plexus weights sample weights animal cocktail sex age body no. no. weight cerebellum brain stem CP LV CP 4V [y] [kg] [g] [g] [g] [mg] [mg] 1 1 m m m m f f mean SD LV=lateral ventricle (containing also 3rd ventricle choroid plexus) 4V=fourth ventricle Choroid plexus (CP) weights refer to the sample weights from both hemispheres, whereas, cerebellum and brain stem weights refer to one hemisphere used for protein quantification Table S2. Pharmacokinetic parameters in dogs after i.v. bolus administration of cocktails cocktail dose [µmol/kg] N CL [ml/min/kg] Vss [L/kg] MRTdisp [h] T1/2 [h] apafant ± ± ± ± dantrolene ± ± ± ± antipyrine ± ± ± ± quinidine ± ± ± ± 0.27 daidzein ± ± ± ± 0.42 antipyrine ± ± ± ± 0.090

2 Table S3. Loading doses and infusion rates for the four transporter substrates and antipyrine cocktail target C ss loading dose infusion rate [µmol/l] [µmol/kg] [µmol/h/kg] apafant dantrolene quinidine daidzein antipyrine 1 &

3 Table S4. Peptide probes and MRM transitions for human molecules gene Symbol alias St or IS probe sequence number of AA SRM transition (m/z) Q1 Q3-1 Q3-2 Q3-3 Q3-4 ABC transporters ABCB1 P-gp St NTTGALTTR IS NTTGAL*TTR ABCC1 MRP1 St TPSGNLVNR IS TPSGNL*VNR ABCC4 MRP4 St GTYTEFLK IS GTYTEFL K* ABCG2 BCRP St SSLLDVLAAR IS SSLLDVL*AAR SLC transporters SLC22A2 OCT2 St WLISQSK IS WLISQSK* SLC22A8 OAT3 St YSVADLFR IS YSVADLFR* SLC15A2 PEPT2 St WTLQAIR IS WTLQAI*R SLC21A3 OATP1A2 St IYDSTNFR IS IYDSTNFR* SLC21A9 OATP2B1 St YYDHDLLR IS YYDHDLL R* SLC29A1 ENT1 St FEGPGEQETK IS FEGPGEQETK* SLC7A5 LAT1 St VQDAFAAAK IS VQDAFAA*AK

4 SLC16A1 MCT1 St SITVFFK IS SITVFF*K SLC3A2 4F2hc St IGDLQAFQGPR IS IGDLQAFQGPR* SLC2A1 GLUT1 St TFDEIASGFR IS TFDEI A*SGFR Receptors INSR INSR St ESLVISGLR IS ESLVISG L*R LRP1 LRP1 St GDYSVLVPGLR IS GDYSVLVPG L*R TfR1 TfR1 St SSVGTALLLELAR IS SSVGTALLLELA R* Other Na + /K + -ATPase Na + /K + -ATPase St AAVPDAVGK IS AAVPDAV*GK The SRM transitions were determined from MS/MS spectra obtained by direct infusion of 1µM peptide solutions at a flow rate of 5 µl/min with a syringe pump (Harvard) into the mass spectrometer. Doubly charged precursor ions (singly charged for some peptides) were selected (Q1). Four transitions per peptide (Q3-1, -2, -3, and -4), which corresponded to high-intensity fragment ions, were selected. The declustering potentials and collision energies were optimized to maximize signal strength. For internal standard peptides, precursor ions and transitions corresponding to those of the standard peptides were selected, with the same declustering potentials and collision energies as those for the standard peptides. 13 C and 15 N were used for amino acid labeling in internal standard peptides. Bold letters with asterisks indicate amino acid residues labeled with stable isotopes ( 13 C and 15 N). The concentration of each peptide in the authentic solution was determined by means of HPLC-UV with quantitative amino acid analysis. AA, amino acid; St, standard; IS, internal standard 4

5 Table S5. Concentrations of transporter substrates and antipyrine in the frontal, median and caudal parts of the in dogs at steady state compound cocktail no. N frontal [nmol/l] median [nmol/l] caudal [nmol/l] mean [nmol/l] apafant ± ± ± ± 5.3 dantrolene ± ± ± ± 164 antipyrin ± ± ± ± 65 quinidine ± ± ± ± 305 daidzein ± ± ± ± 59 antipyrin ± ± ± ± 113 Data are mean ± SD. Table S6. K p,uu of drugs after steady state infusion of the substance cocktails compound cocktail no. N K p,uu,brain K p,uu,csf ratio K puu,csf /K puu,brain P-gp substrates apafant ± ± ± 0.15 quinidine BCRP substrate ± ± ± 0.13 dantrolene ± ± ± 0.67 daidzein ± ± ± 1.4 no efflux transport control ± ± ± 0.15 antipyrine Data are mean ± SD

6 Analytical validation of protein quantification Data demonstrating the reliability of the analytical assays is given below. The calibration curve was linear over a range of 1 50 fmol on the column with a correlation coefficient of > Between-day inaccuracy and imprecision of the analytical assays was assessed using six aliquots (two samples each day for three separate days). The inaccuracy (coefficient of variation of mean values) was <±15%, and the imprecision (relative error) was <±20%, which were well within the proposed criteria for LC-MS/MS-based protein quantification. 1 As a result, the analysis quality in the present study is considered acceptable. Reliability of analytical assays in standard solution target inaccuracy (deviation %) imprecision (variation %) LQC LQC MQC HQC MQC HQC MRP MRP BCRP P-gp OCT OAT PEPT OATP1A OATP2B ENT LAT MCT F2hc GLUT INSR LRP TfR Na + K + ATPase Intra-assay variability was assessed using three different standard peptide samples (LQC, MQC and HQC); 2.5, 10 or 40 fmol of standard peptides with 20 fmol of corresponding internal standard peptides. Each data point represents mean of three runs (n=6) in four MRM channels.

7 Table S7. Species differences of protein expression amounts of transporters and receptors in lysate of isolated brain capillaries target canine human brain cortex protein expression (fmol/µg protein of lysate; mean ± SD) cynomolgus monkey marmoset mouse middle part of half brain rat MRP1 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ MRP4 <LOQ ± ± ± ± ± 0.16 BCRP 45.2 ± ± ± ± ± ± 0.32 P-gp 6.71 ± ± ± ± ± ± 1.0 OCT2 <LOQ <LOQ not measured <LOQ not measured not measured OAT3 <LOQ <LOQ not measured <LOQ 1.97 ± ± 0.25 PEPT ± 0.50 <LOQ not measured not measured not measured not measured OATP1A2 (Oatp1a4: mouse, <LOQ <LOQ not measured ± ± 0.28 not measured Oatp1a5:rat) OATP2B1 <LOQ <LOQ <LOQ <LOQ not measured <LOQ ENT ± ± not measured ± not measured not measured LAT1 <LOQ ± not measured <LOQ 2.19 ± ± 0.62 MCT1 <LOQ 2.27 ± ± ± ± ± 0.5 4F2hc 24.7 ± ± ± 0.3 not measured 16.4 ± 1.1 <LOQ GLUT1 209 ± ± ± ± ± ± 4.7 INSR <LOQ 1.09 ± ± ± ± ± LRP ± ± 0.26 <LOQ 1.29 ± ± ± 0.12 TfR ± ± 0.76 <LOQ not measured 5.84 ± ± 0.53 Na + K + ATPase 70.5 ± ± ± ± ± ± 5.2 The protein expression amounts were determined by LC-MS/MS with internal standard peptides. Human data (n=7) were taken from Uchida et al. 2, mouse data (n=6) from Kamiie et al. 3, rat (average of SD and Wistar strain) and marmoset data (n=5) from Hoshi et al. 4, and cynomolgus monkey data (n=5) from Ito et al. 5. <LOQ, below limit of quantification. 7

8 Table S8. Protein expression amounts of transporters and receptors in lysate of isolated canine choroid plexus from the present study, compared to literature data in humans and Wistar rats target lateral ventricle relative protein expression to Na + K + ATPase a dog fourth ventricle human b Wistar rat b MRP MRP BCRP NC ( 1 <0.0017) NC ( 1 <0.0017) P-gp OCT2 NC NC NC NC OAT3 NC NC PEPT2 NC NC NC OATP1A2 (Oatp1a5:rat) NC OATP2B1 NC NC NC NC ENT not measured LAT1 NC NC NC NC MCT F2hc GLUT INSR not measured not measured LRP not measured not measured TfR not measured not measured NC: not calculated, expression amounts below LOQ. 1 calculated based on LOQ. a The protein expression amounts were determined by LC-MS/MS with internal standard peptides. The relative protein expressions were calculated as the ratio to the expression level of Na + K + -ATPase. b The data of the human fourth ventricle (n=1) and the rat mixture of lateral, third and fourth ventricles (n=1, pooled 30 animals) were taken from Uchida et al. 6

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10 Figure S1. Location and separation of the choroid plexus after mid-sagittal dissection of the brain (A) Dog brain hemisphere (native tissue) after a midsagittal section. The locations of the choroid plexus (CP) of the 4th ventricle (4V) and the 3rd ventricle (3V) are indicated. (B) and (C): Sequence of photographs taken during sampling of the CP of the lateral and 3rd ventricle (L/3V) and 4V from one hemisphere. (D) Photograph of the CP of the L/3V and the CP of the 4V immersed in saline. Note that the CP of the lateral and 3rd ventricle of each hemisphere is connected as are the ventricles. This is depicted in Figure (E) which shows a dog brain hemisphere after opening of the LV and partly extraction of the LV part of the CP. Photos in (B), (C) and (D) were taken with a Canon EOS70D connected to a Leica OPMI 1 surgical microscope via a LMScope (Graz, Austria) camera adapter. Photos were processed with Adobe Lightroom 5 (noise reduction, exposure adjustment and white balance). Figure S2. Correlation of the individual K p,brain values and expression levels of P-gp (A) or BCRP (B) in (triangle), cerebellum (circle) and brain stem (square). The protein expressions of transporters and receptors in lysates of isolated dog brain capillaries were determined by LC-MS/MS. K p,brain was derived from the drug concentrations measured in plasma and the respective brain regions. Lines indicate linear regressions.

11 Figure S3. Regional differences of protein expression amounts of (A) ABC transporters, (B) SLC transporters, (C) receptors and (D) other proteins in lysates of isolated dog brain capillaries, with statistical comparison of the expression levels in, cerebellum and brainstem. The symbols represent data for individual dogs and the lines with error bars represent mean ± SD,

12 n=4-6. Statistics were calculated with one-way ANOVA; ns = not significant.

13 Figure S4. Differences of protein expression amounts of (A) ABC transporters, (B) SLC transporters, (C) receptors and (D) other proteins in lysate of CP. Statistical comparison of expression levels in the lateral and 4 th ventricles. The symbols represent data for individual dogs, lines with error bars represent mean ± SD, n=5-6. Statistics were calculated with t-test; ns = not significant.

14 References 1. Booth, B.; Arnold, M. E.; DeSilva, B.; Amaravadi, L.; Dudal, S.; Fluhler, E.; Gorovits, B.; Haidar, S. H.; Kadavil, J.; Lowes, S.; Nicholson, R.; Rock, M.; Skelly, M.; Stevenson, L.; Subramaniam, S.; Weiner, R.; Woolf, E. Workshop report: Crystal City V--quantitative bioanalytical method validation and implementation: the 2013 revised FDA guidance. The AAPS journal 2015, 17, (2), 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. Journal of Neurochemistry 2011, 117, (2), Kamiie, J.; Ohtsuki, S.; Iwase, R.; Ohmine, K.; Katsukura, Y.; Yanai, K.; Sekine, Y.; Uchida, Y.; Ito, S.; Terasaki, T. Quantitative atlas of membrane transporter proteins: Development and application of a highly sensitive simultaneous LC/MS/MS method combined with novel in-silico peptide selection criteria. Pharmaceutical Research 2008, 25, (6), 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. Journal of pharmaceutical sciences 2013, 102, (9), Ito, K.; Uchida, Y.; Ohtsuki, S.; Aizawa, S.; Kawakami, H.; Katsukura, Y.; Kamiie, J.; Terasaki, T. Quantitative membrane protein expression at the blood-brain barrier of adult and younger cynomolgus monkeys. Journal of pharmaceutical sciences 2011, 100, (9), Uchida, Y.; Zhang, Z.; Tachikawa, M.; Terasaki, T. Quantitative targeted absolute proteomics of rat blood-cerebrospinal fluid barrier transporters: Comparison with a human specimen. Journal of Neurochemistry 2015, 134, (6),