2.1.1. DNA constructs and cloning.

For targeting the Mdr1a gene locus a basic vector containing (1) a Hygromycin expression cassette flanked by frt sites, (2) a 1.1 kb genomic sequence upstream of the translational start ATG of the mouse Mdr1a gene on exon 2 and a 6.2 kb genomic sequence downstream of exon 2 used as targeting arms for homologous recombination and (3) a cDNA of human MDR1 fused to the mouse translational start ATG and followed by a polyadenylation motif was constructed in pBluescript (pBS) as depicted in Fig 1. The human MDR1 cDNA was generated by TaconicArtemis GmbH (Cologne, Germany) by subcloning of PCR fragments amplified from different human cDNA libraries. The human MDR1 cDNA used for the final targeting vector was corrected by TaconicArtemis for polymorphic nucleotide changes, such that it codes for a protein that matches the human P-glycoprotein reference sequence (http://www.uniprot.org/uniprot/P08183).

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Fig 1. Stages for the development of P-gp hMDR1 mice described in different steps during which both Mdr1a and Mdr1b were knocked out from mouse DNA and replaced by human MDR1.

https://doi.org/10.1371/journal.pone.0118638.g001

For targeting the Mdr1b gene locus the same human MDR1 cDNA was fused to the translational start ATG of mouse Mdr1b. In this case the basic targeting vector also contained a 5.2 kb genomic sequence upstream of the translational start ATG of the mouse Mdr1b gene on exon 2 and a 6.9 kb genomic sequence downstream of exon 2 used as targeting arms for homologous recombination, as well as a Neomycin expression cassette flanked by f3 sites and a thymidine kinase expression cassette located outside of the targeting arms (Fig 1).

2.1.2. Generation and molecular characterization of targeted embryonic stem cells.

Culture and targeted mutagenesis of embryonic stem (ES cells) were carried out by TaconicArtemis as described previously [12]. Mdr1a: The targeting vector was linearized with NotI and electroporated into a C57BL/6 mouse ES cell line. Of 367 hygromycin resistant ES cell colonies screened by standard Southern blot analyses, six correctly targeted clones were identified, expanded and further analyzed by Southern blot analyses with 5’ and 3’ external probes and an internal hygromycin probe. All six clones were confirmed as correctly targeted at both homology arms without additional random integrations (data not shown). Mdr1b: The targeting vector was linearized using FseI and electroporated into one of the correctly targeted Mdr1a ES clone described above. Of 662 G418 and gancyclovir resistant ES cell colonies screened by standard Southern blot analyses 13 correctly targeted clones were identified, eight of which were expanded and further analyzed by Southern blot as described above. All of these eight clones were confirmed as correctly targeted at both homology arms without additional random integrations (data not shown).

2.1.3. Generation and molecular characterization of MDR1 humanized mice.

For the generation of hMDR1 mice, four ES cell clones correctly targeted at the mouse Mdr1a and Mdr1b gene locus (described above), were expanded, injected into BALBc-blastocysts and transferred into foster mothers by TaconicArtemis as described previously [12]. Litters from these fosters were inspected visually and chimerism was determined by hair color. Highly chimeric animals were used for breeding to an efficient flipase (Flpe) deleter strain carrying a transgene that expresses Flpe in the germ line in order to delete the hygromycin and neomycin expression cassettes in the offspring (Fig 1E). The Flpe-deleter strain was generated by TaconicArtemis on a C57BL/6 genetic background. Offspring from chimeras derived from two of the Mdr1a and 1b targeted ES cells were analyzed by PCR in order to determine the genotype of these mice. The following PCR primer pairs were used for the detection of the different alleles: (1) WT mouse Mdr1a allele: 5’-GTGATGGAACTTGAAGAGGACC-3’ and 5’-CACACACATTTGAAGGACTGC-3’, resulting in a 236 bp PCR fragment; (2) WT mouse Mdr1b allele: 5’-CCCCACAAACCAGACATAAGAGC-3’ and 5’-ATCTCTCTTAGCACCAGTCCAG-3’, resulting in a 396 bp PCR fragment; (3) Humanized Mdr1a allele: 5’-GGACTGTAACTGACTGCCTTGC-3’ and 5’-CGAAAGTGAAACAATCTGGCTAC-3’, resulting in a 490 bp PCR fragment and (4) Humanized Mdr1b allele: 5’-GGACTGTAACTGACTGCCTTGC-3’ and 5’-CCCTTTGCTTATCCTCGCC-3’, resulting in a 518 bp PCR fragment. The Mdr1a and Mdr1b targeted alleles segregated independently in offspring from chimeras derived from one of the two clones, suggesting that targeting had occurred on different alleles. In case of the other ES cell clone (A-F1) the targeted alleles co-segregated, indicating that the same alleles were targeted. Heterozygous mice derived from ES cell clone A-F1 were crossed by TaconicArtemis to generate homozygous hMDR1 mice. Homozygosity was determined by the presence of the corresponding humanized alleles and absence of the WT alleles using the PCR primers described above.

The hMDR1 mice were generated by a knock-in strategy as depicted in Fig 1, such that a cDNA of human MDR1 followed by a polyA motif was introduced at the ATGs of both the mouse Mdr1a and Mdr1b genes by two consecutive rounds of targeting in mouse embryonic stem (ES) cells, resulting in double targeted ES cells (Fig 1A–1D). Transgenic mice from correctly targeted ES cells were generated subsequently. By further crosses with a mouse line expressing Flp-recombinase in the germ line, the hygromycin and neomycin expression cassette were deleted via recombination at the Flp recombinase recognition sites frt and f3, respectively (Fig 1E). Homozygous hMDR1 mice obtained by breeding appeared normal, could not be distinguished from WT animals, and had normal liver and body weights, survival rates and fertility (data not shown).

2.4.1. BBB penetration studies.

The P-gp substrates digoxin, verapamil and docetaxel, related to different chemical and therapeutic classes of drugs, were administered to study possible functional difference between human and mouse P-gp. Oxycodone was administered as an expected non-P-gp substrate (Boström et al., 2005). Brain to plasma total concentration ratios (K_p) of the substances were measured for each animal. Unbound volume of distribution in brain (V_{u, brain}) was measured with the brain slice method [14] and fraction unbound in plasma (f_{u, p}) was measured with high throughput equilibrium dialysis.

All the substances were administered subcutaneously except docetaxel, which was given intravenously in the tail vein as a slow injection over five minutes due to its tissue irritating character. The doses administered were 1 mg/kg for digoxin, verapamil and oxycodone, respectively. The dose of docetaxel was 10 mg/kg. The volume administered of all substances was 0.01 ml/g_body weight. All substances other than oxycodone were given to five mice from each group i.e. Mdr1a/1b(-/-), FVB WT, hMDR1 and C57BL/6 WT mice. Oxycodone was given to three mice from each group.

Verapamil and docetaxel were also studied in combination with the P-gp inhibitor cyclosporine A in all four groups of mice. Cyclosporine A was administered intraperitoneally one hour before the drugs at a dose of 100 mg/kg.

The mice were sacrificed under anesthesia (2% isoflurane, nitrous oxide 1 L/min and medical air 1.5 L/min), one hour after administration of the drug. Blood was collected with a pre-heparinized syringe by cardiac puncture and was centrifuged for 5 minutes at 10000 rpm to obtain plasma. Brain was also sampled. The brain was divided in two equal parts and frozen on dry ice before weighed. All the samples were stored at -80°C until analysis.

2.4.2. Plasma protein binding.

Equilibrium dialysis was performed to measure the plasma unbound fraction of the drugs studied, and to measure possible differences in the plasma protein binding between hMDR1 and C57BL/6 WT mice. A high throughput, 96 well equilibrium dialysis apparatus (Model HTD96b, HTDialysis, Connecticut, USA) [15] with dialysis membrane strips with a molecular weight cut-off of 12–14 kDa was used. The concentrations of the substances were the same as those achieved in plasma during the in vivo experiments. One side of each well used was loaded with 125 μl of plasma spiked with drug and the other side was loaded with 125 μl phosphate buffer saline (PBS). The top surface of the Teflon block was then sealed with an adhesive film. Oxycodone and verapamil were dialyzed for 6 hours while digoxin and docetaxel was dialyzed for 24 hours, all at 37°C with a shaking speed of 115 rpm, on a Thermo Scientific MaxQ 4450 orbital shaker (Thermo Scientific, Waltham, Massachusetts USA). Triplicates of the same concentration for each compound were measured. After the incubation period 80 μl from the buffer side was added into 80 μl of blank mouse plasma and 80 μl from the plasma was added into 80 μl of PBS. Samples were stored at -20 until analysis.

2.4.3. Nonspecific binding to brain parenchyma.

To measure the volume of distribution for unbound drug in brain (V_{u, brain}) in vitro, the high-throughput brain slice method was used [16, 17], with the main difference of mouse brain slices from four C57BL/6 WT mice rather than slices from rat brain was used. In short, six consecutive 300 μm brain slices on a coronal plane starting approximately 1.7 mm anterior from bregma (rostral striatum) up to approximately -0.1 mm anterior from bregma (caudal striatum) were cut. The cut slices were kept in plain ice-cold oxygenated ECF before the incubation. The six brain slices were transferred into one Ø80-mm flat-bottomed glass beaker containing 15 ml of buffer with the drugs digoxin, verapamil, docetaxel and oxycodone in cassette, each compound of 200 nM concentration in the buffer. Immediately after transferring the brain slices into the beaker, 200 μl of buffer was sampled as a reference for the brain slice viability test. The beaker was filled with 100% oxygen over the buffer, lightly covered with a glass lid and placed in a continuously oxygenated box inside a MaxQ4450 incubated shaker (Thermo Fisher Scientific, NinoLab, Sweden) for 5 h set at 37°C, with a rotation speed of 45 rpm. After 5 hours of incubation, the pH at 37°C was measured. A pH lower than 7.25 was one of the criteria of low viability of the brain slices. Before sampling the buffer, the beaker was placed resting for 5 minutes, which was required for sedimentation of the minor debris from the brain tissue. Two hundred μl of the buffer was thereafter aspirated and dispensed in an Eppendorf tube containing 200 μl of blank brain homogenate prepared with 4 volumes of buffer. The brain slices were removed, dried on filter paper, and weighed. Individual homogenization of the brain slices was made in 9 volumes (w/v) of ECF with an ultrasonic processor (VCX-130; Sonics, Chemical Instruments AB, Sweden). One brain slice was used for the detection of the maximum releasable lactate dehydrogenase (LDH) activity from the brain slice which indicates the viability of the cells in a brain slice [18, 19]. Samples were stored at -20°C pending LC-MS/MS analysis.

2.4.4. P-glycoprotein inhibition studies using digoxin as a substrate.

Inhibition of digoxin transport across the BBB by human and mouse P-gp was studied in C57BL/6 WT and hMDR1 mice. The mice were divided into 5 groups, each containing 5 humanized and 5 wild type mice. Digoxin was administered s.c. at a dose of 1 mg/kg as in the previous experiment. Blood and brain were sampled after 2 hours as described above. One group received digoxin only. The other 4 groups were given cyclosporine A 100 mg/kg i.p., quinidine 50 mg/kg i.p., verapamil 3 mg/kg s.c. or elacridar 3 mg/kg s.c., one hour before the digoxin administration. The volumes administered were 0.01 ml/g body weight, apart from elacridar, which was administered in a volume of 0.005 ml/g.

2.4.5. Effect of P-glycoprotein on the BBB transport of oxycodone.

The influence of P-gp on the transport of oxycodone across the BBB was studied in male C57BL/6 WT mice (n = 20) with and without the P-gp blockers cyclosporine A 100 mg/kg i.p, verapamil 3 mg/kg s.c. or elacridar 6 mg/kg s.c. They were divided into 4 groups of 5 mice each. One group was administered oxycodone only. For the other three groups the P-gp blockers were administered one hour before the administration of oxycodone, and sampling of blood was done under anesthesia one hour after the oxycodone administration as described above. Brain and liver were sampled after sacrificing the animal.

2.5.1. Plasma sample treatment.

The plasma samples were thawed in room tempered water. They were mixed by vortex (Uniequip Laborgeräte, Martinsried) and centrifuged for one minute at 10 000 rpm (Force 7, Denver Instrument Company, Denver, USA). Fifty μl of the samples were transferred to polypropylene Eppendorf tubes. The proteins were precipitated by adding 150 μl acetonitrile. For the verapamil analysis, the acetonitrile contained methoxyverapamil as internal standard at a concentration of 200 ng/ml. For the oxycodone analysis, the acetonitrile contained D6-oxycodone as internal standard at a concentration of 30 ng/ml. The samples were vortexed and centrifuged for three minutes at 10000 rpm. The analysis of digoxin, docetaxel, quinidine and elacridar did not contain an internal standard.

For digoxin and docetaxel 100 μl of the supernatant was added to 200 μl ammonium formate buffer and 100 μl ammonium acetate buffer in Eppendorf tubes, respectively. For verapamil and elacridar 50 μl of the supernatant was added to 1000 μl mobile phase, and for quinidine in 500 μl of mobile phase. The samples were vortexed and centrifuged during one minute at 10000 rpm. Fifty μl of the samples were then transferred to vials and covered with parafilm. Twenty μl was injected onto the LC-MS/MS system. For oxycodone and quinidine 40 μl and 25 μl of supernatant was transferred to a vial, covered with parafilm, 20 μl and 10μl respectively were injected directly onto the LC-MS/MS system.

Cyclosporine A was analyzed in whole blood. The samples were vortexed and 50 μl was transferred to Eppendorf tubes. The proteins were precipitated by adding 100 μl acetonitrile. The samples were vortexed and left for five minutes before they were vortexed again and centrifuged during three minutes. Fifty μl of the supernatant was added to 500 μl mobile phase, vortexed and centrifuged. Fifty μl was transferred to a vial, covered with parafilm and 20 μl was injected onto the LC-MS/MS system.

For the analysis of equilibrium dialysis samples similar methods as for the plasma sample treatment were used with some minor volume adjustments.

2.5.2. Brain sample treatment.

The brains were thawed on an ice bath at 0–2°C. A 4-fold volume (w/v) of ice-cold saline was added before homogenization (Heidolph R2R 2020, Schwabach, Germany). The homogenate was again frozen until sample preparation and analysis. The homogenate samples were thawed in a 4°C water bath. They were vortexed and 200 μl of the samples were transferred to Eppendorf tubes that were put in an ice bath. The proteins were precipitated by adding 600 μl ice-cold acetonitrile.

For verapamil and oxycodone the acetonitrile contained internal standard at a concentration of 5 and 6 ng/ml of methoxyverapamil and oxycodone-D6, respectively. The samples were vortexed and put in an ultrasonic bath (Branson 3210, Branson Ultrasonic Corporation, Danbury, USA) for ten minutes. The samples were again vortexed and centrifuged for five minutes at 10000 rpm.

For digoxin and docetaxel the acetonitrile did not contain an internal standard. After precipitation of proteins, 200 μl of the supernatant was transferred to an Eppendorf tube. It was evaporated to dryness under a flow of nitrogen at 40°C and re-dissolved in 200 μl mobile phase. For verapamil 200 μl of the supernatant was added to 200 μl 0.2% formic acid in an Eppendorf tube. The samples were vortexed and centrifuged for one minute before 50 μl was transferred to new vials, covered with parafilm and 20 μl was injected onto the LC-MS/MS system, except for docetaxel where 40 μl was injected. For oxycodone 40 μl of the supernatant was transferred to a vial, covered with parafilm and 20 μl was injected directly onto the LC-MS/MS system.

2.5.3. Drug quantification.

The samples were analyzed with reversed phase liquid chromatography (LC) in combination with tandem mass spectrometry, LC-MS/MS, equipped with a Shimadzu LC-10ADvp pump (Shimadzu, Kyoto, Japan), an SIL-HTc pump (Shimadzu, Kyoto, Japan) and a Quattro Ultima detector (Waters, Milford, MA, USA). The column used for digoxin and verapamil was a HyPurity C18, 3μm particle size, 50 x 4.6 mm + precolumn 10x4 mm. For docetaxel, oxycodone and cyclosporine A the column was a Zorbax Eclipse XDB-CN, 5μm particle size, 150 x 4.6 mm. Analytical column used for quinidine and elacridar was Gemini C18, 3μm particle size, 100 x 4.6 mm (Phenomenex). The mobile phase used for digoxin was acetonitrile (ACN): 5mM ammonium formate buffer pH 3.4 (26: 74), for verapamil it was 40% ACN in 0.2% formic acid, for docetaxel 50% ACN in 2mM ammonium acetate pH 5.0, for oxycodone 45% ACN on 5 mM ammonium acetate and for cyclosporine A ACN: 5 mM ammonium formate buffer pH 3.4 (50:50). Mobile phase used for quinidine was 10 mM ammonium acetate pH 10: ACN (45:55) and for elacridar it was 10 mM ammonium acetate pH 10: ACN (30:70). The MassLynx software (Micromass UK Limited, Manchester, UK) was used to monitor the analysis. The analysis was performed in positive electrospray ionization (ESI+) mode. The conditions for the LC varied for the different substances. For digoxin, verapamil and methoxyverapamil, the flow rate was 0.8 ml/min while for all other drugs it was 1 ml/min. Retention time for digoxin, verapamil, methoxyverapamil, docetaxel, oxycodone, D6 oxycodone, quinidine, elacridar and cyclosporine A were 4.4, 2.4, 2.7, 4.2, 6.4, 6.5, 2.3, 3.2 and 5.2 min, respectively. Tuning was performed to find optimal settings for the MS/MS detector. Source temperature was set to 130 C for verapamil, methoxyverapamil, quinidine, elacridar and cyclosporine A and 120 C for all other compounds. Desolvation temperature was set to 350 C for digoxin, 300 for docetaxel and 450 for the other compounds. For digoxin, verapamil, methoxyverapamil, docetaxel, elacridar, oxycodone, quinidine and cyclosporine A capillary voltage was 3.5, 1.4, 1.4, 3.8, 1.5, 0.9, 0.9 and 3.2 kV while cone voltage was 40, 70, 70, 25, 110, 55, 40 and 140 V, respectively. Desolvation gas (N₂) and cone gas (N₂) were set to 1000 and 200 L/h for all compounds except digoxin and docetaxel for which cone gas (N₂) was set to 250 L/h. Finally the precursor-product ion pair for digoxin, verapamil, methoxyverapamil, docetaxel, oxycodone, quinidine, elacridar and cyclosporine A were m/z 798.35–651.4, 455.2–165.0, 485.3–165.0, 808.25–527.3, 315.8–297.7, 321.8–303.7, 325–306.9, 564.3–251.8 and 1202.8–1185 respectively.

2.7.1. Preparation of brain capillary rich fraction pooled from 10 mice.

10 frozen cerebrums from wild-type C57BL/6 or hMDR1 mice were dissected into 1-mm pieces, and homogenized with a Potter—Elvehjem homogenizer using 20 up and down no-rotated strokes by hand in 5 volumes of solution B (101 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 15 mM HEPES, pH 7.4) per brain weight. The homogenate was centrifuged at 1,000g for 10 min, the pellet was suspended in solution B containing 16% dextran, and the suspension was centrifuged for 15 min at 4500 g. The resulting pellet was suspended in solution A (solution B containing 25 mM NaHCO₃, 10 mM glucose, 1 mM pyruvate and 5g/L bovine serum albumin), passed through a 210 μm nylon mesh, and then washed with 10 mL of solution A. The solution passing through the 210 μm mesh including wash solution was passed through a 85 μm nylon mesh, and then washed with 10 mL of solution A. The solution passing through the 85 μm mesh including wash solution was passed through a 20 μm nylon mesh, and then washed with 40 mL of solution A. The capillaries retained on the 20 μm nylon mesh were immediately collected using solution A, centrifuged at 1000g for 5 min, and then suspended in solution B. The capillaries were again centrifuged at 1000g for 5 min, and then suspended in solution B. The capillaries were again centrifuged at 1000g for 5 min, suspended in hypotonic buffer (10 mM Tris-HCl, 10 mM NaCl, 1.5 mM MgCl₂, pH7.4), sonicated, and then stored at -80°C after measurement of the protein concentration by the Lowry method using the DC protein assay reagent (Bio-Rad, Hercules, CA). All procedures were carried out at 4°C.

2.7.2. Multiplexed SRM/MRM analysis in LC-MS/MS.

Protein expression amounts of the target molecules were simultaneously determined by means of multiplexed selected/multiple reaction monitoring (SRM/MRM) analysis as described previously [1]. Whole tissue lysates of isolated brain capillaries (50 μg protein) were solubilized in 500 mM Tris—HCl (pH 8.5), 7 M guanidine hydrochloride, 10 mM EDTA, and the proteins were S-carbamoylmethylated as described [1]. The alkylated proteins were precipitated with a mixture of methanol, chloroform and water. The precipitates were dissolved in 6 M urea in 100 mM Tris—HCl (pH 8.5), diluted 5-fold with 100 mM Tris—HCl (pH 8.5) and treated with lysyl endopeptidase (LysC; Wako Pure Chemicals, Osaka, Japan) at an enzyme/substrate ratio of 1:100 and 0.05% ProteaseMax (Promega, Madison, WI) at 25°C for 3 h, followed by treatment of TPCK-treated trypsin (Promega) at an enzyme/substrate ratio of 1:100 at 37°C for 16 h. The tryptic digests were mixed with internal standard peptides and formic acid, and then centrifuged at 4°C and 17,360 g for 5 min. The supernatants were subjected to HPLC or microLC-MS/MS analysis.

The HPLC-MS/MS analysis was performed by coupling an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA) to a triple quadrupole mass spectrometer (QTRAP5500; AB SCIEX, Foster City, CA) equipped with Turbo V ion source (AB SCIEX) and 65 μm ID electrode (AB SCIEX). Samples equivalent to 33.3 μg protein were injected onto a Waters XBridge BEH130 C18 (1.0 × 100 mm, 3.5 μm) column together with 500 fmol of internal standard peptides. Mobile phases A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. The peptides were separated and eluted from the column at room temperature using a linear gradient with a 130-min run time at a flow rate of 50 μL/min. The sequence was as follows: (A:B), 99:1 for 5 min after injection, 40:60 at 65 min, 0:100 at 66 min and up to 68 min, 99:1 at 70 min and up to 130 min.

The microLC-MS/MS analysis was performed by coupling an ekspert microLC 200 system (Eksigent, Dublin, CA) to the QTRAP5500 equipped with Turbo V ion source and 25 μm ID electrode (AB SCIEX). Samples equivalent to 6.73 μg protein were injected onto a HALO Fused-Core C18 (0.3 × 100 mm, 2.7 μm) column (Eksigent) together with 50 fmol of internal standard peptides. Mobile phases A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. The peptides were separated and eluted from the column at 40°C using a linear gradient with a 55-min run time at a flow rate of 10 μL/min. The sequence was as follows: (A:B), 99:1 for 2 min after injection, 50:50 at 32 min, 0:100 at 35 min and up to 37 min, 99:1 at 39 min and up to 55 min.

The eluted peptides were simultaneously and selectively detected by means of electro-spray ionization in a multiplexed SRM/MRM analysis. The dwell time was 8 to 25 msec per SRM/MRM transition. Each molecule was monitored with four sets of SRM/MRM transitions (Q1/Q3-1, Q1/Q3-2, Q1/Q3-3, Q1/Q3-4) derived from one set of standard and internal standard peptides. Mouse Mdr1a-specific and mouse Mdr1b-specific peptides were monitored with the SRM/MRM transitions listed in S1 Table. The other molecules were monitored with the peptides and SRM/MRM transitions reported by Kamiie et al.[22], Uchida et al. [1] and Agarwal et al. [23]. Signal peaks detected at the same retention time as an internal standard peptide were defined as positive. When positive peaks were observed in three or four sets of SRM/MRM transitions, the molecules were considered to be expressed in brain capillaries, the protein expression levels were determined as the average of the quantitative values obtained from the different SRM/MRM transitions in three measurements, and then shown as the mean ± S.E.M. The S.E.M. represents the variability of protein expression level determined in different MS/MS transitions in three analyses, but does not represent the inter-individual variability. The limit of quantification was calculated as described previously [1].