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Research ArticleMetabolism, Transport, and Pharmacogenomics

Blood-Brain Barrier Pharmacoproteomics-Based Reconstruction of the In Vivo Brain Distribution of P-Glycoprotein Substrates in Cynomolgus Monkeys

Yasuo Uchida, Kentaro Wakayama, Sumio Ohtsuki, Masato Chiba, Tomoyuki Ohe, Yasuyuki Ishii and Tetsuya Terasaki
Journal of Pharmacology and Experimental Therapeutics September 2014, 350 (3) 578-588; DOI: https://doi.org/10.1124/jpet.114.214536

Abstract

The aim of this study was to investigate whether in vivo drug distribution in brain in monkeys can be reconstructed by integrating four factors: protein expression levels of P-glycoprotein (P-gp)/multidrug resistance protein 1 at the blood-brain barrier (BBB), in vitro transport activity per P-gp molecule, and unbound drug fractions in plasma and brain. For five P-gp substrates (indinavir, quinidine, loperamide, paclitaxel, and verapamil) and one nonsubstrate (diazepam), in vitro P-gp transport activities were determined by measuring transcellular transport across monolayers of cynomolgus monkey P-gp–transfected LLC-PK1 and parental cells. In vivo P-gp functions at the BBB were reconstructed from in vitro P-gp transport activities and P-gp expression levels in transfected cells and cynomolgus brain microvessels. Brain-to-plasma concentration ratios (Kp,brain) were reconstructed by integrating the reconstructed in vivo P-gp functions with drug unbound fractions in plasma and brain. For all compounds, the reconstructed Kp,brain values were within a 3-fold range of observed values, as determined by constant intravenous infusion in adult cynomolgus monkeys. Among four factors, plasma unbound fraction was the most sensitive factor to species differences in Kp,brain between monkeys and mice. Unbound brain-to-plasma concentration ratios (Kp,uu,brain) were reconstructed as the reciprocal of the reconstructed in vivo P-gp functions, and the reconstructed Kp,uu,brain values were within a 3-fold range of in vivo values, which were estimated from observed Kp,brain and unbound fractions. This study experimentally demonstrates that brain distributions of P-gp substrates and nonsubstrate can be reconstructed on the basis of pharmacoproteomic concept in monkeys, which serve as a robust model of drug distribution in human brain.

Introduction

The number of compounds approved for use as new drugs is very small compared with the number of drug candidates that progress from preclinical to clinical trials. The proportion was just 8% for central nervous system (CNS)–acting drugs during the period 1991–2000 (Kola and Landis, 2004), and also no greater during the period 2000–2008 (Yagi and Ohkubo, 2010). One of the major reasons for the high rate of discontinuation has been the unfavorable distribution of drugs into human brain. More than 98% of small molecules do not cross the blood-brain barrier (BBB) and, thus, do not provide pharmacologically active concentrations in brain (Pardridge, 2002). It has been also reported that there are 7638 molecules in the Comprehensive Medicinal Chemistry database but only 387 (5.1%) of these molecules treat CNS diseases (Ghose et al., 1999). Therefore, it is necessary to make a quantitative and accurate prediction of drug distribution in human brain during the preclinical stages. Brain drug distribution depends on permeability rate across the BBB regulated by a variety of transporters expressed in brain capillary endothelial cells. ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp)/multidrug resistance protein 1 (MDR1)/mdr1a/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2 are major gatekeepers for many drugs (Kusuhara and Sugiyama, 2009; Uchida et al., 2011b). Therefore, it is important to quantitatively clarify the molecular functions of these transporters at the human BBB to predict drug distribution in the human brain.

Protein expression levels have been reported to correlate with activities of functional proteins (Dyer et al., 1997; Hoffmeyer et al., 2000; Fukumoto et al., 2002; Shirasaka et al., 2008; Langenfeld et al., 2009; Tachibana et al., 2010). Hence, we anticipated that in vivo functional activities of target transporters could be reconstructed on the basis of their in vitro activities by integrating these activities with the in vivo/in vitro differences in protein expression levels. We developed an absolute protein quantification method for transporters that uses liquid chromatography–tandem mass spectrometry (LC-MS/MS), termed “quantitative targeted absolute proteomics (QTAP)” (Kamiie et al., 2008). Using QTAP, we demonstrated in mouse model that in vivo P-gp/mdr1a function at the BBB was reconstructed by integrating the protein expression levels of P-gp/mdr1a in the in vivo brain capillaries with the transport function per P-gp/mdr1a molecule, which was determined using an in vitro transport experiment and QTAP (Uchida et al., 2011a). This demonstration opened a new field of pharmacoproteomics (PPx) that is an integrated scientific field of proteomics and pharmacokinetics/pharmacodynamics/toxicokinetics/toxicodynamics to quantitatively understand drug absorption, distribution, metabolism, and excretion; pharmacologic effect; and toxicity (Uchida et al., 2014).

However, brain distributions of P-gp substrates, such as [18F]altanserin and [11C]GR205171 [(S)-(2-methyl-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethylamino)-2(S)-phenylpiperidine], significantly differ between humans and rodents (Syvanen et al., 2009). Therefore, it is debatable whether the demonstration of in vitro–to–in vivo reconstruction (IVIVR) in a mouse model alone is sufficiently valid to apply the theory to reconstruction of in vivo P-gp function at the human BBB, although this reconstruction is theoretically applicable regardless of animal species. In contrast, the quantitative protein expression profile of BBB transporters in cynomolgus monkeys is quite similar to that in humans (e.g., only 1.29-fold difference from humans in P-gp protein levels) (Ito et al., 2011; Uchida et al., 2011b).

It is also important to overcome species difference in brain drug distribution. Brain-to-plasma concentration ratios (Kp,brain) of P-gp substrate verapamil and PF-00905556 in monkey are significantly, 10.8- and 12.2-fold, greater than those in mouse and rat, respectively (Hendrikse et al., 1998; Kpakima et al., 2006; Syvanen et al., 2009). Between monkey and rodents, the protein expression levels of P-gp/mdr1a at the BBB differ only by 3- to 4-fold (Kamiie et al., 2008; Ito et al., 2011; Hoshi et al., 2013). Therefore, the remarkable species differences in Kp,brain cannot be completely explained by the differences in protein expression levels. It is necessary to consider the species differences not only in protein expression levels but also in other factors, such as intrinsic transport activity and unbound fractions in plasma and brain. Although we have already succeeded in predicting Kp,brain by integrating all these factors in mouse model (Uchida et al., 2011a), the further validation in monkey would dramatically increase the reliability of prediction in humans.

The purpose of this study was to experimentally demonstrate the reconstruction/prediction theory in cynomolgus monkeys to ensure that the theory is applicable for clarifying in vivo human BBB P-gp function and predicting brain drug distributions in humans. We reconstructed the brain distributions (Kp,brain and Kp,uu,brain) of six model compounds including five P-gp substrates and one nonsubstrate in cynomolgus monkeys on the basis of the theory that was previously established in a mouse model, and compared them with the observed brain distributions determined in an in vivo study to validate whether in vivo P-gp function at the BBB in monkeys could be reliably reconstructed. Furthermore, we analyzed the influence of species differences in four factors (BBB P-gp protein expression levels, intrinsic transport activity per P-gp molecule, and unbound fractions in plasma and brain) on species differences in brain distributions of P-gp substrates between mice and monkeys.

Materials and Methods

Chemicals.

Buspirone hydrochloride, loperamide hydrochloride, and quinidine were purchased from Sigma-Aldrich (St. Louis, MO). Diazepam, paclitaxel, and verapamil hydrochloride were purchased from Wako Pure Chemicals (Osaka, Japan). Indinavir sulfate was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). P-gp peptides with >95% peptide purity were synthesized by Thermo Fisher Scientific (Sedanstrasse, Germany). All of the other chemicals were of reagent grade and were available commercially.

Animals.

Six male adult cynomolgus monkeys were used for the intravenous constant infusion study and the absolute quantification of P-gp protein expression in isolated brain microvessels. The animals were treated as follows: 1) aged 5 years and 2 months with 3.6 kg b.wt. was treated with indinavir; 2) aged 4 years 1 month with 2.6 kg b.wt. was treated with quinidine; 3) aged 6 years 4 months with 5.4 kg b.wt. was treated with loperamide; 4) aged 5 years 4 months with 3.4 kg b.wt. was treated with paclitaxel; 5) aged 4 years 3 months with 3.15 kg b.wt. was treated with diazepam; and 6) aged 4 years 1 month with 3.2 kg b.wt. was treated with verapamil. The six cynomolgus monkeys received intravenous constant infusions of the test compounds after fasting overnight with free access to water at HAMRI, Co., Ltd. (Ibaraki, Japan), after which the right cerebrums were used to determine the compound concentrations in the cerebrums and the left cerebrums were used for the P-gp quantifications in the brain microvessels.

Another male adult cynomolgus monkey, aged 4 years 1 month with 3.3 kg b.wt., was used as a blank control for the intravenous constant infusion study. The control cynomolgus monkey underwent the same procedure as the six cynomolgus monkeys outlined above without compound administration at HAMRI, Co., Ltd. The plasma and cerebrum of the control cynomolgus monkey were used as blank samples for the concentration determinations of the six test compounds in the plasma and cerebrum, respectively. The cerebrum of the control animal was also used for the measurements of the unbound fractions in the cerebrum.

Animal care and experimental procedures for the cynomolgus monkeys were approved by the Animal Care and Use Committee of Banyu Tsukuba Research Institute.

Determination of the Brain Distributions of the Six Test Compounds in Cynomolgus Monkeys.

After fasting overnight with free access to water, the male adult cynomolgus monkeys were fixed to monkey chairs and received constant infusions of the test compounds (indinavir, quinidine, loperamide, paclitaxel, diazepam, or verapamil) via the cephalic or saphenous veins for 3 hours without anesthesia. With the exception of loperamide and paclitaxel, intravenous bolus injections of the test compounds were performed immediately prior to the constant infusion to quickly reach the steady-state plasma concentration; indinavir, quinidine, diazepam, and verapamil were intravenously infused for 3 hours at dose rates of 0.61, 0.21, 0.070, and 0.40 mg/h per kilogram, respectively, after the intravenous bolus injection of 0.50, 0.30, 0.095, and 1.2 mg/kg doses. Loperamide and paclitaxel were intravenously infused for 3 hours at dose rates of 0.20 and 0.94 mg/h per kilogram, respectively, without intravenous bolus injections. One cynomolgus monkey was used for each compound. Prior to administration and at 2, 2.5, and 3 hours after administration, blood samples were collected using syringes containing EDTA–dipotassium salt (2K) without anesthesia from the cephalic or saphenous vein on the side opposite that used for administration. The blood samples were immediately centrifuged at 4°C and 3500g for 10 minutes to obtain plasma. The plasma samples were stored at –80°C prior to LC-MS/MS analyses. Immediately after the blood sampling at 3 hours, the cynomolgus monkeys were sacrificed by exsanguination under anesthesia with isoflurane inhalation or excessive anesthesia with pentobarbital without exsanguination, and the brains were immediately excised, divided into the right and left cerebrums (for the P-gp quantifications in the brain microvessels), frozen in liquid nitrogen, and stored at –80°C prior to the LC-MS/MS analyses. For the LC-MS/MS analyses, the right cerebrums were weighed and homogenized with a 2-fold volume of phosphate-buffered saline (PBS) to obtain a 33.3% brain homogenate. Ten microliters of plasma or brain homogenate was mixed with 100 μl of ethanol and 100 μl of 75% ethanol containing buspirone as an internal standard (210 μl in total). The samples were centrifuged and filtrated at 4°C and 960g for 10 minutes and subjected to LC-MS/MS analyses. On the basis of pharmacokinetic concepts, the brain-to-plasma concentration ratios (Kp,brain) at 3 hours were estimated by dividing the cerebral concentrations by the plasma concentrations.

Transcellular Transport Study across Cynomolgus Monkey P-gp–Transfected LLC-PK1 and Parental LLC-PK1 Cell Monolayers.

Cynomolgus monkey P-gp–transfected LLC-PK1 and parental LLC-PK1 cells were prepared at Merck Research Laboratories (West Point, PA) (Sankaranarayanan et al., 2009). The transcellular transport study was carried out as described previously (Uchida et al., 2011a) with minor modifications. Cynomolgus monkey P-gp–transfected LLC-PK1 and parental LLC-PK1 cells were seeded at a density of 0.15 × 106 cells/insert (0.484 × 106 cells/cm2) on porous (1.0-μm) polyethylene terephthalate membrane filters (cell culture inserts for 24-well plates; BD Biosciences, Franklin Lakes, NJ) that had been coated with BD Matrigel Basement Membrane Matrix (BD Biosciences). The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air, supplemented with fresh culture medium on the second day, and used for the experiments on the fourth day after seeding. Hanks’ balanced salt solution containing HEPES (136.7 mM NaCl, 5.36 mM KCl, 0.952 mM CaCl2, 0.812 mM MgSO4, 0.441 mM KH2PO4, 0.385 mM Na2HPO4, 25 mM d-glucose, 10 mM HEPES, pH 7.2–7.4) was named as a transport buffer and used throughout the transport experiments. The cell monolayers were preincubated in the transport buffer without test compounds at 37°C for approximately 1 hour, and then the transport experiments were initiated by replacing the buffer in each compartment with 0.5 ml of fresh transport buffer with (donor compartment) and without (acceptor compartment) the test compounds (37°C). Six compounds were tested at 0.5 μM (indinavir, quinidine, loperamide, diazepam, and verapamil) or 1 μM (paclitaxel) concentrations. Both the preincubation and transport experiments were performed in a 5% CO2 incubator at 37°C.

For basal-to-apical transport, the short interval (10 minutes) between sampling times (10, 20, and 30 minutes after initiation) was selected to maintain the concentration in the donor compartment sufficiently higher than that in the acceptor compartment to prevent the underestimation of donor-to-acceptor transport. At each sampling time, 100-μl aliquots were taken from the apical side, the culture inserts (apical side) were transferred to new (vacant) wells of a 24-well plate that had been prewarmed at 37°C, the buffer in the insert was completely removed, and 0.5 ml of fresh transport buffer with and without the test compounds (37°C) was added to the basal and apical sides, respectively, to maintain sink conditions. For apical-to-basal transport, 100-μl aliquots were taken from the basal side at each sampling time (30, 60, and 90 minutes), the culture inserts (apical side) were transferred to new (vacant) wells of a 24-well plate that had been prewarmed at 37°C, and 0.5 ml of fresh transport buffer (37°C) was added to the basal side to maintain sink conditions. One hundred microliters of acetonitrile containing buspirone (internal standard) was added to 100 μl of the collected samples, and the mixtures were then subjected to LC-MS/MS analyses to quantify the amounts of the test compounds that had been transported to the acceptor side.

The transported amounts (pmol/well) were plotted against the transport time (min), and then the transport rate (fmol/min per well) was obtained by the linear regression of three sampling time points. The flux ratio was obtained by dividing the transport rate in the basal-to-apical direction by that in the apical-to-basal direction. The flux ratio in the cynomolgus monkey P-gp–transfected LLC-PK1 cells was divided by that in the parental LLC-PK1 cells to obtain the in vitro P-gp efflux ratio. This in vitro P-gp efflux ratio was used as a measure of the in vitro P-gp transport activity. The paracellular flux was monitored in terms of the appearance of dextran Texas Red in the opposite compartment and was less than 2.6% of the total amount of dextran Texas Red.

Determination of Protein Expression Levels of Cynomolgus Monkey P-gp in the Cynomolgus Monkey P-gp–Transfected LLC-PK1 Cell Monolayer and Isolated Cynomolgus Monkey Brain Microvessels.

Cynomolgus monkey P-gp–transfected LLC-PK1 cells were seeded at a density of 2.081 × 106 cells/insert (0.484 × 106 cells/cm2), which is the same density used in the transcellular transport study, on porous (1.0-μm) polyethylene terephthalate membrane filters (cell culture inserts for six-well plates; BD Biosciences) that had been coated with BD Matrigel Basement Membrane Matrix. The cells were cultured under the same conditions as those used in the transcellular transport study, and the cells were used for the experiments on the fourth day after seeding. The apical and basal sides were washed with ice-cold PBS twice, and the cells were harvested from six inserts using 1 ml/insert of ice-cold PBS by scraping and centrifuged at 4°C and 230g for 5 minutes. The cell pellets were dissolved with 400 μl of Tris-sucrose buffer (10 mM Tris-HCl, 250 mM sucrose, pH 7.4) and suspended well using a 1.0-ml syringe with a 27-gauge × 1/2-inch needle to obtain a whole-cell lysate.

Cynomolgus monkey brain microvessels were isolated from the left cerebrums that had been excised from the cynomolgus monkeys used for the in vivo constant infusion study. The microvessels were isolated by using a combination of dextran density gradient separation and size filtration (nylon mesh method). The isolation procedure was the same as that described in Ito et al. (2011).

The protein expression levels of cynomolgus monkey P-gp in the whole-cell lysates of cynomolgus monkey P-gp–transfected LLC-PK1 cells and the whole-tissue lysates of the isolated cynomolgus monkey brain microvessels were determined by using the same procedure as that described in Ito et al. (2011).

Determination of the Unbound Fractions in Cynomolgus Monkey Plasma Using the Equilibrium Dialysis Method.

Cynomolgus monkey plasma (KAC Co. Ltd., Kyoto, Japan) samples were adjusted to pH 7.4 with saturated CO2 prior to sample preparation. Five microliters of 50% acetonitrile-containing test compound (50 μM) was added to 495 μl of the plasma to obtain a 0.5 μM final concentration in the plasma. One hundred twenty microliters of PBS (pH 7.4) and 120 μl of plasma containing the compounds were placed in the dialysate and sample sides of the 96-well microequilibrium dialysis device (HTD 96b; HTDialysis, Gales Ferry, CT), respectively, with HTD 96a/b Dialysis Membrane Strips (molecular mass cut-off 12–14 kDa; HTDialysis). The wells were sealed and rotated in a 10% CO2 incubator at 37°C and 80 rpm with a multishaker (TOKYO RIKAKIKAI Co. Ltd., Tokyo, Japan). After a 6-hour incubation, 5 μl of plasma and 50 μl of dialysate were collected from the sample and dialysate sides, respectively, and transferred to 96-well format polypropylene plates. Fifty microliters of control PBS and 5 μl of control plasma were added to the collected plasma and dialysate samples, respectively. One hundred fifty microliters of acetonitrile-containing buspirone (internal standard) was added (205 μl in total) and vortexed. The samples were centrifuged and filtrated at 4°C and 960g for 10 minutes and subjected to LC-MS/MS analyses. The unbound fractions of the test compounds in the cynomolgus monkey plasma were calculated on the basis of the ratio of the concentrations that was determined from the plasma and dialysate samples.

Determination of the Unbound Fractions in Cynomolgus Monkey Brain Using a Combination of the Homogenate Method and a pH Partition Model.

The unbound brain fractions were determined by equilibrium dialysis using the brain homogenates in combination with a pH partition model, as previously described (Friden et al., 2011) with minor modifications. The frozen cerebrum of the control cynomolgus monkey was used for this experiment. Cellulose membranes with a molecular mass cutoff of 14,000 Da were shaken in distilled water for 30 minutes, washed with extracellular fluid (ECF) buffer (122 mM NaCl, 3 mM KCl, 0.4 mM K2HPO4, 25 mM NaHCO3, 1.4 mM CaCl2, 1.2 mM MgSO4, 10 mM d-glucose, and 10 mM HEPES, pH 7.4), and conditioned in ECF buffer overnight. The cerebrum was diluted 4-fold with ECF buffer, homogenized using a sonic probe on ice, and then spiked with the test compounds. After preincubation at 37°C for 10 minutes, 400 μl of the brain homogenates containing the compounds (260 nM indinavir, 559 nM quinidine, 870 nM loperamide, 3110 nM paclitaxel, 333 nM diazepam, and 117 nM verapamil) were loaded into the chambers of a Sanplatec EC-1 equilibrium dialysis apparatus (Osaka, Japan), mounted with the dialysis membranes, and dialyzed against 700 μl of ECF buffer that had been preincubated at 37°C. The equilibrium dialysis apparatus was incubated in a 37°C incubator for 6 hours with 300-rpm shaking. After 6 hours, the brain homogenates and dialysate samples were collected from the apparatus. One hundred ninety microliters of acetonitrile containing 1% formic acid and buspirone (internal standard) and 5 μl of 50% acetonitrile were added to 10 μl of the brain homogenate sample (205 μl in total). One hundred microliters of acetonitrile containing 1% formic acid and buspirone (internal standard) and 5 μl of 50% acetonitrile were added to 100 μl of the dialysate sample (205 μl in total). After having been shaken for 20 minutes, the samples were centrifuged at 4°C and 17,360g for 5 minutes. The supernatant (180 μl) was then evaporated by centrifugation under vacuum. The residue was then reconstituted in 0.1% aqueous formic acid and centrifuged at 4°C and 17,360g for 5 minutes. The supernatants were subjected to LC-MS/MS analyses. The following equation was used to calculate the unbound fractions in the brain (fu,brain), where D represents the fold dilution of the brain tissue and fu,measured is the ratio of concentrations determined from the dialysate and brain homogenate samples:Embedded Image(1)According to the pH partition model reported in Friden et al. (2011), fu,brain values that were more relevant to the in vivo condition were calculated using the values of fu,brain determined using the homogenate method above (eq. 1) and the reported pKa values of the test compounds (see Table 3 caption).

LC-MS/MS Analyses of the Compounds.

The sample analyses were automated by coupling a triple quadrupole mass spectrometer (API4000 or API5000; AB SCIEX, Framingham, MA) to an Agilent 1200 high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA). The samples were injected onto either an Agilent XDB-C18 column (2.1 × 150 mm, 5 μm) or a Shiseido Capcell Pak UG120 C18 column (2.0 × 150 mm, 5 μm). The compounds were separated and eluted from the columns under linear gradient or isocratic conditions with a flow rate of 0.2–0.3 ml/min. The eluted compounds were detected using electrospray ionization in selected/multiple reaction monitoring mode. Selected/multiple reaction monitoring transitions for indinavir, quinidine, loperamide, paclitaxel, diazepam, verapamil, and buspirone were 614.5/421.3, 325.3/172.3, 477.4/266.0, 854.5/569.3, 285.1/193.5, 455.1/165.3, and 386.3/122.3, respectively.

IVIVR Theory of BBB P-gp Function and the Brain Distributions of P-gp Substrates.

According to the reconstruction theory reported in Uchida et al. (2011a), in vivo P-gp function at the cynomolgus monkey BBB and the brain distributions of P-gp substrates and nonsubstrate in cynomolgus monkeys were reconstructed using the in vitro experimental values.

In vivo P-gp function at the cynomolgus monkey BBB is defined as the Kp,brain ratio, which is the ratio of Kp,brain in P-gp knockout animals to that in wild-type animals. As previously described (Uchida et al., 2011a), the Kp,brain ratio was reconstructed using eq. 2 and the in vitro P-gp efflux ratio and protein expression levels of P-gp in the cynomolgus monkey P-gp–transfected LLC-PK1 cell monolayer and isolated cynomolgus monkey brain microvessels.Embedded Image(2)The Kp,brain in cynomolgus monkeys was reconstructed in eq. 3 using the reconstructed Kp,brain ratio (eq. 2) and the unbound fractions in the plasma (fu,plasma) and brain (fu,brain), which were measured using the homogenate method and the pH partition model.Embedded Image(3)Furthermore, the Kp,uu,brain is defined as follows:Embedded Image(4)The Kp,uu,brain in cynomolgus monkeys was reconstructed as the reciprocal of the reconstructed Kp,brain ratio in eq. 5, which was obtained using eqs. 3 and 4:Embedded Image(5)We were unable to determine the observed Kp,brain ratio in cynomolgus monkeys because we did not have a P-gp knockout cynomolgus monkey. However, the observed Kp,brain and Kp,uu,brain can be determined via in vivo experiments with wild-type cynomolgus monkeys and consist of the Kp,brain ratio, as described in eqs. 3 and 5. Therefore, the reconstructions of the Kp,brain and Kp,uu,brain can be validated by comparing the reconstructed values with the observed values. The reconstruction of the Kp,brain ratio can also be evaluated on the basis of the validation of the Kp,brain and Kp,uu,brain reconstructions, leading to the validation of IVIVR of P-gp function at the cynomolgus monkey BBB.

Quantitative Evaluation of the Effects of Individual Parameters on Species Differences in Kp,brain and Kp,uu,brain between Cynomolgus Monkeys and Mice.

Using eqs. 2 and 3, the Kp,brain is described by four parameters, as follows:Embedded Image(6)where PLp-gp,vivo represents the protein expression levels of P-gp in isolated brain microvessels and TAint,p-gp represents the intrinsic transport activity per P-gp molecule, which is calculated as [(In vitro P-gp efflux ratio) – 1]/[P-gp protein expression levels in P-gp–transfected LLC-PK1 cells]. Therefore, the species differences in the Kp,brain between cynomolgus monkeys and mice are described as follows:Embedded Image(7)Using eqs. 2 and 5, the Kp,uu,brain is described by two parameters, as follows:Embedded Image(8)Therefore, the species differences in the Kp,uu,brain between cynomolgus monkeys and mice are described as follows:Embedded Image(9)In the present study, the contributions of individual parameters to the species differences in the Kp,brain and Kp,uu,brain between cynomolgus monkeys and mice were described as the Impact on Kp,monkey/Kp,mouse and the Impact on Kp,uu,monkey/Kp,uu,mouse, respectively. To quantitatively evaluate the contributions of each parameter, the Impact on Kp,monkey/Kp,mouse and the Impact on Kp,uu,monkey/Kp,uu,mouse were calculated using the following equations:Embedded Image(10)Embedded Image(11)where the Kp,monkey(mouse) consists of three cynomolgus monkey parameters and one mouse parameter. Briefly, the mouse data were used for either one of PLp-gp,vivo,monkey(mouse), TAint,p-gp,monkey(mouse), fu,plasma,monkey(mouse), or fu,brain,monkey(mouse), which was a targeted parameter for the evaluation of contribution, and the cynomolgus monkey data were used for the other three parameters. In the same manner as the Kp,monkey(mouse), only one of the evaluated parameters was derived from the mouse data and the others were derived from the cynomolgus monkey data for the Kp,uu,monkey(mouse). For the cynomolgus monkey data, the experimental values that were determined in the present study were used. For the mouse data, the values reported by Uchida et al. (2011a) were used.

Results

Determination of the Steady-State Brain-to-Plasma Concentration Ratios of Five P-gp Substrates and One Nonsubstrate in Cynomolgus Monkeys.

Indinavir, quinidine, loperamide, paclitaxel, verapamil, and a nonsubstrate, diazepam, were administered to male adult cynomolgus monkeys by continuous intravenous infusion, and the Kp,brain values were determined at steady-state (3-hour) plasma concentrations (Table 1). The plasma concentrations of the six compounds at 3 hours ranged from 0.0331 to 0.541 μM, which were lower than the reported Km values for P-gp that were determined using the ATPase assay (Adachi et al., 2001). The Kp,brain values of the six compounds in cynomolgus monkeys varied by 15-fold (Table 1).

View this table:
TABLE 1

Time profiles of the plasma concentrations and Kp,brain values of the six compounds in male cynomolgus monkeys after intravenous constant infusion

The male cynomolgus monkeys (fasted overnight) were fixed to monkey chairs without anesthesia during compound administration. Indinavir, quinidine, diazepam, and verapamil were intravenously infused for 3 hours at dose rates of 0.61, 0.21, 0.070, and 0.40 mg/h per kilogram, respectively, after the intravenous bolus injection of 0.50, 0.30, 0.095, and 1.2 mg/kg doses. Loperamide and paclitaxel were intravenously infused for 3 hours at dose rates of 0.20 and 0.94 mg/h per kilogram, respectively, without intravenous bolus injections. The right cerebrums were used to determine the brain-to-plasma concentration ratios (Kp,brain) at 3 hours. One cynomolgus monkey was used for each compound.

Reconstruction of In Vivo P-gp Function (Kp,brain Ratio) at the Cynomolgus Monkey BBB.

The Kp,brain ratio is defined as the ratio of Kp,brain in P-gp knockout animals to that in wild-type animals and is a parameter that describes in vivo P-gp function at the BBB. The reconstruction of the Kp,brain ratio from the in vitro experiments has been previously demonstrated in mice (Uchida et al., 2011a). To demonstrate the reconstruction theory in cynomolgus monkeys as well as mice, we reconstructed the Kp,brain ratios for the six model compounds on the basis of the in vitro transport activities and protein expression levels of cynomolgus monkey P-gp according to the theory that was previously demonstrated in mice (eq. 2) as follows:

In the transcellular transport experiments using cynomolgus monkey P-gp–transfected LLC-PK1 cell monolayers and the parental LLC-PK1 cell monolayers shown in Fig. 1, the in vitro P-gp efflux ratio (a parameter reflecting the P-gp–specific transport activities of the test compounds) was determined and was found to range from 0.984 (diazepam) to 8.00 (loperamide) for the six compounds investigated (Table 2).

Fig. 1.
Fig. 1.

Transepithelial transport of five P-gp substrates and one nonsubstrate across cynomolgus monkey P-gp–transfected and parental LLC-PK1 cell monolayers. Six compounds were tested at 0.5 μM [indinavir (A), quinidine (B), loperamide (C), nonsubstrate diazepam (E), and verapamil (F), or 1 μM paclitaxel (D)] concentrations. The ordinate represents the amounts of each compound that were transported from the donor side (500 μl) to the acceptor side (500 μl). Each point represents the mean ± S.D. (n = 3). ●, basal-to-apical transport across the cynomolgus monkey P-gp–transfected LLC-PK1 cell monolayer; ▪, apical-to-basal transport across the cynomolgus monkey P-gp–transfected LLC-PK1 cell monolayer; ○, basal-to-apical transport across the parental LLC-PK1 cell monolayer; □, apical-to-basal transport across the parental LLC-PK1 cell monolayer.

View this table:
TABLE 2

Reconstruction of P-gp activities at the cynomolgus monkey BBB on the basis of the in vitro transport activities and protein expression levels

On the basis of the results shown in Fig. 1, the apical-to-basal (A to B) transport rate, the basal-to-apical (B to A) transport rate, the flux ratio, and the in vitro P-gp efflux ratio across LLC-PK1/cynomolgus monkey P-gp–transfected LLC-PK1 cell monolayers were calculated as described in Materials and Methods. For the quantifications of P-gp expression, cynomolgus monkey P-gp–transfected LLC-PK1 cells were cultured under the same conditions that were used for the transcellular transport experiments, and the expression levels in the transfected cell monolayers were determined using LC-MS/MS–based quantification (triplicate experiments). After the constant infusion of each compound, the cynomolgus monkeys were sacrificed, the cerebrums were collected, and the left cerebrums were used to isolate brain microvessels using the nylon mesh method. P-gp protein expression levels in the brain microvessels of each cynomolgus monkey were determined using LC-MS/MS–based quantification (triplicate experiments for each cynomolgus monkey). Reconstructed Kp,brain ratios were calculated from the in vitro P-gp efflux ratios and the P-gp protein expression levels using eq. 2. Each value represents the mean ± S.E.M. The S.E.M. was calculated according to the law of propagation of error.

The protein expression levels of cynomolgus monkey P-gp were determined using QTAP. The levels were 2.31 fmol/μg protein of whole-cell lysate in cynomolgus monkey P-gp–transfected LLC-PK1 cell monolayers and ranged from 5.05 to 7.07 fmol/μg protein of whole-tissue lysate in the isolated brain microvessels among the six cynomolgus monkeys administered the six compounds respectively, including data taken from the literature (Ito et al., 2011) (Table 2).

Using these data, the Kp,brain ratios for the six compounds were reconstructed using eq. 2. The reconstruction of the Kp,brain ratio for each compound was performed using P-gp protein expression levels in the cynomolgus monkey that had been administered the corresponding compound. The reconstructed Kp,brain ratios of the six compounds ranged from 0.951 (diazepam) to 16.3 (loperamide) (Table 2).

Reconstruction of the Kp,brain of the Five P-gp Substrates and One Nonsubstrate in Cynomolgus Monkeys.

The Kp,brain values for the six compounds in cynomolgus monkeys were reconstructed using eq. 3 and the reconstructed Kp,brain ratios and the unbound fractions in cynomolgus monkey plasma and brain (Table 3). The unbound fractions in the brain were measured using the homogenate method, converted to values that were more relevant to the in vivo condition in combination with the pH partition model, and used for the Kp,brain reconstructions. The reconstructed Kp,brain values were within a 3-fold range of the observed values for all six compounds (Fig. 2A).

View this table:
TABLE 3

Reconstruction of the Kp,brain of the six compounds in cynomolgus monkeys on the basis of the in vitro data

Cynomolgus monkey plasma was spiked with 500-nM concentrations of the compounds and dialyzed against PBS (pH 7.4) at 37°C for 6 hours (n = 3). One-quarter of the diluted brain homogenates of the cynomolgus monkeys was spiked with 260 nM indinavir, 559 nM quinidine, 870 nM loperamide, 3110 nM paclitaxel, 333 nM diazepam, and 117 nM verapamil and dialyzed against ECF buffer (pH 7.4) at 37°C for 6 hours to obtain the homogenate fu,brain (n = 3). The fu,brain (homogenate + pH partition model) was calculated from the homogenate fu,brain in combination with the reported pH partition model (Friden et al., 2011) using the reported pKa values of the compounds (Carvalho-Silva et al., 2004; Friden et al., 2011). The reconstructed Kp,brain values were calculated from the reconstructed Kp,brain ratios (see Table 2) and the fu,plasma and fu,brain (homogenate + pH partition model) values using eq. 3. Each value represents the mean ± S.E.M. The S.E.M. was calculated according to the law of propagation of error.

Fig. 2.
Fig. 2.

Comparison of the observed and reconstructed Kp,brain (A) and Kp,uu,brain (B) for the six compounds. These data were taken from Tables 1 and 3. The solid line passing through the origin represents the line of identity, and the broken lines represent 3-fold differences. Each point represents the mean ± S.E.M.. 1, indinavir; 2, quinidine; 3, loperamide; 4, paclitaxel; 5, diazepam; and 6, verapamil.

Reconstruction of the Kp,uu,brain of the Five P-gp Substrates and One Nonsubstrate in Cynomolgus Monkeys.

Using eq. 5, the Kp,uu,brain values for the six compounds in cynomolgus monkeys were reconstructed as the reciprocals of the reconstructed Kp,brain ratios (Table 4). The in vivo Kp,uu,brain values were estimated using the observed Kp,brain values in vivo and the unbound fractions using eq. 4. The reconstructed Kp,uu,brain values were within a 3-fold range of the estimated in vivo values for all six compounds (Fig. 2B).

View this table:
TABLE 4

Reconstructed and estimated in vivo Kp,uu,brain values of the six compounds in cynomolgus monkeys

The reconstructed Kp,uu,brain values were calculated as the reciprocals of the reconstructed Kp,brain ratios (see Table 2) using eq. 5. The in vivo Kp,uu,brain values were estimated from the fu,plasma and fu,brain (homogenate + pH partition model) values and the observed Kp,brain values in cynomolgus monkeys (see Tables 1 and 3) using eq. 4. Each value represents the mean ± S.E.M. The S.E.M. was calculated according to the law of propagation of error.

Reconstruction of Species Differences in the Kp,brain and Kp,uu,brain between Cynomolgus Monkeys and Mice.

To clarify the species differences in drug distribution in the brain, we cited the observed Kp,brain and Kp,uu,brain values in mice reported in Uchida et al. (2011a), and divided the observed values in cynomolgus monkeys by those in mice (Fig. 3). The Kp,brain values indicated that the differences between species (observed Kp,monkey/Kp,mouse) were within a 0.657- to 10.9-fold range for the six compounds, with loperamide displaying the greatest difference (10.9-fold), followed by verapamil (5.22-fold). The species differences in the Kp,uu,brain values of each of the six compounds (estimated in vivo Kp,uu,monkey/Kp,uu,mouse) were within a 0.510- to 2.44-fold range.

Fig. 3.
Fig. 3.

Comparison of the observed and reconstructed species differences in the Kp,brain (A) and Kp,uu,brain (B) of the six compounds between cynomolgus monkeys and mice. The cynomolgus monkey data were taken from Tables 1, 3, and 4 and then divided by the mouse data cited in Uchida et al. (2011a). The solid line passing through the origin represents the line of identity, and the broken lines represent 3-fold differences. 1, indinavir; 2, quinidine; 3, loperamide; 4, paclitaxel; 5, diazepam; and 6, verapamil.

It is important for establishing the theory to quantitatively predict and overcome the species differences in brain drug distribution. To elucidate whether the species differences in Kp,brain and Kp,uu,brain values could be reconstructed from the in vitro experimental data, we reconstructed the ratios of the cynomolgus monkey values to the mouse values using eqs. 7 and 9 (reconstructed Kp,monkey/Kp,mouse and reconstructed Kp,uu,monkey/Kp,uu,mouse) and then compared these ratios with the observed ratios (Fig. 3). For both the Kp,brain and Kp,uu,brain values, the reconstructed ratios were within a 3-fold range of the observed ratios, although the reconstructed ratios for indinavir were 3.13-fold higher than the observed ratios.

Quantitative Evaluation of the Contributions of Individual Parameters to Species Differences in the Kp,brain and Kp,uu,brain between Cynomolgus Monkeys and Mice.

To understand the mechanisms underlying the remarkable species differences in the Kp,brain for loperamide and verapamil, we estimated the contributions of four parameters to the species differences in the Kp,brain by calculating the “Impact on Kp,monkey/Kp,mouse” using eq. 10 (Table 5). For loperamide, the species differences in fu,plasma and PLp-gp,vivo were estimated to contribute to the 3.37- and 2.68-fold higher Kp,monkey than Kp,mouse, respectively, which were greater contributions than those of TAint,p-gp (0.550) and fu,brain (1.33). For verapamil, the species differences in PLp-gp,vivo, fu,plasma, and fu,brain were estimated to contribute to the 2.38-, 1.92-, and 1.77-fold higher Kp,monkey than Kp,mouse, respectively.

View this table:
TABLE 5

Impact of four parameters on species differences in the Kp,brain and Kp,uu,brain values between cynomolgus monkeys and mice

The contributions of individual parameters (P-gp protein expression levels at the BBB, the intrinsic transport activity per P-gp molecule, and the unbound fractions in the plasma and brain) to the species differences in the Kp,brain and Kp,uu,brain values between cynomolgus monkeys and mice are presented. PLp-gp,vivo represents the protein expression levels of P-gp in isolated brain microvessels. TAint,p-gp represents the intrinsic transport activity per P-gp molecule and is calculated as [(In vitro P-gp efflux ratio) – 1]/[P-gp protein expression levels in P-gp–transfected LLC-PK1 cells]. The values of PLp-gp,vivo, TAint,p-gp, fu,plasma, and fu,brain in cynomolgus monkeys were divided by the corresponding values in mice to obtain the Monkey/Mouse values for each compound. The cynomolgus monkey data were taken from Tables 2 and 3, and the mouse data were cited from Uchida et al. (2011a). The values of the Impact on Kp,monkey/Kp,mouse and the Impact on Kp,uu,monkey/Kp,uu,mouse describe the contributions of individual parameters to the species differences in the brain-to-plasma concentration ratio (Kp,brain) and Kp,uu,brain values between cynomolgus monkeys and mice and were calculated using eqs. 10 and 11, respectively.

For all six compounds, the species differences in PLp-gp,vivo and TAint,p-gp were estimated to contribute to the 2.16- to 2.68-fold (with the exception of diazepam) and 0.550- to 1.22-fold species differences in both the Kp,brain and Kp,uu,brain, respectively (Table 5). The species differences in fu,plasma and fu,brain only affected the Kp,brain, not the Kp,uu,brain, and were estimated to contribute to the species differences in the Kp,brain of 0.369- to 11.1-fold and 0.313- to 1.87-fold, respectively, for the six compounds.

Discussion

The present study is the first to experimentally demonstrate that the Kp,brain values of P-gp substrates and nonsubstrate can be reconstructed in nonhuman primate cynomolgus monkeys, an animal that is similar to humans, by integrating in vitro P-gp transport activity, P-gp protein expression levels, and the unbound fractions in plasma and brain on the basis of eqs. 2 and 3. This study is also the first to experimentally demonstrate that the Kp,uu,brain values can be reconstructed by integrating in vitro P-gp transport activity and P-gp protein expression levels on the basis of eqs. 2 and 5.

In vivo P-gp transport function at the BBB is defined as the Kp,brain ratio, which is the ratio of Kp,brain value in P-gp knockout animals to that in wild-type animals. Because a P-gp knockout monkey does not exist, we were unable to determine the observed value of Kp,brain ratio in monkeys, thereby making it impossible to directly validate the IVIVR of Kp,brain ratio in monkeys. Equation 3 indicates that the accuracy of reconstruction of Kp,brain value is directly influenced by that of Kp,brain ratio. Therefore, the successful reconstruction of Kp,brain value in this study has indirectly demonstrated that the Kp brain ratio can also be precisely reconstructed from in vitro P-gp transport activity and P-gp protein expression levels in monkeys. P-gp protein expression levels in brain microvessels only differ by 1.29-fold from those in humans (Ito et al., 2011; Uchida et al., 2011b), and a good agreement between monkey P-gp and human P-gp has been also reported in the in vitro transport activities for a variety of substrates (Takeuchi et al., 2006). Therefore, the present demonstration of reconstruction theory suggests that our established PPx-based reconstruction would be useful in clarifying in vivo P-gp function at the human BBB.

Significant species differences in Kp,brain values were observed between monkeys and mice, with a maximum difference of 10.9-fold among six compounds (Fig. 3). Syvanen et al. (2009) also reported remarkable species differences in the Kp,brain values of P-gp substrates between humans and rats (e.g., an 8.6-fold difference for [11C]GR205171). On the basis of these results, it is clear that the Kp,brain values measured in rodent experiments cannot reliably predict drug distributions in human brain. Several studies have suggested the usefulness of in vitro P-gp–transfected cells for predictions of in vivo P-gp functions at the BBB and drug distribution in brain (Adachi et al., 2001; Feng et al., 2008). However, it is challenging to make accurate predictions on the basis of in vitro experiments because the extent to which the transport functions and protein expression levels of P-gp differ between in vivo BBB and in vitro transfected cells is unclear. In this study, using monkeys that are similar to humans in terms of drug distribution in brain, we demonstrated that the Kp,brain and Kp,uu,brain values can be predicted with ±3-fold accuracy from in vitro experiments on the basis of the in vitro/in vivo differences in protein expression levels of P-gp (Fig. 2). Furthermore, we demonstrated that the species differences in Kp,brain and Kp,uu,brain values can also be predicted from in vitro studies with ±3-fold accuracy (Fig. 3). Therefore, our established PPx-based reconstruction overcomes the species differences in drug distribution in brain and provides a useful method to rationally predict drug distribution in human brain from in vitro experiments.

One of the advantages of Kp,brain and Kp,uu,brain reconstructions on the basis of several factors is that the contributions of individual factors can be quantitatively evaluated for changes in Kp,brain and Kp,uu,brain values. As shown in Fig. 3, remarkable species differences in Kp,brain values were observed for loperamide and verapamil between monkeys and mice. Table 5 summarizes the contributions of four factors [BBB P-gp protein expression levels (PLp-gp,vivo), intrinsic transport activity per P-gp molecule (TAint,p-gp), fu,plasma, and fu,brain] to the species differences in Kp,brain and Kp,uu,brain values of six compounds. For loperamide, the species differences in fu,plasma and PLp-gp,vivo were 3.37- and 2.79-fold, respectively, and contributed to 3.37- and 2.68-fold species differences in Kp,brain values, respectively. Smaller contributions were observed for the other two factors. These results suggest that major causes of the species differences in Kp,brain values of loperamide are differences in fu,plasma and PLp-gp,vivo between monkeys and mice. For verapamil, 2.71-, 1.92-, and 1.77-fold species differences in PLp-gp,vivo, fu,plasma, and fu,brain were observed, respectively, and these differences contributed to 2.38-, 1.92-, and 1.77-fold greater Kp,brain values in monkeys than in mice, respectively. These data suggest that the 5.22-fold greater observed Kp,brain values for verapamil in monkeys are caused by the species differences in these three factors.

Among the four factors, the species differences in fu,plasma showed the largest variation among six compounds, ranging from 0.369- (quinidine) to 11.1-fold (indinavir) differences between monkeys and mice (Table 5). The Impact on Kp,monkey/Kp,mouse of fu,plasma also varied from 0.369 (quinidine) to 11.1 (indinavir), which was the largest variation among the four factors, suggesting that fu,plasma most significantly contributes to species variations in Kp,brain values. Several studies using a variety of compounds have indicated that there are remarkable species differences in fu,plasma but no large differences in fu,brain (Fuse et al., 1998; Kratochwil et al., 2004; Di et al., 2011). Table 5 shows that fu,brain did not affect the species differences in Kp,brain values of six compounds to as great an extent as fu,plasma. Therefore, unlike fu,plasma, fu,brain would not contribute substantially to species variations in Kp,brain values.

The Kp,uu,brain value is independent of fu,plasma and fu,brain values, as shown in eq. 5 and Table 5, and consequently species differences in Kp,uu,brain values were smaller than those in Kp,brain values (Fig. 3). As shown in eqs. 2 and 5, the Kp,uu,brain value is affected only by PLp-gp,vivo and TAint,p-gp. PLp-gp,vivo differed by 2.00- to 2.79-fold between monkeys and mice, and TAint,p-gp did not differ so much (Table 5). As a result, the Impact on Kp,uu,monkey/Kp,uu,mouse of PLp-gp,vivo ranged from 2.16- to 2.68-fold (with the exception of diazepam, which is not a P-gp substrate) and was greater than that of TAint,p-gp (Table 5). These data suggest that the PLp-gp,vivo is a major cause of the species differences in Kp,uu,brain values. The species differences in P-gp protein expression levels at the BBB are 2- to 3-fold between humans and rodents, as is the case for monkeys and mice (Kamiie et al., 2008; Ito et al., 2011; Uchida et al., 2011b; Hoshi et al., 2013). Therefore, the differences in the Kp,uu,brain values of P-gp substrates between humans and rodents could typically range from approximately 2- to 3-fold, in accordance with the differences in protein expression levels. From this consideration, it is suggested that the Kp,uu,brain values of P-gp substrates differ between humans and rodents but do not remarkably differ when compared with the species differences in Kp,brain values. Therefore, the measurement of Kp,uu,brain value in rodents during drug development would be useful in understanding drug distribution in human brain.

The reconstructed brain distributions of six compounds in monkeys were within a 3-fold range of the observed distributions but were not completely identical (Fig. 2). One possible explanation is that the fu,brain used for the reconstruction was not identical to that in vivo. Brain slice method can provide fu,brain values that are more relevant to in vivo condition (Kakee et al., 1996; Ooie et al., 1997; Friden et al., 2007). However, frozen monkey brains were used in this study to mimic the way of IVIVR in humans, for which the brain is usually obtained in a frozen state. In frozen brain, the cells may be partially ruptured, and the fu,brain may not be accurately determined using the brain slice method. Therefore, we used the homogenate method with a pH partition model, which results in an fu,brain value that is more relevant to in vivo condition than that obtained using the homogenate method alone (Friden et al., 2011). However, this method does not take into account the involvement of active transport on the cell membrane of brain parenchyma, resulting in the possibility that the determined fu,brain differs from the true in vivo value. Another possible explanation is that only one monkey was studied for each compound to determine the observed Kp,brain values. The variability of observed values could not be considered owing to limited resources. Increasing the number of monkeys for each compound would raise the accuracy for the validation of the present IVIVR.

In conclusion, using cynomolgus monkeys as a robust human model, this study experimentally demonstrated that the Kp,brain and Kp,uu,brain values of P-gp substrates and nonsubstrate can be reconstructed by integrating in vitro P-gp transport activity, P-gp protein expression levels, and the unbound fractions in plasma and brain on the basis of BBB PPx. These results also demonstrate that in vivo P-gp transport function at the BBB can be reconstructed on the basis of in vitro P-gp transport activity and P-gp protein expression levels. These demonstrations illustrate the value of our established PPx-based reconstruction model for clarifying in vivo function at the human BBB and predicting brain drug distribution in humans. Because not only P-gp but also BCRP at the BBB limit brain distributions of a number of drugs, further study would be needed in future to demonstrate that in vivo function of BCRP can be reconstructed from in vitro for the prediction of brain distribution for more drugs.

Acknowledgments

The authors thank A. Niitomi, N. Handa, Y. Yoshikawa, and K. Tsukiura for secretarial assistance.

Authorship Contributions

Participated in the research design: Uchida, Wakayama, Ohtsuki, Chiba, Ohe, Ishii, Terasaki.

Conducted the experiments: Uchida, Wakayama.

Contributed new reagents or analytic tools: Uchida, Wakayama.

Performed data analyses: Uchida, Wakayama.

Wrote or contributed to the writing of the manuscript: Uchida, Ohtsuki, Terasaki.

Footnotes

    • Received March 9, 2014.
    • Accepted June 28, 2014.

  • This study was supported in part by four Grants-in-Aid from the Japanese Society for the Promotion of Science (JSPS) for Scientific Research (S) [KAKENHI: 18109002], Scientific Research (A) [KAKENHI: 24249011], Young Scientists (B) [KAKENHI: 23790170], and a JSPS Fellowship [KAKENHI: 207291]. This study was also supported in part by two Grants for the Development of Creative Technology Seeds Supporting Program for Creating University Ventures and the Revitalization Promotion Program (A-STEP) from the Japan Science and Technology Agency (JST).

  • T.T. and S.O. are full professors at Tohoku University and Kumamoto University, respectively, and are also directors of Proteomedix Frontiers Co. Ltd. This study was not supported by Proteomedix Frontiers Co. Ltd., and their positions at Proteomedix Frontiers Co. Ltd. did not affect the design of the study, the collection, analysis, and interpretation of the data, the writing of the manuscript, or the decision to publish, and did not present any financial conflicts. The remaining authors declare no competing financial interests.

  • dx.doi.org/10.1124/jpet.114.214536.

Abbreviations

ABC
ATP-binding cassette
BBB
blood-brain barrier
BCRP
breast cancer resistance protein
ECF
extracellular fluid
fu,brain
unbound fraction in brain
fu,plasma
unbound fraction in plasma
GR205171
(S)-(2-methyl-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethylamino)-2(S)-phenylpiperidine
IVIVR
in vitro–to–in vivo reconstruction
Kp,brain
brain-to-plasma concentration ratio
Kp,uu,brain
unbound brain-to-plasma concentration ratio
LC-MS/MS
liquid chromatography–tandem mass spectrometry
MDR1
multidrug resistance protein 1
PBS
phosphate-buffered saline
PLp-gp,vivo
protein expression level of P-gp in isolated brain microvessels
P-gp
P-glycoprotein
PPx
pharmacoproteomics
QTAP
quantitative targeted absolute proteomics
TAint,p-gp
intrinsic transport activity per P-gp molecule

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Research ArticleMetabolism, Transport, and Pharmacogenomics

IVIVE of Brain Drug Distribution in Nonhuman Primate

Yasuo Uchida, Kentaro Wakayama, Sumio Ohtsuki, Masato Chiba, Tomoyuki Ohe, Yasuyuki Ishii and Tetsuya Terasaki
Journal of Pharmacology and Experimental Therapeutics September 1, 2014, 350 (3) 578-588; DOI: https://doi.org/10.1124/jpet.114.214536
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