Effect of Knockout of Mdr1a and Mdr1b

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Molecular Pharmaceutics

Effect of knockout of Mdr1a and Mdr1b ABCB1 genes on the systemic exposure of a doxorubicin-conjugated block copolymer in mice

Kumiko Sakai-Katoa*, Kunie Nanjoa, Hiroyuki Kusuharab, Nobuhiro Nishiyamac, Kazunori Kataokad,e, Toru Kawanishif, Haruhiro Okudaf and Yukihiro Godaa

a

Division of Drugs, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo

158-8501, Japan b

Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences,

The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan c

Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology,

R1-11, 4259 Nagatsuda, Midori, Yokohama 226-8503, Japan d

Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The

University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan e

Department of Materials Engineering, Graduate School of Engineering, The University of

Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-8656, Japan f

National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan

*Corresponding author 1

ACS Paragon Plus Environment


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Kumiko Sakai-Kato, PhD Division of Drugs, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan Phone: +81-3-3700-9662, Fax: +81-3-3700-9662, E-mail address: [email protected]

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Abstract

We previously elucidated that ATP-binding cassette sub-family B member 1 (ABCB1) mediates the efflux of doxorubicin-conjugated block copolymers from HeLa cells. Here, we investigated the role of ABCB1 in the in vivo behavior of a doxorubicin-conjugated polymer in Mdr1a/1b(-/-) mice. The area under the curve for intravenously administrated polymer in Mdr1a/1b(-/-) mice was 2.2-fold greater than that in wild-type mice. The polymer was mostly distributed in the liver followed by spleen, and less so in the brain, heart, kidney, and lung. The amount of polymer excreted in the urine was significantly decreased in Mdr1a/1b(-/-) mice. The amounts of polymers excreted in the feces were similar in both groups despite of the higher systemic exposure in Mdr1a/1b(-/-) mice. Confocal microscopy images showed polymer localized in CD68+ macrophages in the liver. These results show that knockout of ABCB1 prolonged systemic exposure of the doxorubicin-conjugated polymer in mice. Our results suggest that ABCB1 mediated the excretion of doxorubicin-conjugated polymer in urine and feces. Our results provide valuable information about the behavior of block copolymers in vivo, which is important for evaluating the pharmacokinetics of active substances conjugated to block copolymers or the accumulation of block copolymers in vivo.

Keywords: block copolymer micelles; ABCB1; systemic exposure; clearance

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Abbreviations: ABCB1, ATP-binding cassette sub-family B member 1 (ABCB1);

DBD, 4-[N,N-dimethylsulfamoyl]-2,1,3-benzoxadiazole); mPEG-FITC, methoxyl poly[ethylene glycol] fluorescein PBS, phosphate-buffered saline; AUC, area under the curve; MRT, mean residence time; CLtot, Total body clearance; PEG, poly(ethylene glycol).

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1. Introduction

Drug delivery systems based on functional carriers show promise for the targeted transfer of chemotherapeutic agents, proteins, and nucleic acids to specific tissues [1–3]. Block copolymer micelles are receiving particular attention as targetable carrier systems in which active substances can be stably incorporated by chemical conjugation or physical entrapment. Several block copolymer micelle carrier systems are currently undergoing clinical evaluation for therapeutic efficacy [4–8]. Improving the pharmacokinetics of active substances, which includes their targeting in vivo, is one of the most common objectives of block copolymer micelle formulation design. Therefore, the pharmacokinetics of active substances conjugated to block copolymers or entrapped within block copolymer micelles is actively being investigated from a clinical efficacy point of view [9–12]. The fate of the micelle constituents by themselves in the body has received greater attention from the regulatory point of view [13]. The biodistribution and routes of elimination of the block copolymer can provide valuable information on the safety of the overall block copolymer micelle product, such as its bioaccumulation potential. However, few investigations have been conducted into the pharmacokinetics of the carrier itself. Doxorubicin-conjugated block copolymers composed of poly(ethylene glycol) and 6


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poly(aspartic acid) can form globular micelles via hydrophobic interactions between the conjugated doxorubicin molecules. Doxorubicin was conjugated to the block copolymer to entrap the free doxorubicin which would exert their pharmacodynamics activity after being released from the inner core of the micelles. This micelles system has proceeded into the clinical trials to evaluate the antitumor efficacy [4, 12]. The conjugation was designed not to release doxorubin in vivo [12]. Indeed, we confirmed that the in vitro cytotoxicity of the doxorubicin-conjugated polymers was negligible [14]. We previously investigated the intracellular trafficking and fate of block copolymers and reported that ATP-binding cassette sub-family B member 1 (ABCB1) (also known as multidrug resistance protein 1 [MDR-1] or P-glycoprotein) mediates the efflux of fluorescent dye- or doxorubicin-conjugated block copolymer from HeLa cells [14,15]. ABCB1 acts as an active efflux pump exporting substrates out of cells [16]. Human ABCB1 is present not only in tumor cells but also in normal cells in the kidney, liver, small and large intestine, brain, testis, adrenal gland, and the pregnant uterus [16]. This tissue distribution shows that ABCB1 plays an important role in the excretion of drug products and metabolites into the urine [17,18], bile [19], and intestinal lumen, and in preventing accumulation of medicinal products and metabolites in the brain [20]. Therefore, we hypothesized that ABCB1 may play a key role in the in vivo behavior of the block copolymers. In the present study, we investigated the role of ABCB1 in the in vivo behavior of the 7



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doxorubicin-conjugated block copolymer where the free doxorubicin was not encapsulated. Plasma and tissue concentrations, and elimination, of the doxorubicin-conjugated block copolymer were compared between wild-type mice and Mdr1a/1b(-/-) mice, which do not express ABCB1 [21-24]. Here we show that ABCB1 plays a key role in the pharmacokinetics of a doxorubicin-conjugated block copolymer.

2. Experimental Section

2.1 Drugs and chemicals Doxorubicin hydrochloride and daunorubicin hydrochloride were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Doxorubicinone was purchased from Toronto Research Chemicals, Inc. (North York, Canada). High-performance liquid chromatography (HPLC)-grade acetonitrile and HPLC-grade methanol were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). Zinc sulfate heptahydrate (ZnSO4) was obtained from Sigma-Aldrich Corporation (St. Louis, MO). Block copolymer composed of poly(ethylene glycol) (MW, ca. 5000 Da) and poly(aspartic acid) (polymerization degree, 30) with doxorubicin partially conjugated (ca. 45 mol %) to the side-chain of the poly(aspartic acid) was synthesized by Nippon Kayaku Co., Ltd. (Tokyo, Japan) [12,15] (Fig. 1). Block copolymer composed of poly(ethylene glycol) (MW, ca. 12000 Da) and poly(aspartic acid) (polymerization degree, 35–45) with DBD (4-[N,N-dimethylsulfamoyl]-2,1,3-benzoxadiazole) 8


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partially conjugated to the side chain of the poly(aspartic acid) was also synthesized by Nippon Kayaku Co., Ltd. [15] (Fig. 1). The particle size and, poly diversity index (PDI), and ξ-potential of the micelles were determined using a Zetasizer Nano ZS (Malvern Instrument, Worcestershire, UK). The particle size was expressed as z-averaged diameters. Fluorescent-labeled poly(ethylene glycol) (methoxyl poly[ethylene glycol] fluorescein [mPEG-FITC]; MW, 10000) was obtained from Nanocs, Inc. (New York, NY). Isolated mammalian cell membrane containing human ABCB1 (Cat No: SB-MDR1-K-VT) for use in vesicle transport assays was purchased from Solvo Biotechnology (Szeged, Hungary).

2.2 Animals Female FVB Mdr1a/1b(-/-) double-knockout mutation mice were obtained from Taconic Farms, Inc. (Hudson St, NY). Female FVB wild-type mice were obtained from CLEA Japan (Tokyo, Japan). Mice 6 weeks of age were used in the analysis of the in vivo behavior of the doxorubicin-conjugated polymer. All mice were maintained under standard conditions under a 12-h dark–light cycle. Food and water were available ad libitum. All experiments in this study using animals were performed according to the guidelines provided by the institutional animal care committee and the gene recombinant committee of the National Institute of Health Sciences, Japan.

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2.3 Doxorubicin-conjugated polymer administration and sample collection Doxorubicin-conjugated polymers dissolved in 10 mM phosphate buffer (pH 7) were incubated for 3 h in light-resistant sample tubes for the formation of micelles. The micelle solutions were then diluted in saline and administered intravenously via the tail vein to the mice (doxorubicin-conjugated polymer, 2.0 mg/20 g mouse). At 1 and 48 h after micelle administration, the mice were sacrificed by cervical dislocation and tissue samples were collected. The tissue samples were rinsed with phosphate-buffered saline (PBS) and stored at −80°C until analysis. In other mice, blood samples were collected from the tail vein into heparinized capillaries at 1, 5, and 30 min, and at 1, 3, 6, 24, 48, 72, 96, and 168 h after micelle administration, and immediately centrifuged at 4000g for 15 minutes at 4°C. The blood samples were stored at −80°C until analysis. Feces and urea were collected at 24 and 48 h after micelle administration and stored at −80°C until analysis.

2.4 Sample preparation for HPLC analysis Samples for HPLC analysis were prepared as described previously [14,25,26]. Tissue samples were weighed and homogenized on ice by using a hand homogenizer (MH-1000, As One, Osaka) and suspended in PBS at a concentration of 500 µL/100 mg tissue (liver, brain, lung, kidney, fat, muscle, small intestine, and feces) or 500 µL/50 mg tissue (heart and spleen), 10


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and the homogenates were stored at −80°C until analysis. The homogenates were disrupted with an ultrasonic liquid processor (ASTRASON 3000, Misonix, NY) for 1 min. The suspensions were mixed with MeOH (final concentration, 60% [v/v]) and then vortexed. To hydrolyze the block copolymer, HCl (final concentration, 1 N) was added and the mixture kept at 50°C for 15 h. After cooling to room temperature, the samples were deproteinized with MeOH and centrifuged (Model 3740, Kubota Corp., Tokyo) at 15,000g for 5 min at 4°C. Ammonium buffer (1 N, pH 9.0) was added to neutralize the hydrolysates. MeOH was then added to obtain a final concentration of 35%, and then daunorubicin hydrochloride (internal standard) was added to a final concentration of 0.02 µg/mL. The mixture was passed through a 0.20-µm-pore filter (Millex-LG, Millipore Corp., Tokyo). The filtrates were transferred to autosampler vials for HPLC analysis. Aliquots of 20 µL were injected into the HPLC apparatus for each analysis. Plasma and urine samples were sonicated with an ultrasonic liquid processor (ASTRASON 3000, Misonix, NY) and treated as described previously [25]. Briefly, plasma was mixed with MeOH (final concentration, 60% in water [v/v]) and ZnSO4 (final concentration, 1.9 mg/mL). The mixture was hydrolyzed with HCl (final concentration, 1 N) at 50°C for 15 h, after which, the samples were processed as described above for the tissue samples. To determine the intracellular amounts of doxorubicin-conjugated polymer, the 11



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amount of doxorubicinone released from the polymers by acid hydrolysis was quantified by means of HPLC by using our previously reported method [25, 26] (Supplementary Fig. 1). To evaluate the amount of free doxorubicinone, that is, doxorubicinone not derived from doxorubicin-conjugated polymer, homogenates were treated as described above but without the hydrolysis step. The results were analyzed statistically by using the two-sided Student’s t-test.

2.5 High-performance liquid chromatographic analysis [25,26] High-throughput quantification of the amounts of doxorubicin and doxorubicinone were performed with a Hitachi LaChrom ULTRA system equipped with an L-2160U pump, L-2200U automated sample injector, L-2300 thermostatted column compartment, and L-2485U fluorescence detector (Hitachi, Tokyo, Japan). Samples were analyzed on a Capcell Pak C18 IF column (2.0 × 50 mm; particle size, 2 µm; Shiseido Corp., Tokyo, Japan). The mobile phase consisted of a 50 mM sodium phosphate buffer (pH 2.0) and acetonitrile mixture (65:27 v/v). The mobile phase was delivered at a rate of 300 µL/min, and the column temperature was maintained at 25°C. The fluorescence detector was operated at an excitation wavelength of 470 nm and an emission wavelength of 590 nm. Twenty microliters of sample was injected for each analysis.

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2.6 Pharmacokinetic analysis The plasma concentrations of doxorubicinone were fitted to a pharmacokinetic model by means of the nonlinear least-square method using WinNonlin Professional software (version 6.3; Pharsight Corp., Palo Alto, CA). We used a noncompartmental analysis. Pharmacokinetic variables were calculated by using the following equations (area under the curve [AUC] was calculated by using the trapezoidal rule): ∞

AUCinf = , CLtot = Dose / AUCinf, MRTinf = AUMCinf / AUCinf, Vz = Dose/(λz・AUCinf), and t1/2Z = 0.693 / λz, where AUCinf is the area under the curve from dosing-time extrapolated to infinity, CLtot is the total body clearance, MTRinf is the mean residence time extrapolated to infinity, AUMCinf is the area under the first moment curve extrapolated to infinity, Vz is the volume of distribution based on the terminal phase, λz is the first-order rate constant associated with the terminal portion of the curve, and t1/2Z is the terminal half-life. Data are expressed as mean ± SD.

2.7 Plasma concentration of mPEG-FITC 13



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mPEG-FITC was administered intravenously via the tail vein to female mice (9 weeks of age; doxorubicin-conjugated polymer, 2.0 mg/30 g mouse). Blood samples were collected from the tail vein into heparinized capillaries at 1 min and at 2, 6, 24, and 48 h after administration. The blood samples were immediately centrifuged at 4000g for 15 minutes at 4°C. The plasma (20 µL) was diluted with PBS (final volume: 300 µL), and the fluorescence intensity of mPEG-FITC in the plasma was measured with a fluorescence spectrophotometer (ex/em, 485 nm/522 nm; F-7000, Hitachi High-Technologies, Tokyo, Japan).

2.8 ATP-dependent transport of mPEG-FITC To investigate the ATP-dependent transport of mPEG-FITC, mammalian cell membrane vesicles expressing ABCB1 were used according to the manufacturer’s instructions. Suspensions of vesicles expressing ABCB1 and control vesicles not expressing ABCB1 (50 µL each) were plated on 96-well plates. mPEG-FITC (0.75 µL) at 50 µM and reaction buffer containing MgATP (25 µL) were added to each well, and the plates were incubated at 37°C for 5 min. After washing by centrifugation (1500g, 5 min, 4°C), fluorescence intensity after intravesicular transport was measured with a fluorescence spectrophotometer (ex/em, 485 nm/522 nm). The fluorescence intensity of intravesicular polymer incubated with MgATP was subtracted from that of polymer incubated without MgATP. 14


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2.9 Biodistribution of doxorubicin-conjugated polymers in liver Micelle solutions prepared as described in Section 2.3 were diluted in saline and administered intravenously via the tail vein to the mice (13–14 weeks of age). At 48 h after micelle administration, the mice were sacrificed under anesthesia, perfused with PBS through the inferior vena cava to prevent blood stasis, and the livers were excised, rinsed with PBS, and fixed overnight with 4% paraformaldehyde/phosphate buffer. After series exchange with sucrose, the livers were embedded in OCT compound (Sakura Finetechnochemical, Tokyo, Japan), quickly frozen in pre-cooled acetone, and stored at −80ºC until analysis. For immunohistochemical analysis, 5-µm frozen sections were prepared with a cryostat. After blocking, sections were incubated for 1 h at room temperature with primary antibodies: rat anti-CD31 antibody for endothelial cells (diluted 1:10; BioLegend, San Diego, CA) and rat anti-CD68 antibody for macrophages (Kupffer cells) (diluted 1:20; AbD Serotec, Raleigh, NC). The sections were then incubated with secondary antibody (1:100 dilution; Alexa Fluor® 488-AffiniPure Goat Anti-Rat IgG (H+L), Jackson ImmunoResearch Laboratories,

Inc.

West

Grove,

PA).

Nuclei

were

counterstained

with

4',6-diamidino-2-phenylindole, dilactate (Life Technologies Japan, Tokyo, Japan). Sections were then examined by means of confocal microscopy (Carl Zeiss LSM510, Carl Zeiss Microscopy GmbH, Germany). 15



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3. Results and discussion

3.1 Quantification of doxorubicin-conjugated polymer in biological samples The amount of doxorubicin-conjugated polymer in the biological samples was determined by measuring the amount of doxorubicinone, which was released by HCl-treated acid hydrolysis of doxorubicin-conjugated polymer after sampling (Supplementary Fig.1) [14,25,26]. Doxorubicin-conjugated polymer contained negligible amounts of free doxorubicinone (0.023% w/w) and free doxorubicin (0.017% w/w) in the injection sample to mice. Without acid hydrolysis, the biological samples contained amounts of free doxorubicinone and doxorubicin that were less than 0.11% and 0.083 % in wild-type mice and 0.11% and 0.075 % in Mdr1a/1b(-/-) mice, respectively, of doxorubicinone measured after HCl-treated acid hydrolysis, supporting that the majority of quantified doxorubicinone was released from the doxorubicin-conjugated polymer by HCl-treated acid hydrolysis (Supplementary Fig. 1). We next determined the recovery rate of doxorubicin-conjugated polymer from biological samples. A designated amount of polymer was added to plasma, urine, feces, liver, or brain homogenates obtained from untreated wild-type mice. We were able to recover more than 99% of the polymer based on the recovery of doxorubicin-conjugated polymer from plasma, urine, feces, liver, and brain (Supplementary Table 1), demonstrating the validity of 16


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our method for quantifying the amount of doxorubicin-conjugated polymer in the biological samples.

3.2 Plasma concentration of the doxorubicin-conjugated polymer Figure 2A shows the plasma concentration–time profile of doxorubicin-conjugated polymer after single intravenous administration in wild type FVB and Mdr1a/1b (-/-) mice. From 6 h after administration, elimination of doxorubicin-conjugated polymer from the systemic circulation was significantly delayed in Mdr1a/1b(-/-) mice compared with that in wild-type mice (P < 0.01). The area under the curve (AUC) and the mean residence time (MRT) were increased 2.2-fold and 2.1-fold, respectively, in Mdr1a/1b(-/-) mice compared with those in wild-type mice (Table 1A). Total body clearance (CLtot) in Mdr1a/1b(-/-) mice was decreased to about one-half of that in wild-type mice (Table 1A). Similarly, we examined the plasma concentration–time profile of DBD-conjugated polymer after single intravenous administration because the efflux of DBD-polymer from HeLa cells was also indicated to be mediated by ABCB1 [15]. From 24 h after administration, elimination of DBD-conjugated polymer from the systemic circulation was also significantly delayed in Mdr1a/1b(-/-) mice compared with that in wild-type mice (P < 0.01) (Supplementary Fig. 2). When free doxorubicin, which is a well-known substrate of ABCB1 [14], was 17



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administered intravenously to wild-type and Mdr1a/1b(-/-) mice, the plasma concentration of doxorubicin was significantly higher in Mdr1a/1b(-/-) mice compared with that in wild-type mice, from 3 h after administration (P < 0.01) (Fig. 2B). However, the concentration–time profile of doxorubicin-conjugated polymer was larger compared with that of free doxorubicin (Fig. 2A and B). The terminal half-life (t1/2z) for doxorubicin-conjugated polymer and for free doxorubicin was 51 h and 2.5 h, respectively, in the Mdr1a/1b(-/-) mice, and those in wild-type mice were 31 h and 2.6 h, respectively (Table 1), indicating that the data shown in Figure 2A represent the in vivo behavior of the doxorubicin-conjugated polymer, not that of doxorubicin released in vivo. We also examined the plasma concentration of mPEG-FITC in wild-type mice and Mdr1a/1b(-/-) mice following intravenous administration. We used mPEG-FITC to show that there is no difference in glomerular filtration rate as a function of kidney that is a main organ for the elimination of PEG [27, 28], and the extracellular space volume between wild-type and Mdr1a/1b(-/-) mice. In fact, the plasma concentration of mPEG-FITC in Mdr1a/1b(-/-) mice was identical to that in wild-type mice (Fig. 3A). We further showed that the mPEG-FITC is not a substrate of ABCB1 by conducting an in vitro transport study using ABCB1-expressing membrane vesicles. Unlike with doxorubicin-conjugated polymer [15] and free doxorubicin (Fig. 3B), we observed no difference in the transport of mPEG-FITC between ABCB1-expressing membranes and control membranes (Fig. 3B). Taken together, it 18


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is most likely that the transport of doxorubicin-conjugated polymer by ABCB1 was closely related to the plasma concentration of the doxorubicin-conjugated polymer.

3.3 Tissue distribution of doxorubicin-conjugated polymer We further investigated the tissue distribution of doxorubicin-conjugated polymer after single intravenous administration. At 1 h after administration, greater accumulation of doxorubicin-conjugated polymer was observed in the reticuloendothelial system, that is, liver, and spleen, followed by brain, heart, kidney, and lung (Fig. 4A). The accumulation of polymers in the reticuloendothelial system indicates that the doxorubicin-conjugated polymers circulate in vivo mainly as a micelle form [9, 29]. In fact, the polymers form micelles in PBS with the particle size of 48.5 ± 0.6 nm (PDI, 0.13 ± 0.01) and ξ-potential of -6.7 ± 0.6 mV. In lung (P < 0.05), kidney (P < 0.01), and brain (P < 0.01), the concentration of doxorubicin-conjugated polymer was significantly higher in Mdr1a/1b(-/-) mice than in wild-type mice. At 48 h after administration, the polymer concentrations in the tissues were increased compared with the concentrations observed at 1 h after administration (Fig. 4B); however, the tissue distribution profile remained unchanged. At 48 h after administration, significant increases in the accumulation of the polymer in liver (1.4-fold), kidney (2.3-fold), and brain (1.7-fold), which are tissues that express ABCB1 in wild-type mice (Supplementary Fig. 3) 19



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[30, 31], were seen in Mdr1a/1b(-/-) mice compared with those observed in wild-type mice. Since the magnitudes of increase were consistent with that observed in the plasma (2.2-fold), the increase in tissue concentration is likely due to the prolonged systemic exposure of the doxorubicin-conjugated polymer, not due to an increase in the efficacy of transfer of the polymer from the systemic circulation to the tissues.

3.4 Excretion of doxorubicin-conjugated polymer in the urine and feces We further investigated the role of ABCB1 in the urinary and fecal excretion of doxorubicin-conjugated

polymer.

In

Mdr1a/1b(-/-)

mice,

the

excretion

of

doxorubicin-conjugated polymer into the urine was significantly lower in both the first (2.2-fold) and second 24-h period (2.8-fold) after administration compared with that in wild-type mice, despite the higher systemic exposure (Fig. 5A and C). Doxorubicin-conjugated polymer was also detected in the feces, although the amount detected in the feces was one-tenth of that detected in the urine. The amount of doxorubicin-conjugated polymer excreted in the feces was similar in wild-type mice and Mdr1a/1b(-/-) mice at 24 h after administration; at 48 h after administration, the excretion of doxorubicin-conjugated polymer in the feces was slightly but with statistical significance, higher in Mdr1a/1b(-/-) mice (Fig. 5B). However, this increase in Mdr1a/1b(-/-) mice was smaller than increase in the area under the plasma concentration of doxorubicin-conjugated 20


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polymer for 24 hours (Fig. 5B and C). Together, these results suggest that ABCB1 mediates the excretion of doxorubicin-conjugated polymer via the urine and feces. The fact that the amount recovered in urine and feces for 24 hours were markedly smaller than the amount administered indicates that the other pathway is the major elimination pathway for doxorubicin-conjugated polymer. Since the liver is the major distribution organ in the body, identification of the cells in which the polymer accumulates would

provide

clues

to

understand

the

fate

of

the

polymer

in

the

liver.

Doxorubicin-conjugated polymer was partly co-stained with CD68+ macrophages (Kupffer cells). Therefore, uptake by CD68+ macrophages (Kupffer cells) likely represents one part of the clearance mechanism. However, some doxorubicin-conjugated polymer was co-stained with cells other than CD68+ macrophages (Kupffer cells) and CD31+ blood vessels (Fig. 6). Non-CD68, non-CD31 cells where doxorubicin-conjugated polymer was localized may include hepatocytes, which mediate the metabolism and excretion of xenobiotics from the body in the bile. Hashida et al. demonstrated that a mitomycin C-dextran conjugate is transferred to the bile after intravenous injection in rats, further demonstrating that hepatocytes have the ability to transfer macromolecules into the bile, and finally to the feces [32]. Since ABCB1 is expressed on the canalicular membrane of hepatocytes, and given that hepatocytes mediate the excretion of xenobiotics in the bile [16], it is possible that ABCB1 mediates the canalicular efflux of polymers into the bile. The fact that fecal excretion 21



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clearance accounted for only a limited fraction of the total body clearance (Fig. 5B) indicates a limited contribution of biliary excretion to the total clearance. Further studies are necessary to clarify the clearance of doxorubicin-conjugated polymer in Kupffer cells, and the contribution biliary excretion makes to the systemic elimination of doxorubicin-conjugated polymer in mouse, and to further characterize the importance of ABCB1 in that process.

4. Conclusions

In the present study, we demonstrated that knockout of the gene encoding ABCB1 prolonged systemic exposure of a ABCB1 substrate polymer. ABCB1 may play a key role not only in vitro intracellular trafficking, but also in the in vivo behavior of block copolymers. Our results suggest that ABCB1 mediated the excretion of doxorubicin-conjugated polymer in urine and feces, and further studies are necessary to confirm the contribution biliary excretion and Kupffer cells makes to the systemic elimination of doxorubicin-conjugated polymer in mouse. The results of the present study suggest that the in vivo behavior of block copolymers used as drug carriers is regulated, at least in part, by ABCB1, which is already known to regulate the in vivo behavior of many low-molecular-weight active substances [16]. Our results provide valuable information about the behavior of block copolymers in vivo, which is important for evaluating the pharmacokinetics of active substances conjugated to block 22


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copolymers or the accumulation of block copolymers in vivo.

Acknowledgements

This work was supported in part by the Public-Private Sector Joint Research on Publicly Essential Drugs from the Japan Health Sciences Foundation, and by Research on Development of New Drugs and Health and Labour Sciences Research Grants from the Ministry of Health, Labour and Welfare of Japan. We thank Nippon Kayaku Co. Ltd. for providing the doxorubicin-conjugated block copolymers, and Dr. K. Un and Dr. Y. Abe for assisting with the data acquisition.

Supporting Information Available Recovery of doxorubicin-conjugated polymers from mouse biological samples. Schematic of the structure of doxorubicin-conjugated polymer. Concentration–time profiles of plasma 4-phenyl-1-butanol concentration after a single intravenous injection of DBD-conjugated polymer. Expression profiles of ABCB1 in the indicated tissues of female wild-type and Mdr1a/1b(-/-) FVB mice as assessed by western blotting. This information is available free of charge via the Internet at http://pubs.acs.org/.

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Table 1 Pharmacokinetic variables of doxorubicin-conjugated polymer (A) and free doxorubicin (B) in plasma after intravenous administration to female wild-type and Mdr1a/1b(-/-) doubleknockout-mutation FVB mice.

1 2 3 4 5 6 7 8 9 10 11 (A) Doxorubicin-conjugated polymer 12 Tmax Cmax AUCinf Vz CLtot MRTinf t1/2z 13 14 (ug•h/mL) (h) (ng/mL) (mL) (mL/h) (h) (h) 15 16Wild-type Mean 0.0167 170.0 4116.3 7.54 0.17 38.75 30.73 17 SD 18.4 38.6 0.40 0.002 0.81 1.62 18 19 20 Mdr1a/ Mean 0.0167 193.3 9131.8*** 5.65 *** 0.08 *** 75.10 *** 51.07 *** 21 1b(-/-) SD 12.6 276.2 0.19 0.002 4.20 2.00 22 ***P

Effect of Knockout of Mdr1a and Mdr1b

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