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P-glycoprotein-Mediated Active Efflux of the Anti-HIV1 Nucleoside Abacavir Limits Cellular Accumulation and Brain Distribution

Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota

(Received July 19, 2007; Accepted August 17, 2007)

	Abstract

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P-glycoprotein (P-gp)-mediated efflux at the blood-brain barrier has been implicated in limiting the brain distribution of many anti-HIV1 drugs, primarily protease inhibitors, resulting in suboptimal concentrations in this important sanctuary site. The objective of this study was to characterize the interaction of abacavir with P-gp and determine whether P-gp is an important mechanism in limiting abacavir delivery to the central nervous system (CNS). In vitro and in vivo techniques were employed to characterize this interaction. Abacavir stimulated P-gp ATPase activity at high concentrations. The cellular accumulation of abacavir was significantly decreased by 70% in Madin-Darby canine kidney II (MDCKII)-MDR1 monolayers compared with wild-type cells and was completely restored by the P-gp inhibitors ((R)-4-((1aR,6R,10bS)-1,2-difluoro-1,1a,6,10b-tetrahydrodibenzo(a,e)cyclopropa(c)cycloheptan-6-yl)--((5-quinoloyloxy)methyl)-1-piperazineethanol, trihydrochloride) (LY335979) and N-[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide (GF120918). Directional flux experiments indicated that abacavir had greater permeability in the basolateral-to-apical direction (1.58E-05 cm/s) than in the apical-to-basolateral direction (3.44E-06 cm/s) in MDR1-transfected monolayers. The directionality in net flux was abolished by both LY335979 and GF120918. In vivo brain distribution studies showed that the AUC_plasma in mdr1a(-/-) CF-1 mutant mice was 2-fold greater than the AUC_plasma in the wild type, whereas the AUC_brain in the mutant was 20-fold higher than that in the wild type. Therefore, the CNS drug targeting index, defined as the ratio of AUC brain-to-plasma for mutant over wild type, was greater than 10. These data are the first in vitro and in vivo evidence that a nucleoside reverse transcriptase inhibitor is a P-gp substrate. The remarkable increase in abacavir brain distribution in P-gp-deficient mutant mice over wild-type mice suggests that P-gp may play a significant role in restricting the abacavir distribution to the CNS.

HIV1 can infect the CNS at a very early stage of the disease and can lead to the development of AIDS-dementia complex (Ances and Ellis, 2007). The CNS has been cited as a reservoir for the HIV1 virus due to incomplete suppression of the viral replication (Kerza-Kwiatecki and Amini, 1999). Suboptimal concentrations of anti-HIV1 drugs in the CNS can not only lead to incomplete eradication of the virus but also provide ideal conditions for the selection of more virulent mutants (Lipniacki, 2003). Recent reports have highlighted the compartmentalization and independent evolution of primary, as well as secondary, drug-resistant viral mutations in the CNS as a response to inadequate concentrations of both reverse transcriptase and protease inhibitors (Smit et al., 2004).

The CNS penetration of a number of anti-HIV1 drugs used in ART is limited (Langford et al., 2006). One of the factors implicated in limiting the CNS distribution of anti-retroviral drugs is the action of drug efflux transporters. These are transmembrane proteins belonging to the ABC (ATP-binding cassette) superfamily that use the energy generated from ATP hydrolysis to transport drugs against a concentration gradient. P-glycoprotein (P-gp), a member of this superfamily, has been implicated in conferring resistance against a variety of drugs (Fromm, 2003). It is highly expressed on the luminal surface of the brain microvasculature that forms the blood-brain barrier (BBB) (Cordon-Cardo et al., 1989). P-gp has been shown to modulate the brain distribution of most HIV protease inhibitors (Kim et al., 1998). Inhibition of P-gp at the BBB (Choo et al., 2000), as well as the use of transgenic mouse models lacking P-gp (Kim et al., 1998; Choo et al., 2000), has led to enhanced brain penetration and distribution of protease inhibitors. This highlights the role of P-gp in limiting this class of anti-HIV1 drug distribution to the CNS. Recently, the breast cancer resistance protein (Bcrp1), another member of the ABC superfamily (ABCG2), has been shown to actively efflux nucleoside reverse transcriptase inhibitors (Pan et al., 2007). Similar to P-gp, Bcrp1 is localized on the luminal surface of the BBB, is instrumental in the efflux of drugs, and shares common substrates with P-gp. Bcrp1 has been shown to efflux the NRTIs, zidovudine (3'-azido-3'-deoxythymidine; AZT), lamivudine (Wang et al., 2003; Pan et al., 2007), and abacavir (Pan et al., 2007).

The CNS penetration of abacavir has been shown in many animal models to be similar to that of AZT (Daluge et al., 1997). In situ brain perfusion studies in guinea pig showed nonlinear uptake of ABC into the brain, raising the possibility that a saturable efflux process could be influencing abacavir brain distribution (Thomas et al., 2001). Recent transport competition studies have examined the inhibitory activity of abacavir on both P-gp and Bcrp1. It was found that among all the NRTIs tested, abacavir was the only NRTI that inhibited P-gp-mediated transport in a calcein uptake assay (Storch et al., 2007). Moreover, abacavir and AZT moderately influenced Bcrp1-mediated transport of pheophorbide A, a prototypical Bcrp1 substrate (Weiss et al., 2007).

The goal of this study was to investigate possible mechanisms that may limit the CNS distribution of abacavir. One of these mechanisms could be an efflux transporter, such as P-gp. Therefore, the specific objective was to examine the interaction of abacavir with P-gp using biochemical assays, in vitro cell transport and in vivo brain distribution studies.

	Materials and Methods

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Chemicals. [³H]Vinblastine sulfate, [³H]abacavir, and [¹⁴C]AZT were obtained from Moravek Biochemicals (Brea, CA). Verapamil was obtained from Sigma Chemical Co. (St. Louis, MO). GF120918 (N-[4-[2-(6, 7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]-5-methoxy-9-oxo-10H-acridine-4-carboxamide) was a gift from GlaxoSmithKline (Research Triangle, NC). Fumitremorgin C (FTC; (9'R-(9'-(4S^*(R^*)),9'β))-4-(2-(1-(acetyloxy)-2-methylpropyl)-4-oxo-3(4H)-quinazolinyl)-1',3,4,9'a-tetrahydro-1'-hydroxy-2',2'-dimethylspiro(furan-2(5H),9'-(9H)imidazo(1,2-a)indole)-3',5(2'H)-dione) was obtained from the National Cancer Institute (Bethesda, MD), Ko143 (a fumitremorgin C analog) (Allen et al., 2002) was kindly provided by Dr. Alfred Schinkel (The Netherlands Cancer Institute, Amsterdam, The Netherlands), and LY335979 ((R)-4-((1aR,6R,10bS)-1,2-difluoro-1,1a,6,10b-tetrahydrodibenzo-(a,e)cyclopropa(c)cycloheptan-6-yl)--((5-quinoloyloxy)methyl)-1-piperazineethanol, trihydrochloride) was a gift from Eli Lilly and Co. (Indianapolis, IN). Human P-gp-expressing membranes were purchased from BD Biosciences (San Jose, CA) (Fig. 1). All other chemicals used were HPLC or reagent grade.

View larger version (13K): [in this window] [in a new window]   FIG. 1. Structures of the nucleoside analogs abacavir and AZT, P-gp inhibitors (LY335979 and GF120918), and the BCRP inhibitors (Ko143 and FTC).

Animals. Age-matched wild-type CF1 (Crl:CF1) and naturally mutant CF1 [mdr1a(-/-)] mice (Crl:CF1-Abcb1a), lacking the mdr1a gene, were purchased from Charles River Laboratories (Wilmington, MA). All the mice were male and between 9 and 12 weeks old. Animals were maintained under temperature-controlled conditions with a 12-h light/dark cycle and unlimited access to food and water. All studies were conducted according to the guidelines set by the Principles of Laboratory Animal Care (National Institutes of Health) and were approved by The Institutional Animal Care and Use Committee of the University of Minnesota.

P-gp ATPase Assay. P-gp ATPase activity was evaluated using a suspension of human P-gp-overexpressing membranes obtained from baculovirus-infected insect cells (High Five, BTI-TN5B1-4; BD Biosciences, San Jose, CA) in 50 mM Tris, pH 7.0, containing 50 mM mannitol, 2 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, and 10 mg/ml leupeptin, antipain, chymostatin, and pepstatin A buffer. In brief, a suspension of P-gp-overexpressing membranes was incubated at 37°C for 20 min in the presence of 4 mM ATP with and without 100, 200, and 500 µM abacavir. An identical reaction mixture containing 100 µM sodium orthovanadate was assayed in parallel. Orthovanadate inhibits P-gp by trapping Mg²⁺ADP in the nucleotide-binding site. Thus, the ATPase activity measured in the presence of orthovanadate represents non-P-gp ATPase activity and was subtracted from the total activity measured in the non-orthovanadate-treated case to yield the P-gp-mediated ATPase activity. The reaction was stopped by the addition of 10% SDS, and the liberated inorganic phosphate was detected by a colorimetric reaction of inorganic phosphate with a solution of ascorbic acid and ammonium molyb-date at 650 nm. The nanomoles of phosphate released were determined from the absorbance values, using a standard curve generated from inorganic potassium phosphate standards (0-60 nM range). The same procedure was followed for control membranes (also obtained from BD Biosciences) that do not express P-gp. Verapamil (20 µM) was used as a positive control for P-gp ATPase-stimulatory activity.

Cellular Accumulation Studies in MDCKII Cells. For the accumulation experiments, cells were seeded in clear polyester 12-well plates (TPP cell culture plate; Sigma-Aldrich, St. Louis, MO) at a seeding density of 2 x 10⁵ cells/well. The medium was changed every other day and the cells formed confluent monolayers in 4 days. On the day of the experiment, the medium was aspirated and the confluent monolayer was washed twice with prewarmed (37°C) assay buffer. The cells were preincubated for 30 min with 1 ml of assay buffer, after which the buffer was aspirated and the cells were exposed to a tracer solution of radiolabeled drug in 1 ml of assay buffer per well. The plates were continuously agitated at 60 rpm in an orbital shaker and maintained at 37°C for the duration of the experiment. After a 3-h accumulation period, the supernatant was aspirated and the cells were first washed three times with 2 ml of ice-cold phosphate-buffered saline and then solubilized using 1 ml of mammalian protein extraction reagent (Pierce Biotechnology, Inc., Rockford, IL). A 200-µl sample was drawn from each well in triplicate, and 4 ml of scintillation fluid (ScintiSafe Econo1 cocktail; Fisher Scientific Co., Pittsburgh, PA) was added to each sample and counted using liquid scintillation counting (LS-6500; Beckman Coulter, Inc., Fullerton, CA) to determine the radioactivity associated with the cells. The protein concentration was determined using the BCA protein assay (Pierce Biotechnology, Inc., Rockford, IL) to normalize the radioactivity in each well.

For inhibition studies, the cells were treated with the inhibitor (1 µM LY335979 or 5 µM GF120918 for P-gp; 200 nM Ko143 or 10 µM FTC for Bcrp1) during both the preincubation and the accumulation periods. Drug accumulation in WT and MDR1 cells was expressed as a percentage of the control radioactivity measured in the wild-type cells (dpm) per milligram of protein. The stock solutions for all the inhibitors used were prepared in dimethyl sulfoxide and diluted using assay buffer (122 mM NaCl, 25 mM NaHCO₃, 10 mM glucose, 10 mM HEPES, 3 mM KCl, 1.2 mM MgSO₄ · 7H₂O, 1.4 mM CaCl₂ · H₂O, 0.4 mM K₂HPO₄, pH 7.4) to obtain working solutions such that the final concentration of dimethyl sulfoxide was less than 0.1%.

Directional Flux Across MDCKII Cell Monolayers. The method used in this study was similar to that previously described by Dai et al. (2003). In brief, MDCKII WT or MDR1 cells were seeded at a density of 2 x 10⁵ cells/well on semipermeable membrane supports in six-well Transwells (Corning Inc., Corning, NY). The medium was changed every day and the cells formed confluent polarized epithelial monolayers in 4 days. The transepithelial electrical resistance was 300 ± 8 ohm · cm² in the MDCKII WT monolayers and 275 ± 26 ohm · cm² in the MDCKII MDR1-transfected monolayers. Mannitol flux across the monolayer was also measured to confirm the existence of tight junctions with approximately 1% per hour flux. The monolayers were washed with prewarmed (37°C) assay buffer, and after a 30-min preincubation, a tracer solution of radiolabeled drug in assay buffer was added to the donor side (apical side, 1.5 ml; basolateral side, 2.6 ml). Fresh assay buffer was added to the receiver side and 100 µl of the receiver compartment was sampled at 0, 10, 20, 30, 45, 60, and 90 min. The volume sampled was immediately replaced with fresh assay buffer. Additional samples were drawn at 0 and 90 min in the donor compartment. The amount of radioactivity in the samples was determined using liquid scintillation counting. The apical-to-basolateral (A-to-B) flux was determined by addition of radiolabeled drug solution to the apical compartment and sampling the basolateral compartment, whereas for basolateral-to-apical (B-to-A) flux, the donor was the basolateral compartment and the apical compartment was sampled over time. When an inhibitor was used in the flux study, the cell monolayers were preincubated with the inhibitor (1 µM LY335979 or 5 µM GF120918 for P-gp and 200 nM Ko143 for Bcrp1) for 30 min, followed by determination of the A-to-B and B-to-A flux, with the inhibitor being present in both compartments throughout the course of the experiment.

Permeability Calculation. The effective permeability (P_eff) was calculated by the following equation,

(1)

where Q is the amount of radioactivity transported across the monolayer, t is time, dQ/dt is the mass transport rate, A is the effective surface area of the cell monolayer, and C_o is the initial donor concentration. The efflux ratio (ER) was used as a measure of P-gp-mediated efflux and calculated as the P_eff in the B-to-A direction over P_eff in the A-to-B direction.

LY335979 Dose Dependence Study. Directional flux experiments for abacavir in the B-to-A direction across the MDCKII-MDR1 monolayers were conducted in the presence of varying concentrations of the P-gp inhibitor LY335979 (Fig. 1). The experimental procedure used was the same as described in the previous section. The B-to-A flux of abacavir was determined in the presence of 0, 0.015, 0.03, 0.06, 0.125, 0.25, 0.5, and 1 µM LY335979. The inhibitory sigmoid E_max model with baseline (eq. 2) was used to fit the model to the data using the following equation,

(2)

where E is the B-to-A permeability of abacavir, E₀ is the permeability of abacavir when no inhibitor is present, I_max is the maximal inhibition of abacavir B-to-A permeability by LY335979, IC₅₀ is the concentration of LY335979 that shows half-maximal inhibition, C is the concentration of LY335979, and is the shape factor.

Brain Distribution of Abacavir in MDR1a(-/-)-Deficient and Wild-Type CF1 Mice. All mice were allowed to acclimatize for a minimum of 5 days upon arrival. Ziagen oral solution (abacavir sulfate, 20 mg/ml; Glaxo-SmithKline) diluted with normal saline to 5 mg/ml was administered to the mice at a dose of 10 mg/kg via tail vein injection. The animals were euthanized using a CO₂ chamber at predetermined time points postdose (5, 10, 20, 30, and 40 min, n = 6 for each time point). Blood was collected by cardiac puncture and transferred to heparinized tubes. Plasma was isolated from the blood by centrifugation at 3000 rpm for 10 min at 4°C. Whole brain was immediately removed from the skull, washed in ice-cold PBS to remove extraneous blood, and flash-frozen in liquid nitrogen. All samples were stored at -80°C until further analysis by HPLC.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Stimulation of P-gp ATPase activity by abacavir. The ATPase activity, expressed as nmol phosphate generated/mg protein/min, was significantly stimulated by 500 µM abacavir (**, p < 0.01) over control P-gp membranes. The positive control verapamil (20 µM) also significantly stimulated P-gp ATPase activity (**, p < 0.01); n = 3to7.

Pharmacokinetic Analysis. Noncompartmental analysis (WinNonlin 5.0.1; Pharsight, Mountain View, CA) was used to obtain pharmacokinetic parameters from concentration-time data in plasma and brain. The terminal rate constants for plasma and brain were determined from the last three data points of the respective concentration-time profiles. The areas under the concentration-time profiles for plasma (AUC_plasma) and brain (AUC_brain) from time 0 to infinity were calculated using the trapezoidal method with estimation of the AUC from the last measured time point to infinity accomplished by dividing the last measured concentration by the respective terminal rate constant. In the case of plasma, the AUC from time 0 to the first measured time point was obtained by back-extrapolation of the first two data points to the concentration estimated at time 0. The enhancement in brain exposure of abacavir in the P-gp-deficient mice compared with wild-type mice was represented by the drug targeting index (DTI), calculated as

(3)

Statistical Analysis. Statistical analysis was conducted using SigmaStat, version 3.1(Systat Software, Inc., Point Richmond, CA). Groups were compared by one-way analysis of variance with the Holm-Sidak post-hoc test for multiple comparisons at a significance level of p < 0.01. When groups failed the normality or equal variance test, analysis of variance on ranks with the Tukey post-hoc test was used for multiple comparisons. The brain-to-plasma concentration ratios between wild-type and mutant CF-1 mice were compared using the nonparametric Mann-Whitney rank sum test. Due to the inability to obtain concentrations at multiple time points from a single mouse, destructive sampling was used. A total of six mice were used at each time point to estimate the interanimal variability, and the area under the curve was constructed from the mean plasma concentration at each time point. The variance of the AUC was estimated using the method described by Yuan (1993). This method allows for the determination of the variability in the AUC estimate from the variability about the mean concentration at each time point, assuming the mean at each time point is independent and the terminal rate constant is the same for each animal. It is reasonable to assume that the sample means are independent of each other, and given that the coefficient of variation in the terminal phase of the curve is low, this method was used to statistically compare AUCs. The AUCs were statistically compared using the Z test at a significance level of p < 0.05.

	Results

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P-gp ATPase Assay. Verapamil (20 µM) significantly stimulated the P-gp ATPase activity over control (p < 0.01) as shown in Fig. 2. The test compound, abacavir (Fig. 1), showed an increase in the ATPase assay at 100, 200, and 500 µM, but because of the high variability in the data, statistically significant stimulation of P-gp ATPase activity was seen only at 500 µM (p < 0.01, Fig. 2).

Cellular Accumulation Studies in the MDCKII Cells. [³H]Vinblastine was used as a positive control to verify P-gp function in the cell accumulation assays. As seen in Fig. 3, [³H]vinblastine had a significantly lower accumulation (p < 0.001) in the MDR1 cells compared with wild-type cells. [³H]Abacavir accumulation in the MDR1 transfects was significantly lower (70%; p < 0.001) than in the wild-type cells (expressed as 100%), whereas the accumulation of [³H]AZT was not significantly different between the two cell types. Treatment with the P-gp inhibitors LY335979 (1 µM) and GF120918 (5 µM) completely abolished the efflux action of P-gp (p < 0.001, compared with MDR1 control), resulting in no significant difference in abacavir accumulation between wild-type and MDR1 (Fig. 4). The Bcrp1 inhibitors Ko143 (200 nM) and FTC (10 µM) had no significant effect on [³H]abacavir cellular accumulation in either wild-type or MDR1 transfects (Fig. 5).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Accumulation of [3H]abacavir, [3H]vinblastine (positive control), and [14C]AZT in wild-type (black bar) and MDR1-transfected (gray bar) MDCK cells. Results are expressed as mean ± S.D. (as percentage of wild-type control); n = 6 to 18 (***, p < 0.001, compared with wild-type control group), with significantly lower accumulation of abacavir and vinblastine in the MDR1 cells but no statistical difference for AZT accumulation between wild-type and MDCKII-MDR1 cells.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Accumulation of [3H]abacavir in wild-type (black bars) and MDR1-transfected (gray bar) MDCK cells and the effect of the P-gp inhibitors LY335979 (1 µM) and GF120918 (5 µM). Results are expressed as mean ± S.D. (as percentage of wild-type control); n = 9 (**, p < 0.01, compared with wild-type control group; , p < 0.01, compared with MDR1 control group). [3H]Vinblastine (n = 6) was used as a positive control for P-gp function. Both the inhibitors restored abacavir level in the MDR1 cells such that it was not different from wild-type control.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Accumulation of [3H]abacavir in wild-type (black bar) and MDR1-transfected (gray bar) MDCK cells and the effect of the Bcrp1 inhibitors Ko143 (200 nM) and FTC (10 µM). Results are expressed as mean ± S.D. (as percentage of wild-type control); n = 3 to 6 (**, p < 0.01, compared with wild-type control group). There was no effect of Bcrp1 inhibitors on the cell accumulation of abacavir in MDCKII-MDR1 cells.

View larger version (28K): [in this window] [in a new window]   FIG. 6. Directional flux of [3H]abacavir (A and B) and [3H]AZT (C and D) across the MDCKII monolayers in wild-type (, A- to-B transport; , B-to-A transport) and MDR1-transfected cells (, A-to-B transport; , B-to-A transport). Effective permeability (Peff) for [3H]abacavir and [3H]AZT transport (B and D) across wild-type and MDR1 monolayers (black bars, A-to-B direction; gray bars, B-to-A direction). Results are expressed as mean ± S.D. (n = 3) (***, p < 0.001, compared with wild-type control). Increase in the Peff and amount transported B-to-A and decrease in the Peff and amount transported A-to-B was seen for abacavir in the MDR1 monolayers compared with wild-type cells. No difference in Peff was observed between wild-type and MDR1 cells for AZT.

View larger version (28K): [in this window] [in a new window]   FIG. 7. Directional flux of [3H]abacavir across the MDCKII -MDR1-transfected cell monolayers in the absence (, A-to-B transport; , B-to-A transport) and presence (, A-to-B transport; , B-to-A transport) of P-gp inhibitors LY335979 (A; 1 µM) and GF120918 (C; 5 µM). Arrows indicate the effect of inhibitor on flux in both directions. Effect of the P-gp inhibitors LY335979 (B; 1 µM) and the dual (P-gp and Bcrp1) inhibitor GF120918 (D; 5 µM) on the effective permeability (Peff) of [3H]abacavir. In the presence of inhibitors, the Peff was significantly increased in the A-to-B direction (black bars) and significantly decreased in the B-to-A direction (gray bars). Results are expressed as mean ± S.D. (n = 9) (**, p < 0.01, compared with MDR1 control).

View larger version (19K): [in this window] [in a new window]   FIG. 8. A, directional flux of [3H]abacavir across the MDCKII -MDR1-transfected cell monolayers in the absence (, A-to-B transport; , B-to-A transport) and presence (, A-to-B transport; , B-to-A transport) of the Bcrp1 inhibitor Ko143. B, effect of the Bcrp1 inhibitor Ko143 (200 nM) on the effective permeability (Peff) of [3H]abacavir. There was no significant effect of Ko143 on the Peff in the A-to-B direction (black bars) or B-to-A direction (gray bars), and the B-to-A Peff was significantly greater than the A-to-B Peff in both the Ko143-treated and control cases. **, p < 0.01, n = 3 to 6. Results are expressed as mean ± S.D.

LY335979 Dose-Dependent Inhibitory Effect. The P_eff B-to-A of abacavir across the MDCKII-MDR1 monolayer in the absence of LY335979, i.e., the maximum observable permeability (E_o), was 13E-06 cm/s (Fig. 9). The concentration of LY335979 that decreased the P_eff B-to-A by 50% (IC₅₀) was 0.07 µM. The P_eff value at the highest inhibitor concentration of 1 µM (E_o - I_max) was 8E-06 cm/s. The maximal effect of LY335979 on P-gp-mediated abacavir P_eff was given by the I_max, which was 5E-06 cm/s. The shape factor from the fit of the model to the data was 5.

View larger version (14K): [in this window] [in a new window]   FIG. 9. Effect of the increasing concentration of the P-gp inhibitor LY335979 (µM) on the B-to-A Peff (cm/s) of abacavir. An inhibitory Emax model with baseline effect was fit to the data. LY335979 inhibited the P-gp-mediated B-to-A flux by 50% (IC50) at 0.07 µM. , observed B-to-A Peff values; solid line, predicted Peff values from the model; ---, Peff value for control case where no inhibitor was used. n = 3to9.

View larger version (18K): [in this window] [in a new window]   FIG. 10. Brain distribution of abacavir in the mdr1a (-/-)-deficient and wild-type CF-1 mouse after intravenous dosing. Mdr1a(-/-) and wild-type [mdr1a(+/+)] mice received 10 mg/kg abacavir via tail vein injection. The brain and plasma samples were collected postdose (5, 10, 20, 30, and 40 min; n = 6), analyzed by HPLC, and plotted on rectangular coordinates. A, concentration-time profiles of abacavir in plasma () and brain () wild-type CF1 mice. B, concentration-time profiles in plasma and brain of naturally mutant mdr1a (-/-)-deficient CF1 mice. The brain concentrations in the mdr1a (-/-) CF1 mutant mice were significantly higher than those in the wild-type mice. Data are represented as mean ± S.D.

View larger version (12K): [in this window] [in a new window]   FIG. 11. Comparison of the brain-to-plasma concentration ratios (Cb/Cp) between wild-type CF-1 () and mutant mdr1a(-/-)-deficient CF-1 () at different sampling time points. The (Cb/Cp) ratio for ABC in the mdr1a(-/-)-deficient CF1 mutants was significantly greater than the (Cb/Cp) ratio in the wild-type mice at all time points, (**, p < 0.01, n = 5-6 per time point). Results are expressed as mean ± S.D.

	Discussion

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The interaction of abacavir with P-glycoprotein was investigated using both in vitro and in vivo methods. Initially, the stimulation of P-gp-mediated ATPase activity indicated that there was a functional binding interaction between abacavir and P-gp in isolated P-gp-overexpressing membranes. The use of an isolated system such as P-gp-overexpressing membranes allowed for study of the interaction of drug with P-gp without the effect of other factors (e.g., other influx and efflux transporters, cytosolic components, etc.) that might mask an interaction. Abacavir, at a concentration of 500 µM, stimulated P-gp activity to a degree that was comparable to the positive control, 20 µM verapamil. The high concentration of abacavir required to generate this response may indicate a moderate interaction with P-gp. The moderate response generated by abacavir compared with verapamil could be due to its interaction at a different binding site. There is evidence that P-gp expresses multiple binding sites, and abacavir may be interacting with P-gp at a low-affinity binding site (Pascaud et al., 1998).

In vitro cellular transport studies were conducted because they provide the advantage of a more complete cell-based system (Polli et al., 2001). Cellular accumulation studies of abacavir were conducted in immortalized MDCKII wild-type and the MDCKII-MDR1 cells that stably overexpress human P-gp. Gene expression studies using real-time polymerase chain reaction showed significantly higher mRNA expression for human P-gp in the MDR1 transfects (data not shown). Vinblastine, an excellent P-gp substrate, was used as a positive control to verify the functional expression of P-gp in the MDR1-MDCKII cell line (Fig. 3). The significantly lower accumulation of abacavir in the MDR1 transfects compared with wild-type suggested P-gp-mediated efflux of abacavir (Fig. 3). This is the first direct evidence of P-gp-mediated efflux of an NRTI. By comparison, AZT was not transported by P-gp. The complete restoration of abacavir accumulation in the transfects by the specific P-gp inhibitor, LY335979 (Choo et al., 2000; Dai et al., 2003), and the dual inhibitor of P-gp and Bcrp1, GF120918, further confirmed the role of P-gp in abacavir cellular accumulation (Fig. 4). The slight, but not significant, increase in wild-type accumulation after treatment with inhibitors may be due to their effect on endogenous P-gp in the MDCK cells.

Recently, AZT and abacavir have been shown to be substrates for the drug efflux transporter Bcrp1 (Wang et al., 2003; Pan et al., 2007). Thus, the possibility that endogenous Bcrp1 might confound the results observed in the P-gp cell model was investigated. Ko143 and FTC, both selective inhibitors of Bcrp1, had no effect on the accumulation of abacavir in the MDR1 cell monolayers (Fig. 5). These data indicate that there was no Bcrp1-mediated component to the active efflux of abacavir from these cells.

The effective permeability (P_eff) of abacavir across the MDCKII cell monolayer was measured in the wild-type and MDR1 transfects. Using tracer amounts of a radiolabeled compound, the flux study allows for detection of the existence of directionality across a cell monolayer. Knowledge of the apical membrane localization of P-gp predicts that P-gp-mediated flux in the B-to-A direction will be much greater than in the A-to-B direction. In the MDCKII-MDR1 cells there was increased directionality of abacavir flux compared with the wild-type cells (Fig. 6A), and the efflux ratio was approximately 4 (Fig. 6B) compared with 1.3 in the wild-type cells. The efflux ratio for AZT was not different between the wild-type (1.3) and MDR1 transfects (1.4). The P_{eff B-to-A} was greater than the P_{eff A-to-B} in both cell lines, indicating the possible role of a common transporter that is responsible for efflux (Fig. 6, C and D). AZT has been shown to be effluxed by a probenecid-sensitive transporter, and the baseline efflux seen in this study is possibly mediated by such a transporter (Wang et al., 1997). Moreover, overexpression of P-gp in the MDR1 cells had no effect on either the A-to-B or the B-to-A flux of AZT, indicating a lack of P-gp-mediated efflux. Further evidence of P-gp-mediated flux was provided by treatment of the MDR1 monolayers with LY335979 (1 µM) and GF120918 (5 µM), which completely abolished the directionality of abacavir transport (Fig. 7, A and C) and reduced the efflux ratio to unity (Fig. 7, B and D). Also, the Bcrp1 inhibitor Ko143 had no significant effect on P_eff or the efflux ratio (Fig. 8, A and B). These observations indicate that LY335979 and GF120918 were able to completely inhibit P-gp efflux of abacavir. The P_{eff B-to-A} of [³H]abacavir in the MDR1 monolayers was not affected by addition of up to 1000 µM nonlabeled abacavir in a competition assay (data not shown). This would suggest a passive process or a high-capacity influx system for abacavir uptake and a relatively high capacity related to the P-gp-mediated efflux. This observation is in agreement with previous studies in which the brain uptake of [¹⁴C]abacavir, after in situ brain perfusion in the guinea pig, was not affected by up to 200 µM nonlabeled abacavir (Thomas et al., 2001). The dose-dependent inhibition of P-gp-mediated B-to-A flux by LY335979 (Fig. 9), in the MDR1 monolayers (Fig. 7B), reduced the effective B-to-A permeability by 40% and generated an IC₅₀ of 0.07 µM LY335979, which was in excellent agreement with the previously reported IC₅₀ (59 nM) for LY335979-mediated inhibition of P-gp in an in vitro cell system (Dantzig et al., 1996).

It has been reported that abacavir can distribute to the cerebrospinal fluid (Daluge et al., 1997; McDowell et al., 1999), but at levels significantly lower than in plasma. Capparelli et al. (2005) suggested that the cerebrospinal fluid penetration of abacavir might be sufficient to prevent viral replication of the HIV1 virus. This suggestion is contradicted by evidence of the development of different genotypic viral mutations in the brain compared with plasma, indicating discordant viral evolution (McCoig et al., 2002) and lack of viral inhibition in the brain (Kandanearatchi et al., 2004) under abacavir therapy. This is of particular concern in the case of AIDS-dementia complex, which is a continuing problem even with the advent of ART (Smit et al., 2004). Therefore, the role of P-gp in the brain distribution of abacavir was evaluated in vivo using the CF1 mouse model.

The mdr1a(-/-) CF1 mouse model is a subpopulation naturally deficient in the expression of the murine mdr1a gene product, due to the loss of exon 23 during mRNA splicing (Pippert and Umbenhauer, 2001). These mutant mice lack functional P-gp in tissues such as the brain, intestine, and testis (Umbenhauer et al., 1997) and are thus useful in understanding the role of P-gp in limiting CNS distribution (Kwei et al., 1999). After intravenous administration, the B/P concentration ratio in mdr1a(+/+) CF1 wild-type mice (P-gp-competent) demonstrated poor penetration of abacavir into the brain (Figs. 10A and 11). This is consistent with the previously reported brain distribution of abacavir in various animal models (Daluge et al., 1997; Thomas et al., 2001) such as the guinea pig, rat, and monkey. Compared with the wild-type mice, the mdr1a(-/-) mutants showed significantly enhanced brain distribution with a B/P ratio of up to 2.5 (40 min). This increase in brain distribution became even more remarkable when the B/P ratios in the mutants were compared with that in the wild type, which showed up to a 10-fold increase in brain levels normalized to plasma (Fig. 11).

In the wild-type mouse the plasma clearance of abacavir was high (171 ml/min/kg) and this resulted in the short plasma half-life of about 14 min. Abacavir is a prodrug and is rapidly converted to its active form, carbovir triphosphate, intracellularly in addition to being metabolized in the liver, and these processes could account for the large clearance observed. The decreased plasma clearance in the mutants (91 ml/min/kg) resulted in the doubling of the AUC_plasma and may be due to the absence of P-gp at the liver in these animals (Pereira de Oliveira et al., 2005), resulting in loss of active efflux. It is also possible that the 2-fold increase in plasma AUC in the mutants could be due to a difference in the metabolism of abacavir, even though general hepatic metabolic activity has been shown to be identical in these subpopulations (Kwei et al., 1999).

A two-fold increase in the plasma exposure in the mutant does not explain the 21-fold increase in the brain exposure reflected by the AUC_brain. The absence of P-gp at the BBB appears to be a critical factor determining the brain uptake of abacavir. P-gp function at the brain would impart an additional clearance out of the brain in the wild-type mice, resulting in a more rapid approach to equilibrium (shorter mean transit time in the brain) than would be seen with only passive clearance (Hammarlund-Udenaes et al., 1997). Figure 11 demonstrates this phenomenon, wherein the B/P ratio in the mutants took longer to reach equilibrium than in the wild-type mice. Increased brain penetration might also lead to higher intracellular levels of abacavir, resulting in the brain levels being higher than in plasma. The AUC ratio (1.85) seen in the mutants and the drug targeting index to the brain being greater than 10 strongly suggests that P-gp expression plays a significant role in limiting the brain distribution of abacavir. Altered brain metabolism of abacavir by increasing brain levels in the mutant is unlikely due to the negligible activity of alcohol dehydrogenase, the enzyme that metabolizes abacavir in conjunction with glucuronyl transferase in the liver (Zimatkin et al., 2006).

The efflux transport of NRTIs out of the brain has previously been reported for AZT and 2',3'-dideoxyinosine (Wang and Sawchuk, 1995) by a probenecid-sensitive system (Wang et al., 1997) probably mediated by an organic anion efflux system different from any nucleoside transporter. A recent study reported high levels of Bcrp1 at the BBB and up-regulation of Bcrp1 expression in the CF1 [mdr1a(-/-)] mutant mouse (Cisternino et al., 2004). In conjunction with the recent report of abacavir being a Bcrp1 substrate in vitro (Pan et al., 2007), this finding suggests that P-gp might have an even larger impact on abacavir brain distribution than observed in this study. It has been well established that P-gp is expressed in lymphocytes and can limit the entry of anti-HIV1 agents into this important site of action (Jorajuria et al., 2004). There have also been reports of increased expression of P-gp after treatment with anti-HIV agents (Ford et al., 2003). Thus, inhibition of P-gp, not only at the blood-brain barrier but also at other sites of P-gp action that are important in anti-HIV1 therapy, might improve the distribution of drug to these restricted sites and lead to inhibition of viral resistance. This study shows, both in vitro and in vivo, that P-gp is involved in the active efflux of abacavir and significant therapeutic advantage might be gained by influencing the active transport of abacavir at critical sites.

	Acknowledgments

 
We thank Dr. Alfred H. Schinkel from the Netherlands Cancer Institute (Amsterdam, The Netherlands) for generously providing Ko143, Eli Lilly and Company (Indianapolis, IN) for kindly providing us with LY335979, GlaxoSmithKline (Research Triangle, NC) for kindly providing us with GF120918, and the National Cancer Institute (Bethesda, MD) for providing us with FTC.

	Footnotes

 
This project was supported by National Institutes of Health Grant NS42549.

doi:10.1124/dmd.107.017723.

ABBREVIATIONS: NRTI, nucleoside reverse transcriptase inhibitor; CNS, central nervous system; ART, active retroviral therapy; P-gp, P-glycoprotein; ABC, ATP-binding cassette; AUC, area under the concentration-time curve; HPLC, high performance liquid chromatography; BBB, blood-brain barrier; ER, efflux ratio; Bcrp1, breast cancer resistance protein 1; AZT, zidovudine (3'-azido-3'-deoxythymidine); FTC, fumitremorgin C; LY335979, (R)-4-((1aR,6R,10bS)-1,2-difluoro-1,1a,6,10b-tetrahydrodibenzo(a,e)cyclopropa(c)cycloheptan-6-yl)--((5-quinoloyloxy)methyl)-1-piperazineethanol, trihydrochloride; WT, wild-type; MDCKII, Madin-Darby canine kidney II; GF120918, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; A-to-B, apical-to-basolateral; B-to-A, basolateral-to-apical; B/P, brain-to-plasma concentration ratio.

Address correspondence to: Dr. William F. Elmquist, Department of Pharmaceutics, University of Minnesota, 308 Harvard St. SE, Room 9-125, Weaver-Densford Hall, Minneapolis, MN 55455. E-mail: elmqu011{at}umn.edu

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