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A comparison of the effects of p-glycoprotein inhibitors on the blood–brain barrier permeation of cyclic prodrugs of an opioid peptide (DADLE)
A Comparison of the Effects of P-Glycoprotein Inhibitors on the Blood–Brain Barrier Permeation of Cyclic Prodrugs of an Opioid Peptide (DADLE) HUI OUYANG,1 THOMAS E. ANDERSEN,2 WEIQING CHEN,1 REBECCA NOFSINGER,1 BENTE STEFFANSEN,2 RONALD T. BORCHARDT1 1
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047
2
Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark
Received 15 April 2008; revised 12 August 2008; accepted 27 August 2008 Published online 14 October 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21585
ABSTRACT: The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the blood–brain barrier (BBB) permeation of cyclic prodrugs of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). The BBB permeation characteristics of these prodrugs and DADLE were determined using an in situ perfused rat brain model and in vitro cell culture model (MDCK-MDR1 cells) of the BBB. The activities of Pgp in these models were characterized using a known substrate (quinidine) and known inhibitors [cyclosporine A (CyA), GF-120918, PSC-833] of P-gp. Cyclic peptide prodrugs exhibited very poor permeation in both models. Inclusion of GF-120918, CyA, or PSC-833 in the brain perfusion medium or the cell culture medium significantly increased the permeation of these cyclic prodrugs. The order of potency of these P-gp inhibitors, as measured using the cyclic prodrugs as substrates, was, by in vitro MDCK-MDR1 cells: GF-120918 ¼ CyA PSC-833; and by in situ rat brain perfusion: GF-120918 > CyA ¼ PSC-833. In conclusion, P-gp in the BBB is the major factor restricting the brain permeation of these cyclic prodrugs. MDCK-MDR1 cells can predict the order of potencies of the investigated P-gp inhibitors to enhance the rat BBB permeation of quinidine and the cyclic prodrugs. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2227–2236, 2009
Keywords: blood–brain barrier; brain perfusion; cell culture; in vitro; in vitro–in vivo correlation; MDCK-WT cells; MDCK-MDR1 cells; P-glycoprotein; quinidine
Abbreviations used: AOA, acyloxyalkoxy; AP, apical; BBB, blood–brain barrier; BL, basolateral; BCRP, breast cancer resistant protein; CA, coumarinic acid; CyA, cyclosporin A; CNS, central nervous system; DADLE, H-Tyr-D-Ala-Gly-PheD-Leu-OH; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution; OMCA, oxymethyl-modified coumarinic acid; MDCK cells, Madin–Darby canine kidney cells; MRP2, multidrug resistant associated protein-2; Papp, BL-to-AP, BL-to-AP permeability coefficient; Papp, AP-to-BL, AP-to-BL permeability coefficient; Papp max, maximum apparent permeability; P-gp, P-glycoprotein. Hui Ouyang’s present address is Allergan, Inc., 2525 Dupont Dr., Irvine, CA 92612. Weiqing Chen’s present address is Pharmacopeia, Inc., 3000 Eastpark Boulevard, Cranbury, NJ 08512. Correspondence to: Hui Ouyang (Telephone: 714-246-6802; Fax: 714-246-5850; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 98, 2227–2236 (2009) ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association
INTRODUCTION Our laboratory has a long-standing interest in the design and synthesis of cyclic prodrugs of DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), an analog of the opioid peptide [Leu5]-enkaphalin.1–3 These cyclic prodrugs have the potential to enhance DADLE’s oral bioavailability and blood–brain barrier (BBB) permeation and thus improve its clinical utility as an analgesic.1–3 Structures of these cyclic peptide prodrugs are shown in Figure 1. When the cell permeation characteristics of these cyclic prodrugs were determined using in vitro cell culture models of the intestinal
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Figure 1. Chemical structures of the cyclic prodrugs of DADLE.
mucosa and the BBB (e.g., Caco-2 cells and MDCK-MDR1 cells, respectively), all three cyclic prodrugs were shown to be poorly permeable. This poor permeation resulted from their substrate activities for apically polarized efflux transporters [e.g., P-glycoprotein (P-gp, MDR1); multidrug resistant associated protein-2 (MRP2)].4–8 Preliminary in vivo pharmacokinetic experiments indicated that AOA-DADLE, CA-DADLE, and OMCA-DADLE are not well absorbed after oral administration to rats (unpublished data) and poorly permeate the brain after IV administration.9 Recently, our laboratory conducted mechanistic biopharmaceutical studies in an attempt to better understand what factors, in addition to substrate activity for P-gp, restrict the oral absorption of these cyclic prodrugs.10,11 Using an in situ perfused rat ileum model of the intestinal mucosa, we showed that PSC-833, a known inhibitor of P-gp, significantly increased the intestinal mucosal permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE.11 Surprisingly, cyclosporine A (CyA) and GF-12918, also potent P-gp inhibitors, were either inactive or substantially less active than PSC-833 in increasing the intestinal mucosal permeation of these cyclic prodrugs.11 In contrast, GF-120918, CyA, and PSC-833 were found to be equally effective as inhibitors of the polarized efflux of these cyclic prodrugs in Caco-2 cells.11 Based on these observations, several hypotheses were put forward to explain the different effects that P-gp inhibitors could have on the permeation of the cyclic prodrugs in the Caco-2 cell model versus their effects on the permeation of these molecules in the perfused rat ileum model of the intestinal mucosa. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
One hypothesis, which arose from the observations made by Ouyang et al.,10 was that AOADADLE, CA-DADLE, and OMCA-DADLE are substrates of a metabolic enzyme(s) (e.g., cytochrome P-450 s, esterases, phenol sulfotransferases, glucuronyltransferases) present in the rat intestinal mucosa but not expressed in Caco-2 cells. Further, it was hypothesized that this enzyme(s) plays an important role in limiting the intestinal mucosal absorption of these cyclic prodrugs. To explain the different effects that the P-gp inhibitors have on the permeation of the cyclic prodrugs in the in situ perfused ileum model,11 PSC-833 would have to be a more potent inhibitor of this metabolic enzyme than GF120918 or CyA. Ouyang et al.10 recently disproved this hypothesis by showing that, while these cyclic prodrugs of DADLE are very good substrates for cytochrome P450 3A, this oxidative pathway of metabolism is not important in determining the intestinal mucosal absorption of AOA-DADLE, CA-DADLE, and OMCA-DADLE. Therefore, we are left with the hypothesis that AOA-DADLE, CA-DADLE, and OMCA-DADLE are substrates of multiple efflux transporters [e.g., P-gp, MRP2, breast cancer resistant protein (BCRP)] in the rat intestinal mucosa and that these transporters are differentially inhibited by PSC-833, CyA, and GF-120918, with PSC-833 being the most potent inhibitor of the efflux transporter(s) that controls the intestinal permeation of these cyclic prodrugs. Thus, the lack of a correlation between the order of potencies observed for these P-gp inhibitors, when measured in Caco-2 cells (PSC-833 ¼ CyA ¼ GF-120918) versus the in situ perfused rat ileum (PSC833 CyA ¼ GF-120918), arises because of different levels of expression of efflux transporters in these models of the intestinal mucosa (i.e., Caco-2 cells express predominantly P-gp, whereas the rat intestinal mucosa express P-gp, MRP2, BCRP, and perhaps other efflux transporters). Based on these observations in the rat intestinal mucosa versus Caco-2 cells, we decided to compare the effects of PSC-833, CyA, and GF-120918 on the permeation characteristics of AOA-DADLE, CADADLE, and OMCA-DADLE using an in vitro cell culture model (MDCK-MDR1 cells) and an in situ perfused rat brain model of the BBB. In earlier studies, our laboratory determined the effects of GF-120918 and CyA on the polarized efflux of these cyclic prodrugs using MDCK-MDR1 cells6–8 and an in situ perfused rat brain.5 Both MDCKMDR1 cells12 and the in situ perfused rat DOI 10.1002/jps
EFFECTS OF P-GP INHIBITORS ON BBB PERMEATION
brain13,14 are widely used models of the BBB. From these studies, we conclude that GF-120918 and CyA can inhibit the efflux transporter (presumably P-gp) that restricts the permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE in MDCK-MDR1 cells4–8 and GF-120918 can inhibit P-gp in the rat brain BBB.5 The objectives of the experiments described in this manuscript were: (i) to compare the effects of PSC-833 and CyA on the permeation of AOA-DADLE, CADADLE, and OMCA-DADLE using the in situ perfused rat brain model and the effects of PSC833 on the permeation of these prodrugs in MDCK-MDR1 cells; and (ii) to compare the effects of GF-120918, CyA, and PSC-833 on the permeation of quinidine, a known substrate of P-gp, in these models of the BBB. The results of these studies should allow us to establish whether P-gp is in fact the major efflux transporter involved in limiting the BBB permeation of the cyclic prodrugs and determine the predictability of the MDCK-MDR1 cell culture model of BBB.
EXPERIMENTAL Materials Dulbecco’s phosphate-buffered saline, Hank’s balanced salts (HBSS) (modified), DADLE, [Leu5]-enkephalin, and CyA were purchased from Sigma–Aldrich (St. Louis, MO). L-glutamine 200 mM (100), penicillin (10,000 U/mL), streptomycin (10,000 mg/mL), and nonessential amino acids [10 mM (100) in 85% saline] were obtained from Gibco BRL, Life Technologies (Grand Island, NY). Dulbecco’s modified Eagle’s medium (DMEM) and trypsin/EDTA solution [0.25% and 0.02%, respectively, in Ca2þ- and Mg2þ-free HBSS] were purchased from JRH Bioscience (Lenexa, KS). Rat-tail collagen (type I) was obtained from Collaborative Biomedical Products (Bedford, MA), and fetal bovine serum (FBS) from Atlanta Biologicals (Norcross, GA). D-1-[14C]-mannitol (specific activity 2.07 Gbq/mmole) was purchased from Moravek Biochemicals (Brea, CA). [14C]Sucrose (specific activity 14.8 Gbq/mmole) was purchased from Perkin Elmer Life Sciences (Boston, MA). [3H]-quinidine (1 mCi/mL) was purchased from American Radiolabeled Chemicals (St. Louis, MO). GF-120918 was a gift from Dr. Kenneth Brouwer (GlaxoSmithKline, Research Triangle Park, NC). PSC-833 was a gift from Dr. Stephan Ruetz (Novartis, Basel, SwitzerDOI 10.1002/jps
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land). Prodrugs of DADLE and [Leu5]-enkephalin were synthesized in our laboratory following procedures described previously.2,3,15 All other chemicals were high grade and were purchased from Aldrich Chemical Co., (Milwaukee, WI) or Acros Organics distributed by Fisher Scientific (Houston, TX).
In Situ Perfused Rat Brain Compliance Statement for Animal Study This study complied with all requirements of the United States Department of Agriculture (USDA), and all regulations issued by the USDA implementing the Animal Welfare Act, 9 CFR, Parts 1, 2, and 3. The animal procedures that were used have been approved by The University of Kansas’ Animal Care and Use Committee (AACUC).
Perfusion Procedures The surgical procedures for the in situ rat brain perfusion technique were similar to those described elsewhere5 For perfusion experiments, the left hemisphere of the rat brain was perfused with the perfusion buffer containing test compound at a flow rate of 10 mL/min through the left common carotid artery catheter that was connected to the infusion syringes via a switching valve (Hamilton, Reno, NV). The perfusion buffer (378C, pH 7.4) consisted of bicarbonate-buffered physiological saline (NaH2PO4, KCl, NaHCO3, NaCl, CaCl2, MgSO4, and D-glucose) and was oxygenated with a mixture air of 95% O2 and 5% CO2 before perfusion. The perfusion was started immediately after the rat heart was cut open. The perfusion protocol, which was controlled by the switching valve, consisted of a 20-s preperfusion wash (saline only or saline with inhibitor), a 240-s perfusion (saline containing test compound or saline containing test compound and inhibitor), and a 5-s postperfusion wash (saline only or saline with inhibitor). The perfusion was terminated by decapitation of the animal. The perfused rat brain was removed from the skull and dissected on ice.
Preparation of In Situ Perfused Rat Brain Samples When perfusion studies were conducted using [3H]-quinidine (1 mCi/mL), brain tissue samples collected from left cortex were weighed and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
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digested in Sovable (Packard Bioscience, Meriden, CT) at 378C for 24 h. When perfusion studies were conducted using DADLE and its prodrugs (20 mM), brain tissue samples were collected from the left frontal, parietal, and occipital cortex as well as the hippocampus and weighed. Internal standards were added to the collected brain tissues before further preparation. The internal standard used for quantification of DADLE was [Leu5]-enkephalin and those for cyclic prodrugs of DADLE were the cyclic [Leu5]-enkephalin containing the same linkers as their DADLE counterparts. Brain tissues were then homogenized (30 strokes) in ice-cold saline (1:4, w/v) using a glass homogenizer (Wheaton, Philadelphia, PA). A capillary depletion method16 was used to remove the drugs that were bound to the brain vasculature. After centrifugation of the homogenate at 4500g (model 59A Micro-Centrifuge; Fisher Scientific, Pittsburgh, PA) at 48C for 15 min, the pellet was discarded and the supernatant containing both the lipid and the aqueous phases was collected and stored at 708C until further extraction. For sample extraction, brain samples were mixed with acetonitrile (1:3, v/v) and centrifuged at 21,000g for 15 min. The supernatant after centrifugation was evaporated to dryness using a Centrivap concentrator (Labconco, Kansas City, MO). The final residue was dissolved in 0.1 mL of 10% acetonitrile and centrifuged again at 10,000g and 48C for 10 min. Thirty microliters of the supernatant was injected for high-performance liquid chromatography analysis with tandem mass spectrometric detection (LC/MS/MS).
Analysis of In Situ Perfused Rat Brain Samples [3H]-Quinidine brain perfusion samples were counted for radioactivity using a dual-label scintillation spectrometer (LS 6000 IC; Beckman Coulter, Inc., Fullerton, CA). To determine the original radioactivity level in the perfusate, duplicate perfusate samples (50 mL) were also collected, mixed with Sovable, and counted. To determine the concentrations of DADLE and its cyclic prodrugs in the perfusates, HPLC with UV detection was performed using an LC-10A gradient system (Shimadzu, Tokyo, Japan) consisting of two LC-10AS pumps, an SCL-10A system controller, and an SIL-10A auto injector with a sample cooler. Sample separation was performed using a C18 reversed-phase column JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
˚ , 250 mm 4.6 mm i.d.; Vydac, Hesperia, (300 A CA) equipped with a C18 guard column (Vydac). Gradient elution was performed at a flow rate of 1 mL/min from 26% to 58% (v/v) acetonitrile in water with 0.1% (v/v) trifluoroacetic acid. The eluents were detected by UV detection (l ¼ 214 nm). The chromatographic data were acquired and analyzed using CLASS-VP version 4.2 Chromatography Data System (Shimadzu). To determine the contents of DADLE and its prodrugs in perfused brain tissue samples, LC/MS/MS was performed using a Micromass Quattro Micro triple quadrupole mass spectrometer (Micromass, Beverly, MA). The analytical method was described extensively elsewhere.17 Briefly, the liquid chromatography was conducted using a 2690 HPLC System (Waters, Milford, MA). A reversed-phase C18 column (50 mm 1.0 mm i.d., 5 mm; Vydac) was used as the analytical column and the column temperature was kept at 258C. Two mobile phases were used to generate a linear gradient to allow simultaneous analyses of DADLE and its prodrugs in a single run. Mobile phase A was water with 0.1% (v/v) formic acid, and mobile phase B was acetonitrile with 0.1% (v/v) formic acid. The HPLC system was interfaced to the mass spectrometer via an electrospray interface. For sample recording, multiple reaction monitoring of several channels was used with 0.1 s dwell time and interchannel delay set at 0.03 s so that several compounds could be monitored simultaneously. Data acquisition and analysis were performed using MassLynx version 3.4 software (Micromass).
Calculations of Papp Values from In Situ Rat Brain Perfusion Experiments Brain uptake of a test drug was analyzed using a simple, linear two-compartment model (the vascular blood and the brain parenchyma). The unidirectional transfer coefficient Kin (mL/s/g) from blood to brain was estimated using the following equations: Kin ¼
Qbr =Cpf Vvasc t
(1)
where Qbr is the amount of drug in the brain parenchyma (ng/g), Cpf is the drug concentration in the perfusion fluid (ng/mL), Qbr/Cpf is defined as the apparent brain distribution volume (Vbr, mL/ g), t is the net perfusion time (s), and Vvasc is the brain intravascular volume. A capillary depletion DOI 10.1002/jps
EFFECTS OF P-GP INHIBITORS ON BBB PERMEATION
method was then used for sample preparation to minimize the residual intravascular tracer remaining in the brain parenchyma. By using the postperfusion wash and the capillary depletion step, Vvasc could be excluded from Eq. (1). The Kin was determined as the slope from the linear regression of the measured Vbr values at multiple time points. Eq. (2) was used for single time point analysis, in which the perfusions were conducted for a single time period. The Kin was determined by dividing the Vbr value by perfusion time. The Kin values were converted to PA values using the Crone–Renkin model of capillary transfer:5 Kin PA ¼ F ln 1 (2) F where F is the regional cerebral perfusion fluid flow, which was determined using [3H]-diazepam in separate experiments (0.025 mL/s/g);5 P is the apparent permeability coefficient of the BBB for a testing compound; and A is the rat brain capillary surface area (130 cm2/g), which was reported previously.18 P was calculated by dividing PA by A. All data are presented as mean SD for at least three rats. A Student’s t-test was used to compare individual means.
MDCK-MDR1 Cell Culture Experiments Cell Culture Conditions MDCK cells, which were transfected with human MDR1 gene (MDCK-MDR1), were obtained as a gift from Drs. Raymond Evers and Piet Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands). As described previously,6,8 cells were grown in a controlled atmosphere of 5% CO2 and 90% relative humidity at 378C in 150 cm2 culture flask using a culture medium consisting of DMEM supplemented with 10% heat-inactivated FBS, 1% nonessential amino acids, 100 mg/mL streptomycin, 100 U/mL penicillin, and 1% L-glutamine. When approximately 80% confluence (i.e., 3–5 days) was reached, cells were detached from the plastic support by partial digestion using trypsin/EDTA solution and either subcultured in new flasks or plated on collagencoated polyester membranes (Transwell1, 0.4 mm pore size, 24.5 mm diameter) at a density of 7.9 104 cells/cm2. Cells were fed with culture medium every other day for 6 days until transport experiments were performed apical (AP) volume 1.5 mL; basolateral (BL) volume 2.6 mL]. DOI 10.1002/jps
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Transport Experiments Cell monolayers were grown on collagen-coated polyester filters (Transwell1). All studies were performed in triplicate in a shaking water bath at 378C. The integrity of each batch of cells was tested by measuring [14C]-mannitol flux in representative monolayers. Cell monolayers were washed three times with prewarmed HBSS, pH 7.4, and incubated with blank HBSS or with HBSS in the presence of various concentrations of inhibitor (GF-120918, CyA, or PSC-833) for 20 min. The [3H]-quinidine in HBSS with various concentrations of P-gp inhibitor was applied to the donor compartment (AP: 1.5 mL). Blank HBSS was added to the receiver compartment (BL: 2.5 mL) and samples were taken at intervals up to 90 min (donor: 10 mL; receiver: 100 mL). Samples were then counted for radioactivity using a duallabel scintillation spectrometer (LS 6000 IC; Beckman Coulter, Inc.). Permeability coefficients ( Papp) of the compounds were calculated by: Papp ¼
DQ=Dt A C0
(3)
where DQ/Dt is the linear appearance rate of mass in the receiver solution, A is the cross-section area (4.71 cm2), and C0 is the initial concentration of the donor side at t ¼ 0. The concentrations of the P-gp inhibitor (GF120918, CyA, or PSC-833) and the corresponding permeability coefficients of quinidine were fitted into a sigmoidal dose–response curve with variable slope, and Papp max and IC50 values were calculated using GraphPad Prism1 3.0.
RESULTS Permeation Characteristics of Quinidine, a Known P-gp Substrate, in the In Situ Perfused Rat Brain and MDCK-MDR1 Cell Culture Models of the BBB [3H]-Quinidine, a known P-gp substrate,19 was used as a marker to monitor the functional P-gp activity in the in situ perfused rat brain and MDCK-MDR1 cell culture models of the BBB. GF120918, CyA, or PSC-833, all known inhibitors of P-gp, were co-administered with [3H]-quinidine in the above-mentioned models of the BBB, and the Papp values of this marker compound were calculated. The concentrations of the P-gp inhibitors and the Papp values of [3H]-quinidine were then plotted as dose–response curves (data not shown) and the maximum intrinsic permeability JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
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values ( Papp max) of [3H]-quinidine and the IC50 values of the P-gp inhibitors were calculated by curve fitting. In this study, the maximum concentrations of CyA and PSC-833 were limited to 10 mM by their solubility in the experimental buffer. For GF-120918, a concentration of 10 mM, determined using the in situ perfused brain model, has been shown by Chen et al.5 to completely inhibit the P-gp activity in the BBB. The Papp max values of quinidine in the absence and presence of GF-120918, CyA, and PSC-833 and the IC50 values of these P-gp inhibitors determined using the in situ perfused rat brain and MDCK-MDR1 cell culture models are summarized in Table 1. The Papp values of quinidine generated in the in situ perfused rat brain model increased from 15 107 cm/s (without a P-gp inhibitor) to 240 107 cm/s ( Papp max) in the presence of the P-gp inhibitors. Similarly, the Papp values of quinidine generated in the MDCKMDR1 cell culture model increased from 18 107 to 120 107 cm/s ( Papp max) in the presence of the P-gp inhibitors. The Papp max values of quinidine in the presence of the three Pgp inhibitors were significantly higher in the in situ perfused rat brain model than the corresponding Papp max value in the MDCKMDR1 cell culture model ( p < 0.05). The in situ perfused rat brain model afforded an IC50 value for GF-120918 that was significantly lower than the IC50 values observed for CyA ( p < 0.05) and PSC 833 ( p < 0.1). These results lead to a potency order of GF-120918 > CyA ¼ PSC-833. In contrast, there were no significant differences among the IC50 values of the three P-gp inhibitors in the MDCK-MDR1 cell culture model, leading to a potency order of GF-120918 ¼ CyA ¼ PSC-833.
Permeation Characteristics of DADLE, AOADADLE, CA-DADLE, and OMCA-DADLE Across MDCK-MDR1 Cells The permeability ( Papp) of AOA-DADLE, CADADLE, and OMCA-DADLE across MDCKMDR1 cell monolayers was determined in the presence and absence of 10 mM of P-gp inhibitor (GF-120918, CyA, or PSC-833). In the absence of P-gp inhibitor, three cyclic peptide prodrugs exhibited high efflux ratio (16.8 for AOA-DADLE, >35 for CA-DADLE and OMCA-DADLE, respectively) across MDCK-MDR1 cell monolayers, indicating that P-gp limits the permeation of these prodrugs. In the presence of 10 mM of P-gp inhibitor, the efflux ratios of all cyclic peptide prodrugs were significantly reduced (<1.18 for AOA-DADLE, <2.11 for CA-DADLE, and <3.91 for OMCA-DADLE, respectively). GF-120918, CyA, and PSC-833 were equally potent in inhibiting the efflux of prodrugs across MDCK-MDR1 cell monolayers. Permabilities are summarized in Table 2. The efflux ratios of AOA-DADLE across MDCK-MDR1 cell monolayers in the presence of GF-120918 and CyA were less than unit value probably due to experimental variations in the determination of Papp values.
Permeation Characteristics of DADLE, AOADADLE, CA-DADLE, and OMCA-DADLE in the In Situ Perfused Rat Brain Model of the BBB In an earlier study,5 our laboratory showed that GF-120918 had no effect on the brain permeation (as measured by the Papp value) of DADLE using the in situ perfused rat brain model of the BBB. However, this P-gp inhibitor significantly
Table 1. The Maximum Apparent Permeability Coefficients ( Papp max) of Quinidine Across In Situ Perfused Rat Brain, MDCK-WT Cells, and MDCK-MDR1 Cells in the Presence of P-gp Inhibitors (GF-120918, CyA, and PSC-833), and the IC50 Values of These P-gp Inhibitors on Reducing Quinidine Efflux Rat Brain Perfusion Papp Control GF-120918 Cyclosporin A PSC-833
max
a
MDCK-WT
(107 cm/s) IC50 (mM) Papp
14.46 203.47 241.10 254.73
(3.77) (15.28) (26.47) (66.75)
— 0.41 (0.06) 1.72 (0.34)b 1.53 (0.55)b
max
MDCK-MDR1
(107 cm/s) IC50 (mM) Papp
60.74 133.17 109.17 114.97
(2.72) (32.53) (15.70) (7.34)
— 2.49 (2.89) 0.38 (0.62) 0.35 (0.33)
max
(107 cm/s) IC50 (mM)
18.05 115.15 126.00 121.23
(0.52) (17.76) (30.01) (15.22)
— 0.15 (0.079) 0.17 (0.09) 0.58 (0.37)
Results were presented as mean SD of triplicate determinations. The concentrations of P-gp inhibitor (GF-120918, CyA, or PSC-833) and the corresponding permeability coefficients of quinidine were fitted into a sigmoidal dose–response curve with variable slope, and both Papp max and IC50 were calculated using GraphPad Prism1 3.0. a Papp max is also the intrinsic permeability of tested compound (e.g., quinidine). b The IC50 value is statistically different from the IC50 value of GF-120918 ( p < 0.1) in rat brain perfusion model. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
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Table 2. Papp Values of AOA-DADAE, CA-DADLE and OMCA-DADLE Across MDCK-MDR1 Cell Monolayer in the Presence and Absence of Known P-gp Inhibitors (10 mM) Papp (106 cm/s) Substrate AOA-DADLE
CA-DADLE
OMCA-DADLE
a
Papp
AP-to-BL
Inhibitor
AP-to-BL
BL-to-AP
— GF120918 CyA PSC-833 — GF120918 CyA PSC-833 — GF120918 CyA PSC-833
0.095a 10.7 1.5 20.4 9.7 6.5 2.6 0.011a 13.5 2.5 17.3 2.0 2.3 0.1 0.013a 11.3 0.63 82.0 2.3 2.3 0.1
1.6 0.23 5.3 1.5 11.7 1.3 7.6 3.9 43.0 5.0 13.8 1.0 15.5 3.5 9.0 0.7 21.8 3.0 16.7 0.84 173 113 9.0 0.7
>16.8 0.50 0.58 1.18 >35 1.02 0.90 3.91 >35 1.48 2.11 3.91
calculated using lowest detectable limits.
increased the Papp values of AOA-DADLE, CADADLE, and OMCA-DADLE. To compare the enhancing effects of GF-120918 to CyA and PSC833 on the brain permeation of these cyclic prodrugs, similar experiments were conducted with CyA and PSC-833, and the resulting data are shown in Table 3. The experimental protocols (e.g., P-gp inhibitor concentration ¼ 10 mM; single pass perfusion for 240 s) used to generate the new data for CyA and PSC-833 shown in Table 3 were identical to the protocols used by Chen et al.5 to determine the effects of GF-120918. Table 3 summarizes the published5 and newly generated Papp ¼ values for DADLE, AOADADLE, CA-DADLE, and OMCA-DADLE in the absence and presence of GF-120918, CyA, or PSC833. In the absence of a P-gp inhibitor (w/o inhibitor), the Papp values observed for both DADLE and its prodrugs were very low (between 0.4 107 and 1.2 107 cm/s). Inclusion of GF120918, CyA, or PSC-833 did not significantly change DADLE’s Papp value ( Papp values varied between 0.5 107 and 0.9 107 cm/s). However, inclusion of 10 mM of GF-120918, CyA, or PSC-833 significantly increased the permeation of all three cyclic prodrugs. Increases in the Papp values of AOA-DADLE, CA-DADLE, and OMCA-DADLE were as follows: GF-120918, 50-, 460-, and 170fold, respectively; CyA, 10-, 50-, and 40-fold, respectively; and PSC-833, 7-, 50-, and 60-fold, respectively. The Papp values observed for the three cyclic prodrugs in the presence of the P-gp inhibitors were statistically different from the DOI 10.1002/jps
Ratio ( Papp BL-to-AP/ Papp AP-to-BL)
Papp values in the absence of the P-gp inhibitors (GF-120918, p < 0.05; CyA, p < 0.05; PSC-833, p < 0.1). Based on these data, the order of potency for the ability of the P-gp inhibitors to enhance the BBB permeation of these cyclic prodrugs would be as follows: GF-120918 > CyA ¼ PSC-833.
DISCUSSION Our laboratory4,6,20,21 has used Caco-2 cells in an attempt to predict the in situ and in vivo permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE across the intestinal mucosa. The resulting Caco-2 cell data showed that all three cyclic prodrugs of DADLE exhibit polarized efflux that can be inhibited by P-gp inhibitors.11 These data suggest that the in vivo and in situ intestinal mucosal permeation of these cyclic prodrugs would be very poor because of their substrate activity for P-gp, which is known to be expressed in intestinal mucosal cells. Recently, our laboratory confirmed this hypothesis by determining the intestinal mucosal permeation of these cyclic prodrugs using an in situ perfused ileum model.11 Ouyang et al.11 also attempted to use Caco-2 cell data to rank order P-gp inhibitors for their abilities to inhibit the polarized efflux of AOADADLE, CA-DADLE, and OMCA-DADLE. The results of these experiments showed that 10 mM concentrations of GF-120918, CyA, or PSC-833 almost completely inhibited the polarized efflux of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
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Table 3. The Effect of P-gp-Inhibitors (GF-120918, CyA, and PSC-833) on the Permeability of DADLE and its Cyclic Prodrugs (AOA-DADLE, CA-DADLE, and OMCA-DADLE) Across the In Situ Perfused Rat Brain Model Papp (107 cm/s) Compounds DADLE AOA-DADLE CA-DADLE OMCA-DADLE
w/o Inhibitor 0.5 1.2 0.4 0.7
(1.4) (1.0) (0.7) (0.6)
GF120918 0.6 60.5 185 119
(0.1) (25.6)a (68.3)a (12.8)a
Cyclosporin A 0.9 11.8 20.0 31.0
(0.2) (2.9)b (5.6)b (11.3)b
PSC833 0.6 8.7 21.9 41.0
(0.2) (3.4)c (9.4)c (18.4)c
Rat brain was perfused with buffer containing 10 mM of P-gp inhibitor and 20 mM of DADLE or its prodrugs for 4 min. Brain samples were collected and processed by a capillary depletion method. After liquid phase exaction with acetonitrile, samples were analyzed by LC/MS/MS. The Papp values were calculated using single time point analysis and presented as mean SD (n 3) and the statistical significance of the data points were evaluated using paired Student’s t-tests. a The data point showed statistical difference ( p < 0.05) from control value (in the absence of inhibitor) and statistical difference ( p < 0.1) from the Papp value with the presence of CyA or PSC-833. b The data point showed statistical difference ( p < 0.05) from control value (in the absence of inhibitor). c The data point showed statistical difference ( p < 0.1) from control value (in the absence of inhibitor).
these cyclic prodrugs in Caco-2 cell monolayers. The maximum concentrations of CyA and PSC-833 were limited to 10 mM by their solubility in the experimental buffer. When these P-gp inhibitors were actually tested at 10 mM concentrations for their abilities to enhance the intestinal mucosal absorption of the cyclic prodrugs using the in situ perfused ileum model, surprising and unexpected results were observed. For example, PSC-833 was found to increase significantly the intestinal permeation of all three prodrugs, whereas CyA and GF-12918 were either inactive or substantially less active than PSC-833.11 Based on these results, the order of potency of these P-gp inhibitors in the in situ perfused ileum model is PSC-833 CyA ¼ GF-120913. Thus, the data generated in Caco-2 cell model did not correctly rank order these P-gp inhibitors for their abilities to enhance the intestinal mucosal permeation of the cyclic prodrugs. After additional experiments were conducted to eliminate some possible explanations (e.g., metabolism) for these differences, we concluded that that AOA-DADLE, CA-DADLE, and OMCA-DADLE were substrates for multiple efflux transporters (e.g., P-gp, MRP2, BCRP) in the rat intestinal mucosa and that these transporters were differentially inhibited by PSC-833, CyA, and GF-120918, with PSC-833 being the most potent inhibitor of the efflux transporter(s) that controls the intestinal permeation of these cyclic prodrugs. In contrast, in Caco-2 cells, these cyclic prodrugs and these P-gp inhibitors interact with only one efflux transporter (i.e., P-gp). Our laboratory6–8 has also shown that: (i) AOADADLE, CA-DADLE, and OMCA-DADLE undergo significant polarized efflux in MDCK-MDR1 cells, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
a model of the BBB; and (ii) this polarized efflux can be inhibited by GF-120918 and CyA. These data suggest that the BBB permeation of these cyclic prodrugs when measured in vivo or in situ would also be very low because of their substrate activities for P-gp since this efflux transporter is known to be expressed in the brain microvessel endothelial cells that make up the BBB. This hypothesis was confirmed by conducting in vivo pharmacokinetic experiments9 and in situ perfused rat brain experiments.5 The in vivo pharmacokinetic experiments9 showed that after IV administration of these cyclic prodrugs negligible amounts of the prodrugs and DADLE were found in brain tissue. Similarly, the in situ perfused rat brain experiments5 showed that perfusion of medium that contained a cyclic prodrug into the brain vascular system afforded negligible amounts of the prodrug and DADLE in brain tissue. However, if a P-gp inhibitor were added to the perfusion medium, the amounts of the prodrug and DADLE in brain tissue increased significantly (e.g., CA-DADLE/GF-120918, 460fold increase). In the experiments described in this manuscript, we determined whether the MDCK-MDR1 cell culture model of the BBB could predict the ability of P-gp inhibitors (i.e., GF-120918, CyA, PSC-833) to enhance the in situ rat brain permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE. As shown in Table 2, inclusion of GF-120918, CyA, or PSC-833 at concentrations of 10 mM produced nearly complete inhibition of the polarized efflux of these cyclic prodrugs in MDCK-MDR1 cells. The equipotent effects of GF120918, CyA and PSC-833 on inhibiting the DOI 10.1002/jps
EFFECTS OF P-GP INHIBITORS ON BBB PERMEATION
polarized efflux of these cyclic prodrugs in MDCKMDR1 cells are consistent with the effects of these P-gp inhibitors on the efflux of quinidine in this same cell culture system (Tab. 1). As shown in Table 3, when 10 mM concentrations of CyA or PSC-833 were included in the perfusion medium with AOA-DADLE, CADADLE, or OMCA-DADLE, the amounts of the prodrugs and DADLE in brain tissue increased significantly (CyA, 10-, 50-, and 40-fold, respectively; and PSC-833, 7-, 50-, and 60-fold, respectively. When these data for PSC-833 and CyA are compared to the data observed for GF-120918 (GF-120918, 50-, 460-, and 170-fold, respectively; Tab. 3 5), one derives the following rank ordering of potencies: GF-120918 > CyA ¼ PSC-833. The rank ordering of potencies of these P-gp inhibitors observed using the in situ rat brain perfusion model (Tab. 3) is in good agreement with the rank ordering of potencies derived at from MDCKMDR1 cell experiments (Tabs. 1 and 2). This agreement suggests: (i) P-gp in the rat brain microvessel endothelial cells is the major factor restricting BBB permeation of these cyclic prodrugs; and (ii) MDR1 and rat P-gp have similar substrate specificities for these cyclic prodrugs and these P-gp inhibitors. The results described in this manuscript for the effects of GF-120918, CyA and PSC-833 on the BBB permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE are very different than the results observed for these P-gp inhibitors on the intestinal mucosal permeation of these cyclic prodrugs.11 These differences probably reflect the more complex nature of the intestinal mucosal barrier and the fact that P-gp is not the only efflux transporter limiting the oral absorption of these cyclic prodrugs. In conclusion, the data presented in this manuscript support the hypothesis that P-gp is the major factor restricting the BBB permeation of these cyclic prodrugs. In addition, data generated using MDCK-MDR1 cells can not only predict the permeation characteristics of the cyclic prodrugs but this BBB cell culture model can also predict the order of potencies of P-gp inhibitors to enhance the BBB permeation of quinidine and the cyclic prodrugs.
ACKNOWLEDGMENTS The authors thank Dr. Kenneth Brouwer (GlaxoSmithKline, Research Triangle Park, NC) for a DOI 10.1002/jps
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generous gift of GF-120918 and Dr. Stephan Ruetz (Novartis, Basel, Switzerland) for a generous gift of PSC-833. This work was supported by a grant from the United States Public Health Service (DA-09315).
REFERENCES 1. Wang B, Gangwar S, Pauletti GM, Siahaan TJ, Borchardt RT. 1997. Synthesis of a novel esterase-sensitive cyclic prodrug system for peptides that utilizes a ‘‘trimethyl lock’’-facilitated lactonization reaction. J Org Chem 62:1363–1367. 2. Bak A, Siahaan TJ, Gudmundsson OS, Gangwar S, Friis GJ, Borchardt RT. 1999. Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using an (acyloxy)alkoxy linker. J Pept Res 53: 393–402. 3. Ouyang H, Vander Velde DG, Borchardt RT, Siahaan TJ. 2002. Synthesis and conformational analysis of a coumarinic acid-based cyclic prodrug of an opioid peptide with modified sensitivity to esterasecatalyzed bioconversion. J Pept Res 59:183–195. 4. Bak A, Gudmundsson OS, Friis GJ, Siahaan TJ, Borchardt RT. 1999. Acyloxyalkoxy-based cyclic prodrugs of opioid peptides: Evaluation of the chemical and enzymatic stability as well as their transport properties across Caco-2 cell monolayers. Pharm Res 16:24–29. 5. Chen W, Yang JZ, Andersen R, Nielsen LH, Borchardt RT. 2002. Evaluation of the permeation characteristics of a model opioid peptide, H-Tyr-D-AlaGly-Phe-D-Leu-OH (DADLE), and its cyclic prodrugs across the blood-brain barrier using an in situ perfused rat brain model. J Pharmacol Exp Ther 303:849–857. 6. Ouyang H, Tang F, Siahaan TJ, Borchardt RT. 2002. A modified coumarinic acid-based cyclic prodrug of an opioid peptide: Its enzymatic and chemical stability and cell permeation characteristics. Pharm Res 19:794–801. 7. Tang F, Borchardt RT. 2002. Characterization of the efflux transporter(s) responsible for restricting intestinal mucosa permeation of an acyloxyalkoxybased cyclic prodrug of the opioid peptide DADLE. Pharm Res 19:780–786. 8. Tang F, Borchardt RT. 2002. Characterization of the efflux transporter(s) responsible for restricting intestinal mucosa permeation of a coumarinic acidbased cyclic prodrug of the opioid peptide DADLE. Pharm Res 19:787–793. 9. Yang JZ, Chen W, Borchardt RT. 2002. In vitro stability and in vivo pharmacokinetic studies of a model opioid peptide, H-Tyr-D-Ala-Gly-Phe-D-LeuOH (DADLE), and its cyclic prodrugs. J Pharmacol Exp Ther 303:840–848. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
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10. Ouyang H, Chen W, Andersen T, Steffansen B, Borchardt RT. 2008. Factors that restrict the intestinal cell permeation of cyclic prodrugs of an opioid peptide (DADLE): Part (II) Role of metabolic enzymes in the intestinal mucosa. J Pharm Sci 2008. DOI: 10.1002/jps.21424. 11. Ouyang H, Chen W, Andersen T, Steffansen B, Borchardt RT. 2008. Factors that restrict the intestinal cell permeation of cyclic prodrugs of an opioid peptide (DADLE): Part (I) Role of efflux transporters in the intestinal mucosa. J Pharm Sci 2008. DOI: 10.1002/jps.21428. 12. Polli JW, Serabjit-Singh CJ. 2004. In vitro cell based assays for estimating the effects of efflux transporters on cell permeation. In: Borchardt RT, Kerns EH, Lipinski CA, Thakker DR, Wang B, editors. Pharmaceutical profiling in drug discovery for lead optimization, 1st edition Arlington, VA: AAPS Press. pp 235–356. 13. Takasato Y, Rapoport SI, Smith QR. 1984. An in situ brain perfusion technique to study cerebrovascular transport in the rat. Am J Physiol 247:H484– H493. 14. Smith QR. 1996. Brain perfusion systems for studies of drug uptake and metabolism in the central nervous system. Pharm Biotechnol 8:285– 307. 15. Wang B, Wang W, Camenisch GP, Elmo J, Zhang H, Borchardt RT. 1999. Synthesis and evaluation of novel coumarin-based esterase-sensitive cyclic prodrugs of peptidomimetic RGD analogs with
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improved membrane permeability. Chem Pharm Bull (Tokyo) 47:90–95. Triguero D, Buciak J, Pardridge WM. 1990. Capillary depletion method for quantification of bloodbrain barrier transport of circulating peptides and plasma proteins. J Neurochem 54:1882–1888. Yang JZ, Bastian KC, Moore RD, Stobaugh JF, Borchardt RT. 2002. Quantitative analysis of a model opioid peptide and its cyclic prodrugs in rat plasma using high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection. J Chromatogr B Anal Technol Biomed Life Sci 780:269–281. Metzer H, Heuber-Metzer S, Steinacker A, Struber J. 1980. Staining PO2 measurement sites in the rat brain and quantitative morphometry if the surrounding capillary. Pfluegers Arch 388:21–27. Kusuhara H, Suzuki H, Terasaki T, Kakee A, Lemaire M, Sugiyama Y. 1997. P-Glycoprotein mediates the efflux of quinidine across the bloodbrain barrier. J Pharmacol Exp Ther 283:574–580. Gudmundsson OS, Nimkar K, Gangwar S, Siahaan T, Borchardt RT. 1999. Phenylpropionic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeation. Pharm Res 16:16–23. Gudmundsson OS, Pauletti GM, Wang W, Shan D, Zhang H, Wang B, Borchardt RT. 1999. Coumarinic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeability. Pharm Res 16:7–15.
DOI 10.1002/jps
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047
2
Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark
Received 15 April 2008; revised 12 August 2008; accepted 27 August 2008 Published online 14 October 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21585
ABSTRACT: The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the blood–brain barrier (BBB) permeation of cyclic prodrugs of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). The BBB permeation characteristics of these prodrugs and DADLE were determined using an in situ perfused rat brain model and in vitro cell culture model (MDCK-MDR1 cells) of the BBB. The activities of Pgp in these models were characterized using a known substrate (quinidine) and known inhibitors [cyclosporine A (CyA), GF-120918, PSC-833] of P-gp. Cyclic peptide prodrugs exhibited very poor permeation in both models. Inclusion of GF-120918, CyA, or PSC-833 in the brain perfusion medium or the cell culture medium significantly increased the permeation of these cyclic prodrugs. The order of potency of these P-gp inhibitors, as measured using the cyclic prodrugs as substrates, was, by in vitro MDCK-MDR1 cells: GF-120918 ¼ CyA PSC-833; and by in situ rat brain perfusion: GF-120918 > CyA ¼ PSC-833. In conclusion, P-gp in the BBB is the major factor restricting the brain permeation of these cyclic prodrugs. MDCK-MDR1 cells can predict the order of potencies of the investigated P-gp inhibitors to enhance the rat BBB permeation of quinidine and the cyclic prodrugs. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2227–2236, 2009
Keywords: blood–brain barrier; brain perfusion; cell culture; in vitro; in vitro–in vivo correlation; MDCK-WT cells; MDCK-MDR1 cells; P-glycoprotein; quinidine
Abbreviations used: AOA, acyloxyalkoxy; AP, apical; BBB, blood–brain barrier; BL, basolateral; BCRP, breast cancer resistant protein; CA, coumarinic acid; CyA, cyclosporin A; CNS, central nervous system; DADLE, H-Tyr-D-Ala-Gly-PheD-Leu-OH; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution; OMCA, oxymethyl-modified coumarinic acid; MDCK cells, Madin–Darby canine kidney cells; MRP2, multidrug resistant associated protein-2; Papp, BL-to-AP, BL-to-AP permeability coefficient; Papp, AP-to-BL, AP-to-BL permeability coefficient; Papp max, maximum apparent permeability; P-gp, P-glycoprotein. Hui Ouyang’s present address is Allergan, Inc., 2525 Dupont Dr., Irvine, CA 92612. Weiqing Chen’s present address is Pharmacopeia, Inc., 3000 Eastpark Boulevard, Cranbury, NJ 08512. Correspondence to: Hui Ouyang (Telephone: 714-246-6802; Fax: 714-246-5850; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 98, 2227–2236 (2009) ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association
INTRODUCTION Our laboratory has a long-standing interest in the design and synthesis of cyclic prodrugs of DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), an analog of the opioid peptide [Leu5]-enkaphalin.1–3 These cyclic prodrugs have the potential to enhance DADLE’s oral bioavailability and blood–brain barrier (BBB) permeation and thus improve its clinical utility as an analgesic.1–3 Structures of these cyclic peptide prodrugs are shown in Figure 1. When the cell permeation characteristics of these cyclic prodrugs were determined using in vitro cell culture models of the intestinal
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Figure 1. Chemical structures of the cyclic prodrugs of DADLE.
mucosa and the BBB (e.g., Caco-2 cells and MDCK-MDR1 cells, respectively), all three cyclic prodrugs were shown to be poorly permeable. This poor permeation resulted from their substrate activities for apically polarized efflux transporters [e.g., P-glycoprotein (P-gp, MDR1); multidrug resistant associated protein-2 (MRP2)].4–8 Preliminary in vivo pharmacokinetic experiments indicated that AOA-DADLE, CA-DADLE, and OMCA-DADLE are not well absorbed after oral administration to rats (unpublished data) and poorly permeate the brain after IV administration.9 Recently, our laboratory conducted mechanistic biopharmaceutical studies in an attempt to better understand what factors, in addition to substrate activity for P-gp, restrict the oral absorption of these cyclic prodrugs.10,11 Using an in situ perfused rat ileum model of the intestinal mucosa, we showed that PSC-833, a known inhibitor of P-gp, significantly increased the intestinal mucosal permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE.11 Surprisingly, cyclosporine A (CyA) and GF-12918, also potent P-gp inhibitors, were either inactive or substantially less active than PSC-833 in increasing the intestinal mucosal permeation of these cyclic prodrugs.11 In contrast, GF-120918, CyA, and PSC-833 were found to be equally effective as inhibitors of the polarized efflux of these cyclic prodrugs in Caco-2 cells.11 Based on these observations, several hypotheses were put forward to explain the different effects that P-gp inhibitors could have on the permeation of the cyclic prodrugs in the Caco-2 cell model versus their effects on the permeation of these molecules in the perfused rat ileum model of the intestinal mucosa. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
One hypothesis, which arose from the observations made by Ouyang et al.,10 was that AOADADLE, CA-DADLE, and OMCA-DADLE are substrates of a metabolic enzyme(s) (e.g., cytochrome P-450 s, esterases, phenol sulfotransferases, glucuronyltransferases) present in the rat intestinal mucosa but not expressed in Caco-2 cells. Further, it was hypothesized that this enzyme(s) plays an important role in limiting the intestinal mucosal absorption of these cyclic prodrugs. To explain the different effects that the P-gp inhibitors have on the permeation of the cyclic prodrugs in the in situ perfused ileum model,11 PSC-833 would have to be a more potent inhibitor of this metabolic enzyme than GF120918 or CyA. Ouyang et al.10 recently disproved this hypothesis by showing that, while these cyclic prodrugs of DADLE are very good substrates for cytochrome P450 3A, this oxidative pathway of metabolism is not important in determining the intestinal mucosal absorption of AOA-DADLE, CA-DADLE, and OMCA-DADLE. Therefore, we are left with the hypothesis that AOA-DADLE, CA-DADLE, and OMCA-DADLE are substrates of multiple efflux transporters [e.g., P-gp, MRP2, breast cancer resistant protein (BCRP)] in the rat intestinal mucosa and that these transporters are differentially inhibited by PSC-833, CyA, and GF-120918, with PSC-833 being the most potent inhibitor of the efflux transporter(s) that controls the intestinal permeation of these cyclic prodrugs. Thus, the lack of a correlation between the order of potencies observed for these P-gp inhibitors, when measured in Caco-2 cells (PSC-833 ¼ CyA ¼ GF-120918) versus the in situ perfused rat ileum (PSC833 CyA ¼ GF-120918), arises because of different levels of expression of efflux transporters in these models of the intestinal mucosa (i.e., Caco-2 cells express predominantly P-gp, whereas the rat intestinal mucosa express P-gp, MRP2, BCRP, and perhaps other efflux transporters). Based on these observations in the rat intestinal mucosa versus Caco-2 cells, we decided to compare the effects of PSC-833, CyA, and GF-120918 on the permeation characteristics of AOA-DADLE, CADADLE, and OMCA-DADLE using an in vitro cell culture model (MDCK-MDR1 cells) and an in situ perfused rat brain model of the BBB. In earlier studies, our laboratory determined the effects of GF-120918 and CyA on the polarized efflux of these cyclic prodrugs using MDCK-MDR1 cells6–8 and an in situ perfused rat brain.5 Both MDCKMDR1 cells12 and the in situ perfused rat DOI 10.1002/jps
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brain13,14 are widely used models of the BBB. From these studies, we conclude that GF-120918 and CyA can inhibit the efflux transporter (presumably P-gp) that restricts the permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE in MDCK-MDR1 cells4–8 and GF-120918 can inhibit P-gp in the rat brain BBB.5 The objectives of the experiments described in this manuscript were: (i) to compare the effects of PSC-833 and CyA on the permeation of AOA-DADLE, CADADLE, and OMCA-DADLE using the in situ perfused rat brain model and the effects of PSC833 on the permeation of these prodrugs in MDCK-MDR1 cells; and (ii) to compare the effects of GF-120918, CyA, and PSC-833 on the permeation of quinidine, a known substrate of P-gp, in these models of the BBB. The results of these studies should allow us to establish whether P-gp is in fact the major efflux transporter involved in limiting the BBB permeation of the cyclic prodrugs and determine the predictability of the MDCK-MDR1 cell culture model of BBB.
EXPERIMENTAL Materials Dulbecco’s phosphate-buffered saline, Hank’s balanced salts (HBSS) (modified), DADLE, [Leu5]-enkephalin, and CyA were purchased from Sigma–Aldrich (St. Louis, MO). L-glutamine 200 mM (100), penicillin (10,000 U/mL), streptomycin (10,000 mg/mL), and nonessential amino acids [10 mM (100) in 85% saline] were obtained from Gibco BRL, Life Technologies (Grand Island, NY). Dulbecco’s modified Eagle’s medium (DMEM) and trypsin/EDTA solution [0.25% and 0.02%, respectively, in Ca2þ- and Mg2þ-free HBSS] were purchased from JRH Bioscience (Lenexa, KS). Rat-tail collagen (type I) was obtained from Collaborative Biomedical Products (Bedford, MA), and fetal bovine serum (FBS) from Atlanta Biologicals (Norcross, GA). D-1-[14C]-mannitol (specific activity 2.07 Gbq/mmole) was purchased from Moravek Biochemicals (Brea, CA). [14C]Sucrose (specific activity 14.8 Gbq/mmole) was purchased from Perkin Elmer Life Sciences (Boston, MA). [3H]-quinidine (1 mCi/mL) was purchased from American Radiolabeled Chemicals (St. Louis, MO). GF-120918 was a gift from Dr. Kenneth Brouwer (GlaxoSmithKline, Research Triangle Park, NC). PSC-833 was a gift from Dr. Stephan Ruetz (Novartis, Basel, SwitzerDOI 10.1002/jps
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land). Prodrugs of DADLE and [Leu5]-enkephalin were synthesized in our laboratory following procedures described previously.2,3,15 All other chemicals were high grade and were purchased from Aldrich Chemical Co., (Milwaukee, WI) or Acros Organics distributed by Fisher Scientific (Houston, TX).
In Situ Perfused Rat Brain Compliance Statement for Animal Study This study complied with all requirements of the United States Department of Agriculture (USDA), and all regulations issued by the USDA implementing the Animal Welfare Act, 9 CFR, Parts 1, 2, and 3. The animal procedures that were used have been approved by The University of Kansas’ Animal Care and Use Committee (AACUC).
Perfusion Procedures The surgical procedures for the in situ rat brain perfusion technique were similar to those described elsewhere5 For perfusion experiments, the left hemisphere of the rat brain was perfused with the perfusion buffer containing test compound at a flow rate of 10 mL/min through the left common carotid artery catheter that was connected to the infusion syringes via a switching valve (Hamilton, Reno, NV). The perfusion buffer (378C, pH 7.4) consisted of bicarbonate-buffered physiological saline (NaH2PO4, KCl, NaHCO3, NaCl, CaCl2, MgSO4, and D-glucose) and was oxygenated with a mixture air of 95% O2 and 5% CO2 before perfusion. The perfusion was started immediately after the rat heart was cut open. The perfusion protocol, which was controlled by the switching valve, consisted of a 20-s preperfusion wash (saline only or saline with inhibitor), a 240-s perfusion (saline containing test compound or saline containing test compound and inhibitor), and a 5-s postperfusion wash (saline only or saline with inhibitor). The perfusion was terminated by decapitation of the animal. The perfused rat brain was removed from the skull and dissected on ice.
Preparation of In Situ Perfused Rat Brain Samples When perfusion studies were conducted using [3H]-quinidine (1 mCi/mL), brain tissue samples collected from left cortex were weighed and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
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digested in Sovable (Packard Bioscience, Meriden, CT) at 378C for 24 h. When perfusion studies were conducted using DADLE and its prodrugs (20 mM), brain tissue samples were collected from the left frontal, parietal, and occipital cortex as well as the hippocampus and weighed. Internal standards were added to the collected brain tissues before further preparation. The internal standard used for quantification of DADLE was [Leu5]-enkephalin and those for cyclic prodrugs of DADLE were the cyclic [Leu5]-enkephalin containing the same linkers as their DADLE counterparts. Brain tissues were then homogenized (30 strokes) in ice-cold saline (1:4, w/v) using a glass homogenizer (Wheaton, Philadelphia, PA). A capillary depletion method16 was used to remove the drugs that were bound to the brain vasculature. After centrifugation of the homogenate at 4500g (model 59A Micro-Centrifuge; Fisher Scientific, Pittsburgh, PA) at 48C for 15 min, the pellet was discarded and the supernatant containing both the lipid and the aqueous phases was collected and stored at 708C until further extraction. For sample extraction, brain samples were mixed with acetonitrile (1:3, v/v) and centrifuged at 21,000g for 15 min. The supernatant after centrifugation was evaporated to dryness using a Centrivap concentrator (Labconco, Kansas City, MO). The final residue was dissolved in 0.1 mL of 10% acetonitrile and centrifuged again at 10,000g and 48C for 10 min. Thirty microliters of the supernatant was injected for high-performance liquid chromatography analysis with tandem mass spectrometric detection (LC/MS/MS).
Analysis of In Situ Perfused Rat Brain Samples [3H]-Quinidine brain perfusion samples were counted for radioactivity using a dual-label scintillation spectrometer (LS 6000 IC; Beckman Coulter, Inc., Fullerton, CA). To determine the original radioactivity level in the perfusate, duplicate perfusate samples (50 mL) were also collected, mixed with Sovable, and counted. To determine the concentrations of DADLE and its cyclic prodrugs in the perfusates, HPLC with UV detection was performed using an LC-10A gradient system (Shimadzu, Tokyo, Japan) consisting of two LC-10AS pumps, an SCL-10A system controller, and an SIL-10A auto injector with a sample cooler. Sample separation was performed using a C18 reversed-phase column JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
˚ , 250 mm 4.6 mm i.d.; Vydac, Hesperia, (300 A CA) equipped with a C18 guard column (Vydac). Gradient elution was performed at a flow rate of 1 mL/min from 26% to 58% (v/v) acetonitrile in water with 0.1% (v/v) trifluoroacetic acid. The eluents were detected by UV detection (l ¼ 214 nm). The chromatographic data were acquired and analyzed using CLASS-VP version 4.2 Chromatography Data System (Shimadzu). To determine the contents of DADLE and its prodrugs in perfused brain tissue samples, LC/MS/MS was performed using a Micromass Quattro Micro triple quadrupole mass spectrometer (Micromass, Beverly, MA). The analytical method was described extensively elsewhere.17 Briefly, the liquid chromatography was conducted using a 2690 HPLC System (Waters, Milford, MA). A reversed-phase C18 column (50 mm 1.0 mm i.d., 5 mm; Vydac) was used as the analytical column and the column temperature was kept at 258C. Two mobile phases were used to generate a linear gradient to allow simultaneous analyses of DADLE and its prodrugs in a single run. Mobile phase A was water with 0.1% (v/v) formic acid, and mobile phase B was acetonitrile with 0.1% (v/v) formic acid. The HPLC system was interfaced to the mass spectrometer via an electrospray interface. For sample recording, multiple reaction monitoring of several channels was used with 0.1 s dwell time and interchannel delay set at 0.03 s so that several compounds could be monitored simultaneously. Data acquisition and analysis were performed using MassLynx version 3.4 software (Micromass).
Calculations of Papp Values from In Situ Rat Brain Perfusion Experiments Brain uptake of a test drug was analyzed using a simple, linear two-compartment model (the vascular blood and the brain parenchyma). The unidirectional transfer coefficient Kin (mL/s/g) from blood to brain was estimated using the following equations: Kin ¼
Qbr =Cpf Vvasc t
(1)
where Qbr is the amount of drug in the brain parenchyma (ng/g), Cpf is the drug concentration in the perfusion fluid (ng/mL), Qbr/Cpf is defined as the apparent brain distribution volume (Vbr, mL/ g), t is the net perfusion time (s), and Vvasc is the brain intravascular volume. A capillary depletion DOI 10.1002/jps
EFFECTS OF P-GP INHIBITORS ON BBB PERMEATION
method was then used for sample preparation to minimize the residual intravascular tracer remaining in the brain parenchyma. By using the postperfusion wash and the capillary depletion step, Vvasc could be excluded from Eq. (1). The Kin was determined as the slope from the linear regression of the measured Vbr values at multiple time points. Eq. (2) was used for single time point analysis, in which the perfusions were conducted for a single time period. The Kin was determined by dividing the Vbr value by perfusion time. The Kin values were converted to PA values using the Crone–Renkin model of capillary transfer:5 Kin PA ¼ F ln 1 (2) F where F is the regional cerebral perfusion fluid flow, which was determined using [3H]-diazepam in separate experiments (0.025 mL/s/g);5 P is the apparent permeability coefficient of the BBB for a testing compound; and A is the rat brain capillary surface area (130 cm2/g), which was reported previously.18 P was calculated by dividing PA by A. All data are presented as mean SD for at least three rats. A Student’s t-test was used to compare individual means.
MDCK-MDR1 Cell Culture Experiments Cell Culture Conditions MDCK cells, which were transfected with human MDR1 gene (MDCK-MDR1), were obtained as a gift from Drs. Raymond Evers and Piet Borst (The Netherlands Cancer Institute, Amsterdam, The Netherlands). As described previously,6,8 cells were grown in a controlled atmosphere of 5% CO2 and 90% relative humidity at 378C in 150 cm2 culture flask using a culture medium consisting of DMEM supplemented with 10% heat-inactivated FBS, 1% nonessential amino acids, 100 mg/mL streptomycin, 100 U/mL penicillin, and 1% L-glutamine. When approximately 80% confluence (i.e., 3–5 days) was reached, cells were detached from the plastic support by partial digestion using trypsin/EDTA solution and either subcultured in new flasks or plated on collagencoated polyester membranes (Transwell1, 0.4 mm pore size, 24.5 mm diameter) at a density of 7.9 104 cells/cm2. Cells were fed with culture medium every other day for 6 days until transport experiments were performed apical (AP) volume 1.5 mL; basolateral (BL) volume 2.6 mL]. DOI 10.1002/jps
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Transport Experiments Cell monolayers were grown on collagen-coated polyester filters (Transwell1). All studies were performed in triplicate in a shaking water bath at 378C. The integrity of each batch of cells was tested by measuring [14C]-mannitol flux in representative monolayers. Cell monolayers were washed three times with prewarmed HBSS, pH 7.4, and incubated with blank HBSS or with HBSS in the presence of various concentrations of inhibitor (GF-120918, CyA, or PSC-833) for 20 min. The [3H]-quinidine in HBSS with various concentrations of P-gp inhibitor was applied to the donor compartment (AP: 1.5 mL). Blank HBSS was added to the receiver compartment (BL: 2.5 mL) and samples were taken at intervals up to 90 min (donor: 10 mL; receiver: 100 mL). Samples were then counted for radioactivity using a duallabel scintillation spectrometer (LS 6000 IC; Beckman Coulter, Inc.). Permeability coefficients ( Papp) of the compounds were calculated by: Papp ¼
DQ=Dt A C0
(3)
where DQ/Dt is the linear appearance rate of mass in the receiver solution, A is the cross-section area (4.71 cm2), and C0 is the initial concentration of the donor side at t ¼ 0. The concentrations of the P-gp inhibitor (GF120918, CyA, or PSC-833) and the corresponding permeability coefficients of quinidine were fitted into a sigmoidal dose–response curve with variable slope, and Papp max and IC50 values were calculated using GraphPad Prism1 3.0.
RESULTS Permeation Characteristics of Quinidine, a Known P-gp Substrate, in the In Situ Perfused Rat Brain and MDCK-MDR1 Cell Culture Models of the BBB [3H]-Quinidine, a known P-gp substrate,19 was used as a marker to monitor the functional P-gp activity in the in situ perfused rat brain and MDCK-MDR1 cell culture models of the BBB. GF120918, CyA, or PSC-833, all known inhibitors of P-gp, were co-administered with [3H]-quinidine in the above-mentioned models of the BBB, and the Papp values of this marker compound were calculated. The concentrations of the P-gp inhibitors and the Papp values of [3H]-quinidine were then plotted as dose–response curves (data not shown) and the maximum intrinsic permeability JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
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values ( Papp max) of [3H]-quinidine and the IC50 values of the P-gp inhibitors were calculated by curve fitting. In this study, the maximum concentrations of CyA and PSC-833 were limited to 10 mM by their solubility in the experimental buffer. For GF-120918, a concentration of 10 mM, determined using the in situ perfused brain model, has been shown by Chen et al.5 to completely inhibit the P-gp activity in the BBB. The Papp max values of quinidine in the absence and presence of GF-120918, CyA, and PSC-833 and the IC50 values of these P-gp inhibitors determined using the in situ perfused rat brain and MDCK-MDR1 cell culture models are summarized in Table 1. The Papp values of quinidine generated in the in situ perfused rat brain model increased from 15 107 cm/s (without a P-gp inhibitor) to 240 107 cm/s ( Papp max) in the presence of the P-gp inhibitors. Similarly, the Papp values of quinidine generated in the MDCKMDR1 cell culture model increased from 18 107 to 120 107 cm/s ( Papp max) in the presence of the P-gp inhibitors. The Papp max values of quinidine in the presence of the three Pgp inhibitors were significantly higher in the in situ perfused rat brain model than the corresponding Papp max value in the MDCKMDR1 cell culture model ( p < 0.05). The in situ perfused rat brain model afforded an IC50 value for GF-120918 that was significantly lower than the IC50 values observed for CyA ( p < 0.05) and PSC 833 ( p < 0.1). These results lead to a potency order of GF-120918 > CyA ¼ PSC-833. In contrast, there were no significant differences among the IC50 values of the three P-gp inhibitors in the MDCK-MDR1 cell culture model, leading to a potency order of GF-120918 ¼ CyA ¼ PSC-833.
Permeation Characteristics of DADLE, AOADADLE, CA-DADLE, and OMCA-DADLE Across MDCK-MDR1 Cells The permeability ( Papp) of AOA-DADLE, CADADLE, and OMCA-DADLE across MDCKMDR1 cell monolayers was determined in the presence and absence of 10 mM of P-gp inhibitor (GF-120918, CyA, or PSC-833). In the absence of P-gp inhibitor, three cyclic peptide prodrugs exhibited high efflux ratio (16.8 for AOA-DADLE, >35 for CA-DADLE and OMCA-DADLE, respectively) across MDCK-MDR1 cell monolayers, indicating that P-gp limits the permeation of these prodrugs. In the presence of 10 mM of P-gp inhibitor, the efflux ratios of all cyclic peptide prodrugs were significantly reduced (<1.18 for AOA-DADLE, <2.11 for CA-DADLE, and <3.91 for OMCA-DADLE, respectively). GF-120918, CyA, and PSC-833 were equally potent in inhibiting the efflux of prodrugs across MDCK-MDR1 cell monolayers. Permabilities are summarized in Table 2. The efflux ratios of AOA-DADLE across MDCK-MDR1 cell monolayers in the presence of GF-120918 and CyA were less than unit value probably due to experimental variations in the determination of Papp values.
Permeation Characteristics of DADLE, AOADADLE, CA-DADLE, and OMCA-DADLE in the In Situ Perfused Rat Brain Model of the BBB In an earlier study,5 our laboratory showed that GF-120918 had no effect on the brain permeation (as measured by the Papp value) of DADLE using the in situ perfused rat brain model of the BBB. However, this P-gp inhibitor significantly
Table 1. The Maximum Apparent Permeability Coefficients ( Papp max) of Quinidine Across In Situ Perfused Rat Brain, MDCK-WT Cells, and MDCK-MDR1 Cells in the Presence of P-gp Inhibitors (GF-120918, CyA, and PSC-833), and the IC50 Values of These P-gp Inhibitors on Reducing Quinidine Efflux Rat Brain Perfusion Papp Control GF-120918 Cyclosporin A PSC-833
max
a
MDCK-WT
(107 cm/s) IC50 (mM) Papp
14.46 203.47 241.10 254.73
(3.77) (15.28) (26.47) (66.75)
— 0.41 (0.06) 1.72 (0.34)b 1.53 (0.55)b
max
MDCK-MDR1
(107 cm/s) IC50 (mM) Papp
60.74 133.17 109.17 114.97
(2.72) (32.53) (15.70) (7.34)
— 2.49 (2.89) 0.38 (0.62) 0.35 (0.33)
max
(107 cm/s) IC50 (mM)
18.05 115.15 126.00 121.23
(0.52) (17.76) (30.01) (15.22)
— 0.15 (0.079) 0.17 (0.09) 0.58 (0.37)
Results were presented as mean SD of triplicate determinations. The concentrations of P-gp inhibitor (GF-120918, CyA, or PSC-833) and the corresponding permeability coefficients of quinidine were fitted into a sigmoidal dose–response curve with variable slope, and both Papp max and IC50 were calculated using GraphPad Prism1 3.0. a Papp max is also the intrinsic permeability of tested compound (e.g., quinidine). b The IC50 value is statistically different from the IC50 value of GF-120918 ( p < 0.1) in rat brain perfusion model. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
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Table 2. Papp Values of AOA-DADAE, CA-DADLE and OMCA-DADLE Across MDCK-MDR1 Cell Monolayer in the Presence and Absence of Known P-gp Inhibitors (10 mM) Papp (106 cm/s) Substrate AOA-DADLE
CA-DADLE
OMCA-DADLE
a
Papp
AP-to-BL
Inhibitor
AP-to-BL
BL-to-AP
— GF120918 CyA PSC-833 — GF120918 CyA PSC-833 — GF120918 CyA PSC-833
0.095a 10.7 1.5 20.4 9.7 6.5 2.6 0.011a 13.5 2.5 17.3 2.0 2.3 0.1 0.013a 11.3 0.63 82.0 2.3 2.3 0.1
1.6 0.23 5.3 1.5 11.7 1.3 7.6 3.9 43.0 5.0 13.8 1.0 15.5 3.5 9.0 0.7 21.8 3.0 16.7 0.84 173 113 9.0 0.7
>16.8 0.50 0.58 1.18 >35 1.02 0.90 3.91 >35 1.48 2.11 3.91
calculated using lowest detectable limits.
increased the Papp values of AOA-DADLE, CADADLE, and OMCA-DADLE. To compare the enhancing effects of GF-120918 to CyA and PSC833 on the brain permeation of these cyclic prodrugs, similar experiments were conducted with CyA and PSC-833, and the resulting data are shown in Table 3. The experimental protocols (e.g., P-gp inhibitor concentration ¼ 10 mM; single pass perfusion for 240 s) used to generate the new data for CyA and PSC-833 shown in Table 3 were identical to the protocols used by Chen et al.5 to determine the effects of GF-120918. Table 3 summarizes the published5 and newly generated Papp ¼ values for DADLE, AOADADLE, CA-DADLE, and OMCA-DADLE in the absence and presence of GF-120918, CyA, or PSC833. In the absence of a P-gp inhibitor (w/o inhibitor), the Papp values observed for both DADLE and its prodrugs were very low (between 0.4 107 and 1.2 107 cm/s). Inclusion of GF120918, CyA, or PSC-833 did not significantly change DADLE’s Papp value ( Papp values varied between 0.5 107 and 0.9 107 cm/s). However, inclusion of 10 mM of GF-120918, CyA, or PSC-833 significantly increased the permeation of all three cyclic prodrugs. Increases in the Papp values of AOA-DADLE, CA-DADLE, and OMCA-DADLE were as follows: GF-120918, 50-, 460-, and 170fold, respectively; CyA, 10-, 50-, and 40-fold, respectively; and PSC-833, 7-, 50-, and 60-fold, respectively. The Papp values observed for the three cyclic prodrugs in the presence of the P-gp inhibitors were statistically different from the DOI 10.1002/jps
Ratio ( Papp BL-to-AP/ Papp AP-to-BL)
Papp values in the absence of the P-gp inhibitors (GF-120918, p < 0.05; CyA, p < 0.05; PSC-833, p < 0.1). Based on these data, the order of potency for the ability of the P-gp inhibitors to enhance the BBB permeation of these cyclic prodrugs would be as follows: GF-120918 > CyA ¼ PSC-833.
DISCUSSION Our laboratory4,6,20,21 has used Caco-2 cells in an attempt to predict the in situ and in vivo permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE across the intestinal mucosa. The resulting Caco-2 cell data showed that all three cyclic prodrugs of DADLE exhibit polarized efflux that can be inhibited by P-gp inhibitors.11 These data suggest that the in vivo and in situ intestinal mucosal permeation of these cyclic prodrugs would be very poor because of their substrate activity for P-gp, which is known to be expressed in intestinal mucosal cells. Recently, our laboratory confirmed this hypothesis by determining the intestinal mucosal permeation of these cyclic prodrugs using an in situ perfused ileum model.11 Ouyang et al.11 also attempted to use Caco-2 cell data to rank order P-gp inhibitors for their abilities to inhibit the polarized efflux of AOADADLE, CA-DADLE, and OMCA-DADLE. The results of these experiments showed that 10 mM concentrations of GF-120918, CyA, or PSC-833 almost completely inhibited the polarized efflux of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
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Table 3. The Effect of P-gp-Inhibitors (GF-120918, CyA, and PSC-833) on the Permeability of DADLE and its Cyclic Prodrugs (AOA-DADLE, CA-DADLE, and OMCA-DADLE) Across the In Situ Perfused Rat Brain Model Papp (107 cm/s) Compounds DADLE AOA-DADLE CA-DADLE OMCA-DADLE
w/o Inhibitor 0.5 1.2 0.4 0.7
(1.4) (1.0) (0.7) (0.6)
GF120918 0.6 60.5 185 119
(0.1) (25.6)a (68.3)a (12.8)a
Cyclosporin A 0.9 11.8 20.0 31.0
(0.2) (2.9)b (5.6)b (11.3)b
PSC833 0.6 8.7 21.9 41.0
(0.2) (3.4)c (9.4)c (18.4)c
Rat brain was perfused with buffer containing 10 mM of P-gp inhibitor and 20 mM of DADLE or its prodrugs for 4 min. Brain samples were collected and processed by a capillary depletion method. After liquid phase exaction with acetonitrile, samples were analyzed by LC/MS/MS. The Papp values were calculated using single time point analysis and presented as mean SD (n 3) and the statistical significance of the data points were evaluated using paired Student’s t-tests. a The data point showed statistical difference ( p < 0.05) from control value (in the absence of inhibitor) and statistical difference ( p < 0.1) from the Papp value with the presence of CyA or PSC-833. b The data point showed statistical difference ( p < 0.05) from control value (in the absence of inhibitor). c The data point showed statistical difference ( p < 0.1) from control value (in the absence of inhibitor).
these cyclic prodrugs in Caco-2 cell monolayers. The maximum concentrations of CyA and PSC-833 were limited to 10 mM by their solubility in the experimental buffer. When these P-gp inhibitors were actually tested at 10 mM concentrations for their abilities to enhance the intestinal mucosal absorption of the cyclic prodrugs using the in situ perfused ileum model, surprising and unexpected results were observed. For example, PSC-833 was found to increase significantly the intestinal permeation of all three prodrugs, whereas CyA and GF-12918 were either inactive or substantially less active than PSC-833.11 Based on these results, the order of potency of these P-gp inhibitors in the in situ perfused ileum model is PSC-833 CyA ¼ GF-120913. Thus, the data generated in Caco-2 cell model did not correctly rank order these P-gp inhibitors for their abilities to enhance the intestinal mucosal permeation of the cyclic prodrugs. After additional experiments were conducted to eliminate some possible explanations (e.g., metabolism) for these differences, we concluded that that AOA-DADLE, CA-DADLE, and OMCA-DADLE were substrates for multiple efflux transporters (e.g., P-gp, MRP2, BCRP) in the rat intestinal mucosa and that these transporters were differentially inhibited by PSC-833, CyA, and GF-120918, with PSC-833 being the most potent inhibitor of the efflux transporter(s) that controls the intestinal permeation of these cyclic prodrugs. In contrast, in Caco-2 cells, these cyclic prodrugs and these P-gp inhibitors interact with only one efflux transporter (i.e., P-gp). Our laboratory6–8 has also shown that: (i) AOADADLE, CA-DADLE, and OMCA-DADLE undergo significant polarized efflux in MDCK-MDR1 cells, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 6, JUNE 2009
a model of the BBB; and (ii) this polarized efflux can be inhibited by GF-120918 and CyA. These data suggest that the BBB permeation of these cyclic prodrugs when measured in vivo or in situ would also be very low because of their substrate activities for P-gp since this efflux transporter is known to be expressed in the brain microvessel endothelial cells that make up the BBB. This hypothesis was confirmed by conducting in vivo pharmacokinetic experiments9 and in situ perfused rat brain experiments.5 The in vivo pharmacokinetic experiments9 showed that after IV administration of these cyclic prodrugs negligible amounts of the prodrugs and DADLE were found in brain tissue. Similarly, the in situ perfused rat brain experiments5 showed that perfusion of medium that contained a cyclic prodrug into the brain vascular system afforded negligible amounts of the prodrug and DADLE in brain tissue. However, if a P-gp inhibitor were added to the perfusion medium, the amounts of the prodrug and DADLE in brain tissue increased significantly (e.g., CA-DADLE/GF-120918, 460fold increase). In the experiments described in this manuscript, we determined whether the MDCK-MDR1 cell culture model of the BBB could predict the ability of P-gp inhibitors (i.e., GF-120918, CyA, PSC-833) to enhance the in situ rat brain permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE. As shown in Table 2, inclusion of GF-120918, CyA, or PSC-833 at concentrations of 10 mM produced nearly complete inhibition of the polarized efflux of these cyclic prodrugs in MDCK-MDR1 cells. The equipotent effects of GF120918, CyA and PSC-833 on inhibiting the DOI 10.1002/jps
EFFECTS OF P-GP INHIBITORS ON BBB PERMEATION
polarized efflux of these cyclic prodrugs in MDCKMDR1 cells are consistent with the effects of these P-gp inhibitors on the efflux of quinidine in this same cell culture system (Tab. 1). As shown in Table 3, when 10 mM concentrations of CyA or PSC-833 were included in the perfusion medium with AOA-DADLE, CADADLE, or OMCA-DADLE, the amounts of the prodrugs and DADLE in brain tissue increased significantly (CyA, 10-, 50-, and 40-fold, respectively; and PSC-833, 7-, 50-, and 60-fold, respectively. When these data for PSC-833 and CyA are compared to the data observed for GF-120918 (GF-120918, 50-, 460-, and 170-fold, respectively; Tab. 3 5), one derives the following rank ordering of potencies: GF-120918 > CyA ¼ PSC-833. The rank ordering of potencies of these P-gp inhibitors observed using the in situ rat brain perfusion model (Tab. 3) is in good agreement with the rank ordering of potencies derived at from MDCKMDR1 cell experiments (Tabs. 1 and 2). This agreement suggests: (i) P-gp in the rat brain microvessel endothelial cells is the major factor restricting BBB permeation of these cyclic prodrugs; and (ii) MDR1 and rat P-gp have similar substrate specificities for these cyclic prodrugs and these P-gp inhibitors. The results described in this manuscript for the effects of GF-120918, CyA and PSC-833 on the BBB permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE are very different than the results observed for these P-gp inhibitors on the intestinal mucosal permeation of these cyclic prodrugs.11 These differences probably reflect the more complex nature of the intestinal mucosal barrier and the fact that P-gp is not the only efflux transporter limiting the oral absorption of these cyclic prodrugs. In conclusion, the data presented in this manuscript support the hypothesis that P-gp is the major factor restricting the BBB permeation of these cyclic prodrugs. In addition, data generated using MDCK-MDR1 cells can not only predict the permeation characteristics of the cyclic prodrugs but this BBB cell culture model can also predict the order of potencies of P-gp inhibitors to enhance the BBB permeation of quinidine and the cyclic prodrugs.
ACKNOWLEDGMENTS The authors thank Dr. Kenneth Brouwer (GlaxoSmithKline, Research Triangle Park, NC) for a DOI 10.1002/jps
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generous gift of GF-120918 and Dr. Stephan Ruetz (Novartis, Basel, Switzerland) for a generous gift of PSC-833. This work was supported by a grant from the United States Public Health Service (DA-09315).
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DOI 10.1002/jps