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

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 HUI OUYANG,1 WEIQING CHEN,1 THOMAS E. ANDERSEN,2 BENTE STEFFANSEN,2 RONALD T. BORCHARDT1 1

Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047

2

University of Copenhagen, Faculty of Pharmaceutical Sciences, Copenhagen, Denmark

Received 21 January 2008; revised 6 March 2008; accepted 30 March 2008 Published online 6 June 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21428

ABSTRACT: The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the intestinal mucosal permeation of cyclic prodrugs (AOA-DADLE, CA-DADLE, and OMCA-DADLE) of the opioid peptide DADLE (H-Tyr-D-Ala-GlyPhe-D-Leu-OH). In the Caco-2 cell model, the high Papp,BL-to-AP/Papp,AP-to-BL ratios of AOA-DADLE, CA-DADLE, and OMCA-DADLE (71–117) were significantly decreased by including known P-gp inhibitors, GF-12098, cyclosporine (CyA), or PSC-833, in the incubation media, suggesting that P-gp is restricting the AP-to-BL permeation of these cyclic prodrugs. In the in situ perfused rat ileum model, AOA-DADLE, CA-DADLE, and OMCA-DADLE were shown to exhibit very low permeation into the mesenteric blood ( PB ¼ 0.40, 0.56 and 0.42  107 cm/s, respectively). PSC-833 was found to increase significantly the PB values for all three prodrugs. In contrast, CyA and GF-12918 were either inactive or substantially less active than PSC-833 in increasing the PB values of these prodrugs. These data suggest that, while P-gp plays a role, other factors (e.g., substrate activity for other efflux transporters and/or for metabolic enzymes) may contribute to restricting the permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE across the rat intestinal mucosa. ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:337– 348, 2009

Keywords: efflux transporter; Caco-2 cell; in situ perfused rat ileum; intestinal mucosa; oral absorption; peptide delivery; prodrugs; P-glycoprotein

Abbreviations used: AOA, acyloxyalkoxy; AP, apical; BBB, blood-brain barrier; BL, basolateral; BCRP, breast cancer resistant protein; CA, coumarinic acid; CyA, cyclosporin A; DADLE, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH; DMEM, Dulbecco’s modified Eagle medium; FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution; MRP2, multidrug resistant associated protein; OMCA, oxymethyl-modified coumarinic acid; PB, mesenteric apparent permeability coefficient; PL, luminal apparent permeability coefficient; Papp,BL-to-AP, BL to AP apparent permeability coefficient; Papp,AP-to-BL, AP to BL apparent 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, 337–348 (2009) ß 2008 Wiley-Liss, Inc. and the American Pharmacists Association

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INTRODUCTION In an attempt to improve the oral bioavailability as well as the blood-brain barrier (BBB) permeation of DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), an analog of [Leu5]-enkaphalin, our laboratory has designed and synthesized cyclic prodrugs of this opioid peptide using various promoiety linkers, that is, an acyloxyalkoxy (AOA) linker, a coumarinic acid (CA) linker, and an oxymethyl-modified coumarinic acid (OMCA) linker (Fig. 1).1–3 The resulting cyclic prodrugs, AOA-DADLE, CADADLE, and OMCA-DADLE, are uncharged, relatively lipophilic, and exist in unique solution structures that favor their permeation across cell membranes by the transcellular pathway.2,4,5 However, in vitro cell culture studies (e.g., Caco-2, MDCK-MDR1, and MDCK-MRP2 cells) and in situ rat brain perfusion studies have shown that the permeation of all three cyclic prodrugs of DADLE is restricted by their substrate activity for efflux transporters in polarized cells.4–9 The polarized efflux of these cyclic prodrugs in Caco-2, MDCK-MDR1, and MDCK-MRP2 cells can be inhibited by GF-120918 and/or cyclosporin A (CyA).4–9 GF-120918 and CyA have also been shown to increase the brain permeation of these prodrugs using an in situ perfused rat brain model.10 GF-120918 is a known inhibitor of p-glycoprotein (P-gp) and breast cancer resistant protein (BCRP) 11,12 and MRP proteins.13 CyA is a known inhibitor of P-gp and multidrug resistant associated protein (MRP2).14 Preliminary in vivo pharmacokinetic studies conducted in our laboratory indicate that AOADADLE, CA-DADLE, and OMCA-DADLE are poorly absorbed after oral administration to rats

Figure 1. Chemical structures of DADLE and its cyclic prodrugs (AOA-DADLE, CA-DADLE, and OMCA-DADLE). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 1, JANUARY 2009

(unpublished data). Since efflux transporters (e.g., P-gp, BCRP, MRP2), which are expressed on the apical membrane of the intestinal epithelial cells, are known to contribute to the poor oral absorption of some therapeutic agents,15,16 the substrate activity for one or more of these efflux proteins could be contributing to the poor oral absorption of AOA-DADLE, CA-DADLE, and OMCA-DADLE. In an attempt to determine the role of efflux transporters in the poor oral absorption of AOADADLE, CA-DADLE, and OMCA-DADLE, we describe here the results of permeation experiments conducted using an in situ perfused rat ileum model. These experiments were designed to characterize the intestinal mucosal permeation of these cyclic prodrugs in the absence and presence of inhibitors (GF-120918, CyA, and PSC-833) of those efflux transporters, which are known to be expressed in the intestinal mucosa.17 Permeation experiments were also conducted using quinidine, a known P-gp substrate,18 as a molecular probe to evaluate the functional activity of this efflux transporter in this model of the intestinal mucosa. The permeation characteristics of AOADADLE, CA-DADLE, OMCA-DADLE, and quinidine across Caco-2 cell monolayers were also determined in an attempt to assess whether this cell culture model would have predicted the effects of efflux transporters and inhibitors of these efflux transporters on the intestinal mucosal permeation of these compounds.

MATERIALS AND METHODS Dulbecco’s phosphate buffered saline, Hank’s balanced salts (HBSS) (modified), DADLE, [Leu5]-enkephalin, [14C]-antipyrine (5.4 mCi/ mmol), and CyA were purchased from Sigma– Aldrich (St. Louis, MO). L-glutamine [200 mM (100)], penicillin (10000 U/mL), streptomycin (10000 (g/mol), and nonessential amino acids [10 mM (100) in 85% saline] were obtained from Gibco BRL, Life Technologies (Grand Island, NY). Dulbecco’s modified Eagle 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 (2.07 Gbq/mmol) was purchased from Moravek DOI 10.1002/jps

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Biochemicals (Brea, CA). [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, Switzerland). All other chemicals were high grade and were purchased from Aldrich Chemical Co. (Milwaukee, WI) or Acros Organics distributed by Fisher Scientific (Houston, TX). AOA-DADLE, CA-DADLE, OMCA-DADLE, and [Leu5]-enkephalin were synthesized in our laboratory following procedures described previously.1–3,19

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conco, Kansas City, MO). The final residues were dissolved in 0.1 mL of acetonitrile/ water (1:1) and subjected to filtration through Millipore Ultrafree-MC centrifugal filters at 7000g centrifugation for up to 3 h. Thirty microliters of the filtrates were injected for LCMS/MS analysis. When perfusion studies were conducted using radioactive compounds (e.g., [3H]-quinidine, [14C]antipyrine, or [14C]-mannitol), the same perfusion procedures as described above were followed. The blood samples were centrifuged at 1800g for 5 min. These plasma and perfusate samples were counted for radioactivity using a dual-label scintillation spectrometer (LS 6000 IC; Beckman Coulter, Inc., Fullerton, CA).

In Situ Perfused Rat Ileum Procedures 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). Surgical Procedures The surgical procedures used to prepare the perfused rat ileum are similar to those described elsewhere with slight modifications.20 After surgery, the perfusate (57.9 mM NaH2PO4, 79.5 mM Na2SO4, pH 6.5, 378C), which contained test compound and/or a P-gp inhibitor (e.g., GF120918, CyA, or PSC-833) was infused at a flow rate of 0.2 mL/min into the ileum segment and collected from the outlet cannula after a single pass perfusion at 5-min intervals from 30 to 60 min. The mesenteric blood and perfusate were collected simultaneously. Blood samples collected from the mesenteric vein were centrifuged at 1800g for 5 min to provide the plasma fractions before the internal standards were added. The internal standard used for quantification of DADLE was [Leu5]-enkephalin and those for cyclic prodrugs of DADLE were the cyclic [Leu5]enkephalins containing the same linkers as their DADLE counterparts. The plasma samples were then mixed with acetonitrile (1:3, v/v) and centrifuged at 21000g for 15 min. After centrifugation, the supernatants were evaporated to dryness using a Centrivap Concentrator (LabDOI 10.1002/jps

Analysis of In Situ Perfused Rat Ileum Samples To determine the concentrations of DADLE and its cyclic prodrugs in the perfusates, HPLC analysis with UV detection was performed using an LC-10A gradient system (Shimadzu, Tokyo, Japan) consisting of two LC-10AS pumps, an SCL10A system controller, and a SIL-10A autoinjector with a sample cooler. Sample separation was performed using a C18 reversed-phase column ˚ , 250  4.6-mm i.d.; Vydac, Hesperia, CA) (300 A 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 quantities of DADLE and its prodrugs in plasma samples, LC/MS/MS analysis was performed using a Micromass Quattro Micro triple quadrupole mass spectrometer (Micromass, Beverly, MA). The liquid chromatography was conducted using a 2690 HPLC System (Waters, Milford, MA) with a reversed-phase C18 column (50  1.0-mm i.d., 5 mm; Vydac). 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 reactions 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 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 1, JANUARY 2009

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performed using MassLynx version 3.4 software (Micromass).

Calculations of PL and PB Values from In Situ Rat Ileum Perfusion Experiments At steady state, the luminal apparent permeability coefficients ( PL) of the cyclic prodrugs were determined based on the disappearance of the compounds from the luminal perfusate. The PL value was calculated using the following equation: PL ¼

Q C0  ln 2prl Cl

where Q is the fluid flow rate (mL/s); r is the luminal radius of the intestine; l is the length of the perfused ileum segment; and C0 and Cl are the drug concentrations of the perfusates at the inlet and the outlet of the perfused ileum segment, respectively. The outlet concentrations were corrected for water absorption monitored using nonabsorbable [14C]-PEG 4000 as described elsewhere.21 The apparent mesenteric permeability coefficient on the blood side ( PB) was determined based on the appearance rate of the compound in the blood. The PB value was calculated using the following equation: PB ¼

ðDMB =DtÞ ð2prl < C >Þ

where MB was the cumulative amount of drugs in the blood with time t and was the logarithmic mean concentration of the drug within the segment, which was calculated using the equation: < C >¼

C0 ½1  ðCl =C0 Þ lnðC0 =Cl Þ

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 sub-cultured in new flasks or plated on collagen-coated polyester membranes (Transwell1, 3 mm pore size, 24.5 mm diameter) at a density of 8.0  104 cells/cm2. Cells were fed with culture medium every other day for 21 days until transport experiments were performed [apical (AP) volume ¼ 1.5 mL; basolateral (BL) volume ¼ 2 mL]. Transport Experiments Caco-2 cell monolayers were grown on collagencoated polyester filters (Transwell1). All studies were performed in triplicate in a shaking water bath at 378C. The integrity of the cell monolayers was determined by periodically measuring the flux of the paracellular marker, [14C]-mannitol. Cell monolayers were washed three times with prewarmed HBSS, pH 7.4 and incubated with blank HBSS or with HBSS containing the test compound in the absence or presence of various concentrations of inhibitors of efflux transporters (e.g., GF-120918, CyA, or PSC-833) for 20 min. For example, [3H]-quinidine in HBSS with various concentrations of P-gp inhibitors was applied to the donor compartment (AP: 1.5 mL). Blank HBSS with or without P-gp inhibitors 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 dual-label scintillation spectrometer (LS 6000 IC; Beckman Coulter, Inc.). Permeability coefficients ( Papp) of the compounds were calculated by: Papp ¼

Caco-2 Cell Culture Experiments Cell Culture Conditions Caco-2 cells were obtained from American Type Culture Collection (Rockville, MD). As described previously,7 cells were grown in a controlled atmosphere of 5% CO2 and 95% relative humidity at 378C in 150 cm2 culture flask using a culture medium consisting of DMEM supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids, 100 mg/mL streptomycin, 100 U/mL penicillin, and 1% L-glutamine. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 1, JANUARY 2009

ðDQ=DtÞ ðD  C0 Þ

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 min.

Determination of IC50 Values of GF-120918, CyA, and PSC-833 in Inhibiting the Efflux of Quinidine [3H]-Quinidine, a known P-gp substrate,18 was used as a marker to monitor the functional P-gp DOI 10.1002/jps

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activity in rat ileum and Caco-2 cells. GF-120918, CyA, or PSC-833, known inhibitors of P-gp, were coadministered with [3H]-quinidine following above-described procedures in Caco-2 cell and perfused rat ileum models. The permeability coefficients of this marker compound ( PL and PB values in the in situ perfused rat ileum model; Papp values in Caco-2 cell model) were determined. The concentrations of the GF-120918, CyA, or PSC833 were from 1 nmol to 10 mM. In this study, the maximum concentrations of GF-120918, CyA, and PSC-833 were limited to10 mM by their insolubility in aqueous media. The PB values (or Papp values) of [3H]-quinidine were then plotted as dose-response curves. The maximum intrinsic permeability values ( Papp,max) of [3H]-quinidine and the IC50values of the P-gp inhibitors were calculated by curve fitting using GraphPad Prism software (San Diego, CA).

RESULTS Intestinal Mucosal Models Used for Permeation Studies Two models of the intestinal mucosa were employed in the present studies, that is, an in vitro cell culture model consisting of Caco-2 cell monolayers grown onto microporous membranes22 and a well-validated model of the intestinal mucosa. Both models permit determination of passive diffusion of solutes as well as transporter mediated (e.g., efflux transporters) difussion of solutes.23 Earlier studies8,9 have shown that our stock of Caco-2 cells express reasonable levels of P-gp, very low levels of MRP2 and undetectable levels of BCRP. These results are qualitatively consistent with those reported by Taipalensuu et al.17 The functional activity of the human P-gp (MDR1) in our stock of Caco-2 cells and the effects of inhibitors on the activity of this efflux transporter were characterized more completely in this study and the results are described below. The second model system that was employed in our studies was the in situ perfused rat ileum, which has been utilized extensively by other investigators to study intestinal mucosal drug transport.20,24 Prior to conducting permeation experiments using DADLE and its cyclic prodrugs, we validated this model by determining the permeation of two markers: [14C]-antipyrine as an intestinal transcellular permeable marker to DOI 10.1002/jps

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Figure 2. The appearance of [14C]-antipyrine and [14C]-mannitol in the mesenteric blood during perfusion of these solutes through a segment of the rat ileum. The length of in situ perfused rat ileum used in these studies was 15 cm and the inner perimeter was 1.2 cm. Blood samples were collected at 5-min intervals for 30 min after reaching steady-state. After removal of the red blood cells by centrifugation, the plasma samples were analyzed using a scintillation counter as described in the Materials and Methods Section.

calibrate the effective permeability of the tested segment of the perfused rat ileum; and [14C]mannitol as an intestinal transcellular impermeable marker to determine the integrity of the perfused rat ileum.25 As shown in Figure 2, both antipyrine and mannitol exhibited linear intestinal permeation during a 60 min perfusion. The mesenteric permeability coefficients ( PB) of antipyrine and mannitol were 4.26 0.6  105 and 5.43 1.5  107 cm/s, respectively. These values were repeatable throughout the course of our studies. The low permeation of mannitol is indicative of only paracellular diffusional transport across an intact mucosal barrier. The functional activity of P-gp and the effects of inhibitors on the activity of this efflux transporter in the rat perfused ileum model were more completely characterized in this study, and the results are described below.

Permeation Characteristics of Quinidine, a Known P-gp Substrate As shown in Table 1, GF-120918 and PSC-833 significantly increased (13.4- and 15-fold, respectively) ( p < 0.05) the [3H]-quinidine permeation in the in situ perfused rat ileum model. CyA was less effective in increasing (3.7-fold) the [3H]-quinidine permeation in this model. In the Caco-2 cell model, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 1, JANUARY 2009

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Table 1. The Maximum Apparent Permeability Coefficients ( Papp,max) of Quinidine Across Rat Intestinal Mucosa and Caco-2 Cell Monolayers in the Presence of P-gp Inhibitors (GF120918, Cyclosporin A, and PSC833), and the IC50 Values of These P-gp Inhibitors in Reducing Quinidine Efflux

In Situ Perfused Rat Ileum Papp,max (107 cm/s)a Control GF120918 Cyclosporin A PSC833

0.73 9.81 2.69 10.94

(0.27) (2.62) (1.05) (0.57)

Caco-2 Cell Monolayers

IC50 (mM) — 1.03 (0.73) 3.59 (1.72) 0.49 (0.15)

Papp,max (107 cm/s) 20.22 91.06 116.23 76.25

IC50 (mM)

(3.22) (8.31) (5.69) (10.98)

— 0.25 (0.13) 2.97 (1.16) 0.072 (0.069)

Results were presented as average (SD) of triplicate determinations.

The concentrations of P-gp inhibitor (GF120918, cyclosporin A, or PSC833) 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 quinidine.

Permeation Characteristics of AOA-DADLE, CA-DADLE, and OMCA-DADLE as Determined in the Caco-2 cell Monolayer Model

all three P-gp inhibitors significantly increased the [3H]-quinidine permeation (GF-120918, 4.5fold; CyA, 5.7-fold; PSC-833, 3.77-fold) ( p < 0.05). PSC-833 was found to be the most potent of the three P-gp inhibitors tested (in situ perfused rat ileum, IC50 ¼ 0.49 0.15 mM; Caco-2 cell model, IC50 ¼ 0.072 0.069 mM). In both models of the intestinal mucosa, the order of potency of Pgp inhibition was: PSC-833 > GF-120918 > CyA. Finally, the following observations are noteworthy: the Papp,max value of [3H]-quinidine as measured in the perfused rat ileum was significantly lower (27.7-fold) than the Papp,max value as measured in Caco-2 cell monolayers (Tab. 1); and GF-120918 and PSC-833 were much more potent inhibitors of P-gp than CyA, as measured by their IC50 values in Caco-2 cells versus the in situ perfused rat ileum model (Tab. 1).

In studies published earlier by our laboratory,7–9 AOA-DADLE, CA-DADLE, and OMCA-DADLE were shown to undergo polarized efflux in Caco-2 cell monolayers and that this polarized efflux could be inhibited by addition of CyA and/or GF-120918. Because a third P-gp inhibitor, PSC833, is employed in the in situ perfused rat ileum studies described below, its inhibitory effects on the polarized efflux of these prodrugs in Caco-2 cells were determined. PSC-833s inhibitory effects were then compared to those of CyA and GF-120918. Consistent with our earlier data,7–9 AOA-DADLE, CA-DADLE, and OMCA-DADLE were shown in this study to undergo extensive

Table 2. The Apparent Permeability Coefficients ( Papp) of the Cyclic Prodrugs Across Caco-2 Cell Monolayers in the Absence and Presence of 10 mM of P-gp Inhibitors (GF120918, Cyclosporin A, and PSC833) Papp (107cm/s) Compound AOA-DADLE

CA-DADLE

OMCA-DADLE

Inhibitor

A–B

None GF120918 CysA PSC833 None GF120918 CysA PSC833 None GF120918 CysA PSC833

0.46 (0.08) 2.17 (0.49) 1.70 (0.32) 2.08 (0.69) 1.81 (0.22) 6.02 (1.71) 4.64 (1.19) 5.09 (0.88) <0.10 1.64 (0.41) 2.10 (0.68) 5.09 (1.84)

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B–A 45.59 5.08 5.51 3.27 129.03 21.06 10.52 9.11 116.56 20.23 15.05 12.50

(3.45) (0.24) (0.31) (0.33) (13.8) (1.53) (0.46) (1.71) (13.59) (0.72) (0.57) (0.52)

Ratio 99.0 2.3 3.2 1.6 71.0 3.5 2.3 1.8 >116.6 12.3 7.2 2.5 DOI 10.1002/jps

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polarized efflux in Caco-2 cells in the absence of P-gp inhibitors (Tab. 2). In the presence of the P-gp inhibitors (GF-120918, CyA, or PSC-833), the Papp,AP-to-BL values for these prodrugs increased significantly and the Papp,BL-to-AP values decreased significantly (Tab. 2). As expected these changes in Papp values resulted in significant changes in the BL-to-AP Papp/AP-to-BL Papp ratios for all three prodrugs (Tab. 2). However, it is worth noting that these ratios never reach unity. In addition, irrespective of whether one looks at the effects of the P-gp inhibitors on the Papp,AP-to-BL values, the Papp,BL-to-AP values or the Papp,BL-to-AP/ Papp,AP-to-BL ratios, GF-120918, CyA and PSC833 appear to be equipotent in inhibiting the polarized efflux of AOA-DADLE, CA-DADLE, and OMCA-DADLE in this assay.

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due to the intestinal metabolism and/or nonspecific adsorption of cyclic peptide prodrugs. Since PB values are most relevant to the drug absorption process, we focus on PB values in the following discussion. The PB values of AOADADLE, CA-DADLE, and OMCA-DADLE in the absence of P-gp inhibitors were significantly lower than the PB value of DADLE (4.4- to 6.15-fold) ( p < 0.05). Inclusion of PSC-833 in the perfusion media significantly increased the PB values of all three prodrugs. As shown in Figure 3 and Table 3, PSC 833 increased the PB values of AOA-DADLE, CA-DADLE, and OMCA-DADLE by 7.6-, 13.4-, and 15.7-fold, respectively ( p < 0.05). In contrast, CyA increased only the PB values of OMCADADLE (4.8-fold; p < 0.05) and GF-120918 increased only the PB values of CA-DADLE (6.2-fold; p < 0.1) (Tab. 3, Fig. 3).

Permeation Characteristics of AOA-DADLE, CA-DADLE, and OMCA-DADLE as Determined in the In Situ Perfused Rat Ileum Model

DISCUSSION

The permeation characteristics of DADLE and its cyclic prodrugs (AOA-DADLE, CA-DADLE, and OMCA-DADLE) in the in situ perfused rat ileum were determined by monitoring both the disappearance rate ( PL) of the test compound from the luminal perfusion buffer and the appearance rate ( PB) of the test compound in the mesenteric blood. Samples were collected at 5 min interval from 30 to 60 min of perfusion. The amounts of DADLE and the cyclic prodrugs were added together to determine the permeability coefficients. As shown in Table 3, there are significant differences between PL and PB values probably

Experimental Model Selection and Validation Among the various models of the intestinal mucosa, the in situ perfused rat intestine bridges the gap between in vivo animal models and in vitro cell culture models. The in situ perfused rat intestine provides a biological barrier (e.g., intestinal mucosa) for drug transport studies, which is identical to that found in vivo in rat without the complications of billary secretions and food substances in the lumen of the gut. The in situ perfused rat intestine model has been widely used to confirm experimental data gener-

Table 3. Apparent Luminal ( PL) and Mesenteric Blood ( PB) Permeability Coefficients of DADLE, AOA-DADLE, CA-DADLE, and OMCA-DADLE in the Absence and Presence of GF120918, Cyclosporin A (CyA), and PSC 833

PB (107 cm/s)a

PL (107 cm/s) Compound DADLE AOA-DADLE CA-DADLE OMCA-DADLE

Control 452 71 194 269

(71) (21)b (71)b (46)

GF120918

CyA

— 167 (46) 204 (46) 64 (1)

— 141 (17) 379 (182) 75 (1)

PSC833 551 293 123 288

(11) (77) (15) (121)

Control 2.46 0.40 0.56 0.42

(0.64) (0.17)b (0.25)b (0.10)b

GF120918

CyA

— 0.29 (0.18) 3.46 (1.43) 0.69 (0.35)

— 0.62 (0.27) 0.45 (0.70) 2.03 (0.53)

PSC833 2.72 3.02 7.52 6.61

(2.39) (1.43) (1.38) (1.25)

The rat ileum was perfused with 20 mM of each compound in the absence and presence of 10 mM of P-gp inhibitor (GF120918, cyclosporin A, or PSC 833). The perfusate and the mesenteric blood were sampled after steady-state at 5-min intervals for 30 min. Luminal samples were analyzed using HPLC-UV detection. The blood samples were extracted with acetonitrile and analyzed using LC/MS/MS as described in Materials and Methods Section. The PL values were determined based on the disappearance of the compounds in the luminal perfusate. The PB values were determined based on the appearance rate of the compound in the mesenteric blood. Results are presented as mean ( SEM) with n ¼ 3–8. a In the case of AOA-DADLE, CA-DADLE, and OMCA-DADLE, PB values are based on the sum of prodrug and DADLE present in the blood. b The value is significantly different from the corresponding value of DADLE in the absence of P-gp inhibitors.

DOI 10.1002/jps

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Figure 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 intestinal mucosa using the in situ perfused rat ileum model. A segment of rat ileum was perfused with buffer containing 10 mM of P-gp inhibitor and 20 mM of DADLE or its prodrugs for 60 min. The blood samples taken from the mesenteric vein were extracted with acetonitrile and analyzed using LC/MS/MS as described in the Materials and Methods Section. The PB values were calculated based on the appearance rates of the compound in the mesenteric blood. Results are presented as mean ( SEM) with n ¼ 3–8. The statistical significance of the data points were evaluated using paired Student t tests. ‘‘ ’’ indicates that the difference between the data point and the control Papp value (in the absence of P-gp inhibitor) is significant ( p < 0.1); ‘‘

’’ indicates that the difference between the data point and the control Papp value (in the absence of P-gp inhibitor) is significant ( p < 0.05).

ated using in vitro cell culture models.26,27 Lennernas28 has concluded that the permeability coefficients determined from in situ perfused rat intestine experiments predict very well the absorption of drugs in human. With the proper use of reference compounds, this in situ model is now being used to classify compounds according to the biopharmaceutical classification system (BCS).28 For our studies, the perfused rat ileum was chosen because of the ease of canulating the mesenteric vein that drains this segment of the rat small intestine. The in situ perfused rat ileum model was first validated in our laboratory by determining the permeability characteristics of two markers: antipyrine and mannitol. Our measured mesenteric permeability coefficient for antipyrine ( PB ¼ 4.26 0.6  105 cm/s) was JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 1, JANUARY 2009

found to be threefold higher than the luminal permeability coefficient ( PL ¼ 1.30 0.23  105 cm/s) reported by Fagerholm et al.29 PB value for antipyrine was not reported in this paper. In spite of this difference, the PB value observed for antipyrine in our study is consistent with it being classified as a ‘‘highly permeable solute.’’ It should be noted that differences between the PB and PL values for the same solute are not unexpected since test compounds often ‘‘disappear’’ from the lumen for reasons (e.g., metabolism, adsorption) other than transit across the intestinal mucosa. Such differences in PB and PL values can also be attributed to subtle differences in experimental protocols (e.g., flow rate).24 The mesenteric permeability coefficient for mannitol ( PB ¼ 5.43 1.5  107 cm/s) determined in our study was also slightly lower than the reported literature value ( PB ¼ 11 1  107 cm/s).25 In spite of this difference, our observed PB value for mannitol is consistent with it being classified as a ‘‘low permeability solute.’’ From mannitol’s PB value, one can conclude that the physical integrity of the rat ileum mucosa was not compromised during the perfusion experiments (Fig. 2). Sometimes, investigators who have used this in situ intestinal perfusion model have simply measured the disappearance of solutes from the perfusate29,30 as a first approximation of drug absorption. However, this assumption is not always correct, especially when there is significant metabolism of the solute during the intestinal mucosal absorption process or significant adsorption of the drug in the intestinal mucosa. Therefore, since our prodrugs are prone to metabolism by esterases, as well as oxidative metabolism, we chose to sample both the luminal perfusate as well as the mesenteric blood, which allows the calculation of both luminal permeability coefficients ( PL) and mesenteric permeability coefficients ( PB). Both PL and PB value of DADLE, AOA-DADLE, CA-DADLE, and OMCADADLE are reported in Table 3. As demonstrated in Table 3, there are significant differences between PL and PB values of peptide prodrugs. These differences might arise from the hydrolysis of peptide prodrugs in the intestinal mucosa due to the presence of ester and amide bonds in these molecules. Other factors, such as the hydrodynamics condition (i.e., the stirring state of the model) and the physiological condition (i.e., beating of microvilli, the chemical composition of the mucus layer) could also lead to significant adsorption of peptide in the intestinal DOI 10.1002/jps

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tissues. In this study, we primarily use PB values as the indication of permeability and we did not further investigate the reasons causing the significant differences between PL and PB.

Efflux Transporters Restrict the Intestinal Absorption of Cyclic Prodrugs Numerous efflux transporters are expressed in the small intestinal mucosa. Some of these transporters play key roles in restricting the absorption of xenobiotics into the mesenteric blood flow.15 For example, P-gp and MRP2 are located on the apical side of intestinal epithelial cells and they contribute to the efflux of cationic drugs and anionic drugs, respectively.31–33 Similarly, BCRP has been shown to be localized on the apical side of intestinal epithelial cells and it contributes to limiting the absorption of certain drugs (e.g., topotecan).34,35 Other membraneassociated efflux transporters have been shown to be expressed in intestinal, but their physiological roles have yet to be fully characterized e.g., lung resistance protein (LRP);36 and a transporter associated with antigen processing (TAP)37]. Among the efflux transporters expressed in the intestinal mucosa, P-gp’s role in restricting the oral absorption of drugs has been most extensively characterized.34,38 Therefore, in this study, we focused on the role of P-gp in restricting the intestinal absorption of the cyclic prodrugs and the ability of three P-gp inhibitors (GF120918, CyA, and PSC833) to potentially enhance their oral absorption. As shown in Table 3, the mesenteric permeability coefficients of the cyclic prodrugs were lower ( PB values ¼ 0.40 to 0.52  107 cm/s) than that for DADLE ( PB value ¼ 2.46 0.64  107 cm/s). The PB values for the cyclic prodrugs do not correlate well with their more lipophilic characteristics.4–7 The low PB values observed for the cyclic prodrugs and DADLE in this in situ perfused rat ileum model did correlate well with the low AP-to-BL Papp values observed for these solutes in the Caco-2 cell model (Tab. 2). Considering the higher lipophilicities of the cyclic prodrugs compared to DADLE, these result lead to the hypothesis that the passive permeation of the cyclic prodrugs across the cell membranes in both models of the intestinal mucosa is being restricted in part by their substrate activity for efflux transporters (e.g., P-gp). The effects of P-gp inhibitors (GF-120918, CyA, and PSC-833) on DOI 10.1002/jps

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the Caco-2 permeation of these cyclic prodrugs is consistent with this hypothesis (Tab. 2).

GF120918, CyA, and PSC833 Demonstrated Differential Effects on Enhancing the Permeability of Cyclic Prodrugs Across Intestinal Mucosa Models The effects of these P-gp inhibitors on the permeation of the cyclic prodrugs were determined using the in situ perfused rat ileum model. In these experiments, PSC-833 was shown to significantly increase the mesenteric permeability ( PB) of all three cyclic prodrugs (7.6-, 13.4-, and 15.7-fold for AOA-DADLE, CA-DADLE, and OMCA-DADLE, respectively) (Tab. 3). In contrast, GF120918 and CyA were found to be significantly less effective than PSC-833 (i.e., GF-120918 only increased the PB value for OMCA-DADLE whereas CyA only increased the PB value for CA-DADLE) (Tab. 3). These results are quite different from the results observed in the Caco-2 cell experiments (Tab. 2), where GF120918, CyA, and PSC-833 all totally inhibited the polarized efflux of these cyclic prodrugs at 10 mM concentrations. Four hypotheses were considered to explain these differences: (i) the human form of P-gp (MDR1) and the rat equivalent of this efflux transporter have different substrate (e.g., AOA-DADLE, CA-DADLE, and OMCA-DADLE) specificities; (ii) the human form of P-gp (MDR1) and the rat equivalent of this efflux transporter have different inhibitor (e.g., GF-120918, CyA, PSC-833) specificities; (iii) AOA-DADLE, CADADLE, and OMCA-DADLE are substrates of other efflux transporters (e.g., MRP2, BCRP) that play important roles in limiting the intestinal mucosal absorption of these cyclic prodrugs and these transporters are differently expressed in rat intestine and the applied Caco-2 cell model and thus apparent permeability in the two models are differentially inhibited by PSC-833, CyA, and GF-120918; and (iv) AOA-DADLE, CA-DADLE, and OMCA-DADLE are substrates for metabolic enzymes (e.g., cytochrome P-450s, esterases, phenol sulfotransferases, glucuronyltransferases) that play important roles in limiting the intestinal mucosal absorption of these cyclic prodrugs and these enzymes are differentially inhibited by PSC833, CyA, and GF-120918. The first hypothesis (i.e., species differences in P-gp substrate specificity) is unlikely for the following reasons. If rat P-gp were able to differentiate the three cyclic prodrugs as subJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 1, JANUARY 2009

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strates, one would have expected to see these differences reflected in their PB values in the absence of an inhibitor of this efflux transporter. In fact, the PB values observed for AOA-DADLE, CA-DADLE, and OMCA-DADLE in the absence of a P-gp inhibitor are essentially identical (Tab. 3). Furthermore, when similar experiments were done to determine the BBB permeability characteristics of these prodrugs using an in situ perfused rat brain model, AOA-DADLE, CADADLE, and OMCA-DADLE exhibited essentially identical BBB Papp values.10 However, it should be noted that species differences in the substrate specificity of P-gp are possible, and these differences can lead to significant differences in the disposition of these prodrugs in vivo (i.e., OMCA-DADLE disposition in guinea pigs vs. rats39). The second hypothesis (i.e., species differences in P-gps inhibitor specificity) is also unlikely based on data generated earlier by our laboratory. Our earlier experiments were designed to determine the effects of GF-120918, CyA and PSC-833 on the BBB permeability characteristics of AOADADLE, CA-DADLE, and OMCA-DADLE using an in situ perfused rat brain model.10 The data resulting from these experiments showed that all three P-gp inhibitors could significantly increase the BBB Papp values of all three prodrugs. Some differences in the potencies of these P-gp inhibitors were observed leading to the conclusion that GF-120918 was slightly more potent in enhancing the BBB Papp values of the cyclic prodrugs than PS-833 and CyA, which were equally potent. This potency order for enhancing BBB permeation in rat is quite different than the potency order observed for the effects of these P-gp inhibitors on enhancing rat intestinal mucosal permeation (i.e., PSC-833  GF-120918 ¼ CyA). To further investigate this second hypothesis, we determined the functional P-gp activity in the in situ perfused rat ileum model using quinidine as a P-gp specific marker of this efflux transporter. The effects of GF-120918, CyA and PSC-833 on quinidine’s PB values were then determined. As shown in Table 1, GF-120918 and PSC-833 are equally effective in enhancing quinidine’s PB values whereas CyA is slightly less effective. In terms of their potencies, as measured by their IC50 values, the rank order is similar (i.e., PSC833 GF120918 > CyA). Very similar results were obtained when the effects of GF-120918, CyA, and PSC-833 on quinidine Papp,AP-to-BL values were assessed using the Caco-2 cell model JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98, NO. 1, JANUARY 2009

(Tab. 1). From these data, one can conclude that, when quinidine is used as a P-gp substrate, GF-120918, CyA, and PSC-833 are all potent inhibitors of MDR1 in Caco-2 cells and of P-gp in rat intestinal mucosa and BBB. To date, our knowledge about the substrate activity of AOA-DADLE, CA-DADLE, and OMCADADLE for specific efflux transporters is somewhat limited. Using MDCK-MDR1 and MDCK-MRP2 cells, our laboratory recently characterized the substrate activity of these cyclic prodrugs for MDR1 and MRP2, respectively.7–9 From these studies, it was concluded that AOADADLE is primarily a substrate for MDR18 while CA-DADLE and OMCA-DADLE are substrates for both MDR1 and MRP2.7–9 At present we have no knowledge about the substrate activity of these cyclic prodrugs for BCRP. When one considers the fact that the stock of Caco-2 cells in our laboratory express high levels of MDR1,8,9 it was not surprising to observe that AOA-DADLE, CA-DADLE, and OMCA-DADLE all exhibited significant polarized efflux in this cell line and that this efflux could be inhibited by GF120918, CyA, and PSC-833 (Tab. 2). Granted, our Caco-2 cells also express measurable amounts of MRP2 but the expression level of this efflux transporter is extremely low; thus, it is unlikely that compounds having cosubstrate activity for MDR1/MRP2 could be differentiated,8,9 Similarly, BCRP is undetectable in our stock of Caco-2 cells (unpublished data). Therefore differentiation of compounds that are cosubstrates for MDR1/BCRP, MRP2/BCRC, or MDR1/BCRP/ MRP2 would be unlikely. Based on the information provided above about the ‘‘efflux transporter profile’’ of our stock of Caco-2 cells, it was probably not unexpected for us to observe a lack of correlation of Caco-2 cell permeation data and rat intestinal mucosal permeation data for some compounds, particularly those that exhibit substrate activity for multiple efflux transporters. As mentioned above, this lack of correlation can not be explained by species differences in the substrate/inhibitor specificity of P-gp. Instead, it would appear that the differences in permeation of the cyclic prodrugs between the two models in the presence of efflux inhibitors could arise from the prodrugs being substrates for yet unidentified efflux transporters expressed in rat intestinal mucosa but not in Caco-2 cells (Hypothesis #3). Interesting, these differences do not manifest themselves in the absence of efflux inhibitors. They only manifest DOI 10.1002/jps

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themselves in the presence of inhibitors of efflux transporters. This might suggest an alternative explanation, that is, that GF-120918, CyA and PSC-833 have broader inhibitory specificities for efflux transporters than what has been described to date in the literature. Finally, based on the data presented in this article, we cannot rule out hypothesis #4 that AOA-DADLE, CA-DADLE, and OMCADADLE are substrates of metabolic enzymes (e.g., cytochrome P-450s, esterases, phenol sulfotransferases, glucuronyltransferases) that play important roles in limiting the intestinal mucosal absorption of these cyclic prodrugs and that these enzymes are differentially inhibited by PSC-833, CyA, and GF-120918. However, in an accompanying paper, we specifically address this hypothesis and show that, while AOA-DADLE, CA-DADLE, and OMCA-DADLE are excellent substrates for cytochrome P-450 enzymes, this metabolic pathway does not contribute significantly to the low oral absorption of these compounds. Instead, it appears that the major factor limiting the oral absorption of AOA-DADLE, CA-DADLE, and OMCA-DADLE is their substrate activity for efflux transporters in the rat intestinal mucosa.

ACKNOWLEDGMENTS The authors thank Dr. Kenneth Brouwer (GlaxoSmithKline, Research Triangle Park, NC) for a generous gift of GF120918 and Dr. Stephan Ruetz (Novartis, Basel, Switzerland) for a generous gift of PSC833. This work was supported by a grant from the United States Public Health Service (DA09315).

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