Drug Metabolism and Disposition Fast Forward
First published on October 11, 2007; DOI: 10.1124/dmd.107.016865
0090-9556/08/3601-87-94$20.00
DMD 36:87-94, 2008
Investigation of Regional Mechanisms Responsible for Poor Oral Absorption in Humans of a Modified Release Preparation of the 
-Adrenoreceptor Antagonist, 4-Amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4 tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline (UK-338,003): The Rational Use of ex Vivo Intestine to Predict in Vivo Absorption
A. Collett,
R. H. Stephens,
M. D. Harwood,
M. Humphrey,
L. Dallman,
J. Bennett,
J. Davis,
G. L. Carlson, and
G. Warhurst
Gut Barrier Group, Faculty of Medical and Human Sciences, University of Manchester, Hope Hospital, Salford, UK (A.C., R.H.S., M.D.H., G.L.C., G.W.); and Pharmaceutical Sciences (M.H., L.D., J.B.) and Clinical Pharmacology (J.D.), Pfizer Global Research and Development, Sandwich, Kent, UK
(Received May 25, 2007;
Accepted October 9, 2007)
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Abstract
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Modified release (MR) formulations are used to enhance the safety and compliance of existing drugs by improving their pharmacokinetics. Predicting the likely success of MR formulations is often difficult before clinical studies. A systematic in vitro approach using mouse and human tissues was adopted to rationalize the in vivo pharmacokinetics of 9- and 15-h MR formulations of an 
-adrenoreceptor antagonist, 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4 tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline (UK-338,003). Immediate release UK-338,003 was well absorbed in humans consistent with moderate Caco-2 cell monolayer permeability. In contrast, 9- and 15-h modified release formulations showed marked reductions in Cmax (47.1 and 68.9%) and AUC0–72 (32.6 and 54.0%). Colonic intubation resulted in 81.3 and 73.8% reductions in Cmax and AUC0–72. Mechanistic studies in isolated mouse tissues showed that colonic UK-338,003 permeability (Papp < 0.5 x 10-6 cm/s) was at least 40 times lower than that for ileum with marked asymmetry. UK-338,003 was found to be a substrate for P-glycoprotein (PGP) with a weaker interaction for multidrug resistance-associated protein-type transporters in mouse intestine. PGP inhibition dramatically increased colonic UK-338,003 permeability to the levels observed in ileum. Low UK-338,003 apical to basolateral permeability was also observed in ex vivo human distal intestine, but both the asymmetry and increase in permeability after PGP inhibition were significantly lower. In conclusion, the poor absorption of MR UK-338,003 in humans can be explained by a combination of PGP-dependent efflux and low intrinsic permeability in the lower bowel. Regional permeability studies in ex vivo tissues used during drug development can highlight absorption problems in the distal bowel and assess the feasibility of developing successful MR formulations.
Oral modified release (MR) formulation can improve the pharmacokinetic and pharmacodynamic properties of a drug and thus lead to both improved patient compliance and safety (Conley et al., 2006
). However, predicting the success of MR formulations for a given drug has proved difficult, particularly for drugs with moderate intestinal permeability, and often the success or failure may only become apparent during clinical trials. One of the problems associated with prediction of absorption from a typical 8- to 24-h MR dosage form is that, unlike for immediate release (IR) forms, the drug is released throughout the GI tract with a significant proportion delivered to the distal intestine. As a result, regional differences and, in particular, the efficiency of colonic absorption will be a critical consideration. For a variety of reasons, drug absorption in the colon appears to be more challenging than absorption in the proximal bowel. In physical terms, the colon is a viscous, low-mixing environment with a lower surface area for absorption compared with the proximal bowel. Both paracellular and transcellular permeabilities appear to be lower in the colon, although these are highly compound-specific with some incidences of transcellular compounds showing higher permeability in the colon than in the upper small intestine, at least in animal models (Ungell et al., 1998). Using ex vivo tissues from different intestinal regions in the mdr1a(-/-) [P-glycoprotein knockout (PGP-KO)] mouse, (Stephens et al., 2002b) demonstrated that PGP expression increased from the proximal to the distal bowel, accounting for the low digoxin permeability in the colon. There is also direct evidence that active efflux can limit the absorption of drugs in the colon of rats (Abushammala et al., 2006); however, data showing that this occurs in humans are equivocal (Makhey et al., 1998). The lower bowel is also known to express several other drug efflux transporters (Zimmerman et al., 2005), although their potential impact on the performance of MR formulations has not been considered. However, there is a significant potential for xenobiotic transporters to affect the absorption of MR formulations because they are delivered to the lower intestine where there is significant transporter expression. In addition, the drug is released more slowly and so its concentration at the site of absorption is lower than that for IR formulations, thus reducing its potential to saturate transporters.
More effective in vitro prediction of the feasibility of developing successful MR formulations would allow MR to be considered at a much earlier stage in exploratory drug development, which might help to reduce the number of failures (Thombre, 2005; Eichenbaum et al., 2006). Basic permeability screens such as Caco-2 cell monolayers may be used to provide an initial assessment of the suitability of a drug candidate for MR development (Thombre, 2005), but there remains some uncertainty about their validity as a model of colonic absorption. Indeed, almost all of the extensive literature on the suitability of Caco-2 monolayers for prediction of oral absorption in humans deals with IR oral administration (for a recent review see Shah et al., 2006), as exemplified by Lennernas (1997), who demonstrated a good correlation between Caco-2 monolayer and human jejunal absorption. However, one study has proposed Caco-2 monolayers as a reasonable model of colonic permeability (Rubas et al., 1996). In terms of specifically predicting the role of transporters in limiting drug absorption in different regions of the intestine the relevance of Caco-2 monolayers is unclear. For example, of three recent studies comparing patterns of transporter expression in Caco-2 monolayers with those in different regions of the intestinal tract, two concluded that Caco-2 cell monolayers resemble the small intestine (Englund et al., 2006, Seithel et al., 2006), whereas a third suggested that the Caco-2 cell line is a more appropriate model of colonic transporters (Calcagno et al., 2006). Such uncertainties highlight the potential limitations of use of Caco-2 and similar cell models for predicting successful MR candidates and suggest the need for additional in vitro models that in terms of inherent permeability and transporter profile may more closely mimic the distal gut in vivo. Here we describe the use of alternative in vitro models applied to UK-338,003, an orally active 1-adrenoceptor antagonist, a class of drugs that are often developed as MR formulations to improve cardiovascular safety (Van Kerrebroeck, 2001; Kirby et al., 2005).
The aim of the present study was 2-fold: first to investigate the in vivo performance of MR dosage forms of the development compound UK-338,003, reporting human pharmacokinetic data that compare absorption of the compound from the upper and lower GI tract; and second, to investigate the use of ex vivo human and mouse intestine, including tissues from PGP-KO mice to determine the relative impact of inherent permeability and interaction with efflux transporters on the absorption of UK-338,003 in different regions of the GI tract, providing a rationalization of the in vivo data based on these mechanistic observations.
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Materials and Methods
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Materials. UK 338,003 (Fig. 1) has a molecular mass of 517 Da and was synthesized at Pfizer Global Research Ltd. (Sandwich, Kent, UK). GF 120918 was a gift from GlaxoSmithKline (Stevenage, UK). [3H]Theophylline and [14C]mannitol were from NEN Life Science Products (Hounslow, UK). MK571 was from Calbiochem (San Diego, CA). All other compounds were obtained from either BDH (Dorset, UK) or Sigma-Aldrich (Poole, UK). Wild-type FVB [mdr1a (+/+)] mice were obtained from local barrier maintained stock. Mdr1a(-/-) (PGP-KO) mice that had been back-crossed for at least seven generations onto the FVB background were obtained from Taconic Farms (Germantown, NY).
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FIG. 1. Chemical structure of 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4 tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline (UK-338,003)
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Human Pharmacokinetic Studies. Two open randomized studies were performed to investigate the pharmacokinetics of UK-338,003 in healthy male subjects. The studies were conducted according to the Association of the British Pharmaceutical Industry guidelines and the revised Declaration of Helsinki and were approved by a local ethics review committee. Study 1 compared the PK of an oral solution with that of two MR formulations after an overnight fast, using a randomized crossover design. Written informed consent was obtained from 18 healthy male subjects aged 21 to 44 years and weighing between 62 to 100 kg, with a body mass index of between 18 and 28 kg/m2 using Quetelet's index. Two subjects withdrew their consent for personal reasons, so 16 subjects received all three treatments. The oral solution consisted of 1 mg of UK-338,003 dissolved in 50 ml of water with 0.1 M hydrochloric acid. The modified release formulations contained 3 mg of UK-338,003 in a modified release tablet with either a 9- or 15-h release profile.
Study 2 compared oral administration with colonic intubation, using an open randomized design in 9 healthy male subjects. The oral solution formulation used in study 1 was administered and compared with colonic administration of UK-338,003 (1 mg) dissolved in 50 ml of 0.9% NaCl containing 0.01M hydrochloric acid (infused at a flow rate of 10 ml/min via an enteric tube with its tip placed in the terminal ileum). Written informed consent was obtained from nine healthy male subjects aged 19 to 42 years and weighing between 67 to 94 kg, with a body mass index of between 18 and 28 kg/m2 using Quetelet's index. A triple lumen tube for drug administration (external diameter 3.5 mm) was supplied by Dentsleeve International Ltd (Mississauga, ON, Canada), with a latex balloon at its tip. The tube was positioned in the duodenum using a flexible fiberoptic pediatric endoscope, approximately 24 h before administration of UK-338,003. The balloon was inflated with 10 ml of saline, and a continuous infusion of saline (10 ml/h) maintained the patency of the tube. On two occasions, the positioning of the tube was visualized by installation of contrast material (Omnipaque, 2–3 ml) via one of the infusion channels. Once the tube was correctly positioned in the terminal ileum, the balloon was deflated to avoid further progression.
In both studies, blood samples (5 ml) were collected in heparinized tubes before dosing and at time periods up to 72 h postdose. Samples were centrifuged (approximately 1500g at 4°C for 10 min) within 60 min of sample collection, and the plasma was removed and stored at -20°C. A minimum of 7 days of washout was applied between dosing periods.
Animal Tissues. Intestinal tissues were removed from nonfasting male PGP-KO mice (10–16 weeks, 20–36 g) or age-matched FVB mice killed by cervical dislocation. The ileum (the segment stretching from 1 to 13 cm proximal to the ileocecal junction) or colon was immediately removed and flushed with ice-cold, bicarbonate-buffered Ringer's solution containing 146 mM-1 Na+, 4.2 mM-1 K+, 1.2 mM-1 Ca2+, 1.2 mM-1 Mg2+, 126 mM-1 Cl-, 27 mM-1 HCO3-, 1.4 mM-1 HPO4-, and 10 mM D-glucose (MBR), which had been equilibrated to pH 7.4 by bubbling with 5% CO2/95% O2. Tissues were mounted intact in modified Ussing chambers (0.52 cm2 cross-sectional area) without removal of the serosal muscle layer as described previously (Stephen et al., 2002a,b). Mounting was completed within 30 min of removal from the animal. All procedures involving animals conformed to current UK Home Office regulations.
Human Tissues. Human distal colon or terminal ileum was obtained with informed consent after local ethical committee approval and according to the Declaration of Helsinki guidelines from patients undergoing surgery for benign or malignant tumors. Immediately after resection, macroscopically normal tissues, at least 5 cm from the tumor margin, were used for drug permeability studies. After removal from the abdomen, tissue specimens were immediately immersed in ice-cold oxygenated human colonic bicarbonate-buffered Ringer's solution containing 135 mM-1 Na+, 4.0 mM-1 K+, 1.2 Ca2+, 1.2 Mg2+, 105 mM-1 Cl-, 5 mM-1 SO4-, 26 mM-1 HCO3-, 1.2 mM-1 HPO4-, 0.2 mM-1 H2PO4, and 10 mM D-glucose (HCR), which had been equilibrated to pH 7.4 by bubbling with 5% CO2/95% O2. After removal of the underlying muscle layers by blunt dissection, mucosal pieces (1 cm2) were mounted in Ussing chambers within 40 min of the tissue being removed from the patient.
Caco-2 Studies. Caco-2 cells (American Type Culture Collection, Manassas, VA) were seeded in 24-well Falcon multiwell plates (polyethylene terephthalate membranes, pore size 1.0 µm) at 4.0 x 104 cells/well. The cells were grown in minimum essential medium containing 20% fetal bovine serum, 1% nonessential amino acids, 2 mM L-glutamine, and 1 mM sodium pyruvate. The culture medium was replaced three times every week, and the cells were maintained at 37°C, with 5% CO2 and 90% relative humidity. Permeability studies were conducted when the monolayers were 24 days old and at passages 36 to 43.
UK-338,003 was prepared at 10 µM in Hanks' balanced salt solution containing 20 mM HEPES to maintain the pH at 7.4. Transport studies were performed in triplicate in both the apical to basolateral (A-B) and basolateral to apical (B-A) directions. After 2 h of incubation at 37°C, samples were removed, concentrations were determined by liquid chromatography-mass spectrometry, and apparent permeability (Papp) values were calculated. Monolayer integrity was determined by examination of the flux of [14C]mannitol with a cutoff of 1.5 x 10-6 cm/s.
Permeability Studies. Drug transport across intestinal tissues was measured as described previously (Stephens et al., 2002a,b). Intestinal mucosa was bathed on the mucosal (apical) and serosal (basolateral) surfaces with 5 ml of MBR (mouse tissues) or HCR (human tissues), pH 7.4, at 37°C. Spontaneous tissue open-circuit potential difference, short-circuit current, and transepithelial electrical resistance were monitored periodically throughout the experiment; otherwise tissues were maintained under open-circuit conditions. A 20-min equilibration period allowed for stabilization of electrical parameters. Human and mouse tissues were excluded in cases where potential difference values fell below 2 mV at any point during the experiment. Asymmetric permeability of 20 µM UK-338,003 was measured in either the A-B or B-A direction in different tissue segments. GF120918 (20 µM) was used as a PGP inhibitor, and MK571 (20 µM) was used as an inhibitor of MRP-family transporters where indicated. For permeability studies in the mouse, 1-ml samples were removed from the receiver chamber at t = 0 and after each of six 30-min flux periods; in each case this volume was replaced with fresh MBR. In human tissues, drug permeability was measured over two 60-min flux periods. The mean rate of drug appearance over the time course of the experiment was used to calculate the Papp value in each case. Some human studies were performed with the addition of the paracellular permeability marker mannitol as a further check (in addition to monitoring of electrical parameters) of tissue integrity. In these cases, [14C]mannitol (
3 kBq/ml) was added to the donor chamber with unlabeled mannitol (100 µM) added to both donor and receiver chambers. Permeability of the high-permeability transcellular marker theophylline was investigated in human and mouse tissue by addition of 20 µM spiked with 6 kBq/ml of [3H]theophylline. For both mannitol and theophylline studies additional 1-ml samples were removed from the receiver chamber at each time point for liquid scintillation counting and replaced with fresh MBR or HCR as appropriate. Where transport inhibitors were used, inhibitors were added to donor and receiver chambers, at the indicated concentration, 60 min after the start of the first flux period.
In all experiments, 100-µl aliquots were taken from the donor chamber at the beginning of the first period and at the end of the final flux period experiment to monitor any changes in donor drug concentrations during the experiment and ensure mass balance. Drugs were added as stock solutions in dimethyl sulfoxide, giving a final solvent concentration of 0.01 to 0.3%.
Values of unidirectional transepithelial apparent permeability (Papp)incentimeters per second were calculated by
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where dQ/dt is the rate of appearance of compound in the receiver chamber, C is the substrate concentration in the donor chamber, and A is the cross-sectional area of the tissue (0.52 cm2 in mouse and 1 cm2 in human tissues). Values of Papp were averaged for control flux periods (before the addition of the inhibitor) and for inhibitor flux periods (after addition of the inhibitor) to yield baseline and postinhibition values. For analysis, the resulting Papp data from several experiments were then pooled (see Kinetic and Statistical Analysis below).
Papp values shown are either unidirectional: A-B or B-A or net Papp (Papp B-A - Papp A-B). Where values for net Papp are shown, positive values represent net secretion in the B-A direction (i.e., B-A > A-B), whereas negative values represent net absorption in the A-B direction (i.e., B-A < A-B).
Analysis of UK-338,003. An API 2000 liquid chromatograph/tandem mass spectrometer (Applied Biosystems, Foster City, CA) fitted with a turbospray ion source was used for the analysis of UK-338,003, with an HP 1100 liquid chromatograph (Agilent Technologies, Palo Alto, CA) consisting of a degasser, binary pump, autosampler, and column oven. The compound was retained on a C18 analytical column. The analysis was performed using a gradient elution profile as shown in Table 1 at a flow rate of 1.0 ml/min and an injection volume of 100 µl. Under these conditions UK-338,003 has a retention time of approximately 5.8 min.
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TABLE 1 Composition of mobile phase and gradient conditions A: HPLC eluant mix MF3 comprising 2 mM ammonium acetate, 0.027% formic acid in 90% water-10% methanol (Romil, Cambridge, UK). B: HPLC eluant mix MF2 comprising 2 mM ammonium acetate, 0.027% formic acid in 10% water-90% methanol (Romil).
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Electrospray ionization was performed in the positive ion mode at unit resolution. Nitrogen was used as the auxiliary, nebulizer, curtain, and collision-assisted dissociation gas and was set at 35, 60, 35, and 2, respectively. The source temperature was kept at 400°C. The pause time was 5 ms, and the dwell time was 500 ms. The detection and quantitation of UK-338,003 were performed in the multiple reaction monitoring mode. The ion transition monitored was m/z 507 
130. The transition ion was selected on the basis of the predominant fragmentation pathway. The operations of the mass spectrometer, the liquid chromatography system, data acquisition, and data analysis were carried out using Analyst v1.4 (Applied Biosystems).
Kinetic and Statistical Analysis. Plasma pharmacokinetic parameters for UK-338003 were calculated using standard noncompartmental techniques (WinNonlin version 4; Pharsight Corporation, Mountain View, CA). Kinetic and statistical analyses for the in vitro experiments were performed using PRISM 2.01 (GraphPad Software Inc., San Diego, CA).
Kinetic values [half-maximal effective concentration (EC50) and maximal net flux rate (Jmax)] for substrate dose-effect relationships were calculated by nonlinear regression with the method of least-squares, fitting for a logistic sigmoid using the Hill equation:
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where J is net flux, C is donor concentration, P is a constant (the Hill slope), and EC50 is the concentration at which half-maximal flux was achieved. The error values associated with the kinetic parameters are asymptotic standard errors returned by the regression routine and are a measure of the certainty of the best fit value. To take account of this, differences in kinetic parameters returned by the regression routine were compared using an unpaired Student's t test but with a higher threshold for significance of 0.01. Statistical comparisons of all other data (i.e., effects of inhibitors on substrate fluxes) were determined using an unpaired Student's t test with a significance level of 0.05.
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FIG. 2. Mean plasma concentration of UK-338,003 after oral administration of a 1-mg solution or 3 mg of 9- and 15-h modified release (CR) matrix tablets in man. The plasma concentration is corrected to a 1-mg dose and represents mean data for the same cohort of 16 to 18 individuals for each dosage form.
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Results
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UK-338,003 Exhibits Poor Absorption from MR Preparations and After Colonic Administration of a Solution in Humans. The mean plasma concentration profiles from a randomized crossover study comparing the pharmacokinetics of a 1-mg solution of UK-338,003 with 3 mg of 9- and 15-h modified release formulations in fasted males are shown in Fig. 2, and the main pharmacokinetic parameters are summarized in Table 2. Compared with the solution, there was a significant reduction in Cmax and AUC for both the 9- and 15-h formulations after dose normalization (Table 2); the greatest reduction was observed in the 15-h formulation (AUC was 46% and Cmax was 31.1% of that observed for the IR formulation). The Tmax occurred much later for the MR formulations (4.4 and 4.7 h) compared with the oral solution (1.7 h) with no difference between the 9- and 15-h preparations. These data suggest that UK-338,003 is well absorbed in the upper GI tract, but that its absorption is significantly restricted as the drug becomes exposed to the lower bowel, particularly the colon. To explore this finding further, a second crossover study was performed to investigate the pharmacokinetics of a 1-mg solution of UK-338,003 delivered directly to the distal bowel compared with oral administration (Fig. 3). Absorption was markedly impaired after colonic administration with Cmax and AUCt being 18.7 and 26.2% of values for oral administration (Table 3). The observed increase in Tmax from 1.2 to 3.1 h is consistent with a slower rate of absorption from the colon. Colonic administration was also associated with a greater intersubject variability (coefficient of variation for AUCt. was 84.5% after colonic administration compared with 31.4% after oral dosing). These observations show that UK-338,003, despite being well absorbed orally, was not a suitable candidate for administration as a once-a-day MR dosage form primarily because of low colonic absorption in humans. Further in vitro studies were performed to understand, at a mechanistic level, the reasons for the low absorption of UK-338,003 and to determine the usefulness of in vitro models for rationalizing and predicting its MR pharmacokinetics.
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TABLE 2 UK-338,003 plasma pharmacokinetics after IR or MR formulations in fasted individuals All calculations for the 3-mg 9- and 15-h formulations are dose-adjusted to 1 mg. Comparisons of each MR formulation against the IR formulation for Cmax and AUC were quantified as the ratio of the means expressed as a percentage and for Tmax and t1/2 as the difference in means. The 90% confidence limit is shown in parentheses.
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FIG. 3. Mean plasma concentration of UK-338,003 after oral or colonic administration in humans. A 1-mg solution of UK-388,003 was administered either orally or via an enteric tube to the terminal ileum. Mean data are shown for the same nine individuals in each group.
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TABLE 3 Comparison of UK-338,003 plasma pharmacokinetics after colonic and oral administration Comparisons Cmax and AUClast were quantified as the ratio of the means expressed as a percentage and for Tmax and t1/2 as the difference in means. The 90% confidence limit is shown in parentheses.
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UK-338,003 Exhibits Marked Regional Differences in Permeability in Mouse Intestine in Vitro and Is a Substrate for Efflux Transporters. Analysis of UK-338,003 permeability at a concentration of 20 µM in gut tissues isolated from FVB mice and mounted in Ussing chambers shows a moderate permeability in the A-B direction across the proximal ileum (Papp 4.2 ± 1.3 x 10-6 cm/s) but minimal permeability (<0.5 x 10-6 cm/s, i.e., below the limit of detection) across colonic tissues (Fig. 4). In both regions, there was a marked asymmetry in UK-338,003 permeability with B-A/A-B ratios of 8.5 and >70 in ileum and colon, respectively indicating that the drug is a substrate for efflux transporters. The transport inhibitor, GF120918 (20 µM), which is a potent inhibitor of PGP at this concentration, reduced but did not abolish the net efflux and increased the A-B permeability of UK-338,003 in both regions although to a different extent. In ileum, GF120918 induced a 2.7-fold increase in A-B permeability (to 11.3 ± 2.5 x 10-6 cm/s), whereas in mouse colon the inhibitor dramatically increased A-B permeability by at least 14-fold to a level similar to that observed in the ileum (7.3 ± 1.4 x 10-6 cm/s). These data suggest that drug efflux, most likely mediated by PGP, is the primary reason for the low permeability of UK-338,003 observed particularly in colon. However, the residual efflux ratios of 1.8 and 2.9 in ileum and colon, respectively, which were consistently present after incubation with 20 µM GF120918, raise the possibility that other non-PGP transporters may also influence UK-388,003 permeability. In Caco-2 monolayers, values for UK-338,003 (10 µM) bidirectional permeability appeared to be more consistent with those for mouse ileum than with those for colon. Papp values in the A-B and B-A directions were 7.0 ± 0.2 and 33 ± 1.9 cm/s, giving a B-A/A-B ratio of 4.8 (n = 3).
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FIG. 4. In vitro permeability of UK-338,003 across wild-type FVB mouse ileum and colon. A-B and B-A permeabilities of 20 µM UK-338,003 across mouse tissues mounted in Ussing chambers as described under Materials and Methods are shown. Papp is shown in the absence (control) or presence of 20 µM GF120918 (GF). *p < 0.05 B-A Papp significantly greater than A-B Papp either for control or after addition of GF120918; #, p < 0.05 A-B Papp with GF120918 compared with A-B Papp in control. The A-B flux across the colon was below the limit of detection for UK-338,003 (<0.5 x 10-6 cm/s). Data are shown as mean ± S.E.M for n = 3 to 6 experiments in each group.
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Both Active Efflux and Lower Intrinsic Permeability Limit UK-338,003 Absorption across Human Distal Intestine in Vitro. The permeability characteristics of UK-338,003 across ex vivo tissues from human distal intestine are shown in Fig. 5. Because of the relatively small number of tissues available, combined data from terminal ileum and colon are shown. The A-B permeability of UK-338,003 in these tissues was low with an A-B Papp of 0.38 ± 0.16 x 10-6 cm/s, and there was clear evidence of drug efflux with a B-A/A-B ratio of 15.3. It is notable that B-A permeability in the human tissues was 80% lower than that observed in the mouse colon, suggesting that UK-338,003 may have a lower intrinsic permeability in humans. This theory was borne out by the effects of inhibiting transport with 20 µM GF120918, which significantly increased A-B Papp in human tissue (to 2.8 ± 1.0 x 10-6 cm/s) but to a lesser extent than in mouse colon in which UK-338,003 permeability increased to 7.3 ± 1.4 x 10-6 cm/s on addition of GF120918 (Fig. 5). In line with mouse tissues, there was a residual efflux ratio of 3.0 after addition of GF120918. These data imply that both active transport processes and low intrinsic permeability to UK-338,003 are likely factors in the poor absorption of UK-338,003 in human distal gut.
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FIG. 5. In vitro permeability of UK-338,003 across human distal intestine. A-B and B-A Papp of 20 µM UK-338,003 across human distal intestine (a combined group of terminal ileum and colonic tissues) is measured either in the absence (control) or in the presence of 20 µM GF120918 (GF). *, p < 0.05, control B-A Papp significantly greater than A-B Papp; #, p < 0.05, A-B Papp in the presence of GF120918 significantly greater than control A-B Papp. The dotted line indicates the limit of detection for UK-338,003 (<0.5 x 10-6 cm/s). Data are mean ± S.E.M. for n = 6 pieces of intestine from four individuals.
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Multiple Transporters Limit Intestinal Permeability of UK-338,003 in Mouse Intestine. As reported above there was a residual B-A/A-B ratio for UK-338,003 in both mouse and human tissues in the presence of the PGP inhibitor GF120918, raising the question as to whether other transporters are involved in limiting UK-338,003 permeability across the distal gut. To investigate this possibility further, UK-338,003 permeability was analyzed in ileum isolated from mdr1a(-/-) (PGP-KO) mice that express no functional PGP in the bowel (Fig. 6) (Schinkel et al., 1994). The A-B permeability of UK-338,003 in ileum from PGP-KO mice was almost 2-fold higher (7.1 ± 1.5 x 10-6 cm/s) than that in ileum from wild-type controls (Figs. 4 and 6), consistent with a role for PGP as described above. Interestingly, however, despite the lack of functional PGP, a significant efflux of UK-338,003 (efflux ratio 3.5) remained in PGP-KO tissues. This was abolished by addition of MK571 (20 µM), a selective inhibitor of MRP-type transporters (Gekeler et al., 1995), resulting in a further increase in UK-338,003 A-B permeability to 12.6 ± 1.5 x 10-6 cm/s. These data are consistent with UK-338,003 permeability being limited, at least in part, by interaction with multiple efflux transporters. The use of ileum from PGP-KO mice allowed a detailed study of the kinetics with which UK-338,003 interacts with PGP and MRP in the mouse gut to be undertaken. Figure 7 shows the net efflux flux of UK-338,003 versus concentration in the ileum isolated from wild-type or PGP-KO mice. The efflux of the compound in wild-type mice, which is dominated by PGP, showed an EC50 of 31.5 ± 3.8 µM with a Jmax of 10.4 ± 0.8 nmol/h/cm2. A similar analysis of the residual efflux in PGP-KO mice, likely to be due to an MRP transporter, showed a markedly higher EC50 (97.3 ± 13.3 µM) and lower Jmax (5.9 ± 0.6 nmol/h/cm2), implying that the MRP transporter saturates at a much higher concentration of UK-338,003 than does PGP and has a 50% lower transport capacity.
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FIG. 6. In vitro permeability of UK-338,003 across PGP-KO mouse ileum. Data show A-B Papp and B-A Papp of 20 µM UK-338,003 in mdr1a(-/-) mouse ileum either in the absence or presence of 20 µM MK571. *, p < 0.05, B-A Papp significantly greater than A-B Papp. Data are mean ± S.E.M. for n = 4 in each group.
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FIG. 7. Concentration dependence of net efflux of UK-338,003 in ileum from wild-type (WT) FVB or mdr1a(-/-) (PGP-KO) mice. Values show mean ± S.E.M. n = 3 to 5 observations at each concentration. Calculated EC50 and Jmax values are 31.5 ± 3.8 mM and 10.4 ± 0.8 nmol/h/cm, respectively, for wild-type control tissues and 97.3 ± 13.3 mM and 5.9 ± 0.6 nmol/h/cm, respectively, for PGP-KO tissues.
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Theophylline Permeability Is High in All Regions of Wild-Type Mouse and Human Tissues. As a further test of the validity of ex vivo human and mouse intestine for predicting the likely performance of candidates for MR, the permeability of theophylline, a compound that is well absorbed as a MR formulation in man (González and Straughan, 1994) was tested. This phosphodiesterase inhibitor is a high-permeability, transcellular compound whose absorption is not limited by PGP or other efflux transporters (Saitoh and Aungst, 1995). Theophylline exhibited high permeability across mouse ileum and colon with no difference between the two regions (Papp values of 30 ± 10 and 28 ± 2.9 x 10-6 cm/s, for ileum and colon, respectively). Theophylline also exhibited significant permeability across human intestine although values (14.8 ± 1.8 x 10-6 cm/s) were approximately 50% lower than those in the mouse. Theophylline A-B permeability was markedly higher than that for UK-338,003 in both species even after inhibition of efflux of UK-338,003 with GF120918 (Table 4). These data indicate that ex vivo preparations of human and mouse intestine show discrimination between compounds that have had differing success as MR formulations.
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TABLE 4 In vitro permeability of UK-338,003 (20 µM) and theophylline (20 µM) in human and mouse intestine Data are from Caco-2 monolayers, wild-type mouse ileum, or colon and distal human intestine. The latter are a combined group of colon and terminal ileum tissues. Values are given as mean ± S.E.M. for Papp (x 10–6 cm/s) in the A-B and B-A directions for UK-338,003 and for A-B only with theophylline.
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Discussion
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Prediction of the likely oral pharmacokinetics of drugs in MR formulations is more difficult than that for IR formulations given our poor understanding of the factors determining absorption in the distal bowel. We have used a systematic in vitro approach, based on the use of human and mouse tissues, to investigate the impact of colonic permeability and efflux transporters on the in vivo performance of a MR formulation of the -adrenoreceptor antagonist UK-338,003 in humans. In vivo data show that UK-338,003, although well absorbed orally from IR preparations, exhibits poor absorption when administered as an MR formulation with a 9- or 15-h release profile. Tmax values for 9- and 15-h formulations of 4.4 and 4.7 h, respectively, in fasted individuals are similar to the small intestinal transit time (Davis et al., 1986; Wilding et al., 2001). This finding suggests that, even with the 15-h matrix, the small intestine is responsible for a significant proportion of UK-338,003 absorption, despite the fact that the drug will be present in the colon for long periods. Intubation studies confirmed the poor colonic absorption of UK-338,003 with 81 and 73% reductions in Cmax and AUC0–72, compared with oral IR administration. The poor absorption appeared to result from poor colonic permeability as there was no evidence of significant solubility issues at the concentrations used. On a mechanistic level, there are likely to be two major influences on UK-338,003 colonic absorption: the intrinsic permeability of the colonic membrane and interaction with drug efflux transporters or a combination of these.
In normal mouse tissues, UK-338,003 shows considerable differences in regional permeability with colonic A-B permeability being at least 10-fold lower than that in proximal ileum but with both tissues showing marked asymmetry indicative of active efflux. GF120918 dramatically increased colonic permeability of UK-338,003, suggesting that its absorption in the colon is severely restricted by PGP, although this compound can also inhibit the transporter breast cancer resistance protein (Tan et al., 2000). GF120918 also raised the A-B permeability of UK-338,003 in the mouse ileum to a level similar to that observed in the colon; however, the greater fold increase in A-B flux elicited by GF120918 in the colon appears consistent with previous evidence that the large bowel has a higher functional level of PGP (Stephens et al., 2002b). Interestingly, even at the relatively high concentration of 20 µM, GF-120918 did not completely abolish net secretion of UK-338,003 in either colon or ileum. This finding suggests that UK-338,003 may be interacting with other intestinal transporters. PGP-KO mouse tissues showed definitively that although the majority of UK-338,003 efflux is accounted for by PGP, a significant non-PGP component is present that was abolished by MK571, a selective inhibitor of MRP-type transporters. A similar approach has been used to identify etoposide as a mixed PGP/MRP substrate (Stephens et al., 2002a). Comparative studies of the dose dependence of UK-338,003 secretion in normal and PGP-KO ileum indicate that the kinetics of the UK-338,003 interaction with PGP and MRP is quite different. The interaction with PGP occurs at lower concentrations (EC50 31.5 ± 3.8 µM) than those for MRP (EC50 97.3 ± 13.3 µM) with PGP also having approximately twice the transport capacity for UK-338,003. These data are consistent with PGP being the primary transporter responsible for UK-338,003 secretion but suggest that as this transporter becomes saturated by higher drug concentrations, an MRP-type transporter takes over as a secondary barrier to UK-338,003 absorption. Evidence suggests that the expression pattern of intestinal MRP transporters is complementary to that of PGP (Zimmermann et al., 2005). The present data are consistent with these transporters acting in concert to limit the absorption of UK-338,003 in the distal gut, resulting in an adverse in vivo kinetic profile for MR formulations.
Although access to tissues from the human terminal ileum and colon was limited, in general terms these show absorption characteristics for UK-338,003 similar to those for mouse intestine with low A-B permeability and a high efflux ratio (15.3), indicative of active transport. However, even though PGP inhibition by GF120918 markedly increased UK-338,003 permeability in human tissues by more than 7-fold, it remained significantly lower than that observed in mouse colon after GF120918 addition. A likely explanation is that UK-338,003 permeability in human distal intestine is limited by a combination of active PGP-mediated efflux and lower intrinsic permeability. In contrast, mouse has higher intrinsic permeability for UK-338,003 with active efflux being the major factor limiting absorption. The much lower B-A permeability of UK-338,003 in human compared with mouse is consistent with this interpretation. Additional efflux transporters, such as MRP, may be involved in limiting human UK-338,003 permeability, and further studies will be needed to address this issue. However, the relatively low B-A permeability of UK-338,003 in human tissues suggests this involvement is unlikely to be a major factor. Our conclusion is that whereas PGP inhibition would probably increase UK-388,033 absorption in human colon, the effect may be relatively small compared with that in the mouse primarily because of the overriding effect of a lower intrinsic UK-338,003 permeability in humans. Comparative studies of etoposide efflux in rat and human intestine by Makhey et al. (1998) lend some support to this view. The B-A etoposide permeability was reported as being 5 times higher than the A-B permeability in rat colon, whereas in the human colon, there was no significant difference between the A-B and B-A directions. Despite the apparent lack of efflux in human colon, etoposide permeability remained low.
Where they occur, interactions with colonic transporters may have adverse effects on the PK of MR formulation drugs including nonlinear absorption (Tubic et al., 2006), food interactions (Wagner et al., 2001), coadministered drugs (Lin and Yamazaki, 2003), and other xenobiotics (Abu-Qare et al., 2003). Interactions with colonic transporters may also increase interindividual variation in PK because of different levels of transporter expression and the impact of transporter polymorphisms (Dey, 2006). In this respect, it is interesting that intubation of UK-338,003 into the distal bowel resulted in a greater interindividual variation in PK compared with oral administration in the same cohort.
The present study raises interesting questions about how in vitro models can be used to assess absorption and the influence of transporters in the lower bowel and thereby predict the likely suitability of a compound as a MR formulation. Caco-2 monolayers are widely used to screen the absorption potential of new drugs and their possible interactions with PGP and other transporters (Bohets et al., 2001). The data for UK-338,003 illustrate problems with the direct extrapolation of data from Caco-2 monolayers to different regions of the human GI tract. UK-338,003 exhibited moderate permeability and a modest efflux ratio in Caco-2 monolayers consistent with the favorable oral PK observed in humans. However, UK-338,003 permeability in Caco-2 monolayers was significantly higher than that observed in human or mouse colonic tissues in vitro, which was consistent with the low absorption seen on colonic intubation and the poor pharmacokinetic properties of the MR formulation in humans. In the case of this compound, therefore, ex vivo colon preparations from both human and mouse appear to be useful predictors of the performance of the drug in the lower bowel. However, whereas the overall UK-338,003 profile of low permeability and significant interaction with transporters is similar in human and mouse colon, there appear to be important mechanistic differences. In mouse colon, the low permeability of UK-338,003 is due almost entirely to its interaction with transporters. However, although the transporter interaction in human colon appears to be similar to that in mouse, the much lower intrinsic membrane permeability of UK-338,003 in human colon is at least as important in determining the level of absorption. The differences between the in vitro models are much less apparent with high-permeability, noneffluxed compounds such as theophylline, which would have been confirmed as a suitable candidate for MR formulation (González and Straughan, 1994) using data from either Caco-2 cells or ex vivo mouse or human tissues. Lower colonic permeability in humans compared with that in rats has been noted previously (Masaoka et al., 2006) and may typify a more generalized permeability difference between humans and rodents (Kim et al., 2006). This difference may have a physiological basis in that the longer colonic transit time in humans (24 h compared with 1 h in mouse) provides greater exposure to potentially toxic substances and thus a significantly lower intrinsic permeability coupled to high levels of efflux transporters is likely to be beneficial.
Other factors such as gut wall metabolism could theoretically contribute to the poor absorption of UK-388,003 but seems unlikely in this case. UK-388,003 is metabolized almost exclusively by CYP3A in humans (Betts et al., 2007), which is localized primarily in the upper GI tract with much lower levels in distal gut (McKinnon et al., 1995). In rodents, UK-388,003 is metabolized exclusively by CYP2C, which has overlapping substrate specificity with CYP3A4 (Betts et al., 2007). Given the evidence for good permeability in the upper intestine, it seems unlikely that gut wall metabolism will be an important factor in reducing UK-388,003 permeability in the distal gut.
In conclusion, we have shown, using the example of an -adrenoreceptor antagonist, UK-338,003, that a complementary approach in which initial assessment of absorption potential in Caco-2 or a similar in vitro model is combined with permeability profiling in ex vivo human and rodent tissues can be more effective in predicting the suitability of compounds for MR development and rationalize pharmacokinetic findings in humans. Ex vivo tissues from normal and PGP-KO mice demonstrated the interaction between UK-338,003 and colonic transporters and their role in limiting UK-338,003 absorption in this region. Complementary studies in ex vivo human tissues confirmed poor UK-338,003 permeability in human colon resulting from a combination of low inherent permeability and active efflux. Such approaches could be used relatively early in the drug development process to highlight potential absorption problems in the distal bowel and assess the feasibility of developing a successful MR formulation. This study illustrates the usefulness of ex vivo human tissues as a tool to elucidate the regional and species-specific factors that determine drug permeability along the gut.
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Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.107.016865.
ABBREVIATIONS: MR, modified release; IR, immediate release; PGP, P-glycoprotein; KO, knockout; GI, gastrointestinal; UK-338,003, 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4 tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline; GF 120918, 9,10-dihydro-5-methoxy-9-oxo-N-[4-[2-(1,2,3,4-tetrahydro-6,7dimethoxy-2-isoquilonyl)ethyl-4-phenyl]-4-acridinecarboxamide; MK571, (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl] phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid; PK, pharmacokinetics; A-B, apical to basolateral; B-A, basolateral to apical; MRP, multidrug resistance-associated protein; AUC, area under the curve.
Address correspondence to: Dr. Geoffrey Warhurst, Gut Barrier Group, Faculty of Medical and Human Sciences, University of Manchester, Hope Hospital, Salford M6 8HD, UK. E-mail: geoffrey.warhurst{at}manchester.ac.uk
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Abstract
Materials and Methods
