Journulo/ Drux Turgeling. Vol. 6. No. I. pp 37-43 Reprints available directly from the publisher Photocopying permitted by license only
t i ' 1998 OPA (Overseas Publishers Association) N.V. Published by license under
the Harwood Academic Publishers imprint. part of The Gordon and Breach Publishing Group.
Printed in Malaysia.
Molecular Weight-Dependent Paracellular Transport of Fluorescent Model Compounds Induced by
Palmitoylcarnitine Chloride across the Human Intestinal Epithelial Cell Line Caco-2
ANTHONY c. CHAO*, MICHELE T. TAYLOR', PETER E. DADDONA, MARY BROUGHALL and JOSEPH A. FIX
A L Z A Tc.chnolog.v Insritutr, Biologicul Scicmc,.s, A L Z A Corporuiion, 950 P u p Mill Roud. P.O. BOT 10950, PUIO A l t o , C A 94303-0802. USA
( Rtwivcvl 4 Deceniher 1996; I n ,finulJorm 23 January 1997)
Long-chain acylcarnitines, such as palmitoylcarnitine chloride (PCC), are endogenous compounds which have been shown to increase intestinal transport of small hydrophilic compounds (including some pharmaceutical agents) through the paracellular pathway. However, the size range of the compounds whose absorption can be improved by PCC has not been fully investigated. In the present study, we systematically examined the effect of PCC on the transport rate of a series of hydrophilic fluorescent model compounds of varying molecular weights (0.3-71.2 kD) across cultured monolayers of the human intestinal epithelial cells Caco-2. Mucosal addition of 100 or 200pM PCC resulted in comparable time-dependent decreases in the transepithelial electric resistance (TI,?, - 15 min). PCC addition induced a striking increase in the transport of sodium fluorescein (Flu-Na; 0.3kD) and a slight or moderate increase in transports of fluorescent compounds of 0.6- 1 I kD. The effect of PCC on transport of compounds with molecular weights of 2 17 kD appeared to be negligible. Examination by confocal laser scanning microscopy clearly revealed dilated paracellular spaces in Caco-2 monolayers which had been mucosally pretreated with PCC, confirming that PCC increases intestinal permeability by opening a paracellular transport pathway. Our results suggest that PCC is particularly effective in enhancing intestinal absorption of small hydrophilic compound like Flu-Na and may also have limited use in promoting the transport of compounds of 5 10 kD.
Krywortls: Acylcarnitines, Caco-2, Confocal laser scanning microscopy, Intestinal transport, Permeation-enhancing agents, TEER
*Corresponding author. Tel.: (650) 237-2706. Fax: (650) 237-2700. 'Prescnt address: Departmcnt of Molecular Pharmacology, Institute of Pharmacology. Roche Bioscience, Neurobiology Unit, Palo
Alto, CA 94304-1397, USA.
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Abhreviurions: PCC, palmitoylcarnitine chloride; Flu-Na, sodium fluorescein; SR-101, Sulforho- damine 101; FD-4, fluorescein isothiocyanate (F1TC)-dextran (MW -4.4 kD); FD-10, FITC-dextran (MW - 11.0 kD); FD-20, FITC-dextran (MW - 17.2 kD); FD-50. FITC-dextran (MW - 50.7 kD); FD-70, FITC-dextran (MW N 71.2 kD); EDTA, ethylenediaminetetraacetate; STDHF, sodium tauro-24,2S-dihydrofusidate; TEER, transepithelial electric resistance; CLSM, confocal laser scanning microscopy
INTRODUCTION
Oral delivery is the most favorable method for drug administration. To improve oral bioavailability of poorly absorbed drugs, efforts have been continu- ously made during the past several decades to search for appropriate formulation agents whose co-administration can enhance intestinal drug absorption without causing significant damage to the epithelial tissues of the GI tract (Fix, 1987; Muranishi, 1990; Lee et al., 1991). In recent years, particular attention has been given to endogenous permeation-enhancing agents which are readily metabolizable by the epithelial cells or biodegrad- able (Muranishi, 1990).
Long-chain (C12-Cl8) acylcarnitines are a class of such endogenous compounds which have been shown to be efficacious in promoting intestinal absorption of small hydrophilic drugs and model compounds (Fix et al., 1986; LeCluyse et al., 1993; Hochman et al., 1994). In particular, palmitoyl (C 16)-carnitine chloride (PCC) was found to be the most effective acylcarnitine in enhancing intestinal permeability (Hochman et al., 1994). Long-chain acylcarnitines are involved in fatty acid transport across mitochondria and are themselves subject to metabolic breakdown in the cell (Bremer, 1983; Fix, 1987). Hence, their absorption-enhancing activity is relatively transient and largely reversible (Hochman et af., 1994). Data have been presented suggesting that PCC enhances intestinal perme- ability by “loosening” the tight junctions of the paracellular pathways (Hochman et al., 1994).
Although PCC has been shown to induce absorption of small hydrophilic molecules across the intestinal epithelial barrier, the size of the putative transport pathway opened by PCC is largely unknown. To determine the size range of molecules whose absorption could be most
improved by PCC administration, we systemati- cally examined and compared the effect of PCC on the transports of a series of fluorescent model compounds of various molecular weights (0.3- 71.2 kD), across cultured monolayers of the Caco- 2 iiitestinal epithelial cells. We have also conducted visualization studies to delineate the transport pathway opened by PCC, by confocal laser scan- ning microscopy (CLSM). Here we demonstrate, for the first time, in a living, un-fixed intestinal preparation that PCC enhances the epithelial permeability mainly by opening a transport route through the dilated paracellular spaces.
MATERIALS AND METHODS
Materials
Palmitoylcarnitine chloride (PCC) and all the fluorescent compounds were purchased from Sigma Chem. Co. (St. Louis, MO). To distinguish the FITC-dextrans of average molecular weight of 50.7kD (“FD-70”; Lot 45H0893) from that of average molecular weight of 7 1.2 kD (“FD-70s”; Lot 105F5029), these two batches of FITC-dex- trans were designated as “FD-50” and “FD-70”, respectively, in the present study. The Medical Antifoam C Emulsion was from Dow Corning Corp. (Midland, MI).
Cell Culture
Caco-2 cells were purchased from ATCC (Rock- ville, MD). Cells (passage 21-42) were maintained in Dulbecco’s Modified Eagle Medium supple- mented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT), non-essential amino acids, L-glutamine, penicillin and strepto- mycin (Gibco BRL, Grand Island, NY). For
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PALMITOYLCARNITINE-INDUCED INTESTINAL TRANSPORT 39
transport studies, cells were plated at a density of 6 x 104cells/cm2 (Hidalgo et d., 1989) on 12-mm Snapwell polycarbonate membranes (surface area: 1 cm’) (Corning Costar, Cambridge, MA) precoated with rat tail collagen (Becton Dickinson, Bedford, MA). The cell monolayers were studied 3-4 weeks following seeding. For optical examination of the cultured Caco-2 monolayer by CLSM, cells were plated on 24-mm collagen-treated Transwell-COL hydrophilized polyfluoroethylene (PTFE) mem- branes (surface area: 4.7 cm’) (Corning Costar) at a lower density of 1 x 104cells/cm2 (Hurni et a/., 1993). To further improve the condition for cell growth, the Transwell-COL PTFE membranes were precoated with rat tail collagen (Becton Dickinson) prior to cell seeding (Hurni et ul., 1993). Cells seeded were allowed to grow to confluency and full differentiation for 3-5 weeks prior to CLSM examination.
Electrophysiology and Transport Experiments
Monolayers of Caco-2 cells cultured on Snapwell filter supports were mounted vertically in the side- by-side acrylic perfusion chamber system (Corning Costar) and bathed on both sides in freshly prepared Kreb’s buffer (pH 7.4) which was con- tinuously oxygenated and mechanically stirred by a 95% 0 2 / 5 % C 0 2 gas-lift system at 37°C. A pair of voltage- and current-sensing electrodes connected to a voltage-clamp amplifier (Physiologic Instru- ments, San Diego, CA) were inserted into the appropriate half-chambers across the cell mono- layer. The transepithelial electric resistance (TEER) of the cell monolayer was monitored essentially as described previously (Chao and Mochizuki, 1992). To initiate mucosal-to-serosal flux, the fluorescent compound in question (100-250 pM) was added to the mucosal (donor) half-chamber at the beginning (time “0”min) of the flux experiment, in the absence or presence of PCC (100 or 200 pM). As the addition of PCC was found to cause excessive foaming in the bathing solution being bubbled by the gas-lift system, PCC was subsequently adminis- tered in the presence of 0.03% (v/v) of Medical
Antifoam C Emulsion (containing 30% simethi- cone). Samples (50 pl) were collected, at 15-min intervals, from the basolateral (receiver) half- chamber and the receiver solution was replenished with an equal volume of fresh Kreb’s buffer, for up to 2h. Samples were subsequently diluted with Milli-Q deionized water in UV grade methacrylate cuvettes (Fisher, Pittsburgh, PA) and assayed by fluorescence spectrophotometry (Hitachi, Tokyo, Japan). For quantitation of FITC-derived com- pounds, the excitation and emission wavelengths were 490 5 10 and 520 5 20 nm, respectively; for quantitation of SR-101, the excitation wavelength was 586 f 10 nm and emitted fluorescence was measured at 607 f 20 nm. The transepithelial transport rate of the fluorescent compounds was assessed by its cumulative percent transport (Hochman et al., 1994), which is the percentage of the initial mucosal-side concentration of the fluorescent compound which is transported to the serosal (receiver) compartment.
CLSM
To delineate the transepithelial route involved in PCC-enhanced intestinal permeability, fluores- cence images of vertical cross-sections of Caco-2 monolayers were examined by CLSM at the Cell Sciences Imaging Facility, Stanford University Medical Center. A MultiProbe 2010 confocal laser scanning system (Molecular Dynamics, Sunnyvale, CA) connected to a Nikon Diaphot 200 inverted microscope (Nikon, Melville, NY) was used. Cell monolayers were first incubated in Hank’s balanced salt solution containing 25 mM glucose and lOmM HEPES (pH 7.4) with the mucosal surface exposed to 10 or lOOpg/ml SR-101 for N 40 min at room temperature. Subsequently, the cell monolayer/PTFE membrane was removed from the plastic support and sandwiched between two round glass coverslips (0.15 mm thickness; Fisher, Pittsburgh, PA) as described by Hurni et a/. (1993). The vertical cross-sections of the cell monolayers were examined in the X - Z scanning mode. The excitation wavelength was at 568 nm
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280 -
n N
E 210 - c! E 6 140 -
pc # 7 0 - +
Y
and emitted fluorescence collected at 2 590 nm. The fluorescence images of cell monolayers, obtained with a Nikon PlanApo 60x (NA 1.4) oil-immersion objective, were displayed on the monitor screen and printed on a dye sublimation printer (Tektronix Phaser 440).
RESULTS
In the initial experiments, the addition of PCC to the mucosal solution in the side-by-side perfusion chamber, which is bubbled by the gas-lift system, caused excessive foaming. Hence, Antifoam C Emulsion was used to prevent foaming. It was found that addition of the antifoam agent alone had little effect on the FITC-dextran transport, but its presence remarkably enhanced the permeation- enhancing activity of PCC (data not shown). How- ever, the antifoam agent did not enhance PCC effectiveness when flux experiments were per- formed in tissue culture cluster wells where the bathing solutions were not bubbled. It appeared that the PCC-induced foaming, which is likely due to detergent-like properties of PCC, reduced the “effective” PCC concentration in the bathing medium. Thus, to fully uncover the effect of PCC, it was administered in the presence of the antifoam agent.
The TEER of the Caco-2 monolayers, an index of the “tightness” of the paracellular pathways, was initially N 260 R . cm2 and decreased promptly upon the mucosal addition of PCC (100 or 200 pM), with a
To examine the molecular weight-dependence of PCC-induced paracellular transport, cumulative percent transepithelial flux of a series of seven fluorescent compounds of different molecular weights (0.3-71.2 kD) was determined in Caco-2 monolayers whose paracellular pathways had been “loosened” by mucosal treatment with PCC. The effect of addition of the antifoam emulsion alone was found to be very slight or absent on the transport rates of all of the compounds tested and, therefore, data collected in the absence and
of - 15 min (Fig. 1).
PCC
U 1 0 0 pM PCC +200 pM PCC
0 : I I I r -30 0 30 60 90
Time (rnin) FIGURE I Effect of mucosal addition of PCC on the time course of TEER of cultured monolayer of Caco-2 cells. PCC was added (100 pM, n = 6; 200 pM, n = 3) where indicated by the arrow.
presence of the antifoam agent (without PCC) were pooled in the control group. In the absence of PCC (control), the cumulative transport of all of the compounds tested was < 0.4% over a 2-h period. PCC was more effective in promoting the absorp- tion of smaller molecular weight compounds, as illustrated by the progressively diminished time- dependent flux of PCC-induced transport of Flu-Na (0.3kD), FD-4 (4.4kD), and FD-20 (17.2 kD) (see Fig. 2). As seen in summarized results given in Fig. 3, PCC was particularly efficacious in promoting the transport of Flu-Na but also seemed to be effective in inducing some enhancement of SR-101, FD-4, and FD-I0 transport.
To elucidate the cellular pathway responsible for PCC-enhanced intestinal permeability, vertical cross-sections of the Caco-2 monolayers pretreated with PCC (200pM) were studied by CLSM. We chose to use SR-IOI as the fluorescent probe because of its small size (- 0.6 kD), extremely high water solubility and its bright and seemingly quench-resistant fluorescence. As shown in Fig. 4, PCC pretreatment noticeably led to penetration of
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PALMITOYLCARNITINE-INDUCED INTESTINAL TRANSPORT 41
A
B
-+Control e l 0 0 p M PCC - 2 0 0 pM PCC
T i 1 k-7- h T/"
0 20 40 60 80 100 120 Time (min)
-Contro l 1 -100 UM PCC T
0 20 40 60 80 100 120 Time (min)
+Contro l e + 1 O O p M PCC T
1.0 I- : I T A
LL 0.0 On5 &
0 20 40 60 80 100 120 Time (min)
FIGURE 2 Effect of mucosal addition of PCC on the time course of cumulative percent transport of Flu-Na (A; n = 4-7 on each condition). FD-4 (B; 11=4 6 each) and FD-20 (C; ti = 3 -5 each) across Caco-2 monolayers.
SR-IOI into dilated paracellular spaces, and enhanced transport of SR-IOI to the serosal space between the cell monolayer and the underlying glass coverslip. In contrast, drastically different images
12 n 0 100 pM PCC
FIGURE 3 Molecular-weight-dependence of PCC-induced transport of hydrophilic fluorescent compounds of 0.3- 70.1 kD across Caco-2 monolayers. Each value shown repre- sents mean * SE of at least three experiments.
were observed in cells pretreated mucosally with saponin (0.04%), a known membrane-active per- meation-enhancing agent, where SR-101 seemed to accumulate intracellularly without penetrating into the paracellular spaces (not shown). These results demonstrate that PCC enhances Caco-2 monolayer permeability by opening up a paracellular route across the cellular tight junctions.
DISCUSSION
As described above (see Results), the effect of PCC seemed to be attenuated by foaming. Thus, to fully reveal the effect of a permeation enhancer which induces excessive foaming, it seems critical to co- administer an appropriate antifoam agent.
Mucosal addition of PCC at 100 or 200pM resulted in a comparable time-dependent decrease in TEER values of the Caco-2 monolayers. The difference in the permeation-enhancing effect of PCC between these two concentrations seems to be small on Flu-Na transport but becomes more conspicuous on transport of compounds of larger molecular weights. Also, as seen in Fig. 2,
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FIGURE 4 PCC widens the paracellular pathways of Caco- 2 cell monolayers. Shown are representative fluorescence images obtained by CLSM of vertical cross-sections of cell monolayers pretreated with SR-101 alone (top) and pretreated with SR-I01 plus 200pM PCC (bottom), respectively, for 2 60 min (n = 4 in each group).
PCC-enhanced transport of Flu-Na seems to reach to a new quasi-steady-state level sooner than that of the larger compounds. In untreated epithelial tissues, TEER values primarily reflect the resistance of the paracellular pathways to the transport of ions. It was suggested recently that, when the structures of the tight junctions are “slightly” affected, TEER values correlate better with changes in the epithelial barrier resistance than the transport rate of non-electrolyte solute like mannitol (Lu et al., 1996). Our data seem to show that, in the cell monolayers treated with l00pM PCC, TEER values correlate more closely to the changes in the epithelial paracellular permeability to smaller hydrophilic molecules (like Flu-Na) than to the changes in permeability to the larger compounds.
Conceivably, PCC at 100 pM opens a transport route which allows Flu-Na and smaller hydrophilic molecules like ions to traverse the epithelial barrier freely and, at 200 pM, “loosens” that pathway somewhat further, rendering it permissive to certain passage of larger compounds. Overall, a seemingly comparable molecular-weight-dependent paracel- Mar transport profile is seen in the cell monolayers treated with either 100 or 200 pM PCC.
PCC was most efficacious in promoting Flu-Na permeation, elevating its cumulative transport over 2 h to - 10% (permeability increased to -0.8 x 1OP4cm/s). PCC also induced a slight or moderate increase in the cumulative absorption of FD-4, FD-I0 and FD-20 (to - 3% over 2 h). These results are consistent with those recently obtained using some non-fluorescent model compounds. For instance, PCC was shown to be more efficacious in enhancing Caco-2 transport of lucifer yellow (MW 0.46 kD) than that of PEG4000 (MW ~ 4 k D ) (Hochman et al., 1994). In studies con- ducted in isolated rat colonic mucosal tissues, PCC was found to be more effective in increasing the absorption of lucifer yellow and calcein (MW 0.6 kD) than that of PEG4000 and a 10-kD dextran (LeCluyse et al., 1993). The results of our systematic examination and comparison of PCC-induced trans- ports of the seven fluorescent compounds seem to indicate that PCC is particularly suitable for pro- moting intestinal absorption of small compounds like Flu-Na as opposed to the larger compounds.
CLSM has recently been shown to be a powerful method for investigating the mechanism of action of permeation-enhancing agents on intestinal perme- ability (Hurni et al., 1993; Sakai et al., 1994), which allows direct optical examination of cross-sections of epithelial tissues with excellent resolution. In this study we showed for the first time in a living, un- fixed intestinal epithelial preparation that PCC exerts its permeation-enhancing effect primarily by opening up a paracellular pathway. These results are in agreement to those recently obtained by Hochman et al. (1994) in fixed Caco-2 cell mono- layers in which PCC-induced dilatation of the pericellular spaces and ultrastructural alterations
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PALM ITOY LCARNITINE-INDUCED INTESTINAL TRANSPORT 43
of the tight junctions. PCC has also been shown to interact with mucosal membranes as suggested by its effect on the cell membrane fluidity (LeCluyse el al., 1991). Whether PCC exerts its effects directly on the tight junctions or PCC-induced alteration in tight junctions is secondary to the changes it initially induced in the cell membranes (and, if so, the mechanisms involved in linking these cellular events) remains to be elucidated.
Besides acylcarnitines like PCC, a number of other “paracellular-acting” permeation-enhancing agents have recently been identified, such as sodium caprate (Anderberg er ul., 1993; Tomita et al., 1995), STDHF, sodium salicylate (Hurni et al., 1993), verapamil (Sakai et ul., 1994) and CaZf chelaters like EDTA (Artursson and Magnusson, 1990). Among these agents, acylcartnitine is the only endogenous compound that is metabolizable in the epithelial cells. At the concentrations used in this study, PCC was shown not to induce discern- ible damages to the intestinal epithelial cells (Hochman rt ul., 1994). CaZf chelating agents like EDTA are known to be toxic to the epithelial tissues. Sodium caprate increases moderately the transport rate of FD-4 across Caco-2, but much higher doses are needed and the effect is attenuated by the presence of ambient Ca*+(Tomita et al., 1995). The permeation-enhancing activities of sodium salicylate and of verapamil appear to be relatively weak. STDHF, a bile acid derivative, is shown to be effective in enhancing intestinal transport of Flu-Na and FD-4 (Hurni et al., 1993). Unfortunately this agent is, to our knowl- edge, still not commercially available. PCC seems to remain optimum “paracellular-acting” perme- ation-enhancing agent identified so far.
Acknowledgements
We wish to thank the members of the mucosal research group (ALZA Technology Institute, ALZA Corporation) for helpful discussions and Dr. Susan L. Palmieri (Cell Sciences Imaging Facility, Stanford University Medical Center) for her expert advice in the CLSM experiments.
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