Ismael J. Hidalgo

Characterization of the Human Colon Carcinoma Cell Line (Caco-2) as a Model System for Intestinal Epithelial Permeability

Caco-2 cells develop morphologic characteristics of normal enterocytes when grown on plastic dishes or nitrocellulose filters. The purpose of this study was to determine whether Caco-2 cells undergo similar differentiation when grown on Transwell polycarbonate membranes, and to study the suitability of Caco-2 monolayers as an intestinal epithelial transport model system. Transepithelial electrical resistance values after confluence were 173.5 omega.cm2 and remained unchanged through day 17. Permeabilities to the water-soluble fluid-phase markers that do not permeate the membrane, Lucifer yellow CH, [14C]inulin, [14C]polyethylene glycol, and [3H] dextran were less than 0.25% of the administered amount per hour after day 10. Qualitative evaluation of uptake and permeability to horseradish peroxidase confirmed the similarity in uptake and barrier properties between this cell system and the small intestinal epithelial layer. We conclude that Caco-2 cells grown on collagen-coated polycarbonate membranes should represent a valuable transport model system for the small intestinal epithelium.

The Use of Cultured Epithelial and Endothelial Cells for Drug Transport and Metabolism Studies

In an effort to develop novel strategies for delivery of drug candidates arising from rational drug design and recombinant DNA technology, pharmaceutical scientists have begun to employ the techniques of cell culture to study drug transport and metabolism at specific biological barriers. This review describes some of the general factors that should be considered in developing a cell culture model for transport studies and metabolism studies. In addition, we review in detail the recent progress that has been made in establishing, validating, and using cell cultures of epithelial barriers (e.g., cells that constitute the intestinal, rectal, buccal, sublingual, nasal, and ophthalmic mucosa as well as the epidermis of the skin) and the endothelial barriers (e.g., brain microvessel endothelial cells).

Transport of a large neutral amino acid (phenylalanine) in a human intestinal epithelial cell line: Caco-2

We have characterized the transcellular transport of a large neutral amino acid (LNAA) in Caco-2 cell monolayers. The apical (AP) to basolateral (BL) transport of phenylalanine (Phe) was approximately 10-times faster than BL-to-AP transport. The evidence for the carrier-mediated AP-to-BL transport of Phe include: (a) temperature dependence and saturability, (b) Phe transport was not affected by a reverse gradient, (c) the activation energy for transport was 12.0 kcal/mol, and (d) an excess amount of unlabeled Phe caused a 75% reduction in transport rate and a delay (lag time) in the appearance of Phe in the BL side. The Vm and Km for Phe transport were 572.4 pmol.mg protein-1.min-1 and 0.56 mM, respectively. Phe transport was decreased in the absence of glucose and in the presence of sodium azide or ouabain. The carrier interacted with LNAAs and with cationic amino acids but not with small neutral or anionic amino acids.

Transport of bile acids in a human intestinal epithelial cell line, Caco-2

The transport of taurocholic acid (TA) across Caco-2 cell monolayers was dependent on time in culture and reached a plateau after 28 days, at which time the apical (AP)-to-basolateral (BL) transport was 10-times greater than BL-to-AP transport. The amounts of TA inside the cells following application of 10 nM [14C]TA to the AP or BL side of the monolayers (30 min) were approximately equal (54.4 +/- 2.7 and 64.6 +/- 2.8 fmol/mg protein, respectively). AP-to-BL transport of TA was saturable and temperature-dependent. Vmax and Km for transport were 13.7 pmol/mg protein per min and 49.7 microM, respectively. The transport of TA had an activation energy of 13.2 kcal.mol-1, required Na+ and glucose. AP-to-BL transport of [14C]TA was inhibited by the co-administration (on the AP side) of either unlabeled TA or deoxycholate, but it was not reduced by the presence of unlabeled TA on the BL side.

CHARACTERIZATION OF THE UNSTIRRED WATER LAYER IN CACO-2 CELL MONOLAYERS USING A NOVEL DIFFUSION APPARATUS

Caco-2 monolayers grown on Transwell polycarbonate membranes have been characterized as a valuable tool in drug transport studies. Despite the clear advantages of this system, the lack of stirring may create an unstirred water layer (UWL) whose resistance may limit the transcellular transport of lipophilic molecules. The objective of this study was to evaluate a novel diffusion cell where the transport buffer is mixed by gas lift and to determine the mixing flow rate needed to reduce the thickness (h) of the UWL adjacent to cell monolayers. The transport of the leakage marker, mannitol, remained at least 15-fold lower than the flux of testosterone, indicating that the stirring flow rates used did not affect the integrity of the monolayers. The permeability (P) of testosterone (log PC 3.13) across monolayers mounted on this diffusion cell was 4.07, 10.90, and 14.18 × 10−5 cm/sec at flow rates of 0, 15, and 40 ml/min, respectively, and the apparent UWLs were calculated to be 1966, 733, and 564µm. P and h in the stagnant Transwell were 3.08 × 10−5 cm/sec and 2597 µm, respectively. On the other hand, h was significantly smaller in the unstirred, cell-free membranes than in their cell-containing counterparts. P was correlated with lipophilicity and, in the case of the more lipophilic compounds, with the mixing flow rate.

Effects of Human Immunodeficiency Virus Protease Inhibitors on the Intestinal Absorption of Tenofovir Disoproxil Fumarate In Vitro

ABSTRACT Human immunodeficiency virus protease inhibitors (PIs) modestly affect the plasma pharmacokinetics of tenofovir (TFV; −15% to +37% change in exposure) following coadministration with the oral prodrug TFV disoproxil fumarate (TDF) by a previously undefined mechanism. TDF permeation was found to be reduced by the combined action of ester cleavage and efflux transport in vitro. Saturable TDF efflux observed in Caco-2 cells suggests that at pharmacologically relevant intestinal concentrations, transport has only a limited effect on TDF absorption, thus minimizing the magnitude of potential intestinal drug interactions. Most tested PIs increased apical-to-basolateral TDF permeation and decreased secretory transport in MDCKII cells overexpressing P-glycoprotein (Pgp; MDCKII-MDR1 cells) and Caco-2 cells. PIs were found to cause a multifactorial effect on the barriers to TDF absorption. All PIs showed similar levels of inhibition of esterase-dependent degradation of TDF in an intestinal subcellular fraction, except for amprenavir, which was found to be a weaker inhibitor. All PIs caused a dose-dependent increase in the accumulation of a model Pgp substrate in MDCKII-MDR1 cells. Pgp inhibition constants ranged from 10.3 μM (lopinavir) to >100 μM (amprenavir, indinavir, and darunavir). Analogous to hepatic cytochrome P450-mediated drug interactions, we propose that the relative differences in perturbations in TFV plasma levels when TDF is coadministered with PIs are based in part on the net effect of inhibition and induction of intestinal Pgp by PIs. Combined with prior studies, these findings indicate that intestinal absorption is the mechanism for changes in TFV plasma levels when TDF is coadministered with PIs.

Enhanced intestinal absorption of an RGD peptide from water-in-oil microemulsions of different composition and particle size

Abstract The fibrinogen receptor antagonist SK&F 106760 is a water-soluble tetrapeptide (Mr634) with very low oil/water partitioning and membrane permeability. Thus, the intraduodenal bioavailability of this peptide in the rat from a saline formulation was found to be less than 1%. Upon formulation, however, in w/o microemulsions of different composition and particle size, the intraduodenal bioavailability of SK&F 106760 was increased up to 29% depending on the microemulsion composition. There was no apparent correlation between the particle size of microemulsions (mean droplet diameter of 0.010–1.0 μm) and enhanced absorption. None of the investigated microemulsions induced gross changes in gastrointestinal mucosa at a dosing volume of 3.3 ml/kg. Lipids containing medium-chain fatty acids play a major role on the observed absorption enhancement. These findings further support the use of microemulsion formulations for oral drug/peptide delivery, provided however, that development issues with these systems are properly addressed.

Uptake and transepithelial transport of the orally absorbed cephalosporin cephalexin, in the human intestinal cell line, Caco-2

Abstract Cephalexin (CPX) uptake in cultures of the human colon adenocarcinoma cell lines, Caco-2 and HT-29 has been shown to involve the di-/tripeptide transporter (DPT). However, little is known about the mechanism mediating the transepithelial (TE) transport of CPX either in vivo or in cultured cells. In this study, uptake and TE transport of CPX were investigated in Caco-2 monolayers grown on microporous membranes. Caco-2 cells did not show net TE transport of CPX when the pH of both apical (AP) and basolateral (BL) bathing solutions was 7.4. When the pH of the AP bathing solution was decreased from 7.4 to 6.0, while maintaining the pH of the BL bathing solution at 7.4, AP-BL transport of CPX (0.1 mM) increased from 0.1 to 0.23% h −1 cm −2 . Reversal of the pH gradient across the monolayer (AP, pH 7.4; BL, pH 6.0) did not alter the BL-AP flux of CPX. Manipulation of the AP or BL pH between 5.5 and 7.4 affected neither the AP-BL nor the BL-AP flux of mannitol (both ~0.1% h −1 cm −2 ), an internal marker of passive, paracellular diffusion. The pH-dependent AP uptake and AP-BL flux of CPX were time-, concentration- and temperature-dependent. Apparent half-maximal transport concentration ( K t ) and maximal transport velocity ( V t ) were 2.9 mM and 1.0 nmol min −1 mg protein −1 for AP uptake, and 4.7 mM and 0.13 nmol min −1 cm −2 (0.30 nmol min −1 mg protein −1 ) for AP-BL transport. The carrier-mediated AP uptake and AP-BL transport of CPX were inhibited by Gly-Pro, Pro-Gly, cephradine, cefadroxil, benzylpenicillin and ampicillin, but not by proline, glycine, valine, lysine or aspartic acid. In addition, CPX uptake was not inhibited by the nucleoside, adenosine, or the bile acid, taurocholic acid, suggesting that the uptake and TE transport of CPX involves the DPT but not other carriers present in the intestinal mucosa. We confirmed that the AP uptake of CPX involves mainly the DPT and conclude the following: (a) AP-BL transport of CPX is predominantly transcellular and involves a carrier, probably the DPT; (b) the rate-limiting step in AP-BL transport of cephalexin appears to be BL efflux and not AP uptake; (c) interaction with the DPT alone may be a poor predictor of substrate transport via this carrier; and (c) the Caco-2 culture system is a good model for studying mucosal uptake and TE transport of small peptides and peptidomimetic drugs.

Use of transporter knockdown Caco-2 cells to investigate the in vitro efflux of statin drugs.

The objective of the present study was to determine the efflux transporters responsible for acid and lactone statin drug efflux using transporter knockdown Caco-2 cells. The bidirectional transport was determined in Caco-2 cell monolayers in which the expression of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), or multidrug resistance associated protein 2 (MRP2) was knocked down by transduction with lentivirus containing human transporter-targeted small hairpin RNAs (shRNAs). Cells transduced with lentivirus containing nontargeted shRNA served as the vector control. Atorvastatin, lovastatin, and rosuvastatin displayed extremely low apical-to-basolateral (A-to-B) transport, which made the Papp,A-B values too unreliable to calculate the efflux ratio. Thus, transport comparisons were performed using the B-to-A permeability (Papp,B-A) values. Presented in the order of vector control, P-gp, BCRP, and MRP2 knockdown Caco-2 cells, the Papp,B-A values (×10−6, cm/s) were 28.1 ± 1.3, 8.6 ± 2.9, 20.3 ± 1.8, and 21.5 ± 1.6 for atorvastatin; 96.1 ± 7.1, 25.3 ± 3.5, 57.3 ± 9.8, and 48.2 ± 2.3 for fluvastatin; and 14.1 ± 1.9, 4.6 ± 1.7, 5.8 ± 0.7, and 6.6 ± 1.8 for rosuvastatin, respectively. Lovastatin and simvastatin showed no efflux in the vector control or knockdown cell monolayers in either lactone or acid forms. Results indicate that atorvastatin, fluvastatin, and rosuvastatin were transported by P-gp, BCRP, and MRP2. On the other hand, neither the lactone nor the resulting acid of lovastatin and simvastatin was transported by P-gp, BCRP, or MRP2. The current study demonstrated that the transporter knockdown Caco-2 cells are useful tools for studying drug-transporter interactions and should help eliminate some of the ambiguity associated with the identification of drug-transporter interactions based on chemical inhibitors alone.

Assessment of the First and Second Generation Antihistamines Brain Penetration and Role of P-Glycoprotein

PurposeThe sedating effect of first generation H1-antihistamines has been associated with their ability to penetrate the blood-brain barrier (BBB) and lack of efflux by P-glycoprotein (Pgp). Second generation H1-antihistamines are relatively free of sedation and their limited brain penetration has been suggested to arise from Pgp-mediated efflux. The objective of this work was to evaluate the role of Pgp in brain penetration of first and second generation antihistamines.MethodsPotential of antihistamines to be Pgp substrates was tested in vitro using Madin Darby canine kidney cells transfected with human Pgp. The role of Pgp in limiting brain penetration of antihistamines was tested by using the in situ brain perfusion technique.ResultsMajority of antihistamines were Pgp substrates in vitro. Following in situ brain perfusion, the first generation antihistamines substantially penetrated into rat brain independently from Pgp function. The second generation antihistamines terfenadine and loratadine, achieved substantial brain penetration, which was further enhanced by Pgp inhibition by cyclosporin A (CSA). In contrast, fexofenadine and cetirizine, penetrated brain poorly regardless of CSA administration.ConclusionsAntihistamines greatly differ in their ability to cross the BBB as well as in the role of Pgp in limiting their transport into the CNS in vivo.