Labrasol® and Salts of Medium-Chain Fatty Acids Can Be Combined in Low Concentrations to Increase the Permeability of a Macromolecule Marker Across Isolated Rat Intestinal Mucosae

In addition to their solubilizing properties, excipients used in lipid-based formulations can improve intestinal permeability of macromolecules. We determined whether admixing of medium-chain fatty acid (MCFA) permeation enhancers with a lipoidal excipient (Labrasol^®) could potentiate transepithelial flux of a poorly permeable macromolecule (fluorescein isothiocyanate dextran 4 kDa [FD4]) across rat intestinal mucosae mounted in Ussing chambers. Low concentrations of sodium caprate (C₁₀), sodium undecylenate (C_11:1), or sodium laurate (C₁₂) combined with Labrasol^® increased the apparent permeability coefficient (P_app) of FD4 to values typically seen with higher concentrations of MCFAs or Labrasol^® alone. For example, combination of C_11:1 (0.5 mg/mL) with Labrasol^® (1 mg/mL) increased the P_app of FD4 by 10- and 11-fold over the respective individual agents at the same concentrations where no enhancement was evident. The increased enhancement ratios seen with the combinations were associated with some perturbation in intestinal histology and with attenuation of an epithelial functional measure, carbachol-stimulated inward short-circuit current. In conclusion, combining three MCFAs separately with Labrasol^® increased the P_app of FD4 to values greater than those seen for MCFAs or Labrasol^® alone. Ultimately, this may permit lower concentrations of MCFA to be used in combination with other excipients in oral formulations of poorly permeable molecules.     

The Effect of β-Turn Structure on the Passive Diffusion of Peptides Across Caco-2 Cell Monolayers

Purpose. To investigate the relationships between the -turn structure of a peptide and its passive diffusion across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. Methods. Linear hydrophilic peptides (Ac-TyrProXaaZaaVal-NH₂; Xaa = Gly, Ile and Zaa = Asp, Asn) and hydrophobic (Ac-YaaPro-XaaIleVal-NH₂; Yaa = Tyr, Phe and Xaa = Gly, Ile: and Ac-PhePro-XaaIle-NH₂; Xaa = Gly, He) peptides were synthesized and their effective permeability coefficients (P_eff) were determined across Caco-2 cell monolayers. The lipophilicities of the peptides were estimated by measuring their partition coefficients (P_o/w) between 1-octanol and HBSS. Two-dimensional NMR (2D-NMR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the solution structures of these model peptides. Results. Using 2D-NMR spectroscopy and CD spectroscopy, the hydrophilic Gly-containing peptides (Ac-TyrProGlyZaaVal-NH₂; Zaa = Asp, Asn) were shown to exhibit a higher degree of -turn structure in solution than the Ile-containing peptides (Ac-TyrProIleZaaVal-NH₂; Zaa = Asp, Asn). CD spectroscopy was used to show that the Gly-containing hydrophobic peptides (Ac-YaaProGlyIleVal-NH₂; Yaa = Tyr, Phe: and Ac-PheProGlyIle-NH₂) exhibited a higher degree of -turn structure in solution than the Ile-containing hydrophobic peptides. The P_eff values of all four hydrophilic peptides across unperturbed Caco-2 cell monolayers were very low and no statistically significant differences were observed between the Gly- and Ile-containing penta-peptides within either the Asp or Asn series. The P_eff values for the hydrophobic Gly-containing peptides were significantly greater than the P_eff values determined for their Ile-containing counterparts. The Gly-containing penta- and tetrapeptides in the Phe series, which exhibited high permeation, were shown to be metabolically unstable. In contrast, the Gly- and Ile-containing pentapeptides in the Tyr series and the Ile-containing penta- and tetrapeptides in the Phe series, which exhibited low permeation, were metabolically stable. Conclusions. Hydrophobic peptides that exhibit significant -turn structure in solution are more lipophilic as measured by log P_o/w and more readily permeate Caco-2 cell monolayers via the transcellular route than hydrophobic peptides that lack this type of solution structure. The ability of these peptides to permeate Caco-2 cell monolayers via the transcellular route also exposed them to metabolism, presumably by cytosolic endopeptidases. Similar secondary structural features in hydrophilic peptides do not appear to sufficiently alter the physicochemical properties fo the peptides so as to alter their paracellular flux through unperturbed Caco-2 cell monolayers.     

The Effect of β-Turn Structure on the Permeation of Peptides Across Monolayers of Bovine Brain Microvessel Endothelial Cells

Purpose. To investigate the effects of the -turn structure of a peptide on its permeation via the paracellular and transcellular routes across cultured bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the blood-brain barrier (BBB). Methods. The effective permeability coefficients (P_eff) of the model peptides were determined across BBMEC monolayers. The dimensions of the aqueous pores in the tight junctions (TJs) of the BBMEC monolayers were determined using a series of hydrophilic permeants. This value and the molecular radius of each peptide were used to calculate the theoretical paracellular (P_P ^*) and transcellular (P_T ^*) permeability coefficients for each peptide. Results. A comparison of the theoretical P_P ^* values with the observed P_eff values was made for a series of model peptides. For the most hydrophobic peptides (Ac-PheProXaaIle-NH₂ and Ac-PheProXaaIleVal-NH₂; Xaa = Gly, Ile), it was concluded that the Gly-containing peptide of each pair more readily permeates BBMEC monolayers via the transcellular pathway than the Ile-containing analog. In addition, the Gly-containing peptides, which exhibit more -turn structure, were shown to be more lipophilic than the Ile-containing peptides as estimated by the log of their l-octanol:HBSS partition coefficients (log P_o/w). However, the three hydrophilic peptide pairs (Ac-TyrProXaaAspVal-NH₂, Ac-TyrProXaaAsnVal-NH₂, and Ac-TyrProXaaIleVal-NH₂; Xaa = Gly, Ile) were found to permeate BBMEC monolayers predominantly via the paracellular pathway. No differences were observed in the P_eff values of the hydrophilic peptides having higher -turn structures as compared to the peptides lacking these structural features. In addition, the Ile-containing peptides exhibited significantly higher log P_o/w values than the Gly-containing hydrophilic peptides. Conclusions. Hydrophobic peptides that exhibit significant -turn structure in solution are more lipophilic as measured by log P_o/w, and more readily permeate BBMEC monolayers via the transcellular route than hydrophobic peptides that lack this type of solution structure. Similar secondary structural features in hydrophilic peptides do not appear to sufficiently alter the physicochemical properties of the peptides so as to alter their paracellular flux through BBMEC monolayers.     

pH-Dependent Bidirectional Transport of Weakly Basic Drugs Across Caco-2 Monolayers: Implications for Drug–Drug Interactions

Purpose. The purpose of this study was to investigate the pH-dependent passive and active transport of weakly basic drugs across the human intestinal epithelium. Methods. The bidirectional pH-dependent transport of weak bases was studied in Caco-2 cell monolayers in the physiologic pH range of the gastrointestinal tract. Results. A net secretion of atenolol and metoprolol was observed when a pH gradient was applied. However, the bidirectional transport of both compounds was equal in the nongradient system. Hence, at lower apical than basolateral pH a change in passive transport caused by an imbalance in the concentration of the uncharged drug species resulted in a false asymmetry (efflux ratio). Furthermore, a mixture of pH-dependent passive and active efflux was found for the P-glycoprotein (P-gp, MDR1, ABCB1) substrates, talinolol and quinidine, but not for the neutral drug, digoxin. However, the clinically important digoxin-quinidine interaction depended on the presence of a pH gradient. Hence, the degree of interaction depends on the amount of quinidine available at the binding site of the P-gp. Conclusions. Active efflux of weak bases can only be accounted for when the fraction of unionized drug species is equal in all compartments because the transport is biased by a pH-dependent passive component. However, this component may take part in vivo and contribute to drug-drug interactions involving P-gp.     

Intestinal Transport of β-Lactam Antibiotics: Analysis of the Affinity at the H+/Peptide Symporter (PEPT1), the Uptake into Caco-2 Cell Monolayers and the Transepithelial Flux

Purpose. This study on the intestinal transport of -lactam antibiotics was undertaken to investigate the correlation between cellular transport parameters and the bioavailability. Methods. Transport of 23 -lactam antibiotics was characterized by measuring their ability to inhibit the uptake of glycylsarcosine into Caco-2 cells, their uptake into the cells and their total flux across the cell monolayers. Results. Ceftibuten and cyclacillin were recognized by PEPT1 with affinity constants comparable to those of natural dipeptides (K_i = 0.3 and 0.5 mM, respectively). Cefadroxil, cefamandole, cephradine, cefaclor, cefuroxime-axetil, cefixime, cephalotin, cephalexin and ampicillin also interacted with PEPT1 (K_i = 7-14 mM). In contrast, cefapirin, cefodizime, cefuroxime, cefmetazole, ceftazidime, benzyl-penicillin, ceftriaxone, cefpirome, cefotaxime, cefepime, cephaloridine and cefsulodin displayed no affinity to the transport system (K_i > 20 mM). The uptake into the cells and the transepithelial flux was highest for those -lactam antibiotics, which showed the strongest inhibition of [¹⁴C]Gly-Sar transport (p < 0.0001). Exceptions were cefuroxim-axetil and cephalotin. Conclusions. The probability of oral bioavailability for -lactam antibiotics is mainly determined by their affinity to PEPT1. A threshold K_i value of 14 mM with respect to Gly-Sar uptake is required.     

Mechanism and Kinetics of Transcellular Transport of a New β-Lactam Antibiotic Loracarbef Across an Intestinal Epithelial Membrane Model System (Caco-2)

Various processes involved in the transcellular transport (TT) of loracarbef (LOR) were studied in the Caco-2 cell monolayer, a cell culture model of the small intestinal epithelium. The results provide support for presence of two AP to BL peptide TT pathways in the intestinal epithelial cell monolayer (Caco-2). The H⁺ gradient-dependent pathway (K_m = 0.789 mM, and J_max = 163 pmol/min per cm²) is relatively high affinity and low capacity compared to H⁺ gradient-independent pathway (K_m = 8.28 mM, and J_max = 316 pmol/min per cm²). In addition, TT of LOR in the presence of a H⁺ gradient was inhibited 77% to 88% (p < 0.05) by 10 mM of cephalexin, enalapril, Gly-Pro and Phe-Pro, while TT of LOR in the absence of a H⁺ gradient was only inhibited 42% to 48% (p < 0.05) by 10 mM of Gly-Pro and Phe-Pro. Since AP uptake is H⁺ gradient-dependent and saturable while the BL efflux is mostly nonsaturable and not driven by a H⁺ gradient, these two transmembrane transport processes must be different, which could be the result of two different peptide carriers. In vivo, these two transport processes must have worked in concert to produce transcellular flux of loracarbef. To explain the differences between kinetic characteristics of AP uptake and TT transport, a cellular pharmacokinetic (PK) model was developed and the results indicate that the PK model appropriately described the kinetics of LOR TT. The use of this PK model may provide an additional advantage to the use of the cell culture model because kinetic parameters at both sides of the intestinal epithelial membrane may be obtained using the same preparation. Taken together, the Caco-2 model system represents an excellent model system for the study of carrier-mediated processes involved in the TT of peptides and peptide-like drugs.     

Non-Polarized Secretion of Mouse Interferon-β from Gene-Transferred Human Intestinal Caco-2 Cells

Purpose. The intestinal epithelium is considered to be a feasible target for somatic gene therapy. To this end, Caco-2 cells derived from human colon carcinoma were transfected with a mouse interferon- (IFN-) expression vector and several stable sublines were established; this hetero-specific cytokine allows unexpected cellular effects to be avoided. Using the highest mouse IFN--producing sublines, the mode of IFN secretion was examined. Methods. The secretion polarity of mouse IFN- in its gene-transduced Caco-2 sublines was studied in a bicameral culture system in which the chambers were separated by microporous filters. Results. Mouse IFN- was secreted to the same extent from both apical and basolateral surfaces of the transduced cells regardless of cell aging. Conclusions. These results suggest that in the intestinal epithelium exogenous gene products such as IFNs can be delivered to both the luminal and blood sides in vivo. Thus, the intestinal epithelium may be suitable for systemic and local delivery of therapeutic proteins by gene transfer.     

On the Mechanism and Dynamics of Uptake and Permeation of Polyether-Copolyester Dendrimers Across an In Vitro Blood–Brain Barrier Model

Dendrimers have emerged as a promising drug delivery system due to their well defined size, tailorability, and multifunctional nature. However, their application in brain delivery is relatively a new area of research. The present study was aimed at evaluating the uptake and permeation of polyether-copolyester (PEPE) dendrimers across the blood–brain barrier model and exploring the underlying mechanisms. Saturation was observed in the uptake of rhodamine B labeled PEPE dendrimers by brain vascular endothelial (bEnd.3) cells at high concentrations. Clathrin and caveolin inhibitors produced partial inhibition of the dendrimer uptake, signifying contribution of both pathways in the uptake process. PEPE dendrimers were able to cross in vitro BBB model in high amounts with P_app of 19.7 ± 1.9 × 10^?6 cm/s and 38.6 ± 4.1 × 10^?6 cm/s for den-1-(G2)-400 and den-2-(G2)-400, respectively; and only 11–14% reduction in transendothelial electrical resistance during initial 4 h. The results of this study suggest that architecture of dendrimers plays a major role not only in influencing the extent and mechanism of uptake by bEnd.3 cells but also permeation across the BBB model. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3748–3760, 2009     

The Effect of Novel Surfactants and Solutol® HS 15 on Paclitaxel Aqueous Solubility and Permeability Across a Caco-2 Monolayer

The effect of novel surfactants on the aqueous solubility and the permeability of paclitaxel across a Caco-2 cell monolayer were examined in this work. The solubility and permeability of paclitaxel was evaluated in the presence of four soft surfactants (SS) KXN441, KXN424, KXN437, and KXN 337 and Solutol® HS15. All surfactants increased the aqueous solubility of paclitaxel. Caco-2 cell membrane integrity in the presence of SS and Solutol® HS15 was assessed by mannitol permeability and LDH release. All surfactants were tested at 0.5 × CMC, 5 × CMC and 1.5 mM concentrations. The effect of SSs on paclitaxel permeability was concentration dependent. At all concentrations tested, KXN 441 and Solutol® HS 15 showed partially inhibition of drug efflux with no discernable change in mannitol permeability or cytotoxicity as observed with LDH release. At these concentrations, other SSs exhibited some partial efflux inhibition along with compromised membrane integrity and increasing mannitol permeability. In conclusion, all SSs were able to increase the aqueous solubility and permeability of paclitaxel across Caco-2 cells monolayer. However, KXN441 and Solutol® HS15 were able to enhance paclitaxel permeability across Caco-2 monolayer without cytotoxicity.     

Transport and Metabolism of the Tea Flavonoid (–)-Epicatechin by the Human Intestinal Cell Line Caco-2

Purpose: Tea flavonoids, including (–)-epicatechin (EC), have been suggested to have chemopreventive properties in cancer. However, there is limited knowledge of the oral bioavailability of these dietary compounds. The purpose of this study was to gain a better understanding of the absorption of EC. Methods: The intestinal epithelial membrane transport of EC was examined using the monolayer of the human Caco-2 cell line grown in Transwells, a common model of intestinal absorption. EC and its metabolites were measured by high performance liquid chromatography with diode array detection. Results: EC showed no apical to basolateral absorption at concentrations ranging from 5 to 50 M. In contrast, EC demonstrated basolateral to apical efflux with a P _app value of 0.67 ± 0.05 × 10^–6 cm/sec, i.e., slightly higher than for mannitol, 0.50 ± 0.30 × 10^–6 cm/sec, a paracellular transport marker. There was a 50% reduction in the efflux of EC in the presence of 50 M MK-571, a competitive inhibitor of the MRP2 transporter expressed in the apical membrane of Caco-2 cells. Most important, the presence of 50 M MK-571 resulted in clearly measurable apical to basolateral absorption of EC with a P _app of 0.31 ± 0.06 × 10^–6 cm/sec. Two polar metabolites, M1 and M2, were formed from EC, both of which appeared exclusively on the apical side. MK-571 (50 M) dramatically inhibited the transport for both of these metabolites. Incubations with inorganic ³⁵SO₄ ^2– and hydrolysis by aryl sulfatase strongly suggested that these metabolites were sulfate conjugates. Conclusions: These results suggest an important role for the multispecific organic anion transporter MRP2 in the bioavailability of EC and possibly other tea flavonoids.     

Prodrug Approach for αΙΙbβ3-Peptidomimetic Antagonists to Enhance Their Transport in Monolayers of a Human Intestinal Cell Line (Caco-2): Comparison of In Vitro and In Vivo Data

Purpose. Different lipophilic derivatives of a potent _b3- antagonist with benzamidino-oxazolidinone structure were investigated with respect to transport and metabolism properties to evaluate their potential as prodrugs with improved absorption behavior. Methods. Intestinal transport and metabolism of the compounds were studied in Caco-2 monolayers under in vitro conditions and quantitated by a reversed-phase HPLC- method. Peroral bioavailability in cynomolgus monkeys and inhibition of platelet aggregation (guinea pig) were compared to in vitro permeability coefficients. Results. N-alkoxycarbonyl- and N-benzoyl- derivatization of the benzamidine-parent drug increased the apparent permeabilities across Caco-2 monolayers by a factor of 25-100 fold. Most prodrugs were transported mainly by passive diffusion, whereas the methoxycarbonyl-derivative EMD 122347 displayed directional transport from basolateral (BL) to apical (AP). This polarized efflux was concentration dependent (saturable kinetics with K_m = 207 M, V_max = 0.275 nmol cm^-2 min^-1) and could be reduced in the presence of verapamil (300 M), an inhibitor of p-glycoprotein. Cell mediated cleavage of the prodrugs was low and showed only slight differences to hydrolysis in buffer solution, indicating a predominantly non enzymatic cleavage. Both peroral bioavailability (monkey) and the inhibition of ex-vivo platelet aggregation (guinea pig) gave the same rank order as the permeability coefficients obtained in Caco-2 monolayers. Conclusions. Alkoxycarbonylamidine- and benzoylamidine promoieties of a RGD mimetic _b3-antagonist considerably increased both effect bioavailabilities in animal experiments as well as in-vitro permeability in cell monolayers, demonstrating the potential of this approach to enhance transport of peptidomimetic drugs.     

Inhibitory Effect of Zinc on PEPT1-Mediated Transport of Glycylsarcosine and β-Lactam Antibiotics in Human Intestinal Cell Line Caco-2

Purpose. The aim of this study was to examine the effects of zinc on the intestinal peptide transporters (PEPT1 and basolateral peptide transporter) and to elucidate the mechanism of the interactions. Methods. Caco-2 cells were pretreated with zinc, and the uptake studies were carried out. Results. Zinc treatment resulted in the inhibition of [¹⁴C]glycylsarcosine (Gly-Sar) uptake via PEPT1 in a concentration-dependent manner, whereas it showed moderate inhibitory effect on the basolateral peptide transporter. Zinc also inhibited the uptake of oral -lactam antibiotics such as ceftibuten and cephradine by PEPT1. Kinetic analysis showed that zinc treatment increased K _m values without affecting V _max values of the [¹⁴C]Gly-Sar uptake. The inhibition of [¹⁴C]Gly-Sar uptake induced by zinc was observed in the presence of an H⁺ gradient but not in the absence of an H⁺ gradient. Conclusions. These results indicate that zinc is a competitive inhibitor of PEPT1. Zinc inhibited the PEPT1 function, possibly by interacting with histidine residues of PEPT1 that are part of an H⁺-binding site. These findings would provide important information for clinical, physiologic, and biochemical aspects of peptide transporters.     

Drug Delivery Studies in Caco-2 Monolayers. Synthesis,Hydrolysis, and Transport of O-Cyclopropane Carboxylic Acid Ester Prodrugs of Various β-Blocking Agents

A series of O-cyclopropane carboxylic acid ester prodrugs of various -blocking agents was synthesized. All prodrugs were hydrolyzed to give their parent compounds in aqueous phosphate buffer of pH 7.4 and in 80% human plasma. The half-lives in buffer solutions varied from 4 hours for the timolol prodrug to about 1 day for the prodrug of alprenolol. In human plasma the half-lives were shorter, ranging from 1 to 7 hours. The formation of the O-cyclopropane carboxylic acid ester derivatives significantly increased the lipophilicities of the -blockers as measured by the distribution coefficient between n-octanol and aqueous phosphate buffer of pH 7.4. To characterize the biomembrane permeability characteristics of the -blockers, transport properties across Caco-2 cell monolayers were investigated. An increase in lipophilicity resulted in a higher permeability of the prodrugs as compared to the parent compounds. Hence, acebutolol experienced an increment of a factor 17 on the apparent permeability coefficient, Papp, whereas Papp for the more lipophilic drug propranolol was increased by a factor of only 1.26. Some conversion of the prodrugs to their parent compounds was observed during the transport and appeared to be due to enzymatic intracellular metabolism.Deseaced.     

Preparation of Asialofetuin-Labeled Liposomes with Encapsulated Human Interferon-γ and Their Uptake by Isolated Rat Hepatocytes

The selective delivery of human recombinant interferon (IFN)- to isolated rat hepatocytes was studied with asialofetuin (AF)-labeled liposomes. AF-liposomes containing buffer solution were initially prepared by the detergent removal method, and IFN- was subsequently encapsulated by the freeze-thawing method without loss of activity. Virtually no free [³²P]IFN- was internalized into isolated rat hepatocytes, whereas AF-liposomes containing [³²P]IFN- were taken up to a significant degree. Liposomal binding to the hepatocytes (estimated at 4°C) was one-fifth of the uptake (estimated at 37°C). Since the uptake was inhibited by the addition of free AF, AF-liposomes may be taken up by the action of galactose-binding protein on the hepatocytic cell surface. The liposome preparation method reported in this paper provides a useful means for the encapsulation of unstable macromolecules into AF-liposomes. AF-liposomes were found effectively to carry IFN- into hepatocytes in vitro.     

Mechanism of L-α-Methyldopa Transport Through a Monolayer of Polarized Human Intestinal Epithelial Cells (Caco-2)

The Caco-2 model system (Hidalgo et al., Gastroenterology, 96:736–749, 1989), which is a monolayer of polarized intestinal epithelial cells grown onto a porous polycarbonate membrane, was used to study the mechanism of transcellular transport of an antihypertensive agent, L--methyldopa (L--MD). The results showed that the transport of L--MD was pH, glucose, concentration, and temperature dependent, and it could be inhibited by metabolic inhibitors (e.g., 2,4-dinitrophenol) and by amino acids (e.g., L-phenylalanine) which have an affinity for the large neutral amino acid (LNAA) carrier. In addition, the apparent kinetic constants describing the transcellular transport of L--MD were altered depending on the time interval between feeding the cells and the transport experiments (postfeeding time, PFT). The apparent maximum carrier flux (J _max) of L--MD was significantly increased (from 155 to 547 pmol/mg protein/min) when PFT was prolonged from 8.5 to 56 hr. These results indicated that the transcellular transport of L--MD through the polarized Caco-2 cell monolayer was carrier mediated via the LNAA carrier. The similarities in the characteristics of L--MD transport exhibited by the Caco-2 model system and other intestinal models in vitro further substantiate the usefulness of this cell culture model for studying the intestinal transport of nutrients and drugs.     

The Effects of Pluronic® Block Copolymers and Cremophor® EL on Intestinal Lipoprotein Processing and the Potential Link with P-Glycoprotein in Caco-2 Cells

Purpose. This investigation was performed to study the effects of Pluronic® block copolymers and Cremophor® EL on intestinal lipoprotein processing and to investigate a potential link between lipoprotein processing and P-glycoprotein. Methods. Caco-2 cells were used to monitor changes in lipoprotein production and secretion following exposure to excipients. Effects on P-glycoprotein were monitored using cyclosporin A as a model substrate. Results. A range of surfactants commonly used as pharmaceutical excipients in lipid-based oral drug delivery systems, including Pluronic® block copolymers L81, P85, and F68 and Cremophor® EL, inhibited intestinal lipoprotein secretion. The effects were concentration dependent and reversible. The mechanism of inhibition appears to be related to the assembly and secretion of lipoproteins rather than to initial intracellular triglyceride synthesis. A strong correlation was found between excipient-mediated inhibition of lipoprotein secretion and inhibition of P-glycoprotein efflux, implying a link between the two biochemical processes. Conclusions. The ability of such bioactive excipients to simultaneously manipulate different cellular processes must be considered in selecting excipients for oral drug delivery systems. Such information is particularly relevant when the drug is lipophilic, a candidate for P-glycoprotein efflux, and where intestinal lymphatic targeting via chylomicron stimulation is desirable.     

Metabolism of Testosterone During in Vitro Transport Across CACO-2 Cell Monolayers: Evidence for β-Hydroxysteroid Dehydrogenase Activity in Differentiated CACO-2 Cells

Testosterone has previously been used as a model compound for the determination of unstirred water layer thickness in the CACO-2 transport model. We have found, however, that testosterone is metabolized during in vitro transport across the CACO-2 cell monolayers. This suggests that testosterone is not an ideal model substance. Testosterone is metabolized to androstenedione, indicating the formation of 17--hydroxysteroid dehydrogenase by differentiated CACO-2 cells. No reverse metabolism is observed, thus androstenedione is considered superior to testosterone for determination of unstirred water layer thickness in the CACO-2 system. Permeability coefficients for testosterone and androstenedione obtained under identical transport conditions were 66 (±7) * 10 ^–6 (n = 26) and 84 (±7) * 10^–6 (n = 9) cm/sec, respectively. The unstirred water layer thicknesses at different agitation rates are determined for the CACO-2 transport model used in our laboratory utilizing androstenedione as a model compound. The system is capable of controlling the water layer thickness from about 200 to 1000 µm.     

Development and Application of an Isolated Perfused Rat Liver Model to Study the Stimulation and Inhibition of Tumor Necrosis Factor-α Production ex Vivo

Purpose. To develop an isolated perfused rat liver (IPRL) model with low baseline levels of tumor necrosis factor (TNF)- in the outlet perfusate to study the effects of immunostimulants and immunosuppressants on the release of TNF- from this organ. Methods. Isolated rat livers were perfused with a buffer containing no albumin or three different bovine serum albumin (BSA) preparations. Using the no-albumin perfusate, the inhibitory effects of methylprednisolone (MP) on lipopolysaccharide (LPS)-stimulated release of TNF- were studied in livers isolated 1 or 5 h after the intravenous administration (5 mg/kg) of MP. The concentrations of TNF- in the outlet perfusates were measured using enzyme-linked immunosorbent assay. Results. In the absence of albumin, the perfusate levels of TNF- were close to zero. However, when the perfusate contained BSA, the TNF- levels in the perfusate reached as high as 1200 pg/ml at steady state. An injection of LPS into IPRLs perfused with a no-albumin perfusate resulted in mean (± SD) TNF- steady-state concentrations of 825 ± 125 pg/ml. The pretreatment of rats with MP before liver harvest attenuated the LPS-induced TNF- release in the livers. However, the attenuation was substantial (>60%) and was statistically significant only 5 h after pretreatment with MP. Conclusions. Perfusates containing BSA may result in nonphysiologically high levels of TNF-. An IPRL with a no-albumin perfusate is more suitable for studies of the stimulation and inhibition of TNF- production by this organ.     

Transport and metabolism of the antitumour drug candidate 2′-benzoyloxycinnamaldehyde in Caco-2 cells

1. The transport and metabolism of the antitumour drug candidate 2′-benzoyloxycinnamaldehyde (BCA) was characterized in Caco-2 cells.

2. BCA disappeared rapidly from the donor side without being transported to the receiver side during its absorptive transport across Caco-2 cells. Its metabolites 2′-hydroxycinnamaldehyde (HCA) and o-coumaric acid (OCA) were formed in both the donor and the receiver sides.

3. HCA, in a separate study, also disappeared rapidly from the donor side, mostly being converted to its oxidative metabolite OCA during its absorptive transport across Caco-2 cells.

4. OCA was transported rapidly in the absorptive direction across Caco-2 cells with a P_app of 25.4?±?1.0?×?10^?6?cm s^?1 (mean?±?standard deviation (SD), n?=?3). OCA was fully recovered from both the donor and the receiver side throughout the time-course of this study.

5. Formation of HCA from BCA was inhibited almost completely by bis(p-nitrophenyl)phosphate (BNPP), a selective inhibitor of carboxylesterases (CES), and phenylmethylsulfonyl fluoride (PMSF), a broad specificity inhibitor of esterases in Caco-2 cells, suggesting that this hydrolytic biotransformation was likely mediated predominantly by CES. Conversion of HCA to OCA was inhibited significantly by isovanillin, a selective inhibitor of aldehyde oxidase (AO). Inhibitors for xanthine oxidase (XO) and aldehyde dehydrogenase (ALDH), which are known to be involved in the oxidation of aldehydes to carboxylic acids, did not have a significant effect on the biotransformation of HCA to OCA in Caco-2 cells.

6. In summary, the present work demonstrates that BCA is hydrolysed rapidly to HCA, followed by subsequent oxidation to OCA, in Caco-2 cells. The results provide a mechanistic understanding of the poor absorption and low bioavailability of BCA after oral administration.