Transepithelial Transport Studies Research Articles

In vitro Bioaccessibility, Bioavailability, and Plasma Protein Interaction of New Oral Anticoagulants in the Presence of Macronutrients.

Over the last years, a new generation of oral anticoagulants (NOACs), including apixaban, rivaroxaban and dabigatran, has been developed for the control of thrombosis and related disorders. The presence of food within the gastrointestinal (GI) tract can potentially affect the oral bioavailability of drugs. In the present paper, we evaluated the stability of these drugs in in vitro GI digestion, with and without the main macronutrients such as proteins, lipids, carbohydrates, and fibers, and their ability to enter into the systemic circulation. In addition, we examined the percentage of the drug binding to plasma proteins, such as human serum albumin, high density- and low density lipoproteins. The NOACs bioaccessibility was evaluated by an in vitro procedure simulating the gastrointestinal enzymatic system, while their bioavailability was studied by cell culture of Caco-2 cells and in vitro study of transepithelial transport. The in vitro transepithelial permeated NOACs were added to plasma protein solutions simulating the average fasting plasma protein concentrations. The NOACs detection was carried out by HPLC-DAD/ESI-MS analysis. GI digestion significantly reduces intestinal bioaccessibility and bioavailability of NOACs, especially as regards apixaban and dabigatran. Interestingly, the co-digestion of fibers led to a strong decrease of NOAC intestinal bioaccessibility and bioavailability, while the effects of the other macronutrients, as well as a low fiber standard meal, had no significant influence in this sense. Dabigatran, rivaroxaban and apixaban may be administered independently of a standard meal, provided that it does not include a high amount of dietary fibers.

The Cell Line NCl-H441 Is a Useful in Vitro Model for Transport Studies of Human Distal Lung Epithelial Barrier

The lack of a well characterized, continuously growing in vitro model of human distal lung epithelial phenotype constitutes a serious limitation in the area of inhalation biopharmaceutics, particularly in the context of transepithelial transport studies. Here, we investigated if a human lung adenocarcinoma cell line, NCl-H441, has potential to serve as an in vitro model of human distal lung epithelium. The development of barrier properties was studied by immunocytochemistry (ICC) against the junction proteins zonula occludens protein 1 (ZO-1) and E-cadherin and measurement of transepithelial electrical resistance (TEER). Moreover, transport studies with the paracellular marker compounds fluorescein sodium and fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights ranging from 4 to 70 kDa were carried out. The expression of P-glycoprotein (P-gp; ABCB1) and organic cation transporters (OCT/Ns; SLC22A1-A5) was investigated by ICC and immunoblot. P-gp function was assessed by monolayer release and bidirectional transport studies using rhodamine 123 (Rh123) and the inhibitors verapamil and LY335979. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was measured, in order to assess organic cation transporter function in vitro. Furthermore, the inhibitory potential of several organic cations on ASP(+) uptake was studied. NCl-H441 cells, when grown under liquid-covered conditions, formed confluent, electrically tight monolayers with peak TEER values of approximately 1000 Ω·cm(2), after 8-12 days in culture. These monolayers were able to differentiate paracellularly transported substrates according to their molecular weight. Presence of P-gp, OCT1, OCT2, OCT3, OCTN1, and OCTN2 was confirmed by Western blot and ICC and was similar to data from freshly isolated human alveolar epithelial cells in primary culture. Rh123 release from NCI-H441 monolayers was time-dependent and showed low, albeit significant attenuation by both inhibitors. In transport studies, Rh123 exhibited net secretion, which again was inhibitable by bona fide P-gp modulators. The uptake of ASP(+) was time- and temperature-dependent with Km = 881.2 ± 195.3 μM and Vmax = 2.07 ± 0.26 nmol/min/mg protein. TEA, amantadine, quinidine, and verapamil significantly inhibited ASP(+) uptake into NCl-H441 cells, whereas the effect of d- and l-carnitine and ergothioneine, two OCTN substrates, was less pronounced. NCl-H441 cells are the first cell line of human distal lung epithelial origin with the ability to form monolayers with appreciable barrier properties. Moreover, drug transporter expression and activity in NCl-H441 cells was consistent with what has been reported for human alveolar epithelial cells in primary culture.

Comparative study of deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol on intestinal transport and IL-8 secretion in the human cell line Caco-2

The effects of the trichothecene mycotoxin deoxynivalenol (DON) and its acetylated derivatives, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) on human intestinal cell Caco-2 were investigated by the studies of transepithelial transport, gene expression, and cytokine secretion. Permeability across a Caco-2 cell monolayer was evaluated by transport study. Transport rates were ranked as DON, 3ADON<15ADON in apical–basolateral direction. 15ADON showed the highest permeability, induced the highest decrease in transepithelial electrical resistance (TEER), and prompted significant Lucifer Yellow permeability. These results showed that 15ADON affect paracellular barrier function extremely. In addition, gene expressions induced by toxins were screened by DNA microarray for investigating cellular effect on Caco-2 cell. The most remarkable gene induced by DON and 15ADON was inflammatory chemokine IL-8 and thus mRNA expression and secretion of IL-8 were analyzed by PCR and ELISA. Both DON and acetylated DONs could induce mRNA expression and production of IL-8. In particular, ELISA assay showed that the ability to produce IL-8 was ranked as 3ADON<DON<15ADON. Our results indicated that 15ADON caused the highest permeability and highest IL-8 secretion among DON, 3ADON, and 15ADON in human intestinal cell.

Targeted lipid based drug conjugates: A novel strategy for drug delivery

A majority of studies involving prodrugs are directed to overcome low bioavailability of the parent drug. The aim of this study is to increase the bioavailability of acyclovir (ACV) by designing a novel prodrug delivery system which is more lipophilic, and at the same time site specific. In this study, a lipid raft has been conjugated to the parent drug molecule to impart lipophilicity. Simultaneously a targeting moiety that can be recognized by a specific transporter/receptor in the cell membrane has also been tethered to the other terminal of lipid raft. Targeted lipid prodrugs i.e., biotin-ricinoleicacid-acyclovir (B-R-ACV) and biotin-12hydroxystearicacid-acyclovir (B-12HS-ACV) were synthesized with ricinoleicacid and 12hydroxystearicacid as the lipophilic rafts and biotin as the targeting moiety. Biotin-ACV (B-ACV), ricinoleicacid-ACV (R-ACV) and 12hydroxystearicacid-ACV (12HS-ACV) were also synthesized to delineate the individual effects of the targeting and the lipid moieties. Cellular accumulation studies were performed in confluent MDCK-MDR1 and Caco-2 cells. The targeted lipid prodrugs B-R-ACV and B-12HS-ACV exhibited much higher cellular accumulation than B-ACV, R-ACV and 12HS-ACV in both cell lines. This result indicates that both the targeting and the lipid moiety act synergistically toward cellular uptake. The biotin conjugated prodrugs caused a decrease in the uptake of [3H] biotin suggesting the role of sodium dependent multivitamin transporter (SMVT) in uptake. The affinity of these targeted lipid prodrugs toward SMVT was studied in MDCK-MDR1 cells. Both the targeted lipid prodrugs B-R-ACV (20.25±1.74μM) and B-12HS-ACV (23.99±3.20μM) demonstrated higher affinity towards SMVT than B-ACV (30.90±4.19μM). Further, dose dependent studies revealed a concentration dependent inhibitory effect on [3H] biotin uptake in the presence of biotinylated prodrugs. Transepithelial transport studies showed lowering of [3H] biotin permeability in the presence of biotin and biotinylated prodrugs, further indicating a carrier mediated translocation by SMVT. Overall, results from these studies clearly suggest that these biotinylated lipid prodrugs of ACV possess enhanced affinity towards SMVT. These prodrugs appear to be potential candidates for the treatment of oral and ocular herpes virus infections, because of higher expression of SMVT on intestinal and corneal epithelial cells. In conclusion we hypothesize that our novel prodrug design strategy may help in higher absorption of hydrophilic parent drug. Moreover, this novel prodrug design can result in higher cell permeability of hydrophilic therapeutics such as genes, siRNA, antisense RNA, DNA, oligonucleotides, peptides and proteins.

Influence of charge on FITC-BSA-loaded chondroitin sulfate-chitosan nanoparticles upon cell uptake in human Caco-2 cell monolayers

Background and methodsChondroitin sulfate-chitosan (ChS-CS) nanoparticles and positively and negatively charged fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA)-loaded ChS-CS nanoparticles were prepared and characterized. The properties of ChS-CS nanoparticles, including cellular uptake, cytotoxicity, and transepithelial transport, as well as findings on field emission-scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy were evaluated in human epithelial colorectal adenocarcinoma (Caco-2) fibroblasts. ChS-CS nanoparticles with a mean particle size of 250 nm and zeta potentials ranging from −30 to +18 mV were prepared using an ionic gelation method.ResultsStandard cell viability assays demonstrated that cells incubated with ChS-CS and FITC-BSA-loaded ChS-CS nanoparticles remained more than 95% viable at particle concentrations up to 0.1 mg/mL. Endocytosis of nanoparticles was confirmed by confocal laser scanning microscopy and measured by flow cytometry. Ex vivo transepithelial transport studies using Caco-2 cells indicated that the nanoparticles were effectively transported into Caco-2 cells via endocytosis. The uptake of positively charged FITC-BSA-loaded ChS-CS nanoparticles across the epithelial membrane was more efficient than that of the negatively charged nanoparticles.ConclusionThe ChS-CS nanoparticles fabricated in this study were effectively endocytosed by Caco-2 fibroblasts without significant cytotoxicity at high nanoparticle concentrations. ChS-CS nanoparticles represent a potential novel delivery system for the transport of hydrophilic macromolecules.

Regulation of brain copper homeostasis by the brain barrier systems: Effects of Fe-overload and Fe-deficiency

Maintaining brain Cu homeostasis is vital for normal brain function. The role of systemic Fe deficiency (FeD) or overload (FeO) due to metabolic diseases or environmental insults in Cu homeostasis in the cerebrospinal fluid (CSF) and brain tissues remains unknown. This study was designed to investigate how blood-brain barrier (BBB) and blood-SCF barrier (BCB) regulated Cu transport and how FeO or FeD altered brain Cu homeostasis. Rats received an Fe-enriched or Fe-depleted diet for 4 weeks. FeD and FeO treatment resulted in a significant increase (+ 55%) and decrease (− 56%) in CSF Cu levels (p < 0.05), respectively; however, neither treatment had any effect on CSF Fe levels. The FeD, but not FeO, led to significant increases in Cu levels in brain parenchyma and the choroid plexus. In situ brain perfusion studies demonstrated that the rate of Cu transport into the brain parenchyma was significantly faster in FeD rats (+ 92%) and significantly slower (− 53%) in FeO rats than in controls. In vitro two chamber Transwell transepithelial transport studies using primary choroidal epithelial cells revealed a predominant efflux of Cu from the CSF to blood compartment by the BCB. Further ventriculo-cisternal perfusion studies showed that Cu clearance by the choroid plexus in FeD animals was significantly greater than control (p < 0.05). Taken together, our results demonstrate that both the BBB and BCB contribute to maintain a stable Cu homeostasis in the brain and CSF. Cu appears to enter the brain primarily via the BBB and is subsequently removed from the CSF by the BCB. FeD has a more profound effect on brain Cu levels than FeO. FeD increases Cu transport at the brain barriers and prompts Cu overload in the CNS. The BCB plays a key role in removing the excess Cu from the CSF.

Multiple KCNQ Potassium Channel Subtypes Mediate Basal Anion Secretion from the Human Airway Epithelial Cell Line Calu-3

Potassium channels play an important role in providing a driving force for anion secretion from secretory epithelia. To investigate the role of KCNQ K+ channels in mediating rates of basal anion secretion across the human airway submucosal gland serous cell model, the Calu-3 cell, we examined the expression, localization and function of these channels. In addition to our previous knowledge that Calu-3 cells express KCNQ1, using reverse transcriptase polymerase chain reaction we determined expression of KCNQ3, KCNQ4 and KCNQ5 mRNA transcripts. Immunoblotting detected KCNQ1, KCNQ3 and KCNQ5 proteins, while KCNQ4 protein was not found. Immunolocalization using polarized Calu-3 cell monolayers revealed that KCNQ1 and KCNQ3 were located in or toward the apical membrane of the cells, while KCNQ5 was detected in the apical and lateral membranes. Transepithelial transport studies revealed a small chromanol 293B-sensitive current at the apical membrane, likely KCNQ1. Application of XE991, an inhibitor of all members of the KCNQ channel family, inhibited the basal short-circuit current when applied to both sides of the cells to a greater extent than 293B, with the largest inhibition seen upon apical application. This result was confirmed using linopiridine, a less potent analogue of XE991, and suggests that functional KCNQ3 and KCNQ5, in addition to KCNQ1, are present at the apical aspect of these cells. These results demonstrate the role of a number of KCNQ channel members in controlling basal anion secretion across Calu-3 cells, while also demonstrating the importance of apically located K+ channels in mediating anion secretion in the airway epithelium.

Tacrine-induced liver damage: an analysis of 19 candidate genes

Tacrine, the first acetylcholinesterase inhibitor used in the treatment of Alzheimer's disease, is associated with transaminase elevation in up to 50% of patients. The mechanism of tacrine-induced liver damage is not fully understood, but earlier studies have suggested that genetic factors may play a role. Our aim was to investigate whether single-nucleotide polymorphisms (SNPs) in 19 candidate genes were associated with tacrine-induced liver damage. Sixty-nine patients of Caucasian origin treated with tacrine for Alzheimer's disease were investigated by genotyping 241 SNPs in 19 candidate genes potentially related to hepatotoxicity. The association with ABCB4 [which encodes MultiDrug Resistance Protein 3 (MDR3)] was explored in transepithelial transport studies using the ABCB4-transfected pig kidney epithelial cell line (LLC-PK1). The strongest association between alanine aminotransferase levels and three SNPs within ATP-binding cassette, subfamily B (MDR/TAP), member 4 (ABCB4) (uncorrected P=0.0005) was not significant after adjusting for multiple testing. No association was demonstrated with ATP-binding cassette, subfamily B (MDR/TAP), member 1 (ABCB1) or carnitine O-octanoyltransferase (CROT) which are located adjacent to ABCB4. Using the transepithelial transport system we failed to show a difference in tacrine accumulation between ABCB4-transfected and parental cell lines. The association with ABCB4 warrants further testing using either another population and/or functional studies.

Functional characterization and cloning of amino acid transporter B0,+ (ATB0,+) in primary cultured rat pneumocytes

Cationic amino acid transport in primary cultured rat pneumocytes exhibiting characteristics of alveolar epithelial type I-like cells are described. Asymmetry and activator ion dependency of (3)H-L-arginine uptake were characterized from the apical or basolateral fluid of pneumocytes grown on permeable support. Substrate specificity of transport was evaluated as a function of (3)H-L-arginine uptake inhibition in the presence of other amino acids. Transepithelial transport studies estimated (3)H-L-arginine flux in the apical-to-basolateral and basolateral-to-apical directions. Full length cDNA of rat amino acid transporter B(0,+) (rATB(0,+)) was cloned and its relative expression level studied. Results indicate that uptake of (3)H-L-arginine from apical fluid is dependent on Na(+) and Cl(-). Zwitterionic and cationic amino acids (excluding L-proline and anionic amino acids) inhibited uptake of (3)H-L-arginine from apical, but not basolateral incubation fluid. Apical-to-basolateral transepithelial flux of (3)H-L-arginine was 20x higher than basolateral-to-apical transport. Kinetic studies of (3)H-L-arginine uptake from apical fluid revealed maximal velocity (V(max)) and Michaelis-Menten constants (K(t)) of 33.32 +/- 2.12 pmol/mg protein/15 min and 0.50 +/- 0.11 mM, respectively, in a cooperative process having a coupling ratio of 1.18 +/- 0.16 with Na(+) and 1.11 +/- 0.13 with Cl(-). Expression of rATB(0,+) mRNA was identified by RT-PCR and Northern analysis. Corresponding cloned 3.2 kb rATB(0,+) cDNA sequence exhibits pronounced homology in deduced amino acid sequence to mouse (95% identity and 97% similarity) and human (89% identity and 95% similarity) ATB(0,+) homologues. We conclude that rat pneumocytes express ATB(0,+), which may partly contribute towards recovering cationic and neutral amino acids from alveolar luminal fluid.

Functional Characterization of Sodium-Dependent Multivitamin Transporter in MDCK-MDR1 Cells and Its Utilization as a Target for Drug Delivery

The objective of this research is to characterize a sodium-dependent multivitamin transporter (SMVT) in MDCK-MDR1 cells (Madin-Darby canine kidney cells transfected with the human MDR1 gene) and to investigate the feasibility of utilizing the MDCK-MDR1 cell line as an in vitro model to study the permeability of biotin-conjugated prodrugs of anti-HIV protease inhibitors. Mechanism of [3H]biotin uptake and transport was delineated. Transepithelial permeability of the biotin-conjugated prodrug, i.e., biotin-saquinavir, was also studied. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to confirm the existence of SMVT in MDCK-MDR1 cells. Biotin uptake was Na+, pH, and temperature dependent, but energy independent. Uptake of biotin was found to be saturable with a Km of 13.0 microM, Vmax 21.5 of pmol min-1 (mg of protein)-1, and Kd of 0.12 microL min-1 (mg of protein)-1. Both apical and basal uptake and transepithelial transport of [3H]biotin showed that SMVT localized predominantly on the apical membrane of MDCK-MDR1 cells. [3H]Biotin uptake was inhibited by excess unlabeled biotin and its structural analogues, i.e., desthiolbiotin and valeric acid, and other vitamins such as lipoic acid and pantothenic acid, but not by acetic acid, benzoic acid, biotin methyl ester, and biocytin. Biotin-saquinavir caused lowering of [3H]biotin uptake, which indicates that it is recognized by SMVT. Apical to basal transport of [3H]biotin was also significantly inhibited in the presence of excess biotin or biotin-saquinavir. Transepithelial transport studies of biotin-saquinavir in MDCK-MDR1, wild type MDCK, and Caco-2 cells revealed that permeability of biotin-saquinavir was similar in all three cell lines. A band of SMVT mRNA at 862 bp was identified by RT-PCR. A sodium-dependent multivitamin transporter, SMVT, responsible for biotin uptake and transport, was identified and functionally characterized in MDCK-MDR1 cells. Therefore, the MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of biotin-conjugated prodrugs such as HIV protease inhibitors.

Evasion of P-gp mediated cellular efflux and permeability enhancement of HIV-protease inhibitor saquinavir by prodrug modification

P-glycoprotein (P-gp) is an efflux pump responsible for limiting oral bioavailability, tissue penetration and increasing metabolism of the HIV protease inhibitor saquinavir (SQV). The objective of this study is to investigate whether prodrug derivatization of SQV to novel dipeptide prodrugs Val–Val–saquinavir (Val–Val–SQV) and Gly–Val–saquinavir (Gly–Val–SQV) targeting peptide transporters can enhance cellular permeability of saquinavir and modulate P-gp mediated efflux. Uptake and transport studies were conducted employing MDCKII-MDRI cell line at 37 °C for 10 min and 3 h, respectively. Uptake of [ 3H]ritonavir and [ 3H]erythromycin, utilized as model P-gp substrates, was carried out in the presence of inhibitory concentration of SQV and its peptide prodrugs. Bidirectional transport studies were conducted on MDCKII-MDR1 cells grown over membrane inserts. Uptake of [ 3H]erythromycin by MDCKII-MDR1 cells exhibited a four-fold increase in the presence of 75 μM SQV. However, equimolar concentrations of Val–Val–SQV and Gly–Val–SQV showed only 2.5-fold increase in [ 3H]erythromycin uptake. Concentration dependent inhibition of [ 3H]glycylsarcosine (Gly-Sar), a model peptide transporter substrate, was observed in the presence of SQV prodrugs. Transepithelial transport studies of Val–Val–SQV and Gly–Val–SQV exhibited an enhanced absorptive flux and reduced secretory flux relative to studies employing SQV. These results are very likely due to decreased efflux of SQV dipeptide prodrugs by P-gp. Peptide prodrug derivatization constitutes an exciting strategy to improve intestinal absorption and oral bioavailability of SQV.

Prodrugs of HIV Protease Inhibitors — Saquinavir, Indinavir and Nelfinavir — Derived from Diglycerides or Amino Acids: Synthesis, Stability and anti‐HIV Activity.

With the aim of improving the pharmacological properties of current protease inhibitors (PIs), the synthesis of various acyl and carbamate amino acid- or diglyceride-containing prodrugs derived from saquinavir, indinavir and nelfinavir, their in vitro stability with respect to hydrolysis and their anti-HIV activity in CEM-SS and MT4 cells have been investigated. L-Leucine (Leu) and L-phenylalanine (Phe) were connected through their carboxyl to the PIs while L-tyrosine (Tyr) was conjugated through its aromatic hydroxyl via various spacer units. Hydrolysis of the prodrug with liberation of the active free drug was crucial for antiviral activity. The Leu- and Phe-PI prodrugs released the active free drug very rapidly (half-lives of hydrolysis in buffer at 37 °C of 3–4 h). The Tyr-PI conjugates with a –C(O)(CH2)4- linker exhibited half-lives in the 40–70 h range and antiviral activities in the 21–325 nM range (from 2 to 22 nM for the free PIs). The chemically very stable carbamate “peptidomimetic” Tyr-PI prodrugs (no hydrolysis detected after 7 days in buffer) displayed a very low anti-HIV activity or were even inactive (EC50 from 2300 nM to >10 µM). A very low antiviral activity was measured for the diglyceride-substituted saquinavir and for all of the disubstituted indinavir and nelfinavir prodrugs. All these prodrugs probably released the active parent PI too slowly under the antiviral assay conditions. These results combined with those from transepithelial transport studies (Rouquayrol et al., Pharm. Res., 2002, 19, 1704–1712) indicate that conjugation of amino acids (through their carboxyl) to the PIs constitutes a most appealing alternative which could improve the intestinal absorption of the PIs and reduce their recognition by efflux carriers.

Phe-Gly dipeptidomimetics designed for the di-/tripeptide transporters PEPT1 and PEPT2: synthesis and biological investigations.

A series of five Phe-Gly dipeptidomimetics containing different amide bond replacements have been synthesized in a facile way from the readily available unsaturated ketoester 1, and their affinities for the di-/tripeptide transporters hPEPT1 (Caco-2 cells) and rPEPT2 (SKPT cells) were tested. The compounds contained the amide bond isosteres ketomethylene (2a), (R)- and (S)-hydroxyethylidene (3a and 4a), and (R)- and (S)-hydroxyethylene (5a and 6a) to provide information on the conformational and stereochemical requirements for hPEPT1 and rPEPT2 affinity. The affinity studies showed that for rPEPT2 there is no significant difference in affinity between the ketomethylene isostere 2a and the natural substrate Phe-Gly (K(i) values of 18.8 and 14.6 microM, respectively). Also the affinities for hPEPT1 are in the same range (K(i) values of 0.40 and 0.20 mM, respectively). This corroborates earlier findings that the amide bond as such is not essential for binding to PEPTX, but the results also reveal possible differences in the binding of ketomethylene isosteres to hPEPT1 and rPEPT2. The trans-hydroxyethylidene and hydroxyethylene isosteres proved to be poor substrates for PEPTX. These results provide new information about the importance of flexibility and of the stereochemistry at the C(4)-position for this class of compounds. Furthermore, the intracellular uptake of 2a-4a in Caco-2 cells was investigated, showing a 3-fold reduction of the uptake of 2a in the presence of the competetive inhibitor Gly-Pro, indicating contribution from an active transport component. No active uptake of 3a and 4a was observed. Transepithelial transport studies also indicated active transport of 2a across Caco-2 monolayers.

Prodrugs of HIV protease inhibitors-saquinavir, indinavir and nelfinavir-derived from diglycerides or amino acids: synthesis, stability and anti-HIV activity.

With the aim of improving the pharmacological properties of current protease inhibitors (PIs), the synthesis of various acyl and carbamate amino acid- or diglyceride-containing prodrugs derived from saquinavir, indinavir and nelfinavir, their in vitro stability with respect to hydrolysis and their anti-HIV activity in CEM-SS and MT4 cells have been investigated. l-Leucine (Leu) and l-phenylalanine (Phe) were connected through their carboxyl to the PIs while l-tyrosine (Tyr) was conjugated through its aromatic hydroxyl via various spacer units. Hydrolysis of the prodrug with liberation of the active free drug was crucial for antiviral activity. The Leu- and Phe-PI prodrugs released the active free drug very rapidly (half-lives of hydrolysis in buffer at 37 degree C of 3-4 h). The Tyr-PI conjugates with a -C(O)(CH(2))(4)- linker exhibited half-lives in the 40-70 h range and antiviral activities in the 21-325 nM range (from 2 to 22 nM for the free PIs). The chemically very stable carbamate "peptidomimetic" Tyr-PI prodrugs (no hydrolysis detected after 7 days in buffer) displayed a very low anti-HIV activity or were even inactive (EC(50) from 2300 nM to >10 microM). A very low antiviral activity was measured for the diglyceride-substituted saquinavir and for all of the disubstituted indinavir and nelfinavir prodrugs. All these prodrugs probably released the active parent PI too slowly under the antiviral assay conditions. These results combined with those from transepithelial transport studies (Rouquayrol et al., Pharm. Res., 2002, 19, 1704-1712) indicate that conjugation of amino acids (through their carboxyl) to the PIs constitutes a most appealing alternative which could improve the intestinal absorption of the PIs and reduce their recognition by efflux carriers.

Dipeptide model prodrugs for the intestinal oligopeptide transporter. Affinity for and transport via hPepT1 in the human intestinal Caco-2 cell line

The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a drug delivery target via increasing the intestinal transport of low permeability compounds by designing peptidomimetic prodrugs. Model ester prodrugs using the stabilized dipeptides d-Glu–Ala and d-Asp–Ala as pro-moieties for benzyl alcohol have been shown to maintain affinity for hPepT1. The primary aim of the present study was to investigate if modifications of the benzyl alcohol model drug influence the corresponding d-Glu–Ala and d-Asp–Ala model prodrugs’ affinity for hPepT1 in Caco-2 cells. A second aim was to investigate the transepithelial transport and hydrolysis parameters for d-Asp(BnO)–Ala and d-Glu(BnO)–Ala across Caco-2 cell monolayers. In the present study, all investigated d-Asp–Ala and d-Glu–Ala model prodrugs retained various degrees of affinity for hPepT1 in Caco-2 cells. These affinities are used to establish a QSAR of our benzyl alcohol modified model prodrugs, aided at elucidating the observed differences in model prodrug affinity for hPepT1; additionally, these data suggest that the hydrophobicity of the side-chain model drug is the major determinant in the compounds affinity for hPepT1. Transepithelial transport studies performed using Caco-2 cells of d-Asp(BnO)–Ala and d-Glu(BnO)–Ala showed that the K m for transepithelial transport was not significantly different for the two compounds. The maximal transport rate of the carrier-mediated flux component does not differ between the two model prodrugs either. The transepithelial transport of d-Asp(BnO)–Ala and d-Glu(BnO)–Ala follows simple kinetics, and the release of benzyl alcohol is pH-dependent, but unaffected by 1 mM of the esterase inhibitor Paraoxon in 80% human plasma and Caco-2 cell homogenate.

Transient alterations in cellular permeability in cultured human proximal tubule cells: implications for transport studies.

Primary cultures of human proximal tubule (HPT) cells possess the characteristics of a tight epithelium and retain the characteristics of in vivo renal function. HPT cells form confluent monolayers when grown on collagen-coated polycarbonate inserts in a hormonally defined serum-free medium. However, initial studies of transepithelial transport observed large bidirectional fluxes of the paracellular probe inulin. The present studies were designed to assess the transformation of HPT cell tight junctions to a "leaky" state and subsequent recovery. The apparent transepithelial electrical resistance of HPT cells at confluence was 952.0 +/- 70.0 ohms*cm2, suggesting a well-developed tight junction-mediated paracellular pathway in this epithelium. However, replacement of the growth media produced an immediate 90% drop in the initial resistance, which was paralleled by an increased flux of inulin and of phenol red. This transient abolition of barrier function spontaneously reestablished over 1-2 h by a process that was dependent on the ionic composition of the added media. Complete recovery of cellular resistance was paralleled by markedly decreased fluxes of inulin and of phenol red. The recovery of cellular barrier function was inhibited by cytochalasin B suggesting an intracellular action, not a physical disruption of the monolayer. These results suggest that the tight junctions in these cells appear to transiently produce a leaky state during removal of the media, but rearrange to a "tight conformation" when incubated in the appropriate media.

Characterization of cell polarity and epithelial junctions in the choriocarcinoma cell line, JAR

In the placenta the trophoblast cell layer separates maternal and fetal circulations and is involved in the active transport of selected substances across this barrier. We have used the JAR choriocarcinoma cell line to study aspects of trophoblast membrane transport. To determine whether JAR cells could be used in studies of vectorial transepithelial transport it was necessary to determine whether these cells were polarized and assembled tight junctions. In the present study we investigated JAR cells using a range of markers for specific cell surface domains combined with confocal laser scanning microscopy. Freshly isolated cells initially formed a confluent epithelial monolayer with recruitment of a tight junction-associated protein, ZO-1, and a cell adhesion molecule, E-cadherin, to the surface at sites of cell-cell contact. They did not, however, display cell surface polarization, as NaK-ATPase was not segregated in the basolateral domain, and a differentiated apical cell surface was not assembled. The monolayer stage was also unstable, as continued proliferation resulted in the formation of multilayered aggregates where ZO-1 and E-cadherin were lost from the cell surface. These results suggest that the JAR cell line is unlikely to be a suitable model for studies of transepithelial transport in the placenta.

Mechanically stripped pigmented and non-pigmented epithelium of the shark ciliary body: Morphology and transepithelial electrical properties

Sections of intact ciliary epithelium and mechanically stripped non-pigmented (NPE) and pigmented (PE) cell layers of adult sharks (Squalus acanthias) were mounted in Ussing-type chambers (area 0.1 cm2). Addition of 10(-5) M forskolin to the aqueous side of intact epithelium significantly increased short-circuit current (Isc) within 15 min and a maximum of approx. 30 microA cm-2 was reached after 45-60 min. Transepithelial potential difference (V) increased from -0.8 mV (aqueous side negative as compared with blood/stromal side) to -1.5 mV, whereas resistance (R) was unchanged (50 omega cm2). Forskolin was without effect when applied to the blood side. In stripped PE preparations (R 15 omega cm2), 10(-5) M forskolin applied to the apical side induced a qualitatively similar change of Isc and V compared with the intact tissue. The forskolin-induced effects were fully reversed by 10(-4) M bumetanide and were not dependent on pretreatment of the tissue with 10(-3) M BaCl2. In stripped NPE preparations resistance was usually less than 10 omega cm2 and was not stable. This is consistent with the morphologic observation that although tight junctions were still demonstrable in stripped NPE cells, the apical membranes were damaged. In preparations taken for light and electron microscopy the stripped PE layer revealed intact epithelial cells. In particular, the basal thirds of the stripped PE cells were in very close contact with each other. These attachment zones may have the appearance of tight junctions. Thus the PE cells of the shark ciliary epithelium can be successfully isolated for transepithelial transport studies. The adenylate cyclase system is present in PE cells, and transepithelial transport of chloride may be regulated by intracellular cAMP.

Characterization of retinal pigment epithelial cells cultured on microporous filters.

Retinal pigment epithelium (RPE) cultured on microporous filter supports was compared to RPE cultured on plastic and evaluated for features characteristic of RPE in vivo. RPE cells grown on filters were cuboidal, formed junctional complex structures between cells, and had elaborate microvilli and basal infoldings similar to RPE in vivo, while RPE grown on plastic also formed intercellular junctions but appeared squamous and had few microvilli and basal infoldings. RPE grown on filters or plastic secreted an extracellular matrix at the basal surface and ingested isolated rat rod outer segments at the apical surface. RPE grown on filters coated with laminin or fibronectin became confluent more rapidly than RPE grown on uncoated filters, while RPE grown at the same density on filters coated with collagen type I did not become confluent. The laminin and fibronectin coatings did not alter the RPE cell morphology; however, cells seeded on collagen-coated filters grew in large disorganized clusters. RPE grown on laminin-coated filters formed functional tight junctions as evidenced by the capacity of RPE monolayers to prevent the bulk flow of medium and the passage of trypan blue across the filter. Radiolabeled sucrose and inulin were used to measure the paracellular flux through the tight junctions between cells. The passage of these tracers was linear over time, with the lower molecular weight tracer, sucrose, passing through the monolayer more readily than inulin. Values for the flux of radiolabeled bovine serum albumin across RPE monolayers fell between values for sucrose and inulin. The results from these studies show that RPE monolayers cultured on laminin-coated filters maintain a morphology similar to that of RPE in vivo, are capable of ingesting rod outer segments, and form a selectively permeable barrier to various tracers. This culture system should be useful for studies of transepithelial transport, secretion, endocytosis and exocytosis that require independent control of the extracellular environment at the apical and basolateral cell surfaces.

Microscopic Analysis of Salt and Water Transport Levels in Toad Urinary Bladder

Several studies correlating a physiologic state with a microscopic appearance have been performed using the urinary bladder of the toad. This tissue is considered ideal for studies of transepithelial transport because of its apparent anatomic simplicity and the ease with which it lends itself to physiologic experimentation. The purpose of this report will be two-fold. First, a brief review of the kinds of studies that have been conducted will be provided; second, some examples of recent studies in structure-function relationships will be discussed.