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The use of pharmaceuticals during pregnancy is often a necessity for the health of the mother. Until recently, the placenta was viewed as a passive organ through which molecules are passed indiscriminately between mother and fetus. In reality, the placenta contains a plethora of transporters, some of which appear to be specifically dedicated to removal of xenobiotics and toxic endogenous compounds. Drug efflux transporters such as P-glycoprotein (P-gp), several multidrug resistant associated proteins (MRPs) and breast cancer resistant protein (BCRP) may provide mechanisms that protect the developing fetus. Bile acid transporters may also play a role in exporting compounds back into the maternal compartment. Steroid hormones directly influence the level of expression and function in some of these transporters. Investigating the link between the hormones of pregnancy and these drug efflux transporters is one possible key in developing strategies to deliver drugs to the mother with minimal fetal risk.

The blood-brain barrier (BBB) effectively prevents microtubule (MT)-stabilizing drugs from readily entering the central nervous system (CNS). A major limiting factor for microtubule-stabilizing drug permeation across the BBB is the active efflux back into the circulation by the overexpression of the multidrug-resistant gene product 1 (MDR1) or P-glycoprotein (P-gp). This study has focused on strategies to overcome P-gp-mediated efflux of Taxol analogs, MT-stabilizing agents that could be used to treat brain tumors and, potentially, neurodegenerative diseases such as Alzheimer's disease. However, taxol is a strong P-gp substrate that limits its distribution across the BBB and therapeutic potential in the CNS. We have found that addition of a succinate group to the C-10 position of paclitaxel (Taxol) results in an agent, Tx-67, with reduced interactions with P-gp and enhanced permeation across the BBB in both in vitro and in situ models. Our studies demonstrate the feasibility of making small chemical modifications to Taxol to generate analogs with reduced affinity for the P-gp but retention of MT-stabilizing properties, i.e., a taxane that may reach and treat therapeutic targets in the CNS.

The activity and inducibility of cytochrome P450 systems (CYP1A1:1A2) of the human placenta were assessed in a representative human trophoblast-like cell line, BeWo. The activity of CYP1A1 and CYP1A2 in microsome preparations from human liver, placenta, primary cultures of human cytotrophoblast, and BeWo cells was measured by O -dealkylation of 7-ethoxyresorufin (EROD) and 7-methoxyresorufin O -demethylation (MROD), respectively. Results indicated high EROD and MROD activity associated with human liver microsomes, sometimes comparable activities in human placenta microsomes prepared from smokers, and relatively low activities in human placenta microsomes from nonsmokers and in the primary cultures of cytotrophoblasts isolated from nonsmokers. Microsomes from BeWo cell monolayers exhibited the lowest EROD and MROD activities relative to all other microsome preparations. However, compared to primary cultures of normal trophoblasts, the EROD activity of the BeWo cells was far more sensitive to typical inducers, 3-methylcholanthrene, 1,2-benzanthracene, and beta-naphthoflavone. EROD activity in BeWo cells was induced approximately 200-fold by 3-methylcholanthrene. Both EROD and MROD activity in BeWo cells was readily induced by 1,2-benzanthracene, 100-fold and 60-fold, respectively. After induction with 1,2-benzanthracene, the CYP1A1 selective inhibitor, alpha-naphthoflavone, and the CYP1A2 selective inhibitor, furafylline, effectively inhibited enzyme activities with IC(50)s of 2.4 microM and 12.8 microM, respectively, in microsomes from both trophoblasts culture systems. These results show that major cytochrome P450 forms present in human placenta are present and inducible in BeWo cells, a potential model for investigation of drug metabolism mechanisms in the human trophoblast.

Poly(ethylene glycol) (PEG) is an excipient commonly used in pharmaceutical formulations to increase the aqueous solubility of drugs intended for oral administration. High concentrations of PEG are often used to solubilize drug candidates for in vitro experiments in cell culture (e.g., Caco-2 cell permeability studies) and/or for in vivo pharmacokinetic and safety studies in animals. Although PEG is often deemed safe in these studies based on gross morphological studies, changes on a molecular level may be overlooked. The purpose of this study was to determine the possible effects of PEG on efflux transporter activity in Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. In these studies, relatively high, yet clinically achievable, concentrations of PEG-300 did not significantly change the passive paracellular or transcellular permeation of model solutes across Caco-2 cell monolayers. More importantly, PEG-300 inhibited efflux transporter activity in Caco-2 cell monolayers, which is probably mediated by P-gp and/or MRP. Such PEG-induced inhibition of efflux transporter activity is most likely caused by changes in the microenvironment of the Caco-2 cell membranes, which perturbs the ability of these transporters to efflux substrates such as taxol and doxorubicin., (Copyright 2002 Wiley-Liss Inc.)

P-glycoprotein (P-gp), a multidrug resistance (MDR) protein encoded by the MDR1 gene in humans, is responsible for the efflux of structurally diverse drugs. Previous studies in our laboratory have shown that excipients such as poly(ethylene)glycol (PEG)-300, Cremophor EL, and Tween 80 inhibit P-gp activity in Caco-2 cell monolayers. The objective of this study was to determine the effects of these excipients in an MDR1- transfected Madin Darby Canine Kidney (MDR1-MDCK) cell line and to compare the results with those obtained from Caco-2 cells. The results presented herein show that PEG-300 (20%, v/v) causes almost complete inhibition of P-gp activity in both Caco-2 and MDR1-MDCK cell monolayers, whereas Cremophor EL (0.1%, w/v) and Tween 80 (0.05%, w/v) only partially inhibit P-gp activity in Caco-2 cells. Cremophor EL (0.1%, w/v) and Tween 80 (0.05%, w/v) were inactive as P-gp inhibitors in MDR1-MDCK cell monolayers. This inability of Tween 80 and Cremphor EL to inhibit P-gp activity in MDR1-MDCK cells may be related to differences in the interactions of the surfactants with these different cell membranes. PEG-induced changes in P-gp activity are probably related to changes in the fluidity of the polar head group regions of cell membranes., (Copyright 2002 Wiley-Liss Inc.)

Purpose: The objective of this work is to evaluate the ability of peptides derived from the bulge (HAV-peptides) and groove (ADT-peptides) regions of E-cadherin EC1-domain to increase the paracellular porosity of the intercellular junctions of Madin-Darby canine kidney (MDCK) cell monolayers., Methods: Peptides were synthesized using a solid-phase method and were purified using semi-preparative HPLC. MDCK monolayers were used to evaluate the ability of cadherin peptides to modulate cadherin-cadherin interactions in the intercellular junctions. The increase in intercellular junction porosity was determined by the change in transepithelial electrical resistance (TEER) values and the paracellular transport of 14C-mannitol., Results: HAV- and ADT-peptides can lower the TEER value of MDCK cell monolayers and enhance the paracellular permeation of 14C-mannitol. HAV- and ADT-decapeptides can modulate the intercellular junctions when they are added from the basolateral side but not from the apical side; on the other hand. HAV- and ADT-hexapeptides increase the paracellular porosity of the monolayers when added from either side. Conjugation of HAV- and ADT-peptides using omega-aminocaproic acid can only work to modulate the paracellular porosity when ADT-peptide is at the N-terminus and HAV-peptide is at the C-terminus; because of its size, the conjugate can only modulate the intercellular junction when added from the basolateral side., Conclusions: Peptides from the bulge and groove regions of the EC1 domain of E-cadherin can inhibit cadherin-cadherin interactions, resulting in the opening of the paracellular junctions. These peptides may be used to improve paracellular permeation of peptides and proteins. Furthermore, this work suggests that both groove and bulge regions of EC-domain are important for cadherin-cadherin interactions.

A study of the fundamental biology of the maternal-fetal interface reveals the complex interactions among multiple cell types and regulatory factors necessary to support a successful pregnancy. Cells of decidua and trophoblast lineages play central roles in creating the maternal-fetal interface and are sources of regulatory factors that can determine the quality and success of pregnancy. The regulatory factors considered here are major placental histocompatibility complex proteins, pregnancy-specific regulatory factors for uterine inflammatory cells, and hormone-controlled placental multidrug-resistant transport systems. Potential targets are discussed and presented as areas where researchers may identify novel pharmacological and immunological strategies that eventually will extend to the clinic to improve the quality and success of pregnancy.

In keeping with the advance of biotechnology, cell culture becomes an important tool for investigating the transport and the metabolism phenomena. A cell line of human origin, the BeWo choriocarcinoma cell line, was used for the study of the transport and metabolism of opioid peptides across the in vitro model of the placental barrier. Opioid peptides, both naturally occurring and their synthetic analogs, are of interest to be developed as potent analgesics and were included in this study. The apparent permeability coefficients (Pe)s of the peptides containing 4-11 amino acid or analog residues were in the range of 0.23-14.6 x 10(-5) cm/s. The (Pe)s of these peptides were comparable to those of sucrose or dextrans, hydrophilic markers. The (Pe)s of low molecular weight (MW) peptides was not dependent on their MW or molecular size, whereas an inversely linear correlation between (Pe)s and molecular size was observed with the larger peptides. Molecular sieving of the BeWo monolayer restricted the transport of the peptides with MW> or =1033 Da or molecular size > or =6.6 A. Membrane partitioning ability and charge of the peptides were also investigated and found to be the minor factors regulating the extent of peptide permeation. Contrasting to the transport of Tyr-[D-pen-Gly-Phe-D-Pen] (DPDPE) peptide analog across the blood-brain barrier, the transport of DPDPE across the BeWo monolayers were not indicated to be via carrier-mediated transport. The major transport pathway of the opioid peptides across the BeWo monolayers was found to be via paracellular route. In metabolism studies, aminopeptidase was found to be a major enzyme type responsible for the degradation of naturally occurring peptides but not for the synthetic analogs. The finding obtained from the present study reveals the applicability of the BeWo cell line as an in vitro model for investigating placental transport and metabolism of opioid peptides.

To date, there are few in vitro models of the human lung that have been used to characterize multidrug resistant (MDR) efflux pump activity. It is expected that the presence of these protein transporter molecules, such as P-glycoprotein (Pgp) and the multidrug resistance protein associated protein-1 (MRP1), might play a role in limiting drug absorption through the pulmonary epithelium, as has been reported for other epithelial drug delivery barriers such as the intestine and brain. To date, the exact role of the lung resistance related protein (LRP) in MDR is unclear. In this article, we have summarized the biochemistry, function and in vitro/in vivo modulation of Pgp and MRP1. These topics are discussed in light of pulmonary delivery of therapeutic agents, with particular emphasis being placed on the bronchial region of human airways.

The transport of substance P (SP) was investigated using the bovine brain microvessel endothelial cell culture model of the blood-brain barrier (BBB). The samples were derivatized precolumn with naphthalene dialdehyde, then analyzed by cyclodextrin-modified micellar electrokinetic chromatography with laser-induced fluorescence detection. SP crossed the BBB in both the apical-to-basolateral and basolateral-to-apical directions through an active transport mechanism. The transport of SP from the apical side was demonstrated to be via transcytosis. The N-terminal (SP(1-4)) and C-terminal (SP(3-11)) fragments were also found to permeate the BBB from the apical side.

Using cultured BeWo cells as a model of human trophoblast, we investigated whether carrier-mediated transport of folic acid occurs. BeWo cells, which were derived from human choriocarcinoma, were cultured on a tissue culture plate or in a permeation chamber. When the cells reached confluence, drug uptake or transport experiments were performed. The uptake of [(3)H]folic acid by BeWo cells occurred at a much lower rate at 4 degrees C than at 37 degrees C. The uptake of [(3)H]folic acid was saturable at higher concentrations and inhibited by typical metabolic inhibitors, sodium azide and 2,4-dinitrophenol. The uptake of [(3)H]folic acid was significantly increased with decreasing pH of the incubation buffer and markedly inhibited by 4,4'-diidothiocyanostilbene-2,2'-disulfonic acid (DIDS). Analogs of folic acid, methotrexate and 5-methyltetrahydrofolate, inhibited the uptake of [(3)H]folic acid by BeWo cells. Kinetic analysis using Lineweaver-Burk plots revealed that methotrexate competitively inhibited the uptake of [(3)H]folic acid and folic acid competitively inhibited the uptake of [(3)H]methotrexate. In transport experiments, the permeation of [(3)H]folic acid from the apical-to-basal side was greater than that from the basal-to-apical side, and the transport of [(3)H]folic acid from the apical-to-basal side was inhibited by an excess of folic acid. The findings obtained in the present study confirm the existence of an asymmetric, carrier-mediated transport system for folic acid and its analog, methotrexate, across BeWo cells, a representative of the human trophoblast., (Copyright 2001 Harcourt Publishers Ltd.)

A relatively simple, widely applicable, and robust in vitro method of predicting blood-brain barrier (BBB) permeability to central nervous system-acting drugs is an increasing need. A cell-based model offers the potential to account for transcellular and paracellular drug diffusional processes, metabolism, and active transport processes, as well as nondefined interactions between a drug and cellular material that may impact upon a membrane's overall permeability profile. Any in vitro BBB cell model to be utilized for the transendothelial BBB permeability screening of potential central nervous system drugs must display reproducible solute permeability, and a number of other general criteria including: a restrictive paracellular barrier; a physiologically realistic cell architecture; the functional expression of key transporter mechanisms; and allow ease of culture to meet the technical and time constraints of a screening program. This article reviews the range of in vitro cell-based BBB models available, including the primary/low passage bovine and porcine brain endothelial cultures as well as the spectrum of immortalized brain endothelial cell lines that have been established. The article further discusses the benefits and limitations of exploiting such systems as in vitro BBB permeability screens., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association)

The purpose of this work was to investigate if P-glycoprotein (Pgp) efflux pump activity could be inhibited in the sub-bronchial epithelial cell line, Calu-3, by glucocorticosteroids and beta-ligands. The Pgp modulation efficiency of each compound was determined by its ability to increase the accumulation of the Pgp substrate rhodamine 123 (Rh123) accumulation in these cells. Pgp inhibition was observed at > or =100 microM steroids and beta-ligand. The modulation effectiveness of the beta-ligands increased with increasing hydrophobicity (logP(octanol/aqueous)) whereas an obvious correlation was not obtained with the complete set of steroids tested. Steroidal Pgp substrates did not affect Rh123 accumulation (e.g. aldosterone, dexamethasone, 11beta,17alpha,21-OH progesterone). In contrast, two hydrophobic non-Pgp steroidal substrates (testosterone and progesterone) displayed different effects on Rh123 accumulation, with progesterone being the more potent modulator. The most hydrophobic beta-ligand, propranolol, a known Pgp substrate, gave the largest increase in Rh123 accumulation in this therapeutic class. The beta-ligand modulation efficiency could also be correlated to Pgp structural recognition elements such as hydrogen bonding potential, the presence of a basic nitrogen and planar aromatic ring. No effect on Rh123 accumulation was observed with the formulation additives tested (ethanol, glycerol and palmitoyl carnitine) at concentrations previously reported to be non-toxic to Calu-3 cells.

Substance P (SP) metabolism was investigated upon exposure to a monolayer of bovine brain microvessel endothelial cells (BBMECs), a cell culture model of the blood-brain barrier. SP was incubated with the BBMECs and its metabolism was followed as a function of time over a 5-h period. The resulting samples were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA)/cyanide, separated, and detected using cyclodextrin-modified electrokinetic chromatography with laser-induced fluorescence detection (CDMEKC-LIF). Upon exposure to the BBMEC monolayer, SP rapidly degraded to produce the N-terminal (1-9), (1-4) and (1-7) and C-terminal (2-11) and (3-11) fragments. These results were compared with those in an earlier report from our laboratory, where SP metabolism was investigated in vivo by microdialysis sampling in rat striatum.

The purpose of this work was to determine whether the in vitro bronchiolar epithelial cell model, Calu-3, possesses efflux pump activity by the multidrug resistance-associated protein-1 (MRP1). Reverse transcription-polymerase chain reaction demonstrated MRP1 gene expression in Calu-3 cells. Indirect fluorescence studies showed a basolateral membrane localization of MRP1 compared with P-glycoprotein (Pgp) that was found on the apical side of these cells. An increase in the rate of accumulation of the MRP1 substrate calcein was observed following treatment with the organic anion/MRP1 inhibitor indomethacin, the Pgp inhibitors cyclosporin A (CsA) and vinblastine, as well as conditions of energy depletion. Total calcein efflux was significantly decreased with the MRP1 inhibitors probenecid and indomethacin, while total efflux was unchanged following treatment with CsA. In the latter case, however, intracellular calcein levels postefflux were significantly greater. Probenecid and indomethacin increased calcein net secretion 2.4- and 3.5-fold, respectively. The efflux of etoposide, a known substrate for both Pgp and MRP1, was shown to be mainly Pgp-mediated by using the multidrug-resistant inhibitors quinidine (mixed Pgp/MRP1), CsA (Pgp), and MK571 (MRP1). Together, these data suggest that Calu-3 cells possess MRP1 functional activity that is subordinate to Pgp efflux. We present here kinetic analysis of calcein efflux from Calu-3 cells to support our findings.

The purpose of this work was to determine if the sub-bronchial epithelial cell model, Calu-3, expresses the functionally active P-glycoprotein (Pgp) efflux pump. Calu-3 cells express lower levels of Pgp than both Caco-2 and A549 cells as determined by Western Blot analysis. In Calu-3 cells, accumulation of the Pgp substrates rhodamine 123 (Rh123) and calcein acetoxymethyl ester (calcein-AM) was increased in the presence of the specific Pgp inhibitors cyclosporin A (CsA), vinblastine, and taxol. Significant inhibition of Pgp activity was not observed until after 2 h in both cell lines. The organic anion/multidrug resistance associated protein-1 (MRP1) inhibitors, probenecid and indomethacin, did not affect Rh123 accumulation, whereas an increase in calcein accumulation was observed by both agents. The metabolic inhibitor sodium azide decreased the efflux of Rh123 out of Calu-3 cells to the same degree as CsA, supporting inhibition of an active, efflux pathway. The basolateral-to-apical transport of Rh123 was significantly higher than that in the reverse direction, indicating a secretory pathway of efflux that was inhibited 25-fold by CsA. Basolateral-to-apical transport of Rh123 was inhibited slightly with both MRP1 inhibitors; however, no significant effect of Rh123 net secretion was observed. Mixed inhibitor studies demonstrated that Rh123 efflux was mainly Pgp mediated. These results support an energy-dependent Pgp efflux pump pathway that is sensitive to inhibition with CsA in Calu-3 cells., (Copyright 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association.)

Purpose: The objective of this work is to understand the sequence specificity of HAV peptides and to improve their selectivity in regulating E-cadherin-E-cadherin interactions in the intercellular junctions., Methods: Peptide 1 was modified using an alanine scanning method to give peptides 2-6. The ability of these peptides to modulate intercellular junctions was evaluated using Madin-Darby Canine Kidney (MDCK) cell monolayers on Transwell membranes from either the apical (AP) or the basolateral (BL) side. Modulation of the intercellular junctions was measured by the ability to lower the transepithelial electrical resistance (TEER) of MDCK monolayers and by the increase in mannitol flux. Molecular docking experiments were performed to model the binding properties of these peptides to the EC1 domain of E-cadherin., Results: Peptides 5 (Ac-SHAVAS-NH2) and 6 (Ac-SHAVSA-NH2) were found to be more effective than the parent peptide 1 in decreasing the resistance of the cell monolayer. Furthermore, comparative studies with the control and the weak inhibitor peptide 2 indicate that peptide 5 displayed a significant increase in mannitol flux. Molecular docking of peptides 1, 2 and 5 to the EC1 domain suggests that peptide 5 has the lowest binding energy., Conclusions: HAV peptides have the ability to modulate E-cadherin-E-cadherin interactions in the intercellular junctions of the MDCK cell monolayer, thus indirectly increasing the permeability of the tight junctions. This observation indicates that residues flanking the HAV sequence are important in the binding selectivity of HAV peptides to E-cadherin. Molecular docking can further aid in the design of peptides with better selectivity to the EC1 domain of E-cadherin.

The blood-brain barrier (BBB) is comprised of the endothelial cells that line the capillaries of the brain. The unique characteristics of this barrier include tight intercellular junctions, a complex glycocalyx, a paucity of pinocytic vesicles, and an absence of fenestra. These properties allow for the selective exchange of substances between the systemic circulation and the extracellular fluid compartment of the brain. It is well established that there are many conditions, including those mediated by nitric oxide (NO), that can lead to an opening of the BBB, eventually leading to vasogenic edema and secondary brain damage. The precise molecular mechanisms mediating NO-induced tissue injury and the breakdown of the BBB are complex and not completely understood. NO is a soluble, easily diffusible gas that is generated by NO synthase. Two of the isoforms of NO synthase are constitutive, calcium-dependent enzymes that modulate many physiological functions, including the regulation of smooth muscle contraction and blood flow. The third isoform is calcium-independent and inducible and can be stimulated by stress, inflammation, and infection. Under these conditions, NO can be generated in large quantities and has detrimental effects on the CNS. NO has been shown to increase permeability of the BBB, allowing substances to enter into the brain passively. This review considers the role of NO and BBB integrity.

The objective of this study was to examine the uptake mechanisms of fluorescent polystyrene microspheres of various diameters and surface chemistry by two human cell lines derived from the respiratory epithelium, A549 and Calu-3. Briefly, A549 and Calu-3 cells were grown to confluence in 12-well cluster plates and the uptake of fluorescent microspheres by the cells was determined at various time points. The amount of microspheres internalized by the cells was determined by correcting for non-specific binding to the cell surface. The data showed that A549 cells appeared to have more phagocytic activity than Calu-3 cells. Albumin-coated microspheres as large as 3 microm diameter can be internalized by A549 cells. The amount of internalization by A549 cells observed for 0.5-microm diameter albumin-coated microspheres was approximately 10-times greater than that observed for 1-microm diameter spheres and approximately 100-times greater than values observed for 2- and 3-microm diameter beads. Transmission electron micrographs confirmed that the microspheres were internalized by the cells. Uptake experiments conducted with Calu-3 cells indicated that albumin-coated microspheres were neither bound nor internalized by the cells. The effect of microsphere surface chemistry on the uptake mechanism indicated that amidine microspheres were internalized more rapidly and to a greater extent by both A549 and Calu-3 cells than carboxylate microspheres and non-coated microspheres. This phenomenon is thought to be attributed to masking of the negative polystyrene core by the positive amidine functional group; this effect was less marked for the carboxylate microspheres. These results suggest that A549 and Calu-3 cells can internalize microspheres and that size and effective charge played an important role in the uptake process.

In vitro studies have shown that conjugation of a model compound [p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivo brain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5,7-dichlorokynurenic acid (DCKA) and D-kyotorphin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.

The objective of this research was to examine the human sub-bronchial gland cell line, Calu-3, and assess its potential as a metabolic and transport model to study drug delivery to the respiratory epithelium. The present studies were conducted using Calu-3 cells grown in Transwells(R) or in multiwell cluster plates. TEER values for Calu-3 monolayers were determined using the World Precision Instrument Voltohmmeter and STX-2 electrode. The results confirmed that Calu-3 cells form tight monolayers and give appreciable TEER values in culture when grown under air-interface conditions. Permeability data for small lipophilic molecules across Calu-3 monolayers suggested that the cell line is a suitable model to examine the transport of low molecular weight substances and xenobiotics. Calu-3 cells were also found to efflux FITC-transferrin (MW 80000) in a polarized manner. The metabolic capacity of Calu-3 cells was also examined. The P4501A1 and P4502B isozymes were determined to be functional, but not inducible, with fluorescent resorufin assays. The data indicated that the Calu-3 cell line may be useful for studying the contributions of bronchial epithelial cells to mechanisms of drug delivery at the respiratory epithelium.

The objective of this study was to investigate the functional expression of the efflux transporter, P-glycoprotein (P-gp), in primary cultures of human cytotrophoblasts and BeWo cell monolayers. Uptake studies with primary cultures of human cytotrophoblasts or BeWo cells were conducted with calcein-AM and vinblastine (P-gp markers) or fluorescein (MRP marker) in the presence of specific P-gp or MRP inhibitors. Results showed that the accumulation of P-gp substrates calcein-AM and vinblastine by BeWo cells or primary cultures of human cytotrophoblasts was significantly enhanced in the presence of a typical P-gp inhibitor, cyclosporin-A, or other inhibitors such as quinidine, verapamil, and dipyridamole. MRP inhibitors had no effect on the accumulation of calcein or fluorescein by BeWo cells. Western blots confirmed the presence of multidrug resistant gene product 1 (MDR1) in both primary cultures of human cytotrophoblasts and BeWo cells. This study demonstrates functional P-gp in term human trophoblasts and further supports the use of primary cultures of human cytotrophoblasts and BeWo cells as in vitro models of the trophoblast to investigate mechanisms regulating drug distribution across the placenta.

The placenta forms a selective barrier that is able to transport nutrients that are of critical use to the fetus. Delivery of essential fatty acids to the fetus is dependent upon transplacental transport and provides the backbone for the biosynthesis of biological membranes, myelin and various signalling molecules. The primary objective of this research was to elucidate the expression patterns of genes that regulate fatty acid transport across the placenta. Several fatty acid transport regulatory genes have been identified in the rat including; cytoplasmic heart fatty acid binding protein (hFABP), plasma membrane fatty acid binding protein (FABPpm), fatty acid translocase (FAT) and fatty acid transport protein (FATP). In this study, we have elucidated temporal and spatial expression patterns for these genes in the rat placenta and in cell culture models of the rat placenta by Northern blot, RT-PCR, Western blot and/or by in situ hybridization analyses. Expression of hFABP was specific to the labyrinth zone, the main barrier and site of transplacental transport in the rat placenta. In addition, the levels of hFABP expression increased with gestational age, suggesting a growing requirement for fatty acid transport with advancing stages of pregnancy. FABPpm, FAT and FATP are expressed in both the junctional and labyrinth zones of the rat placenta. FAT was predominantly localized to the labyrinth zone by in situ hybridization analysis. The placental cell expression patterns of the genes involved in fatty acid transport were supported by our observations of HRP-1 (labyrinth zone) and Rcho-1 (junctional zone) trophoblast cell culture models. Given their cell surface location, we predict that FABPpm, FAT and FATP potentially participate in placental fatty acid uptake. The predominant expression of hFABP and FAT in the labyrinth zone of the chorioallantoic placenta implicates hFABP and FAT in the transplacental movement of fatty acids from maternal to fetal compartments., (Copyright 2000 Harcourt Publishers Ltd.)

The morphological and cytoskeletal reorganization of collagen-sandwiched rat hepatocytes during the de novo formation of complete canalicular networks was examined by phase, fluorescence and electron microscopy. During the initial stages of membrane repolarization, there was a marked accumulation of both microfilaments and microtubules at the sites of canalicular generation. Microtubule-disrupting agents (colchicine, nocodazole) inhibited the localization of actin filaments at cell margins and the initiation and branching of canalicular networks. After removal of microtubule-disrupting agents, microfilaments relocalized to the canalicular borders and microtubules nucleated along the margins of the bile canaliculi at sites distinct from the peri-canalicular actin networks. Microfilament-perturbing agents (cytochalasin D, phalloidin) did not affect the de novo initiation of bile canaliculi and only slightly impaired the development of canalicular lumina into networks. In established cultures with complete canalicular networks, subsequent treatment with microtubule-disrupting agents did not acutely affect the integrity of preformed canalicular networks. In contrast, treatment with microfilament-perturbing agents caused a marked dilation of most canaliculi. These results illustrate the differential role of the cytoskeleton in the regeneration and maintenance of bile canalicular networks by collagen-sandwiched hepatocytes. Moreover, this study shows the utility of this system as an in vitro model for examining the regulation of cell and membrane polarity.

The biochemical mechanisms mediating the rapid distribution of valproic acid across placenta are not precisely known. We have characterized valproic acid transport by the human trophoblast using the human choriocarcinoma cell line, BeWo. The uptake of [14C]valproic acid by BeWo cells was found to be saturable and blocked by pre-exposure to the metabolic inhibitors, sodium azide and 2,4-dinitrophenol. Valproic acid uptake by the BeWo cells was also inhibited by the protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, but not anion exchange inhibitor. Selected monocarboxylic acids inhibited the uptake of [14C]valproic acid by BeWo cells, whereas dicarboxylic acids did not alter the uptake process. Analysis of Lineweaver-Burk plots of valproic acid uptake in the presence of benzoic acid, a marker for the monocarboxylic acid transporter, revealed a competitive process for uptake. In transcellular transport experiments, the permeation of [14C]valproic acid from the apical-to-basal side of the monolayers was significantly greater than the permeation from basal-to-apical side. Additionally, the permeation of [14C]valproic acid from apical-to-basal side was inhibited by monocarboxylic acids and not dicarboxylic acids. The results provide biochemical evidence of a proton-dependent, saturable, and asymmetric transport system, presumed to be a monocarboxylic acid transporter, for valproic acid in a human trophoblast model.

In this paper, the L-glutamate (L-Glu) transport system was targeted to improve the delivery of a model compound, p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD), through the blood-brain barrier (BBB) in vitro cell culture model. D-MOD is an analogue of an antitumor agent D-melphalan. To target the L-Glu transport system, D-MOD was conjugated to L-Glu to give D-MOD-L-Glu conjugate. D-MOD and D-MOD-L-Glu transport properties were evaluated using the bovine brain microvessel endothelial cell (BBMEC) monolayers. The results suggest that D-MOD-L-Glu conjugate permeates through the BBMEC monolayers more readily than the parent D-MOD. The improvement of transport may be due to the recognition of D-MOD-L-Glu by the L-Glu transport system. The transport mechanism was evaluated using several different experiments including: (a) concentration-dependent studies; (b) temperature-dependent studies; (c) substrate inhibition studies; and (d) metabolic inhibitor studies. The D-MOD-L-Glu transport was inhibited by the change of temperature from 37 degrees C to 4 degrees C. At higher concentrations, the transport of D-MOD-L-Glu reached plateau due to saturation. Furthermore, some amino acids (i.e., L-Glu, L-Asp, D-Asp, and L-Gln) inhibited the transport of D-MOD-L-Glu; presumably the conjugate was competing with these amino acids for the same transport system. Metabolic inhibitors (i.e., 2,4-dinitrophenol and sodium azide) suppressed the transport of the conjugate. However, the conjugate was not transported by monocarboxylic acid, dipeptide and neutral amino acid transporters. In conclusion, the L-Glu transport system can be utilized to facilitate a non-permeable drug across the BBB by conjugating the drug with L-Glu amino acid.

The monolayer-forming, human choriocarcinoma cell line, BeWo, was used to study the mechanisms of monocarboxylic acid transport across the human trophoblast. Benzoic acid, acetic acid, and lactic acid were used as markers for monocarboxylic acid carrier-mediated transport. The uptake of benzoic acid by BeWo cells was saturable (K(t) = 0.6 +/- 0.3 mM) at higher concentrations and significantly inhibited by typical metabolic inhibitors, sodium azide and 2, 4-dinitrophenol. A selection of different monocarboxylic acids, including a natural substrate lactic acid, also substantially inhibited the uptake of benzoic acid and acetic acid by BeWo cells, whereas dicarboxylic acids did not affect the uptake of either marker. Monocarboxylic acid uptake was pH-dependent and inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore. Kinetic analysis using Lineweaver-Burk plots revealed that monocarboxylic acids competitively inhibited the uptake of benzoic, lactic, and acetic acid by BeWo cells. In transport experiments, the permeation of benzoic acid from apical-to-basolateral side was greater than the permeation from the basolateral-to-apical side, and the transport of benzoic acid from apical-to-basolateral side was inhibited by monocarboxylic acids. The findings obtained in the present study confirm the existence of an asymmetric, carrier-mediated transport system for monocarboxylic acids across the BeWo cell, a representative of the human trophoblast.

A challenge in modern drug therapy is to develop strategies for safer and more selective targeting of drug delivery in pregnancy. Specifically, approaches are needed that would restrict unnecessary drug exposure to either mother or fetus. There is evidence emerging that indicates the placenta does express natural transport and metabolism processes that function to control drug and nutrient distribution between the mother and fetus. Further, in vitro techniques developed in the past 10 years now provide some of the tools necessary to elucidate transport and metabolism processes typical of the human placenta. As a consequence, pharmaceutical scientists are in a position to contribute significantly to the design and development of drugs for pregnancy.

Advances made in the field of DNA and recombinant technology have led to the emergence of peptides and proteins as an important class of therapeutic compounds. While a significant amount of information exists regarding the transport and metabolism of peptides across different barriers (e.g. gastro-intestinal, nasal, and blood-brain barrier), limited attention has been paid towards their transport and metabolism across the placental barrier. The mechanism of placental transport of peptides is of importance in assessing the exposure of these drugs to the fetus, particularly when the drugs potentially may have adverse effects on the developing fetus.The absence of a well accepted, simple and convenient animal model may be a reason for the limited information available on the placental transport and metabolism of peptides. Although several in vivo models have been utilized to study the transport and metabolism of drugs across the placenta, species differences in the placental physiology and anatomy of the animal models with regard to the human placenta have prevented their widespread use. The in vitro human placental cell culture models are morphologically similar to the trophoblasts and often express the enzymes and carrier systems found in the human placenta. They can provide an easy and rapid method to determine the mechanisms of transport and metabolism of drugs across the placental barrier. These in vitro models have been utilized in the determination of transport mechanisms of drugs of abuse across the placenta.This article overviews the available literature on the placental transport of peptides and describes the application of an in vitro cell culture model (BeWo) to determine the mechanisms of transport of opioid peptides and their analogues across the placenta.

The placenta forms a selective barrier that functions to transport nutrients that are of critical use to the fetus. Nutrient transport across the placenta is regulated by many different active transporters found on the surface of both maternal and fetal facing membranes of the placenta. The presence of these transporters in the placenta has been implicated in the facilitation of nutrient diffusion and proper fetal growth. In this review, recent developments concerning nutrient transporters that regulate glucose, amino acid, fatty acid, and nucleoside transplacental movement are discussed.

Blood-brain barrier transport of the selective serotonin reuptake inhibitor and antidepressant, citalopram, was studied using monolayers of bovine brain microvessel endothelial cells (BMECs). This study provides for the first time, evidence of a transport mechanism for a selective serotonin reuptake inhibitor (SSRI). Carrier-mediated transport, efflux mechanisms, as well as inhibition of metabolizing enzymes of citalopram were investigated. Citalopram transport was saturable and temperature-dependent suggesting that passage of the drug across BMECs was mediated by a carrier mechanism. Since the apical to basolateral and basolateral to apical permeability coefficients were similar and cyclosporin A, a P-glycoprotein inhibitor, does not modify the transport of citalopram, it appeared that no active efflux systems were involved in this transport. Citalopram is only available as a racemic drug and its pharmacological effect resides mainly in the S-(+)-enantiomer. However, the passage of citalopram enantiomers across BMEC monolayers was not stereoselective. Finally, inhibition of the metabolizing enzymes of citalopram and monoamine oxidases did not modify the permeation of citalopram across BMECs. Collectively, our results suggested that citalopram crosses the blood-brain barrier via a non-stereoselective, bidirectional and symmetrical carrier-mediated mechanism without influences of active efflux mechanisms or monoamine oxidases., (Copyright 1999 Elsevier Science B. V.)

The pharmaceutical industry is faced with filtering hundreds of thousands of compounds to identify successful drug candidates. Given these numbers, how does the pharmaceutical industry identify optimal therapeutic agents rapidly, efficiently, economically and successfully, with the ultimate result of the patient receiving the best drug? The conference summarized the present and future requirements for evaluating emerging technologies, integrating that technology into a filter for large and growing numbers of compounds, building and linking diverse knowledge bases, and establishing predictive foundations that will optimize and accelerate drug discovery and development. Specific conference topics focused on organizational and management approaches as well as some of the major technologies and emerging techniques for supporting drug candidate selection and optimization. It is predicted that the pharmaceutical industry will be synthesizing and screening a million or more compounds for multiple therapeutic targets in the near future. Pulling together the resources of current and emerging technology, knowledge, and multidisciplinary teamwork, so that discovery and selection of successful drug candidates from this large pool of compounds can take place rapidly, is a significant challenge. This conference focused on the organizational issues and experimental tools that can provide for a shortening of discovery time, identification of current and future selection techniques and criteria, the linking of technologies and business strategies to reduce risk, and novel processes for optimizing candidates more quickly and efficiently. The conference was directed at industrial scientists involved in all stages along the drug discovery and development interface. This conference was well-attended, with approximately 100 participants.

The endothelial lining of the blood-brain barrier tightly controls the distribution of peptide hormones between the central nervous system and the circulation. By using primary cultures of brain microvessel endothelial cells, an in vitro model of the blood-brain barrier, we report here the uptake and transport of the octapeptide angiotensin II by a specific receptor population. With the angiotensin II antagonists losartan (AT1 specific) and PD 123,319 (AT2 specific), we showed that both the uptake and transport of angiotensin II were mediated by the AT1 receptor. Western blot analysis confirmed the existence of the AT1 receptor in our cell-culture model. Rhodamine 123 studies also suggested that both angiotensin II antagonists, but not angiotensin II, were substrates for the P-glycoprotein efflux system, thus restricting the transport of these compounds. These results suggest an AT1 receptor mediates uptake and transport of angiotensin II at the blood-brain barrier and may contribute to the regulation of cerebrovascular levels of the peptide.

Purpose: This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions., Methods: Hepatocytes isolated from male Wistar rats (230-280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein. Na+/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5., Results: By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was approximately 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na+-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 microM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (3241 microM). In contrast, the Vmax values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were approximately 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film., Conclusions: A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.

Multiple cell types contribute to the pulmonary barrier including Type I and Type II alveolar epithelium. The objective of this research was to establish and characterize an in vitro model of Type II alveolar epithelium using the A549 human lung adenocarcinoma cell line. A549 cells form confluent monolayers with Type II characteristic morphology and tannic acid staining for typical lamellar bodies. A549 cells possess P450 IA1 and P450 IIB6 as determined by Western blots. Both CYPIA1 and CYPIIB6 P450 isozymes were determined to be functional with the fluorescent resorufin assay. Only the IA1 isozyme was observed to be inducible with selected polycyclic hydrocarbons. Uptake and transport experiments were carried out in cluster plates and in Snapwells. Cationized ferritin, a nonspecific absorbtive marker, was found to be taken up by the cells in a concentration-, time-, and temperature-dependent fashion. Lucifer yellow, a fluid-phase marker, was not internalized by the A549 cells. Transferrin, a representative receptor-mediated endocytic marker, was found to be taken up by the cells in a concentration-dependent and competitive fashion. Transport experiments involving fluorescein-transferrin also showed that A549 monolayers were polarized, with a greater amount of intracellular transferrin being transported out of the basolateral side of the cells. The experimental data agree favorably with literature for primary cultures of Type II pulmonary epithelial cells. These results indicated that the A549 cell line may be useful for the studying the metabolic and macromolecule processing contributions of alveolar Type II cells to mechanisms of drug delivery at the pulmonary epithelium., (Copyright 1998 Academic Press.)

The effect of dilution of parenteral vehicles (organic cosolvent and 0.1 M cyclodextrin solutions) on cultured endothelial cells (HUV-EC) were compared in vitro. Cell morphology was observed by phase contrast light microscopy and cell viability by measuring 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction or intracellular lactate dehydrogenase (LDH) activity and total protein. Disruption of the HUV-EC monolayer was observed at dilutions of 1 in 20 for the melphalan and PEP cosolvents, 1 in 100 for an investigational drug cosolvent, and 1 in 10 for 0.1 M dimethyl-beta-cyclodextrin. In comparison, 0.1 M SBE7M- and HP-beta-cyclodextrin caused only minor disruption at a 1 in 5 dilution. MTT reduction, intracellular LDH, and total protein were decreased following exposure to 1 in 10 dilution of the melphalan cosolvent. For other test solutions, intracellular LDH activity and total protein were measured, and reductions were observed following exposure to 1 in 10, 20, and 50 dilutions of the investigational drug cosolvent and 1 in 5 dilution of DM-beta-cyclodextrin (0.1 M). At a dilution of 1 in 10, no delayed toxicity was observed for cosolvents or cyclodextrin solutions. Hence, 0.1 M SBE7M- or HP-beta-cyclodextrin formulations may be less damaging to the venous endothelium at the site of injection than organic cosolvent formulations.

The P-glycoprotein efflux system located on the apical membrane of brain capillary endothelial cells functions as part of the blood-brain barrier. In this study, primary cultures of bovine brain microvessel endothelial cells (BMECs) were investigated for the presence of a P-glycoprotein system and its contribution in regulating ivermectin distribution across the blood-brain barrier. Results of rhodamine 123 uptake studies with cyclosporin A and verapamil as substrates indicated that a functional efflux system was present on BMECs. Immunoblot analysis with the C219 monoclonal antibody to the product of the multidrug resistant member 1(MDR1) gene also confirmed the expression of MDR1 in the BMECs. Unbound ivermectin was shown to significantly increase the uptake of rhodamine 123 in BMECs, however, the drug only modestly enhanced the transcellular passage of rhodamine. The results of these studies affirmed that unbound ivermectin is an inhibitor of the MDR1 efflux system in BMECs.

Angiotensin converting enzyme (ACE) and leucine aminopeptidase (LAP) regulate fetally and maternally generated peptides in the placenta. In this study, ACE-like activity was found to be decreased and LAP-like activity increased with increasing days of gestation in rat placental tissues forming the fetal:maternal interface. Membrane-associated ACE-like and LAP-like activities in the placenta of smokers were also found to be significantly higher than their respective activities in placenta of nonsmokers. Our collective findings suggest that gestational and environmentally-induced changes in placental peptidase activities may account for variable peptide hormone and/or therapeutic peptide metabolism in the placenta.

Angiotensin II (Ang II) uptake and transport across monolayers of bovine brain microvessel endothelial cells (BMECs) was demonstrated. Ang II transport was linear up to 2 h, saturable with a K(m) of 1.7 nM, and tended to be polarized with the apical-to-basolateral transport being greater. [3H]Ang II transport was found to be inhibited by excess unlabeled Ang II, by the Ang II analog sarathrin, and by the endocytic inhibitor phenylarsine oxide. Ang II-(2-8) and-(3-8) were shown to significantly increase the transport of Ang II. These results demonstrate for the first time the receptor-mediated transcytosis of Ang II across brain microvessel endothelium.

The BeWo cell line (b30 clone) has been examined as a potential in vitro system to study transplacental transport. At the light and electron microscope level, the cells were observed to form confluent monolayers on polycarbonate filters in approximately 5 days and morphologically resembled the typical human trophoblast. BeWo monolayers developed a modest transepithelial electrical resistance and a molecular size-dependent permeability to hydrophilic passive diffusion markers, fluorescein, and selected fluorescein-labeled dextrans. Linoleic acid permeation across BeWo monolayers was asymmetric, saturable, and inhibited by low temperature and excess competing fatty acid. Forskolin and 8-bromoadenosine 3',5'-cyclic monophosphate treatments stimulated morphological changes in BeWo cultures and enhanced the asymmetric passage of linoleic acid across the BeWo monolayers while having minimal effects on passive permeability, affirming that the differentiation state of the cells can influence membrane transporters and transmonolayer permeability. The basic permeability properties of the BeWo monolayers suggest that the cells grown on permeable supports may be examined as a convenient in vitro system to evaluate some transplacental transport mechanisms.

The HRP-1 cell line is derived from normal rat placenta and appears morphologically similar to and retains characteristic expression of cellular markers of labyrinthine trophoblast cells. In this study, monolayers of HRP-1 cells grown on permeable supports were evaluated as a potential in vitro system to study trophoblast transport and metabolism. The cell line was shown to express and retain functional activity of the predominant placental cytochrome P450 isozyme, CYP1A1. Additionally, the HRP-1 cells retain functional activity of angiotensin I converting enzyme and carboxypeptidase N-like enzyme, peptidases characteristic of the trophoblast. The permeation of several hydrophilic, inert markers across the HRP-1 monolayers was observed to be dependent on effective molecular size and to be passive in nature. Functional asymmetry of the HRP-1 cells was illustrated by the predominant permeation of linoleic acid in the apical-to-basolateral direction across the monolayers. Transferrin passage across HRP-1 monolayers was concentration-dependent, was bidirectional, and could be inhibited by unlabeled transferrin, features typical of the trophoblast transport system for transferrin. Collectively, these properties suggest that the HRP-1 cell line may provide a useful tool for evaluating some of the permeability and metabolic properties of the trophoblast.

The importance of cell adhesion molecules in maintaining the cellular integrity of the endothelial layer is well recognized, yet their exact participation in regulating the blood-brain barrier (BBB) is poorly understood. Both Ca(2+)-dependent and Ca(2+)-independent cell adhesion molecules are found in endothelial cells. In this study, we used immunofluorescence, ELISA, Western blot and cell adhesion assay to identify a Ca(2+)-dependent cell adhesion molecule, E-cadherin, in bovine brain microvessel endothelial cells (BBMECs). Monoclonal anti-E-cadherin antibody specifically interacted with cultured BBMECs and decorated the cellular junctions with a series of punctate fluorescence spots as seen by indirect immunofluorescence using a confocal microscope. The intensity of these fluorescence spots increased after brief treatment with hIFN-gamma or CPT-cAMP. In the cellular extract of BBMECs, a 120 kDa protein was immunoprecipitated with anti-E-cadherin antibody. BBMECs did not react with anti-N-cadherin antibody, but recognized the FITC-labeled LRAHAVDVNG-NH2, a decapeptide generated from the EC-1 domain of N-cadherin, which decorated the lateral margins of the cells with fluorescence spots. A concentration-dependent binding of this decapeptide was also observed in the flow cytometry assay. BBMECs dissociated with trypsin plus Ca2+ were able to reaggregate only in the presence of Ca2+. However, such cell-cell aggregations of BBMECs were prevented by the presence of either anti-E-cadherin antibody or the decapeptide in the assay medium. These results confirm that BBMECs possess a distinct Ca(2+)-dependent cell adhesion mechanism that can be modulated by the decapeptide. This modulation of cell-cell adhesion in BBMECs by the decapeptide is thought-provoking for creating channels for paracellular drug delivery across the BBB.

A systematic approach was used to demonstrate the quantitative interplay of pH, pKa, lipophilicity, charged and uncharged molecular species, molecular size, aqueous diffusivity, and stirring in passive transport across the aqueous boundary layer, microporous filter support, and transcellular and paracellular barriers in Caco-2 cell monolayers. The relationship of permeability of the aqueous boundary layer and hydrodynamic stirring was elucidated from transmonolayer fluxes of testosterone. Adrenergic receptor antagonists including propranolol (PPL), alprenolol (APL), pindolol (PDL), and atenolol (ATL) represented the model series of structurally similar weak bases with pKa values between 8.8 and 9.65. Although intrinsically lipophilic, their apparent log PC (n-octanol/water) at pH 7.4 and 6.5 ranged from -2.6 to 1.3. Effective permeability coefficients (Pe) correlated with log PC at both pH 7.4 and 6.5 showing a single sigmoidal-like curve: PPL > APL > PDL > or = ATL. The Pe approached a minimum plateau value established by the protonated ATL for the paracellular route (pore radius of 12 A) by molecular size-restricted diffusion within a negative electrostatic field of force. The Pe of the weak bases was delineated into component permeability coefficients of the aqueous boundary layer and porous filter support, the intrinsic permeabilities of charged and uncharged species for the transcellular and paracellular routes, and the extent to which the routes were utilized at each pH. This study emphasized a generally applicable approach to quantitatively analyze passive transport data on weak organic electrolytes and neutral molecules generated using cell culture monolayers.

Selected 21-aminosteroids (U74500A, U74006F, and U74389G) and a 2-(aminomethyl)chromans (U78517F) were tested for their efficacy in preventing arachidonate-induced lipid peroxidation and permeability alterations in brain microvessel endothelial cells (BMECs). The 21-aminosteroids and 2-(aminomethyl)chromans were effective in varying degrees in inhibiting (U74500A = U78517F > U74006F = U74389G) concentration- and time-dependent arachidonate-induced thiobarbituric acid reactive substances (TBARS) production by BMECs. Arachidonate produced a corresponding concentration-dependent increase in BMEC monolayer permeability to the membrane impermeant marker, sucrose. Pretreatment of BMEC monolayers with either the 21-aminosteroids or the 2-(aminomethyl)chromans completely blocked the arachidonate-induced increase in permeability to sucrose. Our results demonstrated that these membrane-associating antioxidants were particularly effective in preventing both arachidonic acid-induced lipid peroxidation and permeability changes in BMEC monolayers. However, concentrations of some antioxidants that only partially inhibited TBARS production, completely inhibited the arachidonic acid-induced enhancement in BMEC monolayer permeability. Therefore, arachidonic acid-induced effects on BMEC permeability were likely due in part to both lipid peroxidation and direct or indirect effects of the fatty acid on membrane integrity. This study provides further support for the application of primary cultures of BMECs as a useful in vitro system to evaluate mechanisms through which mediators of disease or injury states compromise blood-brain barrier integrity.

Leucine enkephalin (YGGFL) effects on markers for transcellular and paracellular permeation across the blood-brain barrier (BBB) were investigated in vitro with bovine brain microvessel endothelial cell (BMEC) monolayers in primary culture. Intact YGGFL, but not metabolites of YGGFL, stimulated BMEC uptake of lucifer yellow (LY), a marker for fluid-phase endocytosis, in a concentration-dependent manner. However, D-[Ala2]-leucine enkephalin (YAGFL), a YGGFL analogue that altered BMEC monolayer permeability, had no effect on LY uptake. In part, these results suggested that YGGFL's effects on fluid-phase uptake might not relate directly to enhanced BMEC transcellular permeability in the presence of the peptide. The measurement of the fluorescence anisotropy of membrane-bound diphenyl-hexatriene probes did not show substantial peptide-induced changes in membrane lipid packing order (i.e., membrane fluidity) and indicated a limited role for membrane perturbations in YGGFL-induced changes in BMEC monolayer permeability. Conversely, the apparent permeability coefficients showed size-dependent YGGL-induced alterations for passage of membrane-impermeant substances across BMEC monolayers. The apparent permeability coefficients of low-molecular-weight (low-mol-wt) molecules (mannitol, sucrose, and fluorescein) were increased on exposure to YGGFL. The apparent permeability coefficients for high-mol-wt molecules, FITC dextran conjugates (4, 20, and 71.6 Kd), were not affected by exposure to YGGFL. Transmission electron micrographs of lanthanum (Stoke's radius, 10 A) exclusion from BMEC intercellular junctions supported these observations. Collectively, results from this study suggest that YGGFL enhanced BMEC permeability either by altering paracellular openings or through formation of a small pore in the monolayers to allow preferential penetration of low-mol-wt or small molecular size (< 10 A) substances.