Transport and metabolism of opioid peptides across BeWo cells, an in vitro model of the placental barrier

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.     

Contribution of efflux pump activity to the delivery of pulmonary therapeutics

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.     

Modulation of P-glycoprotein activity in Calu-3 cells using steroids and beta-ligands

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.     

Fatty acid transport regulatory proteins in the developing rat placenta and in trophoblast cell culture models

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.     

Characteristics of the fetal/maternal interface with potential usefulness in the development of future immunological and pharmacological strategies

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.     

TCP-FA4: A derivative of tranylcypromine showing improved blood-brain permeability

A variety of approaches have been taken to improve the brain penetration of pharmaceutical agents. The amphipathic character of a compound can improve its interaction with the lipid bilayer within cell membranes, and as a result improve permeability. Fatty acid chains or lipoamino acids of various lengths were attached to tranylcypromine (TCP), in an attempt to improve the blood-brain barrier (BBB) permeability by increasing the lipophilicity as well as the amphiphatic character of the drug. TCP-FA4, one of the derivatives containing a four carbon alkyl acid chain, showed the greatest improvement in permeability. This molecule was slightly neuroprotective in a β-amyloid-induced neurodegeneration assay and may also be capable of upregulating brain derived neurotrophic factor (BDNF), as indicated by cell culture assays using human umbilical vein endothelial cells. Since decreased levels of BDNF are observed in many CNS disorders, and direct injection of BDNF is not a viable option due to its poor permeability across the BBB, small molecules capable of regulating BDNF that also cross the BBB may be an interesting treatment option.     

Low-affinity uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) in BeWo cells

Understanding the mechanisms of transport processes in the placenta can improve the safety and efficacy of drug delivery during pregnancy. Functional studies of organic cation transporters (OCTs) are usually carried out using radioactivity, and a fluorescent marker would add flexibility to experimental methods. As a published substrate for OCT1 and OCT2, the fluorescent compound 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) was chosen as a candidate for studying placental OCT function in BeWo cells. The expression of OCT1 and OCT2 was also investigated in BeWo cells, an established human choriocarcinoma trophoblastic cell line frequently used as an in vitro model of the rate-limiting barrier for maternal-fetal exchange of drugs and nutrients within the placenta. 4-Di-1-ASP was taken up into BeWo cells by a low-affinity, carrier-mediated process exhibiting a Km of 580+/-110 microM and Vmax of 97+/-9 nmol/mg protein/30 min, and asymmetric transport was observed, with greater permeability in the apical to basolateral (maternal-to-fetal) direction. However, RT-PCR revealed no expression of OCT1 or OCT2 in either BeWo cells or primary cultured human cytotrophoblast cells, and OCT substrates such as TEA and choline did not inhibit the uptake of 4-Di-1-ASP. Although the uptake of this fluorescent compound in BeWo cells is not mediated by an OCT, the colocalization experiments with fluorescence microscopy and inhibition studies confirmed significant mitochondrial uptake of 4-Di-1-ASP. Transport of 4-Di-1-ASP into the nuclear region of BeWo cells was also observed, which is likely mediated by a nucleoside transporter.