A comparison of commonly used polyethoxylated pharmaceutical excipients on their ability to inhibit P-glycoprotein activity in vitro

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.     

Progress and limitations in the use of in vitro cell cultures to serve as a permeability screen for the blood-brain barrier.

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.     

The Application of Bovine Brain Microvessel Endothelial-Cell Monolayers Grown onto Polycarbonate Membranes in Vitro to Estimate the Potential Permeability of Solutes Through the Blood–Brain Barrier

Previously our laboratory (Rim et aL, Int. J. Pharm. 32:79–84, 1986) described an in vitro blood-brain barrier (BBB) model consisting of cultured bovine brain microvessel endothelial cells (BMECs) grown onto regenerated cellulose acetate membranes. However, the utility of this in vitro BBB model system was limited because the regenerated cellulose acetate membrane and not the monolayer of bovine BMECs was rate limiting for the permeability of very lipophilic compounds. Therefore, in this study we have evaluated polycarbonate membranes as supports for growing bovine BMECs and for conducting in vitro drug permeability studies. Bovine BMECs were cultured on collagen-coated polycarbonate membranes (13-mm diameter, 12-µm pore size) which were then mounted into side-by-side diffusion cells for transport studies. The permeabilities of a series of solutes of varying lipophilicity (progesterone, estrone, testosterone, haloperidol, propranolol, antipyrine, caffeine, urea, acyclovir, ganciclovir, ribavirin, and glycerol) were determined and an excellent correlation (r = 0.97) was established between the permeability coefficients of the solutes and their log partition coefficients (PC)/(MW)^1/2. These results suggest that bovine BMECs cultured onto polycarbonate membranes can be used as an in vitro model system for estimating the potential permeability of a solute through the BBB in vivo.     

Effect of Some Penetration Enhancers on Epithelial Membrane Lipid Domains: Evidence from Fluorescence Spectroscopy Studies

The effect of the penetration enhancers Azone, oleic acid, 1-dodecanol, dodecyl N,N-dimethylaminoacetate (DDAA), and dodecyl N,N-dimethylaminoisopropionate (DDAIP) on epithelial membrane lipids was examined using human buccal cell membranes as a model for epithelial lipid bilayer. Buccal epithelial cells (BEC) were labeled with l,6-diphenyl-l,3,5-hexatriene (DPH), l-(4-(trimethylammonio)phenyl)-6-phenyl-l,3,5-hexatriene (TMA-DPH), and 8-anilino-l-naphthalene sulphonic acid (ANS) fluorophores to characterize enhancer-induced changes in the hydrophobic core, in the superficial polar head region, and on the exterior surface, respectively, with fluorescence anisotropy and fluorescence lifetimes. All the enhancers studied were found to decrease the BEC membrane lipid packing order in a concentration-dependent and time-dependent manner in the deep bilayer region, as shown by a 37–66% decrease in anisotropy. Oleic acid was also found to disrupt membrane lipids strongly in the polar head region, causing at least a 34% decrease in anisotropy values. Azone and DDAA were shown to alter molecular movement on the surface of the bilayers (24 and 19% decrease in anisotropy, respectively). The results suggest that interaction with membrane lipid domains is an important, but not the only, mode of action for the penetration enhancers studied.     

Carrier-Mediated Transport of Baclofen Across Monolayers of Bovine Brain Endothelial Cells in Primary Culture

The mechanism of transport of baclofen, a centrally acting muscle relaxant, across the blood–brain barrier (BBB) was studied using an in vitro model. The model consisted of a monolayer of bovine brain endothelial cells grown in primary culture on a porous regenerated cellulose membrane. The transport of baclofen across the monolayer expressed time and concentration dependency and was saturable. Transport data were corrected for diffusion and fitted to the Michaelis–Menten V _max model: K _m = 58.5 µM, V _max = 0.23 nmol/min. The results validate the use of the in vitro BBB model as described and support the hypothesis that baclofen penetrates the BBB by means of a carrier-mediated transport system.     

Tie-1 and tie-2 define another class of putative receptor tyrosine kinase genes expressed in early embryonic vascular system.

We report the molecular cloning and characterization of two structurally related putative receptor tyrosine kinases, encoded by distinct genes (tie-1 and tie-2) on mouse chromosome 4. Both tie-1 and tie-2 encode receptor proteins possessing unique multiple extracellular domains: two immunoglobulin-like loop domains flanking three epidermal growth factor repeats followed by three fibronectin-type III repeats. Both genes are expressed in early embryonic vascular system and in maternal decidual vascular endothelial cells, where the vasculature undergoes an active angiogenesis. tie-2, but not tie-1, expression was also detected in extraembryonic mesoderm of the amnion. tie-1, but not tie-2, is expressed in an acute myelogenic cell line in vitro. tie-1 and tie-2 may form another class within the receptor tyrosine kinase gene family, and further characterization of these genes and identification of their putative ligands should define the nature of the signal-transduction cascades underlying early vascular system development, as well as their differential roles in mesodermal cells of the amniotic and myeloid lineages.     

Role of P-glycoprotein in transplacental transfer of methadone

Methadone is the therapeutic agent of choice for treatment of the pregnant opiate addict. However, little is known on the factors affecting its concentration in the fetal circulation during pregnancy and how it might relate to neonatal outcome. Therefore, a better understanding of the function of placental metabolic enzymes and transporters should add to the knowledge of the role of the tissue in the disposition of methadone and its relation to neonatal outcome. We hypothesized that the expression and activity of the placental efflux transporter P-glycoprotein (P-gp) would affect the transfer of methadone to the fetal circulation. Data obtained utilizing dual perfusion of placental lobule and monolayers of Be-Wo cell line indicated that methadone is extruded by P-gp. Transfer of methadone to the fetal circuit was increased by 30% in the presence of the P-gp inhibitor GF120918 while the transfer of paclitaxel, a typical substrate of the glycoprotein, was increased by 50%. In the Be-Wo cell line, methadone and paclitaxel uptake was also increased in the presence of the P-gp inhibitor cyclosporin A. Moreover, the expression of P-gp in placental brush-border membranes varied between term placentas. Taken together, these data strongly suggest that the concentration of methadone in the fetal circulation is affected by the expression and activity of P-gp. It is reasonable to speculate that placental disposition of methadone affects its concentration in the fetal circulation. If true, this may also be directly related to the incidence and intensity of neonatal abstinence syndrome (NAS).     

Tetrazole compounds: the effect of structure and pH on Caco-2 cell permeability

A tetrazole ring is often used in drug discovery as a replacement for the carboxylic acid group. Previous work indicates that compounds containing a tetrazole moiety show asymmetric permeability in Caco-2 cells characteristic of an efflux transporter substrate. The aim of this study is to determine which transporters are responsible for polarization of transport of tetrazole-containing compounds in Caco-2 cells. Results indicate that only select compounds with tetrazole moieties display asymmetric transport. Three compounds (two commercial drug products and one druglike structure) were selected for further studies. Losartan appears to be primarily a P-glycoprotein (P-gp) substrate, as previously reported, but MRP inhibitors such as MK-571 and rifampicin also affect the difference between apical to basolateral and basolateral to apical transport. Pemirolast and phenyltetrazole derivative C are sensitive to P-gp inhibition, but transport seems to be mediated by one or more of the MRP family of transporters. Additionally, lowering the pH from 7.4 to 4.0 eliminates the polarization of permeability in Caco-2 cells. These studies indicate that some tetrazole compounds are susceptible to efflux, therefore caution should be used when choosing an appropriate functional group to replace carboxylic acids when synthesizing a drug candidate.