Is PAMPA a useful tool for discovery?

Experimental PAMPA data generated for 40 low molecular weight commercial drugs is reviewed in the context of its utility in the drug discovery process. Several experimental variables that have been introduced in the literature as additions or improvements to the PAMPA were also evaluated. The relationship between PAMPA data and both calculated and measured octanol/water distribution coefficients was examined. From this assessment, it was concluded that the PAMPA yields information about the lipophilicity as measured by the octanol/water partitioning of a compound, but that this same information could be derived in a simpler manner from calculated log D values.

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.

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Permeability measurement of macromolecules and assessment of mucosal antigen sampling using in vitro converted M cells.

INTRODUCTION: M cells are located in the epithelial layer covering the gut-associated lymphoid tissue and are responsible for delivery of macromolecules and microorganisms to the underlying lymphoid cells. It has been shown that the human colonic cell line Caco-2 can be converted to M cells in vitro following coculture with isolated lymphocytes from murine Peyer's patches. Studies were undertaken to evaluate and characterize the transepithelial transport of select macromolecules across these in vitro derived M cells. METHODS: Caco-2 cells were converted to M cells as reported previously. The morphology of Caco-2 cells and M cells was compared by transmission electron microscopy (TEM). The transport properties of macromolecules such as horseradish peroxidase, FITC-conjugated polystyrene beads, and radiolabeled dextrans were examined. The activation of murine antigen-specific T cells following transport of the antigen ovalbumin across the M-cell barrier was assessed by measuring cytokine production. RESULTS: M cells were shown to be irregular in shape and have fewer and shorter microvilli compared to the Caco-2 cell progenitors. These cells were still able to form tight junctions and monolayers on polycarbonate membranes. Time-course studies demonstrated that the transport of polystyrene beads and large-molecular-weight dextrans at physiological temperature across M-cell-containing monolayers was size dependent and more rapid than across Caco-2 cell monolayers. The transport of dextrans was also shown to be temperature and concentration dependent. Befitting the role of the M cell in mucosal defense, protein antigen could be delivered by these cells in order to be processed and presented to antigen-specific CD4+ T lymphocytes. DISCUSSION: The M-cell permeability model is a functional and practical system for evaluating the transport properties of macromolecules and assessing the potential for intestinal mucosal antigen sampling to elicit immunological responses.

Evaluation of an accelerated Caco-2 cell permeability model.

An accelerated 3-7-day Caco-2 cell permeability model was examined and compared to the traditional 21-25-day model. Caco-2 cell permeability coefficients (P(Caco-2)) of 33 structurally diverse small molecular weight compounds from apical to basolateral (AP-->BL) direction in the accelerated model were approximately twice those in the traditional model. As observed with microscopy and transepithelial electrical resistance measurements, this difference was attributed to less confluent and differentiated Caco-2 cell monolayers in the accelerated model. However, there were no significant differences in rank ordering of the compounds. The expression of P-glycoprotein in the accelerated model was shown to be significantly less than that in the traditional model. This resulted in lower permeability directional ratios defined as the ratio between permeability coefficients from BL-->AP and from AP-->BL for compounds that were cellular efflux pump substrates. The accelerated model may not be suitable for studying cellular efflux pumps such as P-glycoproteins. However, it is a feasible alternative to the traditional model for rank ordering of compounds in the process of drug discovery and development by significantly improving the turnover time and labor efficiency. This makes it an excellent Caco-2 cell permeability model for high throughput screening.

In vitro blood-brain barrier permeability of nevirapine compared to other HIV antiretroviral agents.

To combat infection and inhibit viral replication of HIV in the brain, antiretroviral agents must cross the blood-brain barrier (BBB). An in vitro BBB model consisting of bovine brain microvessel endothelial cells grown on porous filters was used to study and compare the transport of nevirapine, a potent and selective nonnucleoside reverse transcriptase inhibitor, with other HIV antiretroviral agents currently in use for the treatment of HIV infection. These included nucleoside reverse transcriptase inhibitors (didanosine, stavudine, zalcitabine, zidovudine), a nonnucleoside reverse transcriptase (delaviridine), and protease inhibitors (indinavir, saquinavir, VX-478). Nevirapine was the most permeable antiretroviral agent studied in the BBB model. The order of in vitro BBB permeability was nevirapine >> VX-478 > didanosine, stavudine, zalcitabine, zidovudine > indinavir > saquinavir. There was an apparent bell-shaped relationship between in vitro BBB permeability and octanol/phosphate-buffered saline distribution coefficient (D) where all lipophilic (log D > 2.5) as well as hydrophilic (log D < -0.5) antiretrovirals were less permeable than nevirapine (log D = 1.8). There were no significant effects on the in vitro BBB permeability of nevirapine in combination with other antiretroviral agents. Saquinavir was the only drug shown to have an affinity for the P-glycoprotein efflux pump, which may have contributed to its very low permeability. The apparent ability of nevirapine to readily permeate the BBB and enter the brain, where it may inhibit replication of HIV, potentially increases its therapeutic value.

P-glycoprotein efflux pump expression and activity in Calu-3 cells.

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.

Characterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery.

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.

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.

Microparticulate uptake mechanisms of in-vitro cell culture models of the respiratory epithelium.

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.

Comparison of the effects of an aqueous extract of Physalis alkekengi fruits and/or various doses of 17-beta-estradiol on rat estrous cycle and uterine glucose 6-phosphate dehydrogenase activity.

Intraperitoneal injections of an aqueous extract of winter cherry fruits (Physalis alkekengi) to adult normal cycling female rats produced 100% diestrus and diminished uterine glucose 6-P dehydrogenase activity (an estrogen-induced protein) by 52%. Daily doses of 1.88, 3.75 and 7.5 micrograms 17-beta-estradiol administered intraperitoneally to adult female rats for a period of 6-8 days prolonged proestrus or estrus and increased uterine glucose 6-P dehydrogenase activity by 11.5%, 26.9% and 82.1%, respectively. Combined intraperitoneal injections of a given dose of the aqueous extract together with the above doses of 17-beta-estradiol for 8 consecutive days shortened the time spent in diestrus proportional to the dose employed and proportionately reduced the uterine glucose 6-P dehydrogenase inhibitory power of the aqueous extract (1.88 micrograms estradiol, 33.9% inhibition; 3.75 micrograms estradiol, 27% inhibition; and 7.5 micrograms estradiol, 6.0% activation). The data obtained clearly demonstrate the presence of an estrogen antagonist in the aqueous extract of Physalis alkekengi fruits.

The effect of diethylene glycol monoethyl ether as a vehicle for topical delivery of ivermectin.

The effect of diethylene glycol monoethyl ether (DGME; Transcutol) on the permeation of ivermectin, a broad-spectrum antiparasitic agent, through bovine skin was evaluated by in vitro permeation experiments followed by serial sectioning of the skin to assess the amount of ivermectin retained in the skin. Ivermectin permeation through bovine skin was enhanced by DGME and this enhancement was DGME-concentration-dependent. Permeation of ivermectin was effectively enhanced in vehicles with low proportions of DGME, but the magnitude of permeation enhancement decreased as the proportion of DGME increased. The permeation was accompanied by the formation of cutaneous depots of ivermectin. Furthermore, the data indicated that the flux and the cutaneous accumulation of ivermectin were sensitive to the concentration gradient of DGME across the skin. This suggested that ivermectin was permeating with DGME, in which it is very soluble. Hence, the enhancing mechanism involves solubilization of the ivermectin by DGME and the transport of DGME itself across the skin. Based on these results, DGME appears to be a potential vehicle for topical delivery of ivermectin by transport through the skin and through formation of cutaneous depots of ivermectin.

Mechanisms of transport and structure-permeability relationship of sulfasalazine and its analogs in Caco-2 cell monolayers.

PURPOSE: To investigate the mechanisms involved in transport of sulfasalazine in Caco-2 cells. METHODS: Permeability coefficients of sulfasalazine and its analogs across Caco-2 cell monolayers were measured as a function of direction of transport, energy and concentration dependence, and in the presence of inhibitors of various cellular efflux pumps and transporters. RESULTS: Permeability coefficients of sulfasalazine across Caco-2 cell monolayers were approximately 342-, 261-, and 176-fold higher from basolateral to apical direction (BL-->AP) than from apical to basolateral direction (AP-->BL) at 100, 200, and 500 microM, respectively. Carrier permeability coefficient, non-saturable membrane permeability coefficient, and Michaelis constant were estimated to be 1.4x10(-5) cm/s, 1.9x10(-8) cm/s, and 369 microM, respectively. The efflux of sulfasalazine was completely blocked at 4 degrees C and in the presence of an uncoupler of oxidative phosphorylation. Using cellular efflux inhibitors, the permeability of sulfasalazine was shown to depend on multidrug resistance-associated protein and anion sensitive transport mechanisms. Structure-permeability studies showed that the affinity of sulfasalazine for the cellular efflux pumps and transporters in Caco-2 cells depended strongly on the carboxylic acid functional group. CONCLUSIONS: The permeability of sulfasalazine across Caco-2 cell monolayer is very low due to its strong interaction with multiple cellular efflux pumps and transporters. This may partially explain its low absorption in vivo.

Note: sucrose transport and metabolism in Clostridium beijerinckii NCIMB 8052.

Sucrose is the major carbon source in molasses, the traditional substrate employed in the industrial acetonebutanol-ethanol (ABE) fermentation by solventogenic clostridia. The utilization of sucrose by Clostridium beijerinckii NCIMB 8052 was investigated. Extracts prepared from cultures grown on sucrose (but not xylose or fructose) as the sole carbon source possessed sucrose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity. Extract fractionation and reconstitution experiments revealed that the entire sucrose Enzyme II complex resides within the membrane in this organism. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose grown cultures. The fructokinase activity, which is required specifically during growth on sucrose, was shown to be inducible under these conditions. A pathway for sucrose metabolism in this organism is proposed.

Multidrug resistance-associated protein-1 functional activity in Calu-3 cells.

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.