Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions.

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5-7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.

Human PEPT1 pharmacophore distinguishes between dipeptide transport and binding.

The human intestinal oligopeptide transporter (PEPT1) facilitates the absorption of dipeptides, tripeptides, and many peptidomimetic drugs. In this study, a large number of peptides were selected to investigate the structural features required for PEPT1 transport. Binding affinity was determined in a Gly-Sar uptake inhibition assay, whereas functional transport was ranked in a membrane depolarization assay. Although most of the peptides tested could bind to PEPT1, not all were substrates. As expected, single amino acids and tetrapeptides could not bind to or be transported by PEPT1. Dipeptide transport was influenced by charge, hydrophobicity, size, and side chain flexibility. The extent of transport was variable, and unexpectedly, some dipeptides were not substrates of PEPT1. These included dipeptides with two positive charges or extreme bulk in either position 1 or 2. Our results identify key features required for PEPT1 transport in contrast to most previously described pharmacophores, which are based on the inhibition of transport of a known substrate.

Carbopol-mediated paracellular transport enhancement in Calu-3 cell layers.

The interaction of Carbopol polymers with mucus producing Calu-3 human bronchial epithelial cells was evaluated to test for potential paracellular transport enhancement. Using desmopressin (1-deamino-8-arginine-vasopressin, DDAVP) as the model peptide, apical treatment with Carbopol polymer gel formulations resulted in molecular size-dependent permeability enhancement with a concomitant drop in the transepithelial electrical resistance (TEER). Permeability enhancement of DDAVP was dependent on the formulation vehicle composition and polymer concentration, was noncytotoxic, and completely reversible. Carbopol 971P displayed the greatest permeability enhancement across Calu-3 cells compared to other more viscous Carbopol polymers 934P and 974P, and other mucoadhesive cellulosic polymers. The greatest enhancement was observed when C971P formulation was prepared in water at a concentration of 0.25% w/v. Enhancement was confirmed in rabbit dosed with intranasal fluorescent dextran 4400. The C(max) and absorption rate each increased by 48% in C971P formulations compared to control, while the relative exposure increased 30%. In conclusion, Carbopol polymers are potentially useful excipients to enhance intranasal peptide absorption. We hypothesize that the permeation enhancement is related to the chelation of extracellular or tight-junctional Ca(2+) by charged polymer carboxylate groups that leads to temporary disruption of tight-junctions, thereby facilitating paracellular transport.

pH-dependent dissolution in vitro and absorption in vivo of weakly basic drugs: development of a canine model.

PURPOSE: The aim of this research was to develop a pH-dependent canine absorption model for studying pH effect on both dissolution in vitro and pharmacokinetics in vivo using the weak bases ketoconazole and dipyridamole as model drugs. METHODS: Ketoconazole and dipyridamole pH-dependent dissolution profiles in vitro were determined by dissolution test at different pH values using USP apparatus II and an Opt-Diss Fiber Optic UV System. In vivo absorption studies for ketoconazole and dipyridamole were performed with crossover design in three groups of beagle dogs under control (no treatment), pentagastrin, and famotidine treatments. Ketoconazole and dipyridamole plasma concentrations were quantified by gradient high performance liquid chromatography mass spectroscopy (HPLC MS/MS). Pharmacokinetic parameters were determined from individual plasma concentration vs. time profiles. RESULTS: Ketoconazole and dipyridamole displayed pH-dependent dissolution. Increasing the pH of the dissolution medium from 1.2 to 6.8 reduced the extent of dissolution of ketoconazole and dipyridamole at 1 h by 96% and 92%, respectively. In vivo studies in dogs under control (no treatment), pentagastrin, and famotidine treatments show marked differences in systemic ketoconazole and dipyridamole exposure. Area under the concentration-time curve (AUC) increased more than 4-fold as compared to control group, whereas it increased nearly 30-fold for ketoconazole and 9-fold for dipyridamole with pentagastrin (gastric pH approximately 2-3) as compared to famotidine (gastric pH approximately 5-7.5) treatment. CONCLUSIONS: This work demonstrates a pH-dependent dissolution in vitro and absorption in vivo for the weak bases ketoconazole and dipyridamole independent of food effects. This model is useful to examine pH-dependent effects on oral drug absorption and for screening formulations to overcome the pH dependency.

In vitro testing of drug absorption for drug 'developability' assessment: forming an interface between in vitro preclinical data and clinical outcome.

Drug 'developability' assessment has become an increasingly important addition to traditional drug efficacy and toxicity evaluations, as pharmaceutical scientists strive to accelerate drug discovery and development processes in a time- and cost-effective manner. The fraction of drug absorbed and the maximum absorbable dose (MAD) can be estimated from in vivo clinical pharmacokinetics, mass balance studies or in vivo drug permeability in humans by different calculation methods. Unfortunately, in vivo data are usually unavailable at the early stages of drug discovery and development, and in vitro screening for the permeability, solubility, activity and toxicity of a drug has become a routine measurement in drug discovery and development. These in vitro data could be used to predict drug 'developability' with different calculation methods before selecting candidates for clinical evaluation. The fraction of drug absorbed in human could be predicted by in vivo human permeability or in vitro Caco2 permeability. For example, if drug permeability in Caco2 cells reaches 13.3 to 18.1 x 10(-6) cm/s, its predicted in vivo permeability in humans would reach 2 x 10(-4) cm/s, and its predicted fraction of drug absorbed would be > 90%, which is defined as highly permeable. The MAD could also be predicted with in vitro permeability, or calculated absorption rate constant. In addition, in vitro solubility and permeability data can also be used for the biopharmaceutics classification system (BCS) and, subsequently, to direct formulation optimization strategies. If drug 'developability' becomes an obstacle for drug delivery based on these in vitro data and predictions at the early stages of drug discovery and development, options such as prodrug approaches could be explored to enhance drug 'developability', in addition to different formulation methods. Therefore, in vitro absorption testing is a highly valuable tool in the decision-making process to select candidates for in vivo clinical studies at early-stage drug discovery and development.

A novel high-throughput pepT1 transporter assay differentiates between substrates and antagonists.

PepT1 is a transporter of proven pharmaceutical utility for enhancing oral absorption. A high-throughput, robust functional assay, capable of distinguishing PepT1 binders from substrates, allowing identification and/or prediction of drug candidate activation was developed. An MDCK epithelial cell line was transfected with rPepT1. The high level of stable rPepT1 expression that was achieved enabled development of a miniaturized PepT1 assay in a 96-well format, which could be scaled to 384 wells. The assay is based on measurement of membrane depolarization resulting from the cotransport of protons and PepT1 substrates. Membrane potential changes are tracked with a voltage-sensitive fluorescent indicator. Control (mock-transfected) cells are used to determine nonspecific membrane potential changes. A variety of fluorescent dyes were tested during initial assay design, including intracellular pH and membrane potential indicators. A membrane potential indicator was chosen because of its superior performance. Upon PepT1 activation with glycylsarcosine, dose-dependent membrane depolarization was observed with an EC50 of 0.49 mM. Maximum depolarization was dependent on the level of PepT1 expression. Testing of 38 known PepT1 substrates, binders, and nonbinders demonstrated that this assay accurately distinguished substrates from binders and from nonbinders. Initial validation of this novel assay indicates that it is sensitive and robust, and can distinguish between transporter substrates and antagonists. This important distinction has been previously achieved only with lower-throughput assays. This assay might also be used to determine substrate potency and establish a high-quality data set for PepT1 SAR modeling.

A novel hPepT1 stably transfected cell line: establishing a correlation between expression and function.

Stably transfected MDCK/hPepT1-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein were established to quantify the relationship between transgene hPepT1 expression levels and its functional kinetics in facilitating peptide and peptide-like drug uptake and transport in vitro. The hPepT1 sequence was amplified from Caco-2 cell mRNA, inserted into the pcDNA3.1 -V5&His TOPO plasmid, and transfected into MDCK cells. Transgene protein levels were quantified by Western Blot analysis utilizing a standard curve generated with a positive control protein containing a V5&His epitope. Three clones expressing different levels of the hPepT1 fusion protein (low, medium, and high) were selected for the functional characterization with [14C]Gly-Sar and [3H]carnosine. The MDCK/hPepT1 cells expressed a novel hPepT1/epitope tag protein with an apparent molecular mass of 110 kDa. The [14C]Gly-Sar uptake in the transfected cells was sodium-independent and pH-dependent, demonstrating enhanced uptake, the rate of which increased significantly from the weakly to strongly expressing hPepT1 MDCK/hPepT1 -V5&His clones as compared to the mock cell line at pH 6.0. The uptake and permeability of [14C]Gly-Sar and [3H]carnosine demonstrated a direct correlation between the hPepT1 level of expression, uptake, and transport capabilities. Molecular and functional characterization of the MDCK/hPepT1-V5&His cell line confirmed a directly proportional relationship between Vmax and Papp versus the molar levels of hPepT1 transgene expression. This stably transfected hPepT1 cell line may serve as a useful in vitro model for screening and quantifying peptide and peptide-like drug transport as a function of hPepT1 expression in drug discovery.

Permeability characteristics of calu-3 human bronchial epithelial cells: in vitro-in vivo correlation to predict lung absorption in rats.

The goal of this study was to evaluate the permeability characteristics of Calu-3, human bronchial epithelial cells to passive and actively transported drugs and to correlate the data with other in vitro models and rat lung absorption in vivo. Air-interface cultured Calu-3 cells grown on collagen-coated permeable filter supports formed "tight" polarized and well differentiated cell monolayers with apical microvilli and tight-junctional complexes. Within 8-10 days, cell monolayers developed trans-epithelial electrical resistance (TEER) > 1000 ohm cm2 and potential difference about 11-16 mV. Solute permeability was dependent on lipophilicity, and inversely related to molecular size. Calu-3 cells actively transported amino acids, nucleosides and dipeptide analogs, but not organic anions, organic cations or efflux pump substrates. The permeability characteristics of Calu-3 cells correlated well with primary cultured rabbit tracheal epithelial cells in vitro (r2 = 0.91), and the rate of drug absorption from the rat lung in vivo (r2 = 0.94). The absorption predicted from the regression equation correlated well with observed values. In conclusion, in vitro-in vivo correlation studies indicate that the Calu-3 cell culture model is a potentially useful model to predict absorption of inhalation delivery drug candidates.