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Cogburn, J. N.; Donovan, Matthew; Schasteen, Charles S.

doi:10.1023/a:1015844104539

Cited by 99 publications

(8 citation statements)

References 13 publications

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“…The different culturing conditions (e.g., collagen-coated versus noncollagen-coated filters, 21-28 days of culture versus 13-16 days of culture, 37°C versus 25°C incubation temperature) could have resulted in different cell densities, which could result in different pore sizes (i.e., a lower cell density could result in a larger pore size, whereas a higher cell density could result in a smaller pore size). Furthermore, the studies by Cogburn et al 20 and Gonzá lesMariscal et al 21 indicate that paracellular permeabilities decrease with lowered temperature.…”

Section: Discussionmentioning

confidence: 97%

Paracellular Diffusion in Caco-2 Cell Monolayers: Effect of Perturbation on the Transport of Hydrophilic Compounds That Vary in Charge and Size

Knipp¹,

Ho²,

Barsuhn³

et al. 1997

Journal of Pharmaceutical Sciences

188

148

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We applied the principles of molecular-size-restricted diffusion within a negative electrostatic field of force to follow the changes in the aqueous pore radius of tight junctions (TJs) induced by perturbants and the accompanying influence on the permeation of neutral (urea and mannitol), cationic (methylamine and atenolol), and anionic (formate and lactate) compounds that vary in size. The perturbants included palmitoyl-DL-carnitine (PC), which opens TJs by an unknown Ca++-independent mechanism, and ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a Ca++ chelator. Mass transfer resistances of the collagen-coated filter support and the aqueous boundary layers were factored out to yield paracellular permeability coefficients (P[P]). As viewed from the P(P) values of urea and mannitol, EGTA exhibited insignificant effects on pore size at low concentrations compared with control, and then caused a dramatic opening of the TJs over a narrow concentration range (1.35-1.4 mM). The P(P) values for urea and mannitol remained constant at >1.4 mM EGTA. However, PC produced dose-dependent responses from O to 0.15 mM that plateaued at >0.15 mM. In general, cations permeated the cellular TJs faster and anions slower than their neutral images. The effects of changes in pore size (4.6 to 14.6 A in effective radius) on the ability of these solutes to permeate the TJs were analyzed by the Renkin molecular sieving function. These studies established an experimental, theoretical, and quantitative template to assess perturbants of the TJ and define the limits, short of detrimental effects, at which the TJs may be sufficiently perturbed for maximal enhancement of permeation of solutes varying in size and charge.

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Section: Discussionmentioning

confidence: 97%

Paracellular Diffusion in Caco-2 Cell Monolayers: Effect of Perturbation on the Transport of Hydrophilic Compounds That Vary in Charge and Size

Knipp¹,

Ho²,

Barsuhn³

et al. 1997

Journal of Pharmaceutical Sciences

188

148

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“…Since they differentiate in a single cell monolayer, they provide an easy model for mechanistic studies in contrast to other methods using ex vivo tissues (e.g., Ussing chambers). 16,17,20,21) Both AP-to-BL and BL-to-AP transport of compounds with or without inhibitors can be performed to identify a transport mechanism though the cell monolayers, active or passive. In this base, we evaluated the carrier-mediated transport of the L-Phe in both models using a single concentration around the K m value of each apical and basolateral transporter at 37°C, since it was demonstrated by others that this compound is a specific substrate of the large neutral amino acid carrier.…”

Section: Discussionmentioning

confidence: 99%

“…Morphological Comparison Electron Microscopy: Caco-2/TC7 cells at passage 22 and 24, respectively, for 21 and fixed in glutaraldehyde 2.5% vol/vol in PBS. They were then treated as described elsewhere for transmission electron microscopy (TEM) using a JEOL JEM-100S.…”

Section: Methodsmentioning

confidence: 99%

Short Term Caco-2/TC7 Cell Culture: Comparison between of Conventional 21-d and a Commercially Available 3-d System

Violante¹,

Zerrouk²,

Richard

et al. 2004

Biological & Pharmaceutical Bulletin

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The Caco-2 cell model is a valuable tool for studying intestinal biotransformation of xenobiotics and to evaluate the potential of human intestinal absorption of new compounds. These properties were evaluated with Caco-2/TC7 cells in accelerated conditions to reduce maturation lag time from 21-d to 3-d in order to increase time and labor efficiency. Transmission electron and fluorescent microscopy were used for morphological characterization. Alkaline phosphatase and lactate dehydrogenase activities were assessed within time. Cytochrome P450 expression was studied by RT-PCR. Apparent permeabilities of a set of passively absorbed molecules across Caco-2/TC7 cell monolayers were determined to evaluate potential of both systems for prediction of human intestinal absorption. Microscopic images revealed that cells under both conditions differentiated as enterocyte-like cells but did so heterogeneously in the 3-d model. TEER values have shown that the 3-d model is a leakier cell system with higher mannitol Papp (cm/s). Biochemical characterization (hydrolase activities, CYP450 expression) suggested that the 3-d model was at a lower maturation level than the 21-d model. Carrier-mediated uptake of L-Phe was lower in the 3-d model suggesting that this model has limited application for mechanistic studies. Reasonable correlation was obtained between the two models (r2=0.88, p>0.01) for 11 passively absorbed compounds with high potential of rank ordering of compounds. Although results suggested that the 3-d cells are under-differentiated, they could be usable to estimate the oral absorption of passively absorbed compounds.

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“…In addition to prediction studies for the membrane permeability using chemical descriptors, validation studies for these screening assays were performed using marketed drugs and reagents mostly with acceptable solubility and/or lipophilicity [12,33,[51][52][53][54][56][57][58][59]. However, because the screenings aim to improve the poor physicochemical properties of compounds, the systems have to be validated based on the data derived from model compounds, partially including insoluble and highly lipophilic derivatives.…”

Section: Pitfalls In High Throughput Screening For Absorptionmentioning

confidence: 99%

Pitfalls in High Throughput Screening for Drug Absorption Optimization in Drug Discovery

Kono¹,

Iwaki

2007

CAC

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Implementation of combinatorial chemistry and high throughput screening to biological targets has led to produce lipophilic and poorly water soluble lead compounds. Since these compounds show poor absorbability, high throughput methods for improving solubility and membrane permeability have been developed. Kinetic solubility of compounds after dilution of dimethyl sulfoxide solution into aqueous buffer is determined using high throughput methods with turbidimetry, nephelometry or UV assays. The kinetic solubility tends to be overestimated compared with the corresponding thermodynamic solubility obtained using a traditional flask shaking method. A new solid-dissolution method providing thermodynamic solubility similar to that in traditional method has been developed using a 96-well plate for equilibrium dialysis. In conventional Caco-2 assays, the membrane permeability (P app ) of certain lipophilic compounds was underestimated due to insolubility in the apical compartment and adhesion to the device, resulting in a poor relationship between the in vivo absorption fraction and the P app values. The addition of a solubilizer into the apical compartment and bovine serum albumin in the basolateral compartment improved the relationship, enabling evaluation of widely diverse compounds. The usefulness of these newly-developed methods for compounds with poor physical properties will be discussed, with reviewing currently available high throughput methods for optimizing absorption.

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Cited by 99 publications

References 13 publications

Paracellular Diffusion in Caco-2 Cell Monolayers: Effect of Perturbation on the Transport of Hydrophilic Compounds That Vary in Charge and Size

Paracellular Diffusion in Caco-2 Cell Monolayers: Effect of Perturbation on the Transport of Hydrophilic Compounds That Vary in Charge and Size

Short Term Caco-2/TC7 Cell Culture: Comparison between of Conventional 21-d and a Commercially Available 3-d System

Pitfalls in High Throughput Screening for Drug Absorption Optimization in Drug Discovery

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