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New Approaches to Studies of Paracellular Drug Transport in Intestinal Epithelial Cell Monolayers
Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmacy.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Studies of intestinal drug permeability have traditionally been performed in the colon-derived Caco-2 cell model. However, the permeability of these cell monolayers resembles that of the colon rather than that of the small intestine, which is the major site of drug absorption following oral administration. One aim of this thesis was therefore to develop a new cell culture model that mimics the permeability of the small intestine. 2/4/A1 cells are conditionally immortalized with a temperature sensitive mutant of SV40T. These cells proliferate and form multilayers at 33°C. At cultivation temperatures of 37 – 39°C, they stop proliferating and form monolayers. 2/4/A1 cells cultivated on permeable supports expressed functional tight junctions. The barrier properties of the tight junctions such as transepithelial electrical resistance and permeability to hydrophilic markers resembled those of the human small intestine in vivo. These cells lacked functional expression of drug transport proteins and can therefore be used as a model to study passive drug permeability unbiased by active transport. The permeability to diverse sets of drugs in 2/4/A1 was comparable to that of the human jejunum for both incompletely and completely absorbed drugs, and the prediction of human intestinal permeability was better in 2/4/A1 than in Caco-2 for incompletely absorbed drugs. The small intestinal-like paracellular permeability of 2/4/A1 thus enables better predictions of drug permeability in the small intestine than does Caco-2.

The studies of the paracellular route and its importance for intestinal drug permeability was also in focus in the second part of this thesis, in which a new principle for tight junction modulation was developed, based on the primary structure of the extracellular tight junction protein occludin. Peptides corresponding to the N-terminus of the first extracellular loop increased the permeability of the tight junctions, but lacked apical effect. This problem was solved by conjugation of one peptide to a lipoamino acid, resulting in two diastereomers with different effects. The L-isomer had a sustained apical effect, while that of the D-isomer was transient. In conclusion, conjugated occludin peptides constitute a new class of tight junction modulators that can enhance the tight junction permeability.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2003. , p. 66
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 285
Keywords [en]
Pharmaceutics, intestinal epithelium, tight junctions, cell culture
Keywords [sv]
Galenisk farmaci
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutics
Identifiers
URN: urn:nbn:se:uu:diva-3388ISBN: 91-554-5582-4 (print)OAI: oai:DiVA.org:uu-3388DiVA, id: diva2:162617
Public defence
2003-04-29, B41, Uppsala Biomedical Centre, Uppsala, 13:00
Opponent
Supervisors
Available from: 2003-04-08 Created: 2003-04-08 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Conditionally Immortalized Epithelial Cell Line for Studies of Intestinal Drug Transport
Open this publication in new window or tab >>Conditionally Immortalized Epithelial Cell Line for Studies of Intestinal Drug Transport
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1999 In: Journal of Pharmacology and Experimental Therapeutics, Vol. 290, no 3, p. 1212–1221-Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90305 (URN)
Available from: 2003-04-08 Created: 2003-04-08Bibliographically approved
2. An Improved Cell Culture Model Based on 2/4/A1 Cell Monolayers for Studies of Intestinal Drug Transport
Open this publication in new window or tab >>An Improved Cell Culture Model Based on 2/4/A1 Cell Monolayers for Studies of Intestinal Drug Transport
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2003 (English)In: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 20, no 3, p. 373-381Article in journal (Refereed) Published
Abstract [en]

PURPOSE:

To improve the viability of the 2/4/A1 cell culture model and to investigate different routes of drug transport in this cell line.

METHODS:

Two approaches were taken to decrease apoptosis. First, rat intestinal 2/4/A1 cells were transfected to overexpress the antiapoptotic protein Bcl-2. Second. normal 2/4/A1 cells were cultivated under conditions that stimulate differentiation and limit apoptosis. The monolayer integrity was investigated by transepithelial electrical resistance, permeability, and microscopy. The expression of drug transporters was investigated by RT-PCR, and transport function was assessed using specific markers.

RESULTS:

Normal 2/4/A1 cells died by apoptosis at 39°C. Bcl-2-expressing 2/4/A1 cells were viable but adopted a morphology of less-differentiated epithelial cells. Optimization of the culture conditions for 2/4/A1 cells inhibited cell death. The integrity was comparable to that of the human jejunum (50 Ω × cm2), making this approach preferable to Bcl-2 overexpression. Transcriptional analysis showed that some (e.g., MDR1), but not all (e.g., PepT1), transporters were found in 2/4/A1 cells. Studies using substrates for PepT1, P-gp, MRP2, and BCRP showed that none of the transporters were functional in 2/4/A1.

CONCLUSIONS:

The improved culture procedure will facilitate the use of 2/4/A1 cells. 2/4/A1 lack several transporters, which makes them a promising alternative to Caco-2 cells and artificial membranes in studies of passive drug transport.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-90306 (URN)10.1023/A:1022643802296 (DOI)12669956 (PubMedID)
Available from: 2003-04-08 Created: 2003-04-08 Last updated: 2018-01-13Bibliographically approved
3. Prediction of the Oral Absorption of Low Permeability Drugs Using Small Intestinal-like 2/4/A1 Cell Monolayers
Open this publication in new window or tab >>Prediction of the Oral Absorption of Low Permeability Drugs Using Small Intestinal-like 2/4/A1 Cell Monolayers
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2003 In: Pharmaceutical Research, Vol. 20, p. 397-405Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-90307 (URN)
Available from: 2003-04-08 Created: 2003-04-08Bibliographically approved
4. The Contribution of the Paracellular Route to The pH-Dependent Epithelial Permeability to Cationic Drugs
Open this publication in new window or tab >>The Contribution of the Paracellular Route to The pH-Dependent Epithelial Permeability to Cationic Drugs
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-90308 (URN)
Available from: 2003-04-08 Created: 2003-04-08 Last updated: 2010-01-13Bibliographically approved
5. A New Principle for Tight Junction Modulation Based on Occludin Peptides
Open this publication in new window or tab >>A New Principle for Tight Junction Modulation Based on Occludin Peptides
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2003 (English)In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 64, no 6, p. 1530-1540Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to investigate whether peptides from the extracellular loops of the tight junction protein occludin could be used as a new principle for tight junction modulation. Peptides of 4 to 47 amino acids in length and covering the two extracellular loops of the tight junction protein occludin were synthesized, and their effect on the tight junction permeability in Caco-2 cells was investigated using [14C]mannitol as a para-cellular marker. Lipopeptide derivatives of one of the active occludin peptides (OPs), synthesized by adding a lipoamino acid containing 14 carbon atoms (C14-) to the N terminus of the peptide, were also investigated. Peptides corresponding to the N terminus of the first extracellular loop of occludin increased the permeability of the tight junctions without causing short-term toxicity. However, the peptides had an effect only when added to the basolateral side of the cells, which could be partly explained by degradation by apical peptidases and aggregate formation. By contrast, the lipopeptide C14-OP90-103, which protects the peptide from degradation and aggregation, displayed a rapid apical effect. The l- and d-diastereomers of C14-OP90-103 had distinctly different effects. The d-isomer, which releases intact OP90-103 from the lipoamino acid, displayed a rapid and transient increase in tight junction permeability. The l-isomer, which releases OP90-103 more rapidly, gave a more sustained increase in tight junction permeability. In conclusion, C14-OP90-103 represents a prototype of a new class of tight junction modulators that act on the extracellular domains of tight junction proteins.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-90309 (URN)10.1124/mol.64.6.1530 (DOI)14645684 (PubMedID)
Available from: 2003-04-08 Created: 2003-04-08 Last updated: 2017-12-14Bibliographically approved

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