Application of an in vitro cell monolayer model for evaluating the transport of a peptide and a mineral nutrient.

Persistent Link:
http://hdl.handle.net/10150/187048
Title:
Application of an in vitro cell monolayer model for evaluating the transport of a peptide and a mineral nutrient.
Author:
Surendran, Narayanan.
Issue Date:
1995
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The overall objectives of this research is two-fold. First, preformulation studies of a novel chemopreventive peptide known as Melanotan-I (MT-I) were performed in order to facilitate the development of suitable formulations of this compound. The studies conducted include (a) the development of a HPLC assay in cell culture transport buffer and human plasma, (b) determination of its partitioning properties and stability kinetics in aqueous solutions, and (c) evaluation of transport properties using an in-vitro cell monolayer model consisting of Caco-2 cells grown as a monolayer on permeable supports. The HPLC method developed here was sensitive, specific and stability-indicating. This assay was successfully applied to the study of MT-I transport across Caco-2 cells. Partitioning of MT-I determined in n-octanol:buffer and isooctane:buffer solvent systems indicated the potential of delivering this peptide via the oral route. MT-I degradation in phosphate buffer exhibited apparent first order kinetics with an estimated shelf life of 40 days at room temperature. It was found to be relatively stable at acidic conditions but had an increased degradation rate at pH > 7.4. Transport of MT-I across Caco-2 cells indicated the degradation of this peptide by the proteases associated with the enterocyte to be the primary obstacle to its oral delivery. MT-I transport was significantly enhanced in the presence of a protease inhibitor (aprotinin). The second objective of this research was to identify enhancers of calcium transport using the in-vitro Caco-2 cell monolayer model. Both medium-chain triglycerides and acylcarnitines were found to enhance the transport of calcium, although to varying degrees. Acylcarnitines were found to be more potent enhancers of calcium transport, but a greater extent of cell damage was observed with their use. In addition, Caco-2 cells were shown to possess L-type calcium channels for the first time, and acylcarnitines were demonstrated to behave like calcium channel agonists similar to that of Bay K 8644 (an established channel agonist). Promotion of both transcellular (membrane perturbation) and paracellular (loosening of tight junctions) pathways of calcium transport were shown to be significant contributors toward the overall enhancement mediated by the acylcarnitines chosen for this study.
Type:
text; Dissertation-Reproduction (electronic)
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Pharmaceutical Sciences; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Blanchard, James

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleApplication of an in vitro cell monolayer model for evaluating the transport of a peptide and a mineral nutrient.en_US
dc.creatorSurendran, Narayanan.en_US
dc.contributor.authorSurendran, Narayanan.en_US
dc.date.issued1995en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe overall objectives of this research is two-fold. First, preformulation studies of a novel chemopreventive peptide known as Melanotan-I (MT-I) were performed in order to facilitate the development of suitable formulations of this compound. The studies conducted include (a) the development of a HPLC assay in cell culture transport buffer and human plasma, (b) determination of its partitioning properties and stability kinetics in aqueous solutions, and (c) evaluation of transport properties using an in-vitro cell monolayer model consisting of Caco-2 cells grown as a monolayer on permeable supports. The HPLC method developed here was sensitive, specific and stability-indicating. This assay was successfully applied to the study of MT-I transport across Caco-2 cells. Partitioning of MT-I determined in n-octanol:buffer and isooctane:buffer solvent systems indicated the potential of delivering this peptide via the oral route. MT-I degradation in phosphate buffer exhibited apparent first order kinetics with an estimated shelf life of 40 days at room temperature. It was found to be relatively stable at acidic conditions but had an increased degradation rate at pH > 7.4. Transport of MT-I across Caco-2 cells indicated the degradation of this peptide by the proteases associated with the enterocyte to be the primary obstacle to its oral delivery. MT-I transport was significantly enhanced in the presence of a protease inhibitor (aprotinin). The second objective of this research was to identify enhancers of calcium transport using the in-vitro Caco-2 cell monolayer model. Both medium-chain triglycerides and acylcarnitines were found to enhance the transport of calcium, although to varying degrees. Acylcarnitines were found to be more potent enhancers of calcium transport, but a greater extent of cell damage was observed with their use. In addition, Caco-2 cells were shown to possess L-type calcium channels for the first time, and acylcarnitines were demonstrated to behave like calcium channel agonists similar to that of Bay K 8644 (an established channel agonist). Promotion of both transcellular (membrane perturbation) and paracellular (loosening of tight junctions) pathways of calcium transport were shown to be significant contributors toward the overall enhancement mediated by the acylcarnitines chosen for this study.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplinePharmaceutical Sciencesen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairBlanchard, Jamesen_US
dc.contributor.committeememberMayersohn, Michaelen_US
dc.contributor.committeememberMartin, Arnolden_US
dc.identifier.proquest9531072en_US
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