Robert A. Conradi

Caco-2 Cell Monolayers as a Model for Drug Transport Across the Intestinal Mucosa

Human colon adenocarcinoma (Caco-2) cells, when grown on semipermeable filters, spontaneously differentiate in culture to form confluent monolayers which both structurally and functionally resemble the small intestinal epithelium. Because of this property they show promise as a simple, in vitro model for the study of drug absorption and metabolism during absorption in the intestinal mucosa. In the present study, the transport of several model solutes across Caco-2 cell monolayers grown in the Transwell ™ diffusion cell system was examined. Maximum transport rates were found for the actively transported substance glucose and the lipophilic solutes testosterone and salicylic acid. Slower rates were observed for urea, hippurate, and salicylate anions and were correlated with the apparent partition coefficient of the solute. These results are similar to what is found with the same compounds in other, in vivo absorption model systems. It is concluded that the Caco-2 cell system may give useful predictions concerning the oral absorption potential of new drug substances.

The Influence of Peptide Structure on Transport Across Caco-2 Cells

The relationship between structure and permeability of peptides across epithelial cells was studied. Using confluent monolayers of Caco-2 cells as a model of the intestinal epithelium, permeability coefficients were obtained from the steady-state flux of a series of neutral and zwitterionic peptides prepared from D-phenylalanine and glycine. Although these peptides ranged in lipophilicity (log octanol/water partition coefficient) from −2.2 to +2.8, no correlation was found between the observed flux and the apparent lipophilicity. However, a strong correlation was found for the flux of the neutral series and the total number of hydrogen bonds the peptide could potentially make with water. These results suggest that a major impediment to peptide passive absorption is the energy required to break water–peptide hydrogen bonds in order for the solute to enter the cell membrane. This energy appears not to be offset by the favorable introduction of lipophilic side chains in the amino acid residues.

The Influence of Peptide Structure on Transport Across Caco-2 Cells. II. Peptide Bond Modification Which Results in Improved Permeability

In order to study the influence of hydrogen bonding in the amide backbone of a peptide on permeability across a cell membrane, a series of tetrapeptide analogues was prepared from D-phenylalanine. The amide nitrogens in the parent oligomer were sequentially methylated to give a series containing from one to four methyl groups. The transport of these peptides was examined across confluent monolayers of Caco-2 cells as a model of the intestinal mucosa. The results of these studies showed a substantial increase in transport with each methyl group added. Only slight differences in the octanol–water partition coefficient accompanied this alkylation, suggesting that the increase in permeability is not due to lipophilicity considerations. These observations are, however, consistent with a model in which hydrogen bonding in the backbone is a principal determinant of transport. Methylation is seen to reduce the overall hydrogen bond potential of the peptide and increases flux by this mechanism. These results suggest that alkylation of the amides in the peptide chain is an effective way to improve the passive absorption potential for this class of compounds.

The relationship between peptide structure and transport across epithelial cell monolayers

Abstract The successful development of orally bioavailable peptides and peptide-like substances as therapeutic agents will require an understanding of how structure influences absorption across the intestinal mucosa. In an attempt to define such relationships, homologous series of peptides were prepared which varied in lipophilicity, chain length and number of polar functionalities, and permeability studies conducted across Caco-2 cell monolayers as a model of the intestinal mucosa. The results suggested that the number of polar groups in the peptide, which presumably require desolvation before transfer of the peptide into the cell membrane, was a principal determinant of transport. Consistent with this hypothesis, two experimental methods of determining desolvation potential were found to correlate well with the observed permeability results for the peptides. The insights gained from these studies were used in an attempt to rationally modify a renin inhibitory peptide, in order to improve its permeability across the intestinal mucosa. Based on the results of this work, it is argued that a peptide must possess a delicate balance of affinity for the aqueous-membrane interface and a reasonably low desolvation energy in order for it to efficiently cross an epithelial cell membrane.

(B) Mechanisms of peptide and protein absorption: (2) Transcellular mechanism of peptide and protein absorption: passive aspects

Abstract A major factor contributing to the poor bioavailability of peptides and proteins after non-parenteral administration is thought to be inefficient transport across cellular barriers. While the transport barrier for less functionalized drugs has been successfully modeled as a homogeneous octanol-like phase, peptide transport results do not fit this model. In this article we reexamine the literature on peptide transport, the architecture of biological membranes and peptide-lipid interactions. Based on these considerations, it is suggested that more realistic models, which incorporate the non-homogeneous nature of real biological membranes, provide a better basis for understanding the transport potential of highly functionalized molecules such as peptides and proteins.

In vitro permeability of peptidomimetic drugs: The role of polarized efflux pathways as additional barriers to absorption

Abstract Cellular efflux pathways function to remove both endogenous and exogenous substances from the cell. In the case of a polarized cellular barrier, such as the epithelium, these pathways serve an excretory or secretory role in transporting solutes out of tissue. Although well recognized in organs typically associated with drug excretion such as liver and kidney, similar transport pathways have been found in other tissues including the intestinal mucosa and the endothelial cells comprising the blood-brain barrier. Current evidence suggests that these systems may act as barriers to drug absorption into the tissues in which they are found. More recent studies have shown that hydrophobic peptides such as cyclosporin A are substrates for polarized efflux. In this review we examine the evidence for these mechanisms as absorption barriers and the use of in vitro transport models for characterizing this phenomenon. The presence of such pathways may help explain the poor membrane permeability of peptides which, along with metabolism, contributes to their poor in vivo performance.

Epithelial cell permeability of a series of peptidic HIV protease inhibitors: Aminoterminal substituent effects

The influence of the aminoterminal substituent in a homologous series of tetrapeptide analogs on transport across Caco-2 cell monolayers was studied. In a series of pyridylcarboxamide regioisomers, the 2-pyridyl isomer was significantly more permeable than either the 3- or 4-congeners. The uniqueness of this peptide was further suggested by examining the partitioning behavior between heptane and ethylene glycol, a system which has been developed as a simple estimate of the desolvation energy or hydrogen bonding potential of a peptide. In this model, the 2-isomer has a much larger partition coefficient than either the 3- or 4-analogs, consistent with its being less solvated than expected based on simple structural considerations. Factors possibly contributing to this decreased effective polarity could be steric interactions or intramolecular hydrogen bonding.

An NMR study of conformations of substituted dipeptides in dodecylphosphocholine micelles: Implications for drug transport

Efficient transport of intact drug (solute) across the intestinal epithelium is typically a requirement for good oral activity. In general, the membrane permeability of a solute is a complex function of its size, lipophilicity, hydrogen bond potential, charge, and conformation. In conjunction with theoretical/computational and in vitro drug transport studies, seven dipeptide (R1–D‐Xaa–D‐Phe–NHMe) homologues were each dissolved in a micellar d38‐dodecylphosphocholine solvent system. In this homologous dipeptide series, factors such as size, lipophilicity, hydrogen‐bond potential, and charge were either tightly controlled or well‐characterized by other methods in order to investigate by nmr how conformational factors relate to transport. Nuclear Overhauser effect spectroscopy experiments and amide‐NH–H2O chemical exchange rates showed that the five more lipophilic dipeptides were predominately associated with micelle, whereas the two less lipophilic analogues were not. Rotating frame nuclear Overhauser effect spectroscopy derived interproton distance restraints for each analogue, along with 3JHH‐derived dihedral restraints, were used in molecular dynamics/simulated annealing computations. Our results suggest that—other factors being equal—flexible dipeptides having a propensity to fold together nonpolar N‐ and C‐terminal moieties allow greater segregation of polar and nonpolar domains and may possess enhanced transport characteristics. Dipeptides that were less flexible or that retained a less amphiphilic conformation did not have comparably enhanced transport characteristics. We suggest that these conformational/transport correlations may hold true for small, highly functionalized solutes (drugs) in general.