Development of high-throughput screening assays for oral peptide drug delivery

Due to non-invasiveness and patient compliance, the oral route is the preferred route of drug delivery. One class of drugs with a great potential is peptides due to their high specificity, high potency, and low toxicity. However, oral delivery of peptide drugs usually result in low bioavailability. This is in part due to the low membrane permeability of peptides which makes it difficult for the peptides to cross the cell membrane of epithelial cells. The membrane impermeability to peptides is so extreme that it constitutes a challenge for peptide uptake despite the very large surface area of the upper part of the small intestine where most substances are absorbed.
The success in delivering peptide drugs orally is limited with the bioavailability of clinically tested oral peptide drugs typically being a few percent or less, Co-formulating drugs with permeation enhancers have in some cases increased their bioavailability. Still, only one linear polypeptide drug has ma been commercialized.
Most permeation enhancers are membrane active and interact with the cell membrane either in a way that increases the permeability of the membrane through fluidization or solubilization of the membrane, or in a way that. Also, some peptides are inherently membrane active either as pore-forming antimicrobial peptides or as cell-penetrating peptides. By studying membrane activity, it is possible to obtain information that can lead to increased bioavailability of oral peptide drugs.
Some permeation enhancers require direct interaction with the peptide drug to enhance permeation, and many permeation enhancers are fat-soluble and have been shown to interact with the bile acids and phospholipids present in the upper part of the small intestine. It has also been shown for at least one combination of permeation enhancer and peptide drug that the presence of bile acids and phospholipids influence the interaction between permeation enhancer and peptide drug. However, how such interactions affect the membrane activity has not previously been reported.
In this PhD thesis it is studied how interactions between permeation enhancers, peptide drugs, and bile components influence the membrane activity of the permeation enhancers, and a method is developed to study membrane activity in a high-throughput manner on individual lipid membranes. The latter part enables that information on subpopulations can be obtained.
In project one, the membrane active permeation enhancers and peptides C10, sodium cholate, dodecyl maltoside, sodium dodecyl sulfate, salcaprozate sodium, melittin, and penetratin are studied with respect to their membrane perturbation, their interactions with the peptide drugs insulin and salmon calcitonin, their interactions with the bile components taurocholate and phospholipids, and how these interactions influence the membrane perturbation. The membrane perturbations was studied using a calcein release assay and dynamic light scattering-based size measurements of liposomes, and the interactions were studied using dynamic light scattering and a hydrophobicity assay, the Nile Red assay. The study thus also underlines the possibility of performing such studies in the presence of bile components by the use of POPC: Cholesterol (9:1 molar ratio)-liposomes. Three distinct mechanisms of actions were identified for the membrane active species, with dodecyl maltoside perturbating the lipid membrane in a two-state mechanism, eventually leading to solubilization. Interactions between several permeation enhancers and peptide drugs were identified, and particularly for C10 these interactions increased the membrane perturbation though the effect seemed to be limited by the presence of bile components. Contrary, the membrane perturbation of sodium dodecyl sulfate was generally limited upon interactions with other species. In summary, this project highlights the importance of carrying out mechanistic studies of permeation enhancers in the presence of peptide drugs and bile components, and choosing the combination of peptide drug and permeation enhancer carefully.
In project two a flow cytometry based method was developed allowing the determination of the mode of action for membrane active peptides in a high-througput label free manner. It was established that POPC:POPS liposomes membrane-labeled with DOPE-Atto655 and encapsulating Alexa488 could be detected using flow cytometry. The mode of action for the membrane active peptides penetratin, Tat magainin-2, macrolittin-70, LL-37, and melittin could be determined to be non-membrane perturbating, membrane perturbating without or membrane perturbating with solubilization. Also, the aggregation of liposomes could be detected using this method. The method was applicable to study single liposomes, and thereby study the simultaneity of the effects. The method was also shown to be suitable to distinguish between modes of actions for liposomes with different charges. The method has potential to be further developed to distinguish between different subpopulations of a sample or to include membrane association of peptides in the study by fluorophore labeling of the peptide. The method developed in this project thus provides a mean to perform high-throughput mechanistic studies of membrane activity contributing to the understanding of permeation enhancement.
Together, this thesis provides insight into the mechanisms of membrane activity and a high-throughput method to study such mechanisms on a single liposome basis. The outcome of this thesis hence provide a basis for faster and more relevant understanding and screening of factors governing permeation enhancement across lipid membranes in a setting relevant for oral drug delivery of peptide.