Abstract by Passant Mohamed Mohamed A M Omar

Oral delivery of therapeutic peptides is challenging because they are susceptible to gastrointestinal (GI) degradation and exhibit limited epithelial permeation due to their high hydrophilicity and large molecular size. Formulation strategies are therefore required to protect peptides during GI transit and enhance epithelial transport. Self-nanoemulsifying drug delivery systems (SNEDDS), oral lipid-based formulations that form nanometer-sized dispersions upon dilution, can protect peptides from luminal degradation and incorporate excipients with permeation-enhancing potential. However, the hydrophilicity of peptides often limits direct loading into SNEDDS. Hydrophobic ion pairs (HIPs) between peptides and amphiphilic counterions address this limitation via reversible electrostatic interactions, reducing apparent aqueous solubility and improving lipid compatibility. Although HIP-loaded SNEDDS are promising, systematic evaluation of how HIP type and SNEDDS composition affect in vitro and in vivo outcomes remains limited, hindering rational formulation selection for oral peptide delivery.

This thesis aimed to investigate how HIP type and SNEDDS composition, including lipid digestion behavior, influence oral peptide delivery. It also evaluated whether computational approaches, including molecular dynamics (MD) simulations and design-of-experiments (DoE) modeling, can assist interpretation of experimental outcomes and inform rational formulation selection. Salmon calcitonin (sCT) and octreotide (OCT) were used as cationic model peptides with low oral bioavailability, representing differences in molecular structure and intrinsic stability. For systematic evaluation in lipid-based carriers, peptides were prepared as HIPs via complexation with surfactant counterions and freeze-dried prior to incorporation into SNEDDS.

The first part of the thesis focused on HIP design variables, including surfactant type and molar ratio, and their influence on complexation efficiency and in vitro performance. For sCT, across all tested counterions (sodium decanoate, C10; sodium docusate, DOC; sodium oleate, OLA; and sodium deoxycholate, DCH), maximal complexation was observed at an sCT:surfactant 1:4 molar ratio, consistent with near-stoichiometric association with the net positive charge of sCT (approximately + 4); however, proteolytic stability depended strongly on counterion type. HIPs formed with counterions containing either branched hydrocarbon chains (DOC) or long unsaturated linear chains (OLA) exhibited significantly higher resistance to proteolytic degradation than HIPs formed with short linear saturated fatty acids such as C10, whereas DCH provided intermediate protection. HIPs are therefore not interchangeable based solely on complexation stoichiometry, as proteolytic stability may depend on specific peptide–surfactant interactions beyond charge neutralization. MD simulations complemented the experimental work by indicating differences between sCT:OLA and sCT:DCH in peptide–surfactant contact persistence and electrostatic coordination to basic sCT residues, providing a molecular-level rationale for their different proteolytic stability. For OCT, OCT:DOC 1:2 molar ratio provided > 98 % complexation efficiency and increased apparent lipophilicity (Log P > 2), consistent with near-stoichiometric association with OCT’s net (+ 2) charge, enabling efficient SNEDDS incorporation.

The second part evaluated how SNEDDS composition and HIP type translate into peptide-relevant in vitro readouts. For sCT, sCT:C10 and sCT:DOC HIPs were incorporated into two SNEDDS that differed only by a 10 % excipient substitution (F1: lysophosphatidylcholine; F2: propylene glycol). Despite comparable dispersion characteristics, F2 provided higher proteolytic stability and higher apparent transport across Caco-2 monolayers, assessed using fluorescein isothiocyanate–dextran (4 kDa) as a paracellular marker, than F1 for the same HIP. In both formulations, sCT:DOC showed higher proteolytic stability than sCT:C10. In F2, additional HIPs (sCT:OLA and sCT:DCH) were further tested for proteolytic stability, and sCT:OLA showed higher proteolytic stability than sCT:DCH, confirming that counterion choice still affected protection. For OCT, OCT:DOC HIP was used to evaluate how SNEDDS composition and lipid digestion influence OCT delivery using a DoE approach. Two design spaces with distinct digestibility were established using DoE approach: a low-digestibility (LD) space enriched in oleoyl polyoxyl-6 glycerides and propylene glycol monocaprylate, and a high-digestibility (HD) space enriched in medium-chain triglycerides and medium-chain mono-diglycerides (C8/C10). DoE modeling was used to quantify composition–response relationships. One formulation from each space (LD₆ and HD₁₀) was selected with matched droplet size and OCT:DOC solubility while showing a clear contrast in digestibility. This digestion contrast was confirmed by dynamic lipolysis and digestion-associated structural evolution observed by in situ small-angle X-ray scattering, confirming that the selected formulations would behave differently in vivo for evaluating OCT absorption.

The third part assessed in vivo translation of the in vitro screening outcomes in rats by evaluating pharmacodynamic (PD) responses for sCT and pharmacokinetic profiles for OCT after oral dosing. For sCT, HIPs without a lipid carrier produced only transient PD effects, whereas incorporation into SNEDDS was required to obtain a significant hypocalcemic response. Within the SNEDDS systems, F2 produced stronger PD responses than F1. Among all sCT HIPs formulated in F2, sCT:DOC showed the most sustained PD response and the lowest plasma calcium level, consistent with its higher proteolytic stability observed in vitro. For OCT, oral administration of OCT:DOC-loaded HD₁₀ produced approximately three-fold higher systemic OCT exposure than LD₆, and co-administration of Orlistat (1 % w/w; a pancreatic lipase inhibitor) minimized this difference, indicating that lipid digestion contributed to the higher OCT absorption observed with HD₁₀.

In conclusion, this thesis demonstrates that HIP type, through counterion selection, and SNEDDS composition, including lipid digestion behavior, influence oral peptide delivery outcomes. MD simulations provided complementary molecular-level insight into peptide–surfactant association within HIPs, while DoE modeling supported rational formulation selection for oral peptide delivery.