Development of a lipid-based drug delivery system formulated with a permeation enhancer to increase oral bioavailability of peptides

<p dir="ltr">Therapeutic peptides play a crucial role in a range of treatment areas such as oncology, urology, metabolic and cardiovascular diseases. For these therapies, oral delivery would be highly advantageous, particularly for chronic diseases that require long-term administration. A key challenge associated with oral delivery of peptide drug actives is poor oral bioavailability (BA) due to pre-systemic degradation and low intestinal permeation attributed to their high molecular weight and hydrophilic properties. Two core approaches employed to date to enable oral peptide delivery are i) peptide modification and ii) formulation optimisation. These approaches have had some commercial success, but as oral BA remains low (<2%), there is demand for novel drug delivery systems (DDS) that lead to improvement in oral peptide absorption.</p><p dir="ltr">The overarching goal of this research was to develop a novel prototype drug delivery system capable of increasing oral peptide BA. The underpinning rationale was that hydrophilic peptides subjected to hydrophobic ion pairing (HIP) might have increased lipophilicity and ultimately exhibit higher passive gastrointestinal (GI) transcellular flux. This reversible structural alteration causes a major decrease in aqueous solubility, but also enables solubilisation of the peptide within a self‐emulsifying drug delivery system (SEDDS), offering protection from peptidases and ensuring efficient dispersion within GI luminal fluids. This approach, when co-presented with an intestinal permeation enhancer (PE) was hypothesised to enhance passive GI epithelial permeation.</p><p dir="ltr">In this thesis, an <i>in vitro</i> screen of novel and established PEs in Caco-2 monolayers identified trehalose 6-monoesters (TEs) with alkyl chain lengths C₈ – C₁₆, as lead candidates for inclusion in the DDS. Furthermore, cytotoxicity analysis identified a modest separation between enhancement action and loss in cell viability for these PE candidates. Trehalose laurate (TE12) was found to be the most effective PE in this screen, demonstrating a 13-fold greater P_app of FD4 than sodium caprate. This result was confirmed on rat colonic mucosae with an 8-fold (at 10 mM) increase in P_app of FD4 coupled with moderate reductions in % TEER and tissue functionality preserved, thus justifying its selection for inclusion in the DDS under development.</p><p dir="ltr">Preliminary HIP development was performed with the model peptide vancomycin and a large panel of potential amphiphilic counterions. This research led to selection of the counterions; sodium docusate, and the novel HIP agent sodium 1-dodecane sulfonate. Following optimisation of the HIP reactions and characterisation of the resultant HIP complexes (HIPs), docusate was selected for HIP with the lead model peptide; leuprolide, resulting in 98.9% precipitation of the peptide and a 3.2-fold increase in LogP_{Octanol/water.} Leuprolide docusate (10 mg/mL) was loaded to a developed SEDDS consisting of 60% Capryol^®90, 30% Kolliphor^®EL and 10% Transcutol^® HP (% <i>v/v</i>). Characterisation of the dispersed SEDDS revealed formation of a nanoemulsion with dispersed droplets of approximate size and PDI; 64.34 nm and 0.2 respectively. These properties, combined with modest protection of leuprolide by HIP and SEDDS against the action of trypsin, indicate favourable characteristics for GI epithelial absorption.</p><p dir="ltr">The complete DDS consisting of leuprolide docusate (10 mg/mL) loaded SEDDS, co-delivered with TE12, caused a 4-fold increase in the P_app of leuprolide across Caco-2 monolayers and a 7-fold increase across rat colonic mucosae <i>ex vivo</i>. A mechanistic assessment of the DDS constituents found that the SEDDS caused epithelial perturbation in Caco-2 monolayers, so it was not possible to confirm if there was passive permeation of leuprolide docusate independent from non-specific perturbation. There was a similar trend in isolated rat colonic mucosae, although TE12 as a standalone PE performed best in this study. The combination of HIPs loaded SEDDS and TE12 attenuated the PE effects of TE12 on isolated rat colonic mucosae, which was attributed to an interaction between TE12 and the SEDDS constituents. Furthermore, no loss of colonic tissue viability was observed upon exposure to the DDS or its components, providing preliminary toxicology data for the DDS.</p><p dir="ltr">Collectively, this study shows the feasibility of HIP as a strategy to hydrophobise ionisable peptides. Solubilisation of the HIPs in SEDDS was a critical step, as low solubility of ion pairs limited their effectiveness. Inclusion of a PE with the HIPs loaded SEDDS improved permeation of leuprolide. <i>In vivo</i> studies are required to determine if this approach can be more effective than traditional PE approaches, which have sub-optimal effectiveness in dynamic<i> in vivo</i> conditions.</p>