Abstract by Daniel Alexander Støvring Laima

Skin and soft tissue infections (SSTIs) are a common cause of hospitalization and may result in severe health conditions. Unfortunately, the stagnated discovery rate of novel antibiotics and the perpetual evolution of drug resistance in bacteria have rendered conventional therapeutics ineffective for treatment of many infectious skin diseases. There is thus a clear need to develop novel antibacterial drugs and to reduce the overall antibiotics consumption. In this view, antimicrobial peptides (AMPs) can be designed to display selective and potent antibacterial properties as they may bypass resistance in bacteria evolved against conventional antibiotics. In addition to antibacterial activity, AMPs may control diverse host physiological functions related to regulating inflammatory and immune responses and promoting angiogenesis for increased wound healing. Despite this, few AMP formulations have successfully passed late stage clinical trials impeded by, e.g., low bioavailability, short serum life-time, poor stability, high cytotoxicity, and insufficient antibacterial potency. Overcoming these challenges for employing AMPs in treatment of bacterial skin infections and chronic skin disorders thus presents a scientific problem currently unmet. Here, localized AMP delivery via the topical route may be of interest due to sustained release profiles, reduced first-pass metabolism, and reduced systemic absorption. However, being relatively large and polycationic the topical delivery of AMPs may be restricted, e.g., due to tight barriers in stratum corneum and binding to anionic components intrinsically present in skin. In this regard, surfactant and oil based microemulsions (MEs) are widely used as topical excipients with well-demonstrated skin permeation enhancement for drugs with various physico-chemical properties. The underlying mechanisms of skin permeation enhancement are generally not well understood, however they are often assumed to be associated with properties related to the structures formed in excipient systems. In this view, ME morphology can be controlled compositionally to form structures ranging from swollen-micelles, to interdispersed bicontinuous systems, to reversed-micelles. Moreover, surfactants may display antibacterial properties on their own which further emphasizes their potential use in treatment of bacterial induced skin disease. Despite this, antibacterial effects of mixed AMP-excipient systems remain relatively unexplored, not only for improving bioavailability, but also for increasing antibacterial potency and selectivity. The aim of this project was to investigate the potential of MEs as topical delivery systems for AMPs in treatment of skin disease. Specifically, we investigated the potential relationship between ME microstructure and skin uptake and permeation enhancement employing a diverse range of active pharmaceutical ingredients (APIs) of relevance for skin disease. The low 15 molecular weight drugs (LMWDs) were: metronidazole (antibiotic), lidocaine (local anaesthetic), and tacrolimus (immunosuppressant). The peptides were: cyclosporine a (immunosuppressant), polymyxin b (AMP), and GRR10W4 (AMP). In doing so, we also investigated if incorporation of these APIs into MEs perturbed ME morphology, and subsequently how this affected the antibacterial properties. In part 1, we formed stable spherical oil-in-water MEs employing non-ionic glucoside surfactants (βCjG1) and emollient isopropyl esters (IPEs) as oils, forming droplets of 5 10 nm in diameter. In doing so, we identified that the aliphatic chain lengths of both compounds were key parameters for controlling ME composition and morphology at the oil saturation limit. Here, the oil dissolving capacity and ME droplet size increased with: (i) increasing length of surfactant aliphatic tail, and (ii) decreasing length of IPE acyl tail. When mixed with the APIs in topically relevant dosages, we did not observe significant perturbation to ME phase and microstructure. In part 2, we observed that the in vitro skin uptake and permeation of metronidazole and lidocaine was significantly increased after 24 h using MEs compared to 50% isopropanol control solution. For metronidazole (hydrophilic), we observed that the permeability increased with decreasing amounts of dispersed oil, while the opposite trend was true for lidocaine (hydrophobic). Ultimately, we did not detect uptake or permeation of tacrolimus or peptides after minimum 24 h of skin treatment. In part 3, we observed substantial combinatory effects on inhibition of Escherichia coli and Staphylococcus aureus when βCjG1/IPM (MEs) were employed with polymyxin B and GRR10W4 (AMPs). These enhanced antibacterial effects on potency and specificity were especially pronounced with βC10G1/IPM and GRR10W4. In summary, our findings demonstrate that βCjG1/IPE form stable o/w MEs, and that their morphology may be controlled through compositional variations in aliphatic tail lengths. These MEs increase permeability in vitro through healthy porcine skin of small APIs exhibiting a significant degree of lipophilicity (metronidazole, lidocaine). In these, the permeability is likely influenced by API solubility in ME rather than by ME morphology. However, the skin permeation enhancement of these MEs are unlikely sufficient to promote the uptake of cationic AMPs to achieve topically relevant local concentrations in healthy porcine skin. Finally, our study demonstrates enhanced antibacterial potency and specificity from combinations of βCjG1/IPM and cationic AMPs. These results emphasize their potential application in antibiotic therapy for treatment of superficial bacterial skin disease.