Abstract by Grzegorz Sebastian Czyrski

Delivery of drug molecules through the skin remains a challenge due to the structural and physicochemical properties of the skin limiting the number of drugs that can permeate the skin at rates sufficient to achieve therapeutic doses. However, drug permeation rates can be increased by altering the barrier function of skin using permeation enhancers. Two main categories of enhancers can be distinguished – chemical permeation enhancers (CPEs) and physical permeation enhancers (PPEs). CPEs facilitate drug permeation by impairing the skin barrier through interactions with stratum corneum (SC) lipid or protein components, or by increasing drug partitioning into the skin. PPEs, on the other hand, enhance drug permeation by physically altering the skin, either through temporary changes in permeability or by removing the SC. However, despite their effectiveness, the permeation-enhancing effects of CPEs and PPEs are often insufficient to ensure the delivery of drugs at therapeutic doses. Thus, new strategies to enhance drug permeation are still in high demand.

Recently, terpenes, a popular group of CPEs, have been shown to form eutectic mixtures (EMs), which are mixtures of two or more components exhibiting lower melting temperatures than each component individually. However, little is known about the role of the molar composition of EMs in defining their permeation-enhancing properties. Additionally, high concentrations of terpenes applied to the skin might cause adverse reactions. Therefore, adding co-solvents like propylene glycol or ethanol to EMs might optimize the permeation-enhancing effect of the formulations while minimizing their potential side effects. However, few studies have investigated the impact of co-solvents on the permeation-enhancing properties of terpene-based EMs. Furthermore, little is known about the potential of terpene-based EMs to enhance permeation of drugs compared to PPEs, such as solid microneedles (MNs). Findings of previous studies suggest that the combined use of CPEs and PPEs might result in greater permeation enhancement than each strategy used individually, yet this has not been tested for the combination of terpene-based EMs and solid MNs, especially in the context of drug lipophilicity.

Based on identified research gaps, the main aims of this thesis were to: 1) investigate the influence of the molar ratio of binary terpene-based EMs on the permeation and retention of the model drug clotrimazole (CLOT); 2) Investigate the influence of adding co-solvents to binary terpene-based EMs on the permeation and retention of CLOT; 3) investigate the influence of adding co-solvents to binary terpene-based EMs on the structure and molecular mobility of SC lipids and proteins; 4) compare the permeation-enhancing effects of terpene-based EMs and MNs, and assess the rationale for their combined use for the highly hydrophilic model drug metronidazole (MET), the moderately lipophilic model drug lidocaine (LID), and the highly lipophilic model drug CLOT.

To address these aims, the effects of the assessed permeation-enhancing strategies on skin permeation and retention of the drugs were tested in an in vitro setup using Franz diffusion cells and porcine ear skin as a model of human skin. The amounts of drugs permeated and retained in the skin were quantified using high-performance liquid chromatography coupled with a UV detector. Moreover, the impact of the permeation-enhancing strategies on skin integrity was assessed by comparing transepidermal water loss of skin samples before and after exposure to the investigated permeation-enhancing strategies. Additionally, the effects of EMs and their blends with co-solvents on the structure and molecular mobility of SC lipid and protein components were assessed using X-ray scattering, Fourier transform infrared spectroscopy, and solid-state nuclear magnetic resonance spectroscopy techniques. Terpene-based EMs had a minor effect on the permeation of CLOT, regardless of their molar ratio. In contrast, the extent of CLOT retained in the skin depended on the molar ratio between terpenes forming the mixtures. However, this was likely due to the different viscosities of the EMs, the content of individual terpenes in the mixtures, and their impact on the skin barrier function, rather than the specific ratio between them. Therefore, the molar ratios between the CPEs forming the EMs can be optimized to achieve improved skin retention of drugs. Adding co-solvents to terpene-based EMs resulted in enhanced permeation and retention of CLOT compared to using EMs and co-solvents alone. Higher content of co-solvents in the blends corresponded to greater permeation of CLOT and, to some extent, greater retention in the skin. All co-solvent:EM blends impaired skin integrity, with blends containing more EMs causing greater disruption of SC lipid packing and stronger increase in molecular mobility of SC lipid and protein components. Additionally, formulations containing co-solvents caused changes in the secondary structure of SC proteins. Thus, the permeation-enhancing effect of co-solvent:EM blends likely resulted from the combined effect of EMs impairing the SC barrier function and co-solvents facilitating CLOT permeation through more hydrophilic skin layers. In conclusion, the effects of EMs on skin permeation and retention of drugs can be optimized by the addition of co-solvents.
The permeation-enhancing effect of EMs and solid MNs depended on the lipophilicity of the model drug tested. The permeation of the highly hydrophilic drug MET was enhanced by EMs and, to a lesser extent, by MNs, whereas only MNs enhanced the permeation of the highly lipophilic drug CLOT. Neither strategy significantly enhanced the permeation of the moderately lipophilic drug LID. Furthermore, the combined application of EMs and solid MNs did not enhance permeation of the drugs to greater extent than each of the strategies used individually. Therefore, for the given setup, the combination of CPEs and PPEs did not enhance skin permeation and retention of drugs more than each strategy used individually.

In summary, the work presented in the thesis demonstrates that while terpene-based EMs and their combinations with co-solvents can enhance drug permeation and retention, their effectiveness varies depending on the drug's lipophilicity and the specific formulation used. The findings highlight the potential for optimizing EM compositions and incorporating co-solvents to improve dermal and transdermal delivery. Additionally, while solid MNs showed promise in enhancing the permeation of certain drugs, combined use with EMs did not result in further enhancement compared to each strategy used individually. This suggests that the choice of permeation-enhancing strategy should be tailored to the specific drug and its properties. However, further research is needed to fully understand the mechanisms responsible for the permeation-enhancing effect of terpene-based EMs and to develop more effective strategies for different types of drugs.